Fig 1.
Schematic of the anaerobic single-cell imaging set-up.
Exponentially growing C. difficile cells in TY medium supplemented with cysteine (TYC) are spotted onto 1.5% agarose pads formed within gas-tight adhesive Gene Frames inside the anaerobic chamber. Up to 6 strains can be spotted onto a pad. The pad is sealed with a coverslip, and the imaging chamber is removed from the anaerobic chamber and transferred to a heated (37˚C) microscope stage. Time-lapse microscopy is used to visualize the growth of individual bacterial cells for 2-6 hrs. The output data is segmented and tracked with the DeLTA Python package (191). An example filmstrip of strain 630 grown on TYC medium over time is shown (Bottom).
Table 1.
Clostridioides difficile clinical isolates used in this study.
Fig 2.
Clade 5 strain M120 elongates more quickly and exhibits cell chaining.
(A) Violin plot of the elongation rates measured during time-lapse microscopy analyses of strains 630 (Clade 1), R20291 (Clade 2), E15 (Clade 3), M68 (Clade 4), and M120 (Clade 5) grown on TY supplemented with cysteine (TYC) agar. Data are from three biological replicates, with the mean of each replicate shown as a point on the violin. (B) Optical density-based analyses of bulk population growth of the indicated strains in TYC or BHIS media. The number in brackets indicates the clade to which a given strain belongs. (C) Violin plot of the cell or chain length measured during time-lapse microscopy for the strains shown in A. Each replicate mean is shown as a point on the violin. Statistical significance for A and B was determined by comparing the mean of the three replicates of strains from Clades 1-4 strains relative to the Clade 5 M120 strain using a Kruskal-Wallis test * p < 0.05. (B) Phase-contrast image from time-lapse microscopy movies. Scale bar is 10 μm.
Fig 3.
C. difficile Clade 5 can form large heterogenous chains.
Large mosaic phase-contrast image of the Clade 5 strain M120. Inset shows chains revealed by staining with the membrane dye FM4-64; septa are highlighted with yellow arrows. The image was stitched from 8 individual fields of view at 63X magnification. Scale bar is 100 μm.
Table 2.
Cell size and growth rate statistics for the studied strains.
Fig 4.
Faster growth is a common feature of Clade 5 strains, but the chaining phenotype is not fully penetrant in Clade 5 strains.
(A) Phase-contrast microscopy images from time-lapse microscopy studies of Clade 5 strains; Clade 1 strain 630 is included for comparison. Fluorescence microscopy was used to visualize the FM4-64 stain incorporated into the agarose pads. Masks generated using DeLTA are shown, with the bottom panel showing division septa identified with our adaptive thresholding approach (red dots). (B) Example image showing the parameters identified using DeLTA combined with our adaptive threshold method for detecting division septa (red dot) within a chain of cells. Following the automated thresholding analysis for detecting septa, the images were manually inspected to ensure that all septa were properly identified. (C) Cell or chain length measured using automated DeLTA analyses (pink violin plot) or DeLTA combined with the adaptive threshold-manual inspection analyses (purple violin plot). The former method is more likely to measure chain length, while the latter method accurately measures cell length. (D) Violin plot of the elongation rates measured based on three biological replicates. Each replicate mean is shown as a point on the violin; statistical significance was determined by comparing the mean of the three replicates using a Kruskal-Wallis test, * p < 0.05.
Fig 5.
Clade 5 strains form chains during logarithmic growth in broth culture.
Representative micrographs showing phase-contrast (top) and peptidoglycan labeling with the fluorescent D-amino acid, HADA, (bottom) following growth in rich broth (BHIS) to mid-logarithmic phase. All strains shown, with the exception of Clade 1 strain 630, are Clade 5 strains. Scale bar, 10 µm. Data are representative of three independent experiments. ADJ indicates that the brightness of the image was enhanced to detect HADA labeling in V48.
Fig 6.
Sporulation levels in Clade 5 isolates grown on 70:30 medium.
Phase-contrast microscopy of the indicated strains ~24 hrs after sporulation induction. All strains shown with the exception of 630 (Clade 1) are Clade 5 strains. The percent heat-resistant spores is indicated below the respective images. The percentage was determined from 20-24 hr sporulating cultures and represent the mean and standard deviation for a given strain based on a minimum of three biological replicates. Statistical significance relative to strain 630 was determined using a one-way ANOVA and Tukey’s test. The scale bar represents 5 µm.
Fig 7.
The orientation of the cmr switch promotes Clade 5 strain cell chaining and surface motility.
(A) Orientation-specific qPCR for detecting the orientation of the cmr switch in the indicated strains. The mean and standard deviation based on one to three biological replicates are shown. Statistical significance relative to strain TAL29996 was determined using a one-way ANOVA and Tukey’s test for strains where data from three independent experiments was obtained. **** p < 0.0001. (B) Representative micrographs of cmrRST CRISPRi knock-down strains compared to a no target control. Phase-contrast (top) and peptidoglycan labeling with the fluorescent D-amino acid, HADA, (bottom) images following growth in rich broth (BHIS) to mid-logarithmic phase. Scale bar, 5 µm. (C) Representative images of surface motility 5 days after exponentially growing liquid cultures of the indicated strains were spotted onto BHIS agar plates.
Fig 8.
Infection and colonization dynamics of Clade 5 strains in mice.
(A) Fecal colony-forming units measured by selective plating and (B) Percentage of weight loss to baseline of infected mice on Days 1-4, 7, and 14. The mean and standard deviation are shown based on the results of two experiments consisting of four mice each (n = 8). (C) Percentage of cmr-ON switch orientation measured in fecal pellets on the indicated days by qPCR. For the strain TAL29996 inoculum, 1% of the spores had the cmr-ON switch orientation, while the TAL29600 inoculum consisted of 32% cmr-ON spores. The mean and standard deviation based on analyses of eight mice are shown, although fecal pellets could not be collected from some of the mice on Day 2. The same mouse exhibited a higher cmr-ON frequency for TAL29600 over time (15%, Day 7 and 27%, Day 14).