BioProject

Isogenic, toxin B (tcdB) mutants of two independently isolated erythromycin-sensitive derivatives (630E and 630Δerm) of the Clostridium difficile strain CD630 have previously been shown to behave differently in the hamster model of infection. More...

Isogenic, toxin B (tcdB) mutants of two independently isolated erythromycin-sensitive derivatives (630E and 630Δerm) of the Clostridium difficile strain CD630 have previously been shown to behave differently in the hamster model of infection. Those based on strain 630E failed to cause disease, whereas those based on 630Δerm did. Here we show that whilst 630Δerm and its derivatives behave in a comparable way to the parental strain CD630 in terms of growth rate, motility, sporulation, toxin production and virulence, strain 630E and its derivatives do not. They grow more slowly, achieve lower final cell densities, exhibit a reduced capacity for spore formation, express lower levels of toxin and are less virulent in the hamster model. Using Next Generation Sequencing, a total of three SNPs were found that were in both CD630 and 630Δerm. In addition to these, 630Δerm had eight unique SNPs compared to CD630 and 630E, while 630E had 11 SNPs not found in the other two strains. The strain-specific SNPs most likely arose during the repeated subculture experiments undertaken to isolate these ermB deletion strains. The relatively large number of mutations present meant that the identification of those SNPs responsible for the altered properties of 630E was not possible. Allelic exchange was used to correct three mutational changes in 630E, but with essentially no effect on phenotype. These were a 150 bp inversion in the promoter region of a flagella operon, the truncation of CD630_12740 (topoisomerase I), and the correction of a SNP that results in the fusion of two components of a glucose phosphor-transferase-system. To complete the analysis we performed RNAseq, comparing the transcriptome of the three strains at three different time points. The finding corroborated the many differences between the strains, but also highlighted that one SNP cannot necessarily be attributed to one particular expression pattern or even phenotype. In conclusion 630Δerm more accurately mirrors the behaviour of the parental strain CD630, hence if genetic-based studies with C. difficile strain CD630 are necessary, then the use of strain 630∆erm should be favoured over strain 630E. At a fundamental level, our data underlines the importance of effective strain curation and the need to genome re-sequence master seed banks wherever possible. Overall design: Total RNA was extracted from 2 independent biological replicates of 630, 630∆erm and 630E C. difficile strains at 3 time points (18 samples). Less...