Deciphering Adaptation Strategies of the Epidemic <i>Clostridium difficile</i> 027 Strain during Infection through <i>In Vivo</i> Transcriptional Analysis

<div><p><i>Clostridium difficile</i> is responsible for a wide spectrum of infection from asymptomatic carriage to severe, relapsing colitis. Since 2003, <i>C</i>. <i>difficile</i> infections have increased with a higher morbidity and mortality due to the emergence of epidemic and hypervirulent <i>C</i>. <i>difficile</i> strains such as those of the epidemic lineage 027/BI/NAP1. To decipher the hypervirulence and epidemicity of 027 strains, we analyzed gene expression profiles of the R20291 027 strain using a monoxenic mouse model during the first 38h of infection. A total of 741 genes were differentially expressed during the course of infection. They are mainly distributed in functional categories involved in host adaptation. Several genes of PTS and ABC transporters were significantly regulated during the infection, underlying the ability of strain R20291 to adapt its metabolism according to nutrient availability in the digestive tract. In this animal model, despite the early sporulation process, sporulation efficiency seems to indicate that growth of R20291 vegetative cells versus spores were favored during infection. The bacterial mechanisms associated to adaptability and flexibility within the gut environment, in addition to the virulence factor expression and antibiotic resistance, should contribute to the epidemicity and hypervirulence of the <i>C</i>. <i>difficile</i> 027 strains.</p></div

Kinetics of sporulation rate in <i>C</i>. <i>difficile</i> associated mice.

<p>Mice were orally challenged with 1x10⁸ CFUs of vegetative cells. Vegetative cells were enumerated on BHI agar plates and spores after a heat shock treatment on BHI containing 0.1% of taurocholate sodium salt. </p

Regulation of sporulation and germination genes during the kinetics of infection.

<p>Regulation of sporulation and germination genes during the kinetics of infection.</p

Validation of microarray data by qRT-PCR.

<p>Fold changes in in vivo gene expression at 4, 6, 14 and 38h post-infection, compared to the in vivo expression at 8h post-infection, were measured by microarray and qRT-PCR. Data are plotted as log₂ ratios of microarrays data (<i>x</i>-axis) compared with those of qRT-PCR (<i>y</i>-axis).</p

Regulation of toxin genes during the kinetics of infection.

<p>Regulation of toxin genes during the kinetics of infection.</p

Flagellar genes differentially expressed <i>in vivo</i> in the <i>fliC</i> mutant.

<p>*From F1 region (late-stage flagellar genes) and **F3 region (early-stage flagellar genes) of the flagellar operon from <i>C. difficile</i> 630 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096876#pone.0096876-Aubry1" target="_blank">[39]</a>.</p

Membrane transport genes highly differentially expressed <i>in vivo</i> in the <i>fliC</i> mutant compared to strain R20291.

<p>Membrane transport genes highly differentially expressed <i>in vivo</i> in the <i>fliC</i> mutant compared to strain R20291.</p

Ability of sporulation of the <i>fliC</i> mutant compared to wild-type R20291 <i>in vivo</i> and <i>in vitro</i>.

<p>(<b>A</b>) Groups of 6 axenic mice were infected by oral gavage route with 1×10⁸<i>C. difficile</i> CFU. The <i>C. difficile</i> faecal vegetative cells and spores were measured by determining the concentration of CFU in faeces at 14 h post-infection by homogenising and plating on appropriated agar medium after heat shock (for spores) or not (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096876#s4" target="_blank">Materials and Methods</a>). Data represent the ratio of spores per vegetative cells. (<b>B</b>) Cultures in BHIS broth in anaerobic conditions were prepared from 2 successive subcultures as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096876#s4" target="_blank">Materials and Methods</a>. After heat shock, spores were quantified (CFU/ml) by performing serial dilutions and spread plating on BHIS agar supplemented with 0.1% bile salt taurocholate to induce germination. Data represent the ratio of spores per total cells. The presented values are the mean of 3 different cultures. Statistically significant difference is indicated by * for p<0,05.</p

Known and putative virulence factor genes differentially expressed <i>in vivo i</i>n the <i>fliC</i> mutant compared to strain R20291.

<p>ND: non determined.</p

Functional clusters of <i>in vivo</i> differentially expressed genes.

<p>Differentially expressed genes in the <i>fliC</i> mutant compared to wild-type R20291 from caeca of 14 h post-infection mice were classified in functional groups according to their involvement in the biological process categories. (<b>A</b>) The number of analyzed genes is represented in green bars and the number of significantly differentially expressed genes is shown in black bars. Percentages of differentially regulated genes are indicated at right. (<b>B</b>) The numbers of up- and down-regulated genes for each cluster are indicated in green and black bars, respectively.</p