Ultrastructure of Clostridium difficile colonies

Anaerobe 24 (2013) 66e70

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Pathogenesis and toxins

Ultrastructure of Clostridium difficile colonies Sa
sa Lipovsek a, b, Gerd Leitinger c, Maja Rupnik a, d, e, * a

Faculty of Medicine, University of Maribor, Slovenia Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Slovenia c Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Austria d Institute of Public Health Maribor, Maribor, Slovenia e Center of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 July 2013 Accepted 29 September 2013 Available online 9 October 2013

The ultrastructural colony architecture of six Clostridium difficile strains belonging to four different ribotypes (001, 027, 078/126 and 036) was studied by scanning electron microscopy (SEM). In 2-day-old colonies several microareas differing in cell length and organization could be differentiated. Some cells at colony edges were extremely long and exhibited invasiveness into the agar. The ultrastructure of 5-dayold colonies is more homogenous and characterized by presence of sporulating cells, spores and extracellular matrix. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Clostridium difficile Biofilm Bacterial colony patterns Scanning electron microscopy Sporulation Motility

1. Introduction Clostridium difficile is a sporogenic, anaerobic, Gram positive rod, and is currently one of the most important hospital and health care associated pathogens. After colonization of the gut it produces two large toxins, toxin A and toxin B, and causes a spectrum of diseases ranging from mild diarrhoea to colitis and pseudomembranous colitis that can be also lethal [1]. Previously, the understanding of C. difficile virulence was simplistic and focused only on the role of the toxins. But the changes in epidemiology (including increased mortality and spread of highly virulent strains) have prompted studies of additional virulence properties such as adhesion, sporulation and biofilm formation [2]. Colony morphology could be a very simple and robust marker for some virulence associated properties. In some Gram negative bacteria variations between smooth and rough colony types are associated with the expression of surface antigens or with motility [3]. In Staphylococcus aureus small colony variants are associated with intracellular survival, which is important in relapsing infections [4]. Colonies are no longer seen as populations of identical bacterial cells originating from a single ancestor cell. Cells within the single colony are clonal but could differ in gene expression and

* Corresponding author. Institute of Public Health Maribor, Prvomajska 1, 2000 Maribor, Slovenia. Tel.: þ386 2 4500 183; fax: þ386 2 4500 193. E-mail addresses: [email protected], [email protected] (M. Rupnik). 1075-9964/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anaerobe.2013.09.014

hence in properties such as expression of certain molecules, sporulation, or morphology [5,6]. Different cell types within the colony can be detected either as different morphotypes or by differences in single cell expression using fluorescent reporter genes. As fluorescent reporter systems for anaerobic bacteria are just becoming available, the present studies on C. difficile colonies are based on morphological observations only. Siani and Baillie have reported two different colony morphotypes within a single strain being associated with presence or absence of the capsule (poster P26 presented at Clospath Conference, 2008, Rome). Also, switching from typical large irregular rough colonies to small smooth colonies in C. difficile as a consequence of mutagenesis of cell wall protease Cwp84 was described [7,8]. C. difficile colony ultrastructure was so far not reported. In this study we have compared colony ultrastructure of six strains belonging to four different PCR ribotypes, groups to which C. difficile strains are typically attributed [9,10]. 2. Material and methods Strains and growth conditions. Six C. difficile strains were used in the study. Three of them belonged to ribotype 027: CD196 (hystoric 027 human isolate) [11] and two recent human isolates from two different geographic origin E4 (Austria) and E20 (France). Two further human isolates were E23 (ribotype 001) and 8864 (ribotype 036/toxinotype X; [12]). A single animal strain, E5 (ribotype 078/ 126) was included in the study.

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Fig. 1. SEM of gross colony morphology of 2- and 5-day cultures (here shown for C. difficile strain E4). The 2-day-old colonies were rough and wrinkled while the 5-day old colonies had smother surface. The growth on filter or directly on agar did not influence the appearance of the colony surface.

Fig. 2. Ultrastructural characteristics of the 2-day-old C. difficile colonies. Two different areas could be observed, defined as the core region in the middle part of the colony and the edge region on its periphery (F). In the core region (A, B) the cells were organized in ordered, often wave-like patterns. The edge regions of the colonies (Fig. B, C, D, E) showed different morphology. In Fig. C, the cells are organized in thin protrusions; the longitudinal axis of individual cells is perpendicular to the edge of the colony. In Fig. D, microarea with very long cells is shown. In Fig. E, area with small and very long cells could be seen.

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Fig. 3. Ultrastructural characteristics of the 5-day-old C. difficile colonies. Ultrastructure is much more uniform than in the 2-day-old colonies. Locally, some cells are still organized in aligned shapes (A). Spores are embedded in the extracellular matrix (B) and could be present throughout the whole colony, (C). In some colonies, the edges of the colony are composed of vegetative cells only (D).

Scanning electron microscopy (SEM). For scanning electron microscopy, 2- and 5-day-old cultures were grown on commercial COH solid medium (bioMerieux) in anaerobic jars. Plates were inoculated from stock spore suspensions either directly on the plate or on nitrocellulose membrane filter (0.45 mm; Whatman). Three to five different colonies were collected with surrounding agar (either with or without the filter) by using razor blades and were fixed in 2.0% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 2 h and rinsed in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 1 h. Following post-fixation in 2% OsO4 for 2 h, the samples were dehydrated through graded ethanol solutions (30%, 50%, 70%, 80%, 90%, 96%, 100%; each 30 min), critical point dried, and gold coated by sputtering. The cells were observed and examined with a digital scanning electron microscope Zeiss DSM 950. 3. Results and discussion Colonies of six different strains from four PCR ribotypes were studied to examine the characteristics in ultrastrastructural morphology such as bacterial cell shapes, their organization, and distribution of spores and sporulating cells within the colony. Most of the strains in this study produced two colony morphotypes on COH agar; large irregular colonies and small round colonies. It is known that these two colony types could differ in at least one

genetic trait, in cell wall protein Cwp84 [7,8]. Due to technical difficulties during the fixation of small colonies only large, typical colonies were studied here with SEM. Gross colony morphology differed between two- and five-dayold culture but not between cultures grown on the filter or in direct contact with agar. Also, none of the PCR ribotypes showed any specific feature in colony ultrastructure. In all studied strains the colonies of 2-day-old cultures were rough and wrinkled while the 5-day-old colonies had a smoother surface (Fig. 1). This morphology was in good correlation with organizational patterns of bacterial cells and with the presence or absence of extracellular matrix in two- or in five-day-old colonies (Figs. 2 and 3). Within one and the same 2-day-old colony several different structural motives were present. None of them was specific for a single ribotype. The most prominent feature of 2-day-old colonies was the distribution of the cells throughout the colony: two well defined areas could be discriminated, defined as the core region and the edge region (Fig. 2). The core region represents the main area of the colony and despite a very dynamic colony surface at lower magnifications (Fig. 1) the bacterial cells within the core region were perfectly organized in curved patterns (Fig. 2A, B). This organization is lost at the colony margins (Fig. 2B, C). Similar patterns of organized cells were reported also for mycobacteria [3] and transition from chaotic distribution in initial inoculums to organization in aligned patterns within 2 h was observed at early stages of

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Fig. 4. Spores and sporulating cells in 5-day-old colonies. Spores and sporulating cells were present throughout the colony, also at the colony edges (A, C). Sporulating cells were short (strain E5; A, B) or longer and organized in chains (CeE; strain E20). Fine fibrillar material (indicated by white arrow) was occasionally present on the surface of the colony, overlying individual cells (B, C).

Fig. 5. Motility of the cells within C. difficile colony. Invasiveness into the agar was observed in single cells (white arrow; A) or in rhizoid outgrows at colony edge (C). Spreading of cells away from the colony indicating a gliding motility was often observed (B, D).

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Escherichia coli colony formation [13]. Such spatial cell organization seems to be important for intracolony differentiation and intercellular communication [14]. At the periphery of 2-day-old colonies different microareas were detected. Some of them were characterized by various distributions of cells (Fig. 2C). Other microareas were characterized by bacterial cells expanding away from the colony and this will be further explained below (Fig. 5). The third type of microareas was characterized by the presence of very small and/or very long cells (Fig. 2D and E). The 2- and 5-day-old colonies differed in the cell types present, distribution of cells and not surprisingly in the presence of spores. In contrast to 2-day-old colonies the ultrastructure of the 5-day-old colonies was much more uniform (Fig. 3). No particular microareas were observed and no variability in cell length was apparent. Occasionally, vegetative cells were still organized in aligned shapes (Fig. 3A). Large regions within the colony were composed of spores and sporulating cells. Spores and vegetative cells were embedded in extracellular matrix characteristic of biofilm formation (Fig. 3B, C). Extracellular matrix could be also absent in larger areas of the colony (Fig. 3A). Biofilm formation is one of the bacterial virulence properties but is only partially studied in C. difficile [15,16]. Our results show that all studied ribotypes are capable of producing extracellular matrix. Also, absence of extracellular matrix in 2-dayold colonies and its presence in 5-day-old colonies are in agreement with increasing amount of matrix present from two to six days old biofilms [15]. Spores were present in all strains at day five. Regions with spores and sporulating cells were sometimes seen throughout the colony or were present only in the core regions while edges were composed only of vegetative cells (Fig. 3B, D; Fig. 4). Sporulating cells could be shorter or long (Fig. 4A, B, C, E). Fibrillar structures, either single or in network, were often associated with the presence of spores (Fig. 4B). Some ultrastructural motives indicating motility were observed. Typical rhizoid colony gross morphology could already indicate a gliding motility. Here we show that also on microscopic level cells expanding from the colony were often seen and this could be due to gliding motility (Fig. 5B, D). Gliding motility was shown as important virulence factor for Clostridium perfringens [17]. In this study, in the periphery of several colonies growth inside the agar was noted (Fig. 5C). Also individual cells could exhibit invasive character (Fig. 5A). Such invasive cells could have a role in penetration through the mucus during the colonization or in translocation through intestinal epithelium. C. difficile is not known as an invasive bacterium, however, cases of C. difficile bacteriaemia have been described [18,19]. Also, in a hamster model C. difficile cells were detected in polimorphonuclear cells, in enterocytes and in muscle cells [20]. In summary, we have for the first time described the ultrastructural morphology of C. difficile colonies and have shown that several populations exist within the colony differing in cell morphology (length), motility (invasiveness) and spatial organization.

Acknowledgements The authors would like to thank M. Medved and E. Bock for technical assistance. MR was supported by Slovenian Ministry of Education, Science, Culture and Sport (ERA-NET PathoGenoMics CDIFFGEN; grant 3211-09-000141).

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