Haem is crucial for medium-dependent metronidazole resistance in clinical isolates of Clostridioides difficile

Abstract

Background: Until recently, metronidazole was the first-line treatment for Clostridioides difficile infection and it is still commonly used. Though resistance has been reported due to the plasmid pCD-METRO, this does not explain all cases.

Objectives: To identify factors that contribute to plasmid-independent metronidazole resistance of C. difficile.

Methods: Here, we investigate resistance to metronidazole in a collection of clinical isolates of C. difficile using a combination of antimicrobial susceptibility testing on different solid agar media and WGS of selected isolates.

Results: We find that nearly all isolates demonstrate a haem-dependent increase in the MIC of metronidazole, which in some cases leads to isolates qualifying as resistant (MIC >2 mg/L). Moreover, we find an SNP in the haem-responsive gene hsmA, which defines a metronidazole-resistant lineage of PCR ribotype 010/MLST ST15 isolates that also includes pCD-METRO-containing strains.

Conclusions: Our data demonstrate that haem is crucial for medium-dependent metronidazole resistance in C. difficile.

Figure 1.

Medium-dependent resistance to metronidazole is independent of base medium. Strain GSK241 was grown and resuspended to a turbidity equivalent to that of a 2.0 McFarland standard and spread on BHI agar, Brucella Blood Agar (BBA) (containing 5% defibrinated sheep blood, 1 mg/L vitamin K and 5 mg/L haemin) and BHI blood agar (BHI agar supplemented with 5% defibrinated sheep blood, 1 mg/L vitamin K and 5 mg/L haemin). Etest strips were applied and plates were incubated for 48 h prior to imaging. Red lines indicate the epidemiological cut-off value for metronidazole as determined by EUCAST and used to indicate resistance in this study. MIC values at the top of the image correspond to the reported MIC values on BHI agar, BBA and BHI blood agar.

Figure 2.

Haem supplementation increases the MICs of metronidazole, but not of vancomycin. (a) Strains GSK64 and GSK241 were resuspended in PBS to a turbidity equivalent to that of a 2.0 McFarland standard and plated on BHI agar and on BHI agar supplemented with 5 mg/L haemin. Etest strips for metronidazole and vancomycin were applied and plates were incubated for 48 h prior to imaging. Red lines indicate the epidemiological cut-off value for metronidazole as determined by EUCAST and used to indicate resistance in this study. For a complete overview of the MICs of metronidazole for these strains on BHI agar and BHI agar supplemented with haemin, please see Table 2. (b) Typical metronidazole (top) and vancomycin (bottom) Etest results for strains GSK64 (orange) and GSK241 (red) and when grown on BHI supplemented with various concentration of haemin (0, 0.5, 1, 2.5, 5, 10 and 15 mg/L). The dashed line indicates the epidemiological cut-off value for metronidazole and vancomycin as determined by EUCAST (2 mg/L).

Figure 3.

TAT-C signature in hsmA correlates to metronidazole resistance in RT010 isolates. The horizontal black bars represent the median MIC. (a) hsmA signature based on SNP analysis in the COMBACTE-CDI clinical isolates. Strains containing the GGCAT signature belong to RT016, RT027, RT176, RT181 and RT198. The TAT-C and TATAC signatures correspond to RT010 in this study, whereas RT002 and RT018 contain sequence TGTAC. (b) hsmA signature based on SNP analysis in the clinical isolates sequenced in the pCD-METRO study. The signature sequence GGCAT is found in RT027, the signature sequences TAT-C and TATAC are found in RT010 and the signature sequence TGTAC is found in RT012 and RT020.

Figure 4.

The C-terminal deletion in hsmA is associated with a lineage of ST15 and ST15-like isolates. When available, metronidazole susceptibility data and pCD-METRO carriage are indicated. Country flags prior to strain names indicate strain origin, when known. Strain names or SRA accession numbers are included. Distances in tree are shown in logarithmic scale. N/A, not available (i.e. no MIC metadata are deposited with the sequences and the isolates were not available for testing).