Characterization of a Stable, Metronidazole-Resistant Clostridium difficile Clinical Isolate

Abstract

Background Clostridium difficile are Gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15–35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking.Methodology/Principal FindingsGenotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production.Conclusions/SignificanceThis is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.

Multidisciplinary Analysis of a Nontoxigenic Clostridium difficile Strain with Stable Resistance to Metronidazole

“…difficile strains showing resistance to metronidazole are rare, and laboratory manipulation of these strains frequently results in a MIC decrease toward the susceptibility range (4,5). Therefore, the investigation of resistance mechanisms in C. difficile has proved difficult.…”

Emerging Metronidazole Resistance in Anaerobes and Mapping Their Susceptibility Behaviour

“…The development of resistance to metronidazole leading to treatment failure during the course of metronidazole therapy in a patient has been reported (Buta et al, 2010;Soki et al, 2013). Numerous studies have demonstrated the incidence of metronidazole resistance in recent years (Alauzet et al, 2010;Lynch et al, 2013;Abdollahi et al, 2011). In a study conducted by Datta et al (2005) reported that 85% strains of Helicobacter pylori were resistant to metronidazole.…”

“…Whereas mechanism for metronidazole resistance in B. fragilis include the presence of nim genes, which encode 5-nitroimidazole reductases that convert metronidazole to a non-toxic amino derivative (Pumbwe et al, 2008), overexpression of the DNA repair protein, RecA (Steffens et al, 2010) and disruption of the electron transport chain (Lynch et al, 2013). 5-nitroimidazole resistance genes, nimA to E, have been identified to confer reduced susceptibility to 5-nitroimidazole antibiotics on species of the B. fragilis group (Hartmeyer et al, 2012).…”

Clostridium difficile Infection

“…For example, the Tn6218 element of C. difficile contains a cfr-like gene that can confer resistance to peptidyl transferase inhibitors such as linezolid 255 and the Tn5397 element carries a tetracycline resistance determinant 256 . Reduced susceptibility or resistance to the commonly used antibiotics vancomycin, metronidazole and fidaxomicin has been noted [257][258][259][260] . Although this is cause for concern, the clinical relevance of resistance to these antibiotics so far is limited.…”