We have presented four phenotypes associated with the stable, metronidazole-resistant CD26A54_R subpopulation: (i) aberrant growth in liquid media, (ii) attenuated cell wall separation (iii) lack of spore production by 48 hours and (iv) heteroresistance or more accurately, a slower growing subpopulation that increases the metronidazole MIC. By standard diagnostic methods CD26A54_S is considered susceptible to metronidazole, however this strain shares some characteristics with its resistant counterpart, specifically attenuated cell separation and heteroresistance against metronidazole thus also distinguishing it from the wild type VLOO13. We will discuss how whole genome sequence data may explain these phenotypes however further investigation is needed to understand the role of these changes in metronidazole resistance and how this impacts pathogenicity.
In BHI broth, CD26A54_R appeared to have a prolonged lag phase followed by reduced cell density, particularly during the late-log to early stationary phases. Even at 24 hours post-inoculation, CD26A54_R never reached the same cell density as either CD26A54_S or VLOO13. This may suggest CD26A54_R encountered a nutrient limitation in the media that was not exogenously required by the other strains, enabling the other two to reach a higher cell density. Genetically, there are two variants that may have contributed to the aberrant growth observed, namely the frameshift mutation in the hemN
gene, whose product is involved haem biosynthesis and thiH
, encoding a protein involved in thiamine biosynthesis. There are additional variants found in CD26A54_R that are associated with electron transport, such as glycerol-3-phosphate dehydrogenase (glyC
) and the pyruvate-flavodoxin oxidoreductase (nifJ
). Disruption of electron transport alters both the energy production and intracellular redox potential which influence the efficiency of metronidazole entry and activation 
Deficiencies leading to disruptions in electron transport have been previously attributed to small colony variants (SCV) in other bacterial genera. Some general characteristics of SCV include smaller colonies on agar, slower growth, decreased respiration, attenuated cell separation and antibiotic resistance; additionally, they are often associated with persistent or recurrent infections 
.We have observed by SEM that both CD26A54_R and CD26A54_S demonstrate elongated cells and incomplete cell separation which is similar to previous reports of Enterococcus faecium
and Staphylococcus aureus
. The shared characteristics of previously described SCV and the CD26A54 subpopulations such as aberrant growth, attenuated cell separation and antibiotic resistance have been demonstrated, however we have not observed smaller colonies on non-selective agar.
Resistance to additional antibiotics was tested to assess possible disruptions in metabolic pathways such as protein or cell wall biosynthesis that could contribute to altered cell morphology and aberrant growth however the MIC values of CD26A54_R were similar to one or both of the metronidazole-susceptible strains. A high prevalence of moxifloxacin and clindamycin resistance among ribotype 027 strains has been reported previously (97.5% and 47.5% respectively), although no observations of metronidazole resistance or altered cell morphology were reported 
. We have a few hypotheses with respect to our observations that CD26A54_R did not produce spores by 48 hours whereas CD26A54_S and VLOO13 both demonstrate an increasing proportion of spores. There are multiple methods in the literature to study sporulation of C. difficile
isolates (reviewed in 
). We did attempt this analysis with various media formulations and methods to shock the culture for spore selection, and although total numbers did vary across replicates and across individual experiments, we observed a consistent trend that no spores were ever counted in the CD26A54_R culture up to 48 hours. Within the CD26A54_R genome there is a point mutation in the stage 0 sporulation protein A gene (spo0A
) which is a major regulator of the sporulation process 
, as well as a nonsynonymous variant in the cspC
gene which encodes a putative germination-specific protease. Further experimentation is needed to investigate whether this data demonstrates a lack or delay of spore production owing to disruption of the spore initiation pathway (via spoOA
mutation) or conversely owing to disruption in the germination process resulting from the cspC
mutation; causing the inability of spores to germinate efficiently. We speculate that disruption of the sporulation process could prolong the vegetative state resulting in prolonged expression of virulence factors during host infection. Importantly however, reduced propensity for spore generation and/or germination should render CD26A54_R less transmissible in hospital or long-term care facility settings as vegetative cells are more susceptible to regular cleaning methods.
In addition to the phenotypic and genotypic changes described above, we identified genetic mutations similar to those of other bacterial genera which have been shown to confer metronidazole resistance. The CD26A54_R strain possesses a point mutation in the fur
gene of which encodes the ferric uptake regulator, Fur. Disruption of the fur
gene has been associated with metronidazole resistance in H. pylori
by altering binding of Fur to superoxide dismutase thereby reducing oxidative stress within the cell 
. Both CD26A54_R and CD26A54_S contain a frameshift mutation in the rsbW
gene which encodes an anti-sigma factor associated with σB
, which plays a central role in stress responses 
. We speculate that alteration of rsbW
may similarly enhance the ability of the bacterium to reduce oxidative stress caused by metronidazole. Both strains also possess a frameshift mutation in a putative nitroreductase gene which may alter the requisite activation of metronidazole in the cell 
. The association between nitroreductases and metronidazole resistance has been described previously for both B. fragilis
and H. pylori
As discussed above, there are numerous possible mechanisms to explain the metronidazole resistance observed in the current study. In the literature there are multiple mechanisms for metronidazole resistance described within a species suggesting resistance can occur via different pathways and can be a multifactorial process. In the present study we presented phenotypic observations and genomic analyses of a stable, metronidazole resistant isolate of C. difficile in the aim of providing a foundation for future experimental investigation to elucidate the mechanisms of resistance and ultimately translate the knowledge to improve clinical diagnosis and treatment of CDI.