title: Isolation of a point mutation associated with altered expression of the CmeABC efflux pump in a multidrug-resistant Campylobacter jejuni population of poultry origin.
abstract: The objective of this study was to investigate the antibiotic resistance phenotype of Campylobacter jejuni isolates from a poultry flock of broiler production in Spain. Isolates were characterised by RFLP-PCR of the flaA gene and multilocus sequence typing. Minimum inhibitory concentrations of quinolones, aminoglycosides, β-lactams, tetracyclines, phenicols, macrolides and lincosamides were determined by Etest. Determinants of resistance and the regulatory region of the cmeABC operon were investigated in all isolates by PCR detection and sequencing. Expression of the CmeABC efflux pump was investigated by quantitative RT-PCR and accumulation assay. Based on their molecular markers, two different populations of C. jejuni were identified: one resistant to quinolones, β-lactams and tetracyclines, considered multidrug-resistant (MDR); and another resistant only to tetracyclines. Both populations possessed the tetO gene, previously associated with tetracycline resistance. The blaOXA-61gene was also present in both populations, although only the MDR population showed β-lactamase activity. In addition, MDR isolates possessed the Thr86Ile mutation in the gyrA gene responsible for quinolone resistance. Moreover, sequencing of the regulatory region of the cmeABC operon revealed the presence of the C-32→T mutation in the MDR isolates, which was accompanied by an increase in cmeA mRNA levels compared with the non-mutant population. In conclusion, this is the first report of the mutation C-32→T in the cmeABC operon in C. jejuni isolates of veterinary origin. This mutation is associated with overexpression of the CmeABC efflux pump in a MDR population and is possibly related to enhanced tolerance to antimicrobials that favours the development of resistance.
authors.affiliation: Unidad de Enterobacterias, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. Electronic address: dperez@externos.msssi.es.
authors.forename: David
authors.initials: D
authors.lastname: Pérez-Boto
authors.affiliation: Unidad de Patología Molecular del Neumococo, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain.
authors.forename: Paloma
authors.initials: P
authors.lastname: Acebo
authors.affiliation: Laboratorio Central de Veterinaria, Dpto. Bacteriología, Algete, Madrid, Spain.
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: García-Peña
authors.affiliation: Cobb-Vantress Spain SA, Alcalá de Henares, Madrid, Spain.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Abad
authors.affiliation: Unidad de Enterobacterias, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: María Aurora
authors.initials: MA
authors.lastname: Echeita
authors.affiliation: Unidad de Patología Molecular del Neumococo, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain; CIBER Enfermedades Respiratorias, Spain.
authors.forename: Mónica
authors.initials: M
authors.lastname: Amblar
keywords: Antimicrobial resistance; Campylobacter jejuni; CmeABC efflux pump; bla(OXA-61); gyrA; tetO