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

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.

Plasmid-mediated metronidazole resistance in Clostridioides difficile.

Metronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been documented, no clear mechanism for metronidazole resistance or a role for plasmids in antimicrobial resistance has been described for CD. Here, we report genome sequences of seven susceptible and sixteen resistant CD isolates from human and animal sources, including isolates from a patient with recurrent CD infection by a PCR ribotype (RT) 020 strain, which developed resistance to metronidazole over the course of treatment (minimal inhibitory concentration [MIC] = 8 mg L-1). Metronidazole resistance correlates with the presence of a 7-kb plasmid, pCD-METRO. pCD-METRO is present in toxigenic and non-toxigenic resistant (n = 23), but not susceptible (n = 563), isolates from multiple countries. Introduction of a pCD-METRO-derived vector into a susceptible strain increases the MIC 25-fold. Our finding of plasmid-mediated resistance can impact diagnostics and treatment of CD infections.

Evaluation of the Liat Cdiff Assay for Direct Detection of Clostridioides difficile Toxin Genes within 20 Minutes.

Clostridioides difficile is the main causative agent of antibiotic-associated diarrhea. Prompt diagnosis is required for initiation of timely infection control measures and appropriate adjustment of antibiotic treatment. The cobas Cdiff assay for use on the cobas Liat system enables a diagnostic result in 20 minutes. A total of 252 prospective (n = 150) and retrospective (n = 102) stool specimens from The Netherlands, France, and Switzerland were tested on the cobas Cdiff assay using the Xpert C. difficile assay as a reference method. The overall positive and negative percent agreement (PPA and NPA, respectively) of the cobas Cdiff assay compared with the Xpert C. difficile assay was 98.0% (100/102; 95% confidence interval [CI], 93.1% to 99.5%) and 94.0% (141/150; 95% CI, 89.0% to 96.8%), respectively. When comparing the PPAs of cobas Cdiff and Xpert C. difficile with culture, the results were 91.7% (55/60; 95% CI, 81.9% to 96.4%) and 85.0% (51/60; 95% CI, 73.9% to 91.9%), respectively. The difference was not statistically significant. The cobas Cdiff assay offers a very rapid alternative for diagnosing C. difficile infection. The 20-minute turnaround time provides the potential for point-of-care testing so that adequate infection control measures can be initiated promptly.

Genome Location Dictates the Transcriptional Response to PolC Inhibition in Clostridium difficile.

Clostridium difficile is a potentially lethal gut pathogen that causes nosocomial and community-acquired infections. Limited treatment options and reports of reduced susceptibility to current treatment emphasize the necessity for novel antimicrobials. The DNA polymerase of Gram-positive organisms is an attractive target for the development of antimicrobials. ACX-362E [N2-(3,4-dichlorobenzyl)-7-(2-[1-morpholinyl]ethyl)guanine; MorE-DCBG] is a DNA polymerase inhibitor in preclinical development as a novel therapeutic against C. difficile infection. This synthetic purine shows preferential activity against C. difficile PolC over those of other organisms in vitro and is effective in an animal model of C. difficile infection. In this study, we have determined its efficacy against a large collection of clinical isolates. At concentrations below the MIC, the presumed slowing (or stalling) of replication forks due to ACX-362E leads to a growth defect. We have determined the transcriptional response of C. difficile to replication inhibition and observed an overrepresentation of upregulated genes near the origin of replication in the presence of PolC inhibitors, but not when cells were subjected to subinhibitory concentrations of other antibiotics. This phenomenon can be explained by a gene dosage shift, as we observed a concomitant increase in the ratio between origin-proximal and terminus-proximal gene copy number upon exposure to PolC inhibitors. Moreover, we show that certain genes differentially regulated under PolC inhibition are controlled by the origin-proximal general stress response regulator sigma factor B. Together, these data suggest that genome location both directly and indirectly determines the transcriptional response to replication inhibition in C. difficile.