Detection of High-Level Rifaximin Resistance in Enteric Bacteria by Agar Screen

This study aimed at determining the prevalence of rifaximin resistance in a large collection of Enterobacterales resistant to third-generation cephalosporins. A simple agar screen was developed to detect high-level resistance. A total of 401 isolates nonsusceptible to third-generation cephalosporins (including 342 Escherichia coli and 39 Klebsiella spp. and 20 Enterobacter spp.) were tested by microdilution for their MICs of rifaximin and rifampicin. Isolates with a confirmed rifaximin minimal inhibitory concentration (MIC) of >64 mg/L and a number of high-level resistant, and susceptible control isolates were tested for growth on Mueller-Hinton agar supplemented with rifaximin or rifampicin at a concentration of 256 mg/L. Amino acid mutations in rpoB and the presence of rifaximin resistance-associated genes arabidopsis response regulator (arr) 2/3 were investigated. Microdilution assays identified rifaximin resistance in nine E. coli and three Klebsiella spp. isolates with complete cross-resistance to rifampicin (MICs of both >64 mg/L). The rifaximin agar screen correctly identified 9/9 clinical E. coli isolates, 2/2 E. coli controls, and 3/3 Klebsiella spp. with high-level rifaximin resistance, and was negative in 45 control clinical isolates with rifaximin MICs ranging between 2 and 32 mg/L according to broth microdilution. All nine high-level rifaximin agar screen-positive E. coli clinical isolates (vs. none of the tested controls) had rpoB mutations or carried arr2/3. Our agar screen test has the potential to detect high-level rifaximin-resistant Enterobacterales. Such strains remain rare among extended spectrum beta-lactamase (ESBL)-positive enteric bacteria, but may emerge among patients receiving rifaximin for prevention of hepatic encephalopathy and spontaneous bacterial peritonitis or among patients receiving rifaximin for other indications.