Validation of the T86I mutation in the gyrA gene as a highly reliable real time PCR target to detect Fluoroquinolone-resistant Campylobacter jejuni

Nereyda Espinoza,Ryan Maves,Máximo Camiña,Drake H Tilley,Jesús Rojas,Simon Pollett,Rina Meza,Nathanael D Reynolds,Manuela Bernal,Lilian Patiño,Matthew Kasper,Manuel Leiva,Mark P Simons

doi:10.1186/s12879-020-05202-4

Abstract

BackgroundCampylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Detection of point mutations at T86I in the gyrA gene by real-time polymerase chain reaction (RT-PCR) is a rapid detection tool that may improve FQ resistance tracking.MethodsC. jejuni isolates obtained from children with diarrhea in Peru were tested by RT-PCR to detect point mutations at T86I in gyrA. Further confirmation was performed by sequencing of the gyrA gene.ResultsWe detected point mutations at T86I in the gyrA gene in 100% (141/141) of C. jejuni clinical isolates that were previously confirmed as ciprofloxacin-resistant by E-test. No mutations were detected at T86I in gyrA in any ciprofloxacin-sensitive isolates.ConclusionsDetection of T86I mutations in C. jejuni is a rapid, sensitive, and specific method to identify fluoroquinolone resistance in Peru. This detection approach could be broadly employed in epidemiologic surveillance, therefore reducing time and cost in regions with limited resources.

Highlights

Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern
Resistance to ciprofloxacin is mediated by mutations in the gyrA gene, with the single-step T86I amino acid change as one of the most common mutations associated with decreased susceptibility in Campylobacter [1, 7,8,9,10]
The molecular detection of the T86I gyrA mutation was observed in 100% (141/141) of ciprofloxacinresistant phenotypes and none of the ciprofloxacinsensitive phenotypes (Table 3)

Summary

Introduction

Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Campylobacter infection is the leading zoonotic cause of foodborne illness in the world, with 400 million acute cases of diarrheal disease reported worldwide annually [1]. Increasing rates of resistance to these drugs have been reported worldwide [11], with significant treatment implications for the management of Campylobacter infections in developing settings with limited access to resources and alternative therapeutic choices [12,13,14]. Alterations of nucleotide 257 (codon 86) from ACA to ATA of the gyrA gene result in a threonine to isoleucine substitution in the GyrA protein and confer high- level resistance to ciprofloxacin [15,16,17,18,19,20]

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