Rifaximin resistance in Clostridioides difficile is associated with specific rpoB alleles and multilocus sequence typing (MLST) clades

Abstract

Background and objectives: Rifaximin (RFX) has recently been suggested as an alternative treatment option for Clostridioides difficile infection. This study reports the survey on RFX susceptibility within a C. difficile test cohort that represents the five clinically relevant phylogenetic clades.

Methods: Agar dilution assays were conducted to determine the minimum inhibitory concentrations (MICs) of RFX for 129 clinical C. difficile isolates from Germany (86), Indonesia (29), and Ghana (14). Genome sequence data were obtained for 50 representative isolates, including all those with a minimum inhibitory concentration MIC[RFX] of ≥ 32.0 µg/mL, to identify the underlying rpoB gene resistance alleles, determine the multilocus sequence typing (MLST) sequence types (STs), and infer phylogenetic relatedness.

Results: 10.1% of the isolates were found to be resistant to RFX. The resistance rate varies by region, with 4.7% in Germany, 27.6% in Indonesia, and 7.1% in Ghana. Three distinct rpoB alleles were associated with RFX resistance. The presence of a specific rpoB allele correlates with the MLST-based ST of the isolate, indicating that the rifaximin-resistant isolates belong to phylogenetic clades 1, 2, and 4. These isolates are represented by six different ribotypes: 010, 017, 027, 046, 084, and 131. Furthermore, we identified seven amino acid substitutions resulting from SNPs in the rpoB gene through alignment analysis. These substitutions are found in both RFX-resistant and susceptible isolates, suggesting that they are neutral mutations in relation to RFX susceptibility. These observations also indicate that RFX resistance arose independently in different clades.

Conclusions: A substantial rate of RFX resistance, particularly among Indonesian isolates, was observed. This may be attributed to the prolonged use of rifampicin, especially in the treatment of tuberculosis. RFX resistance has been linked to specific amino acid substitutions in the β-subunit of RNA polymerase encoded by the rpoB gene. To the best of our knowledge, one of the identified RFX resistance-associated rpoB alleles (H502N, R505K, I750M) has not been previously described, whereupon, the amino acid substitutions I750M as well as I750V, E1037Q, A1205V, N1207A, A1208T, and D1232E were identified as neutral mutations that do not confer resistance to RFX.

Keywords: Clostridioides difficile; rpoB; Antibiotic resistance; Germany; Ghana; Indonesia; Nosocomial diarrhea; Rifaximin.

Fig. 1

Frequency distribution of MIC [RFX]. The epidemiological cut off was determined at 0.5 mg/L, while RFX resistance was determined at an MIC ≥ 32 mg/L

Fig. 2

Midpoint-rooted phylogenetic tree of all detected RFX-resistant C. difficile isolates, based on GBDP distances calculated from genomic sequences. The branch lengths are scaled according to the GBDP distance formula d₅. Numbers above the branches represent GBDP pseudo-bootstrap support values greater than 60% from 100 replications, with an average branch support of 75.3%. C. difficile 630 ∆erm (DSM 28645, CP016318.1) was inculded as reference in this analysis