Characterization of Clostridium difficile biofilm : from the inert support to the digestive colonization

Clostridium difficile est une bactérie enteropathogène responsable d'infections intestinales dont les manifestations cliniques varient d’une simple diarrhée à une colite pseudomembraneuse parfois mortelle. L’une des problématiques majeures rencontrées dans la prise en charge des infections à C. difficile (ICD) est la survenue de récidives. Chez de nombreuses espèces bactériennes, la formation de biofilm est associée à la chronicité de l’infection. Le biofilm est un mode de vie dans lequel les bactéries sont engluées dans une substance polymérique qu'elles secrètent elles-mêmes. L’aptitude de C. difficile à former un biofilm in vitro a été clairement établie depuis 2012. Cependant aucune étude n’a montré jusqu’ici s’il est capable de former un biofilm in vivo. L’objectif de cette thèse était d’une part d’étudier différents paramètres impliqués dans la formation du biofilm in vitro et d’autre part de déterminer si C. difficile est capable de former un biofilm in vivo. Dans un premier temps, nous avons analysé la capacité de différentes souches de C. difficile (souches cliniques et souches de laboratoires modifiées génétiquement) à former un biofilm in vitro. Parmi ces dernières, la souche mutée pour le gène cwp84, codant une protéase de surface responsable de la maturation de la couche S de C. difficile, présente un biofilm particulièrement robuste et épais comparé à la souche parentale 630∆erm. L’activité protéolytique de Cwp84 est donc impliquée dans la formation du biofilm et module certaines propriétés de surface de la bactérie telles que l’hydrophobicité. Nous avons également étudié la composition en sucre de la matrice du biofilm, après une étape de mise au point qui a permis de déterminer les conditions permettant d’obtenir suffisamment de matériel. Les conditions retenues ont été les suivantes : formation de biofilm sur un support en verre, en présence de glucose et en milieu renouvelé. La présence d’un sucre qui possède un profil proche du PSII (polysaccharide associé à la surface des cellules planctoniques de C. difficile) dans la matrice du biofilm a été détecté par spectroscopie infrarouge. Dans un second temps, afin d’étudier l’aptitude de C. difficile à former un biofilm in vivo, différents modèles animaux ont été utilisés : un modèle de souris monoxéniques dans lequel plusieurs souches de C. difficile ont été testées (souches 630∆erm, mutant cwp84, R20291, P30) et un modèle de souris dixénique pour étudier la formation de biofilm mixte (C. difficile/Finegoldia magna et C. difficile/Clostridium scindens). Dans le modèle monoxénique, quelles que soient les souches testées, C. difficile est distribué de manière hétérogène tout au long de la surface du tissu intestinal. Les bactéries sont majoritairement retrouvées isolées sauf pour la souche R20291 qui forme le plus souvent des petits agrégats. Pour cette souche, différents marquages immunohistochimiques réalisés sur des coupes de cecum et de colon ont montré que la majorité des bactéries sont enchâssées dans de petites structures en 3 dimensions adhérentes à la couche du mucus. Le polysaccharide PSII est détecté en grande quantité à l'intérieur de cette structure. Ce composé étant présent dans la matrice de biofilm de C. difficile formé in vitro, ces résultats suggèrent que la souche R20291 pourrait s’organiser en biofilm dans le modèle de souris monoxénique. En modèle de souris dixéniques, nous avons montré que la présence de F. magna n’influe pas sur le niveau de colonisation de C. difficile, alors que l'association avec C. scindens semble être bénéfique aux deux bactéries puisqu'une augmentation de la population globale de deux espèces est observée comparé à la population présente dans chaque modèle mono-espèce. En conclusion, une souche productrice de biofilm in vitro semble être capable de s’organiser en une structure biofilm in vivo. Le rôle du biofilm dans l'étape de colonisation du colon par C. difficile et les rechutes des ICD devra être analysé.Clostridium difficile is an enteropathogenic bacterium responsible for intestinal infections, the clinical symptoms vary from moderate diarrhea to pseudomembranous colitis, sometimes fatal. One of the major problems encountered in the management of C. difficile infections (DCI) is the occurrence of recurrences. In many bacterial species, biofilm formation is associated with the chronicity of infection. Biofilm is a way of life in which bacteria are entrapped in a polymeric substance secreted by the bacteria themselves. The ability of C. difficile to form an in vitro biofilm has been clearly established since 2012. However, no study has so far shown whether it is capable of forming a biofilm in vivo. The objective of this thesis was to analyze different parameters involved in the formation of biofilm in vitro and to determine whether C. difficile is able to form a biofilm in vivo.We first analyzed the ability of different strains of C. difficile to form a biofilm in vitro (clinical strains and strains genetically modified laboratories). Among the latter, the mutant strain for the cwp84 gene, encoding a surface-associated protease responsible for the maturation of the S layer of C. difficile, forms a particularly robust and thick biofilm compared to the 630Δerm parental strain. The proteolytic activity of Cwp84 is involved in the formation of biofilm and modulates certain surface properties of the bacterium such as hydrophobicity. We have also studied the polysaccharide composition of the biofilm matrix, after a development stage that allowed us to determine the optimal conditions for obtaining sufficient material. The conditions retained were the following: formation of biofilm on a glass support in the presence of glucose and in a renewed medium. We were able to determine by infrared spectroscopy the presence of a sugar which has a similar profile than the PSII (polysaccharide associated with the surface of C. difficile planktonic cells) in the matrix of the biofilm.Second, in order to study the ability of C. difficile to form a biofilm in vivo, different animal models were used: a monoxenic mouse model in which we tested several strains of C. difficile (strains 630Δerm, mutant cwp84, R20291, P30) and a dixenic mouse model to study the formation of mixed biofilm (C .difficile/Finegoldia magna and C. difficile/Clostridium scindens). In the monoxenic model, regardless of the strains tested, C. difficile is distributed heterogeneously throughout the intestinal tissue surface. The bacteria are mostly found isolated except for C. difficile R20291 which usually forms small aggregates. For this strain we have shown, thanks to various immunohistochemical labeling performed on cecum and colon sections, that the majority of the bacteria are embedded in a small 3-dimensional structures overlaying the mucus layer. The PSII polysaccharide is present in a large amount in this structure. As this compound has been detected in the in vitro C. difficile biofilm matrix, these results suggest strongly that the R20291 strain could be organized into biofilm structures in the monoxenic mouse model. In the dixenic mouse model, we have shown that the presence of F. magna does not influence the level of colonization of C. difficile, whereas the association with C. scindens seems to be beneficial to both bacteria since an increase of the global population is observed in this model compared to the population present in each single-species model.To conclude, an in vitro biofilm producing strain appears to be able to organize in a biofilm structure in vivo. The role of biofilm in the colonization step of the intestinal tract by C. difficile and in the occurrence of recurrences should be further analyzed

Biofilm Structures in a Mono-Associated Mouse Model of Clostridium difficile Infection

Clostridium difficile infection (CDI) is a major healthcare-associated disease with high recurrence rates. Host colonization is critical for the infectious process, both in first episodes and in recurrent disease, with biofilm formation playing a key role. The ability of C. difficile to form a biofilm on abiotic surfaces is established, but has not yet been confirmed in the intestinal tract. Here, four different isolates of C. difficile, which are in vitro biofilm producers, were studied for their ability to colonize germ-free mice. The level of colonization achieved was similar for all isolates in the different parts of the murine gastrointestinal tract, but pathogen burden was higher in the cecum and colon. Confocal laser scanning microscopy revealed that C. difficile bacteria were distributed heterogeneously over the intestinal tissue, without contact with epithelial cells. The R20291 strain, which belongs to the Ribotype 027 lineage, displayed a unique behavior compared to the other strains by forming numerous aggregates. By immunochemistry analyses, we showed that bacteria were localized inside and outside the mucus layer, irrespective of the strains tested. Most bacteria were entrapped in 3-D structures overlaying the mucus layer. For the R20291 strain, the cell-wall associated polysaccharide PS-II was detected in large amounts in the 3-D structure. As this component has been detected in the extrapolymeric matrix of in vitro C. difficile biofilms, our data suggest strongly that at least the R20291 strain is organized in the mono-associated mouse model in glycan-rich biofilm architecture, which sustainably maintains bacteria outside the mucus layer

The Clostridium difficile Protease Cwp84 Modulates both Biofilm Formation and Cell-Surface Properties

Clostridium difficile is responsible for 15-20% of antibiotic-associated diarrheas, and nearly all cases of pseudomembranous colitis. Among the cell wall proteins involved in the colonization process, Cwp84 is a protease that cleaves the S-layer protein SlpA into two subunits. A cwp84 mutant was previously shown to be affected for in vitro growth but not in its virulence in a hamster model. In this study, the cwp84 mutant elaborated biofilms with increased biomass compared with the parental strain, allowing the mutant to grow more robustly in the biofilm state. Proteomic analyses of the 630 Delta erm bacteria growing within the biofilm revealed the distribution of abundant proteins either in cell surface, matrix or supernatant fractions. Of note, the toxin TcdA was found in the biofilm matrix. Although the overall proteome differences between the cwp84 mutant and the parental strains were modest, there was still a significant impact on bacterial surface properties such as altered hydrophobicity. In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo. Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins

<i>cwp84</i> mutant displays a growth defect only in planktonic culture.

<p>The 630Δ<i>erm</i> (●) and <i>cwp84</i> mutant (□) strains were grown in competition in agitated planktonic culture (Panel A) or recovered from biofilm supernatants (non-settled/escaped bacteria; Panel B), and colony forming units (CFU) enumerated in three independent experiments. The planktonic cultures of both strains are significantly different (<i>p</i><0.05; Student <i>t</i> test) whereas there is no significant difference for bacteria grown in biofilm, except for bacteria from the biofilm supernatant at 24h. Panel C, for biofilm-associated bacteria, colony counts (CFU), biomass after competition (grey bars, in vitro competition 630Δ<i>erm</i> and <i>cwp84</i> mutant strains) or biomass after individual growth (630Δ<i>erm</i>, black bars; <i>cwp84</i> mutant, white bars) were determined. Error bars represent standard deviation of the mean. Biofilms of strains in competition were significantly different to those formed by individual strains (<i>p</i> <0.05; Student <i>t</i> test). Significantly different ratios are indicated by asterisks.</p

Proteins increased in abundance in the 630Δ<i>erm</i> parental strain biofilm proteome compared with the cwp84 mutant biofilm proteome.

<p>Proteins increased in abundance in the 630Δ<i>erm</i> parental strain biofilm proteome compared with the cwp84 mutant biofilm proteome.</p

The <i>cwp84</i> mutant strain forms a robust biofilm.

<p><i>cwp84</i> mutant grown in BHIS + glucose for 72 hours in liquid culture forms macroscopic structures, whereas the 630Δ<i>erm</i> parent and the complemented <i>cwp84</i> mutant strains do not (Panel A). Insets below the centrifuge tubes are magnified views of the box depicted on the tubes (the scale bar corresponds to 0.1 cm). Panel B shows biofilms of the parental 630Δ<i>erm</i> strain, the <i>cwp84</i> mutant and the complemented <i>cwp84</i> mutant strain depicted before (Bi) or after (Bii) crystal violet staining. Panel C depicts biofilm quantitation. Data are representative of at least three independent experiments, each performed in triplicate. The error bars represent standard deviation. Panel D depicts enumeration of biofilm-associated bacteria generated by the various genotypes shown. Significantly different (<i>p</i> < 0.05) ratios are indicated by asterisks (Wilcoxon test for the comparison of Δ<i>erm</i> 630/<i>cwp84</i> and Student <i>t</i> tests for the others).</p

Distribution of surface, matrix and released (secretome) proteins of the 630Δ<i>erm</i> parental strain.

<p>* proteins found in the same quantity in the surface proteome of bacteria in biofilm and in biofilm secretome.</p><p>Distribution of surface, matrix and released (secretome) proteins of the 630Δ<i>erm</i> parental strain.</p

<i>C</i>. <i>difficile</i> biofilms are visualized by CLSM and electron microscopy.

<p>Biofilms elaborated by the 630Δ<i>erm</i> parent strain (A) and the cognate <i>cwp84</i> mutant and complemented strains (B and C respectively) were visualized by CLSM. The bar corresponds to a height of 50μm, captured via z-axis scans. Biofilm thickness averaged 39.2± 6.9μm for the <i>cwp84</i> mutant strain and was significantly higher (<i>p</i><0.01; paired Student <i>t</i> test) than that of the parent and complemented strains (17.7±5.8μm and 18.5±2.8 μm respectively). A top-down view using electron microscopy is shown for the parental and <i>cwp84</i> mutant strains, in lower magnification (D and E respectively) or in higher magnification (F and G respectively). A side-view is shown for the parental and <i>cwp84</i> mutant strains at lower magnification (H and I respectively). The <i>cwp84</i> mutant biofilm (60.3±4 μm thick) is significantly larger than the biofilm formed by the parental strain (11.5±1.6 μm) (p<0.001; paired Student <i>t</i> test). Black bars, 5μm.</p

Biofilm-forming ability is independent of SlpA primary sequence.

<p>Biofilm propensity of different strains of <i>C</i>. <i>difficile</i> (X-axis) was compared using crystal violet staining (Y-axis; filled green bars). SlpA primary amino acid sequence was also determined for these same strains, and percentage identity (filled red dot) compared with that of the 1064 comparator strain. The strains evaluated were P30, 4684/08, 3457, 95–1078, R20291, VPI11186, CD196, 79685, CD4, 630Δ<i>erm</i>, IT1106 and 95–1578 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124971#pone.0124971.t001" target="_blank">Table 1</a> for details). Biofilms formed by 1064, P30, 4684/08 and 3457 (group 1, OD₅₇₀>4.6), 95–1078, R20291, VPI11186, CD196 and 79685 (group 2; 1.5 570< 2.65) and CD4, 630Δ<i>erm</i>, IT1106 and 96–1578 (group 3; OD₅₇₀< 0.95) are significantly different (<i>p</i><0.01; Student <i>t</i> test). However, amino acid sequences between groups are not significantly different.</p

Parental and <i>cwp84</i> mutant strains display similar adhesion to abiotic surfaces.

<p>Percentage initial adhesion of 630Δ<i>erm</i> (black bars), <i>cwp84</i> mutant (white bars), <i>cwp84</i> mutant +<i>cwp84</i> (grey bars) strains to a polypropylene matrix for 15–120 minutes. Error bars represent standard deviation. Data are representative of four independent experiments each performed in triplicate. The observed differences are not significant (Student <i>t</i> test).</p