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 findings: Genotypic 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/significance: This 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.

Figure 1. Growth of C. difficile in BHI broth.

CD26A54_R, the metronidazole resistant strain (□) demonstrated aberrant growth compared to VLOO13 (□) at all time points between 5 and 24 hours (p<0.05*). However, the CD26A54_S strain (□) only had a significantly greater cell density than CD26A54_R during late-log and early stationary time points (7–11 hours, p<0.05**). There was no significant difference observed between VLOO13 and CD26A54_S throughout the growth curve.

Figure 2. Scanning electron microscopy of C. difficile.

SEM images of VLOO13 (A), CD26A54_S (B) and CD26A54_R (C). (D) Histograms of calculated bacterial length are presented; they demonstrate the cell length variation that exists across the three C. difficile specimens that were imaged by SEM. The insets with arrows in panels (B, C) highlight the septum which partially forms between adjacent cells. Cell separation appears to be impaired resulting in the longer phenotype.

Figure 3. Summary of metronidazole susceptibility results by Etest®.

Susceptibility to metronidazole was repeatedly tested for a total of n = 11 independent experiments to monitor the stability of the resistance phenotype.

Figure 4. BLAST atlas of NAP1 genomes.

BLASTn was used to compare NAP1 genomes listed in Table 1. The rings, listed from inner to outer tracks are as follows: Orange peaks represent GC skew while the pink peaks represent GC content. The innermost black track is the R20291 reference genome. Purple tracks are the historical, pre-epidemic strains, CD196 and BI-1. The blue rings consist of Canadian NAP1 genomes, QCD32g58, QCD-97b34, QCD-37×79 and QCD-66c26. The green is the susceptible CD26A54_S while the red is the resistant subpopulation, CD26A54_R. The outermost ring contains the sequences for the two transposable elements, Tn6104 and Tn6106 to confirm their absence in all genomes except R20291.

Figure 5. Variant distribution of both CD26A54_R and CD26A54_S relative to the reference sequence R20291.

The total SNP and indel variants for the CD26A54_R (red diamonds) and the CD26A54_S (green diamonds) are plotted along the R20291 reference genome. The reference sequence tracks include CDS (black bars), GC content (pink peaks) and GC skew (orange peaks).