Molecular Cut-off Values for Aliarcobacter butzleri Susceptibility Testing

Abstract

Aliarcobacter butzleri is an emerging gastrointestinal pathogen found in many countries worldwide. In France, it has become the third most commonly isolated bacterial species from the stools of patients with intestinal infections. No interpretative criteria for antimicrobial susceptibility testing have been proposed for A. butzleri, and most strains are categorized using the recommendations of the Clinical and Laboratory Standards Institute or the European Committee on Antimicrobial Susceptibility Testing for Campylobacter or Enterobacterales. In the present study, the genomes of 30 resistant A. butzleri isolates were analyzed to propose specific epidemiological cut-off values for ampicillin, ciprofloxacin, erythromycin, and tetracycline. The identification of a β-lactamase and the T85I GyrA mutation associated with ampicillin and ciprofloxacin resistance, respectively, allowed us to adjust the disk diffusion (DD) and MIC cut-off values for these molecules. However, epidemiological cut-off values for erythromycin and tetracycline could not be estimated due to the absence of known resistance mechanisms. The present study paves the way for building a consensus for antimicrobial susceptibility testing for this concerning pathogen. IMPORTANCE Aliarcobacter butzleri is an emerging and concerning intestinal pathogen. Very few studies have focused on this particular species, and antimicrobial susceptibility testing (AST) is based on methods that have been mostly developed for Campylobacter spp. In fact, no disk diffusion and E-tests adapted cut-offs for A. butzleri are available which leads to misinterpretations. We have shown here that NGS approach to identify genes and mutations in close relation to phenotypic resistance levels is a robust way to solve that issue and precisely differentiate WT and NWT A. butzleri isolates for frequently used antimicrobials. MIC and DD cut-off values have been significantly adjusted and answer the need for a global consensus regarding AST for A. butzleri.

Keywords: Aliarcobacter; NGS; antimicrobials; susceptibility.

FIG 1

Distributions of percentages of WT and NWT isolates using disk diffusion and Etest methods with recommended EUCAST and adjusted cut-offs. In this study, resistance rates using disk diffusion (A) and Etest (B) were estimated for the following four antimicrobials: ampicillin (AMP), ciprofloxacin (CIP), erythromycin (ERY), and tetracycline (TET). The colors used were as follows: green for WT isolates and red for NWT isolates. EUCAST C. jejuni and C. coli: data interpreted according to the EUCAST ECOFF values proposed for these two species; CNRCH: data interpreted according to the cut-off values (CO_WT) proposed in the present study.

FIG 2

Distributions of ampicillin and ciprofloxacin diameters and MICs according to the resistance mechanism identified for each molecule. Resistance markers are displayed in red or blue, and nonsignificant markers or WT isolates are displayed in yellow or green. Specifically, ampicillin resistance (A and C) is associated with the presence of a bla_{OXA-15/464-like} gene (OM617734), and ciprofloxacin resistance (B and D) is associated with the presence of a single mutation (T85I) in the GyrA protein sequence (OM617736). Moreover, red triangles represent EUCAST epidemiological cut-off values for C. jejuni and C. coli and blue triangles represent CO_wt values proposed in the present study.

FIG 3

Distributions of erythromycin and tetracycline diameters and MICs. MICs in mg/L (A and C) or DD in mm (B and D) values are displayed here. Pink triangles represent EUCAST epidemiological cut-off values for C. jejuni, orange triangles for C. coli and red triangles for both.