Cloning and nucleotide sequence of the Campylobacter jejuni gyrA gene and characterization of quinolone resistance mutations

Abstract

The gyrA gene of Campylobacterjejuni UA580, which encodes the A subunit of DNA gyrase, was cloned and its nucleotide sequence was determined. An open reading frame of 2,589 nucleotides was identified, which could code for a polypeptide of 863 amino acids with a Mr of 97 kDa. Both the nucleotide sequence and the putative amino acid sequence show ca. 50% identity with those of other gyrA genes from gram-positive and gram-negative bacteria. Similar mutations were also identified in ciprofloxacin-resistant isolates of S. aureus (10,27).Gootz and Martin (9) demonstrated that the DNA gyrases from Nalr mutants of C. jejuni UA535 were 100-fold less susceptible than the wild-type enzyme to inhibition by quinolones in the DNA supercoiling reaction. Subunit switching experiments with purified A and B subunits from the wild type and one of the quinolone-resistant mutants indicated that an alteration in the A subunit was responsible for resistance. Here, we report the cloning and nucleotide sequence of the C. jejuni gyrA gene and the location of the gene on both C. jejuni and C. coli chromosomes. Several mutations responsible for quinolone resistance were detected in the gyrA sequence. MATERUILS AND METHODSStrains and culture conditions. The Campylobacter spp. employed in this study were C. jejuni UA67 (Nalr mutant [35]); UA536, UA543, and UA549 (Nalr clinical isolates from H. Lior); UA580 (35); UA58OR1 and UA580R3 (Nalr mutant from UA580 [this study]); and C. coli UA417 (Nalr clinical isolates [35]). E. coli DH5at (23) was also used. The plasmids and phages employed were pUC19, M13mpl8, M13mpl9 (38), pBluescript II SK (Stratagene), pK194 (16), and pT7-5 (30).Campylobacters were grown at 37°C on Mueller-Hinton agar medium containing 7% CO2. E. coli was grown in 2x YT medium or on Luria-Bertani agar (23) at 37°C. When necessary, the medium was supplemented with ampicillin (100 ,ug/ml), kanamycin (15 jig/ml), or nalidixic acid (24 ig/ml).DNA isolation, transformation, and nucleotide sequence analysis. Plasmid DNA was isolated by a modification of the alkaline lysis method of Birnboim and Doly (2) and purified by the "magic miniprep" (Promega) when used for restriction analysis and sequencing. M13 phage DNA was prepared by the method described by Sambrook et al. (23).

Molecular mechanisms of quinolone, macrolide, and tetracycline resistance amongCampylobacterisolates from initial stages of broiler production

“…This mutation is the most prevalent in clinical and veterinary isolates, among other described mutations (Thr86Ala, Thr86Lys, Ala87Pro, Asp90His, Asp90Tyr, etc.) (Wang et al, 1993;Bachoual et al, 2001;Piddock et al, 2003) mostly of in vitro origin (Bachoual et al, 2001). One isolate possessed another mutation in QRDR, Pro104Ser, previously described in isolates of animal and human origin (Piddock et al, 2003;Jesse et al, 2006).…”

“…Among the described mutations, Thr86Ile is the most prevalent (Bachoual et al, 2001), conferring high minimum inhibitory concentration (MIC) values. In addition to this mutation, several other point mutations at gyrA have been described at the same codon-Thr86Val (Piddock et al, 2003), Thr86Lys and Thr86Ala (Wang et al, 1993)-as well as in other codons: Ala87Pro, Asp90His, and Asp90Tyr. These other mutations confer resistance against quinolones with different MICs values.…”

Campylobacter

“…In a study of quinolone-resistant Campylobacter a majority of isolates analysed were shown to possess a common mutation [128]. The predominant genetic alteration responsible for conferring resistance to ciprofloxacin in C. jejuni and C. coli is the result of a mutation in the gyrA gene, whereby many isolates tested demonstrated a Thr-86-Ile substitution in the A-subunit of DNA gyrase [185]. A Mismatch Amplification Mutation Assay (MAMA)-PCR has been successfully applied to the detection of ciprofloxacin resistance in C. jejuni and C. coli, and this protocol is a convenient screening tool among these isolates [196].…”

“…This observation may in turn, lead to Campylobacter-associated deaths among vulnerable members in the community. Resistance to fluoroquinolones arises following a point mutation [ACAATA], which produces a Thr-86-Ile amino acid substitution in the Quinolone Resistance Determining Region (QRDR) of the gyrA subunit-encoding gene [185]. Genetic alterations in this region are often associated with high-level resistance to nalidixic acid (MIC > 64-128 µg/mL) and ciprofloxacin (MIC > 16-64 µg/mL).…”

Detecção de resistência às fluoroquinolonas em Campylobacter isolados de frangos de criação orgânica

“…às fluoroquinolonas (Wang et al 1993, Wilson et al 2000, Bachoual eu al. 2001, Hakanen et al 2002, Qin et al 2011, Iovine 2013, Wieczorek & Osek 2013, Hungaro et al 2015.…”