Azithromycin resistance levels and mechanisms in Escherichia coli

Abstract

Despite azithromycin being used in some countries to treat infections caused by Gram-negative pathogens, no resistance breakpoint for Escherichia coli exists. The aim of this study was to analyse the levels and mechanisms of azithromycin resistance in E. coli. The presence of chromosomal (rplD, rplV and 23S rRNA) mutations, 10 macrolide resistance genes (MRGs) and efflux pump overexpression was determined in 343 E. coli isolates. Overall, 89 (25.9%) isolates had MICs ≥ 32 mg/L to azithromycin, decreasing to 42 (12.2%) when assayed in the presence of Phe-Arg-β-Napthylamide, with 35 of these 42 possessing at least one MRG. Efflux pumps played a role in azithromycin resistance affecting the Minimal Inhibitory Concentration (MIC) levels of 91.2% isolates whereas chromosomal alterations seem to have a minimal role. At least one MRG was found in 22.7% of the isolates with mph(A) being the most commonly found gene. The mph(A) gene plays the main role in the development of azithromycin resistance and 93% of the mph(A)-carrying isolates showed a MIC of 32 mg/L. In the absence of a specific resistance breakpoint our results suggest a MIC of 32 mg/L to be considered in order to detect isolates carrying mechanisms able to confer azithromycin resistance.

Figure 1

Analysis of Minimal Inhibitory Concentration (MIC) of 16 and 32 mg/L to detect the presence of specific macrolide-resistance mechanisms. R: Resistance; R_PAβN: resistance in presence of PAβN. (a) Overall. (b) Isolates in which no sought mechanisms of resistance was found. (c) Isolates carrying the mph(A) gene alone or with a target mutation. (d) Isolates carrying the mph(A) gene together other MRG. (e) Isolates carrying a MRG different that mph(A). (f) Isolates carrying only L4 and/or L22 amino acid changes. ^*The single isolate (isolate 3491) which remains resistant after PAβN addition possesses an unidentified MRG.

Figure 2

Minimal Inhibitory Concentration (MIC) distribution. MRG: Macrolide resistance gene (other than mph(A)); wt: wild type. Any MIC category with ≥5% of the isolates is highlighted in dark grey. If a strain had a L4 and/or L22 mutation(s) and a MRG, then the isolates are included in either the mph(A) or MRG category. ¹One isolate (isolate 3491) in which an unidentified MRG was detected by conjugation.

Figure 3

Minimal Inhibitory Concentration (MIC) cumulative curves (a) MIC cumulative curve in standard clinical conditions (MIC evaluation in absence of PAβN). (b) MIC cumulative curve in presence of 20 mg/L of PAβN. Horizontal lines marks the 50 and 90% of isolates inhibition.