Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human... Show moreClostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrent CDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventing CDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (at a dose of 10(7)) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain (210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas; bacterial populations and cytotoxin production were determined using viable counting and Vero cell cytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculated singly, with RT027 demonstrating high-level cytotoxin (3-5-log(10)-relative units) production. In experiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescent and failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous to CD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked other metronidazole resistance determinants. This study showed that RT027 was unable to elicit simulated infection in the presence of NTCD-E4 following stimulation by four different antimicrobials. These data complement animal and clinical studies in suggesting NTCD offer prophylactic potential in the management of human CDI. Show less
Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenicC. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human... Show moreClostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenicC. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrentCDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventingCDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (ata dose of 107) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain(210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas;bacterial populations and cytotoxin production were determined using viable counting and Vero cellcytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculatedsingly, with RT027 demonstrating high-level cytotoxin (3-5-log10-relative units) production. Inexperiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescentand failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous toCD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked othermetronidazole resistance determinants. This study showed that RT027 was unable to elicit simulatedinfection in the presence of NTCD-E4 following stimulation by four different antimicrobials. Thesedata complement animal and clinical studies in suggesting NTCD offer prophylactic potential in themanagement of human CDI. Show less
Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human... Show moreClostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrent CDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventing CDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (at a dose of 107) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain (210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas; bacterial populations and cytotoxin production were determined using viable counting and Vero cell cytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculated singly, with RT027 demonstrating high-level cytotoxin (3-5-log10-relative units) production. In experiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescent and failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous to CD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked other metronidazole resistance determinants. This study showed that RT027 was unable to elicit simulated infection in the presence of NTCD-E4 following stimulation by four different antimicrobials. These data complement animal and clinical studies in suggesting NTCD offer prophylactic potential in the management of human CDI. Show less
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... Show moreBackground: 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. Show less
The epidemiology of Clostridium difficile infection (CDI) has changed dramatically during this millennium. Infection rates have increased markedly in most countries with detailed surveillance data.... Show moreThe epidemiology of Clostridium difficile infection (CDI) has changed dramatically during this millennium. Infection rates have increased markedly in most countries with detailed surveillance data. There have been clear changes in the clinical presentation, response to treatment, and outcome of CDI. These changes have been driven to a major degree by the emergence and epidemic spread of a novel strain, known as PCR ribotype 027 (sometimes referred to as BI/NAP1/027). We review the evidence for the changing epidemiology, clinical virulence and outcome of treatment of CDI, and the similarities and differences between data from various countries and continents. Community-acquired CDI has also emerged, although the evidence for this as a distinct new entity is less clear. There are new data on the etiology of and potential risk factors for CDI; controversial issues include specific antimicrobial agents, gastric acid suppressants, potential animal and food sources of C. difficile, and the effect of the use of alcohol-based hand hygiene agents. Show less
