Detection of capsule in strains of Clostridium difficile of varying virulence and toxigenicity

Microbial Pathogenesis 1990 ; 9 : 141-146

Detection of capsule in strains of Clostridium difficile of varying virulence and toxigenicity H . A . Davies' and S . P . Borriello 2 * 'Electron Microscopy Support and 'Microbial Pathogenicity Research Groups, Clinical Research Centre, Watford Road, Harrow, Middlesex HA 1 3UJ, U . K. (Received April 23, 1990 ; accepted in revised form June 12, 1990)

Davies, H . A . (Electron Microscopy Support Research Group, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, U .K .) and S . P . Borriello . Detection of capsule in strains of Clostridium difficile of varying virulence and toxigenicity . Microbial Pathogenesis 1990; 9 : 141-146 . Nine toxigenic and six non-toxigenic strains of Clostridium difficile, of varying virulence in the hamster model of antibiotic-associated colitis, were examined for the presence of a capsule . Antibody stabilisation of the capsule with heterologous and/or homologous antiserum fixed in glutaraldehyde, or direct fixation in glutaraldehyde/diamine, were used with added ruthenium red to stain the capsular glycocalyx . All strains possessed a capsule which was either looseknit or compact, sometimes with attached globular masses . Better capsule preservation was achieved in some strains with glutaraldehyde/diamine/ruthenium red fixative than with homologous or heterologous antibody stabilisation . The possession of a capsule following in vitro growth does not appear to correlate with the virulence status of these strains of C. difficile . Key words : capsule ; antibody stabilisation ; Clostridium difficile .

Introduction

Clostridium difficile

is the aetiological agent of antibiotic-associated pseudomembranous colitis (PMC) in man . Toxigenic strains exhibit a range of virulence in the hamster model of the disease,' suggesting that virulence factors other than toxins may be involved . Fimbriae, capsules and hydrolytic enzymes have been described as potential virulence factors in C. difficile.

2-4

In patients with PMC there is a polymorphonuclear leukocyte (PMNL) infiltration into the lamina propria of colonic villi .' Furthermore, in in vitro studies, C. difficile has been shown to be resistant to phagocytosis by PMNLs s and it is suggested that the anti-phagocytic factor may be a capsule on these organisms . Strelau et al.' published indirect evidence indicating the presence of a capsule on four strains of C. difficile. The possession of a capsule by bacterial species is an important virulence factor in

vivo .' There are several published methods available by which to preserve the capsule in its hydrated form for examination by electron microscopy, based on antibody stabilisation,"' less stringent processing" or the use of stains, such as ruthenium red, that react with polysaccharides to yield an electron-dense prod uct . 12 This study was undertaken to examine several well-characterised strains of C .

* Author to whom correspondence should be addressed . 0882-4010/90/080141 +06 $03 .00/0

Cu) 1990 Academic Press Limited

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H . A . Davies and S . P . Borriello

Table 1

Methods of capsule preservation of C . difficile strains of different virulence and toxigenic status Method of capsule preservation Strain B-1 PT 2B P-1 MA SC DD Bat Tra 5/5 M-1 MN PE P1 KN RY

Virulence

Toxigenic status

Antibody stabilisation'

Glutaraldehyde/ lysine

High High High High Low Low Low Low Low None None None None None None

+ + + + + + + + + -

+ ± ± + + + + + + +

+ + + + + + + + + + + + + +

± : A small proportion of cells in the ultrathin section possess capsule . 'Antibody raised to strain M-1 .

difficile, both non-toxigenic and toxigenic, for the presence of a capsule using different methods of preservation .

Results The presence of a layer of electron-dense material on the cell wall in unstained ultrathin sections was taken as demonstrating the presence of a capsule . In general, a more distinct capsule was preserved on more organisms using glutaraldehyde/diamine/ ruthenium red fixative ; capsules in 14 of the 15 strains were preserved using this fixative compared with 10 using antibody stabilisation (Table 1) . However, in strain B-1 capsulate cells were seen only after antibody stabilisation, and in strain 2B only a small number of capsulate cells were found using either method . In strains showing no capsule by a given method, further blocks were sectioned to overcome any sampling error but no capsulate cells were found . In the four strains where capsule stabilisation with homologous antiserum was used, capsule was demonstrated on all the cells of the strains except strain DD . Only a small proportion of cells of this strain possessed a capsule . Antiserum against strain M-1 reacted with two of the three heterologous strains, though the corresponding heterologous antisera did not react with strain M-1 . Thus, M-1 antiserum was chosen for use in antibody stabilisation of the capsule in the remaining 11 strains in the study . Strain DD provides an example of the effectiveness of the different methods . In this strain no capsule was detected after treatment with heterologous antiserum [Fig . 1 (a)], but a capsule was seen on some cells after treatment with homologous antibody [Fig . 1 (b)] . In contrast, using glutaraldehyde/diamine/ruthenium red fixation, capsule was found to be present on all cells of this strain [Fig . 1(c)] . There were two morphological types of capsule in the different strains : (1) dense with or without globular masses [Fig . 1 (c)], and (2) loose-knit and branching up to

Capsule of Clostridium difficile

143

Fig . 1 . Capsule preservation (a) after heterologous antibody stabilisation of strain DD ; (b) after homologous antibody stabilisation of strain DD, both followed by glutaraldehyde/ruthenium red fixation ; (c) after glutaraldehyde/lysine/ruthenium red fixation of strain DD, showing a compact capsule with globular masses (arrows), and (d) after glutaraldehyde/lysine/ruthenium red fixation of strain PI, showing a looseknit branching capsule (arrows) . All sections are unstained . Bar represents 100 nm .

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H . A . Davies and S . P . Borriello

350 nm from the cell wall [Fig . 1 (d)], and both were demonstrated with both methods of capsule preservation .

Discussion and conclusions The capsule/glycocalyx of both prokaryotes and eukaryotes is a highly hydrated carbohydrate-containing macromolecule . The exposed carboxyl groups that are present react with ruthenium red which produces an electron-dense product after reaction with osmium tetroxide . 12 Because of this hydrated nature, the chemical dehydration of bacterial capsules during processing for electron microscopy can cause shrinkage of the capsule onto the cell wall . Methods are available by which the capsule structure can be cross-linked to prevent much of this shrinkage, such as antibody stabilisation, which cross-links the capsule structure by virtue of bound immunoglobulin, 10 or incorporation in the primary fixative of lysine, a diamine, the positively charged amino groups of which cross-link the negatively charged moieties 13 in the capsule . In both of these methods the ruthenium red reaction is used to detect the presence of capsules . In this study these two methods of capsule preservation were used on 15 strains of

C. difficile of varying virulence and toxigenicity and capsules were demonstrated on strains by one or other method, although there was little correlation between possession of capsule and virulence status. Several of the strains in this study were examined previously, using primary and secondary fixation, with and without added ruthenium red and no extracellular material was seen . Using the methods described, we have demonstrated the presence of capsules after LR White resin e mbedding . L R White resin was chosen because preliminary studies on one strain indicated the resulting preserved capsule was of greater diameter than when Spurr resin was used (unpublished data) . There was some variation of capsule production by individual strains that may be due to cultural conditions . Indeed capsule production appeared to be time-dependent in one strain that was examined in detail, with optimal production after 48 h growth (unpublished data) . Five strains revealed a capsule only after diamine cross-linking, which suggests that the heterologous antiserum used did not react with the capsules of these strains . One strain did not reveal a capsule after diamine cross-linking, but one was present after antibody stabilisation, which would suggest a lack or low number, of exposed carboxyl groups . The two morphological types of capsule observed may reflect different degrees of cross-linking ; the branching capsule having greater cross-linking than the compact capsule thus being more resistant to shrinkage, which also reflects the exposed carboxyl groups . The compact capsule sometimes has attached globular masses as seen in Fig . 1 (c), which we regard as condensed capsule and not lysine/glutaraldehyde/ ruthenium red aggregates as they are not found in every strain . Strelau et al.' reported that the presence of a halo around epoxy resin-embedded C . difficile was indicative of capsule . Their preparation of bacterial cells for electron microscopy was based upon that used by Bayer et al." to preserve the polysaccharide capsule domain of Escherichia co/i where the capsule fibrils in an electron-lucent halo were localised using capsule-specific IgG and protein A/gold . Strelau et a/.' have, therefore, only indirectly indicated the presence of capsular material . Because these workers have not localised the capsule specifically, either by staining methods or probing with antibody, we would judge their interpretation to be invalid . Recently, capsule has been demonstrated on another anaerobe, Bacterioides fragilis, where a ruthenium red layer' on the cell wall was shown ." This is the first direct demonstration of capsule on C . difficile in vitro . The possession

Capsule of Clostridium difficile

145

of capsule appears not to correlate with the toxigenicity or virulence status . Future work will attempt to establish whether capsules are present on organisms in vivo .

Materials and methods Bacterial culture . Fifteen strains of C. difficile were grown on Columbia agar (Difco) with 5% horse blood for 48 h at 37°C in an anaerobic cabinet (Forma Scientific, Ohio) . Preparation of antisera . The strains of C. difficile were inoculated into BHI broth (Difco) from Columbia blood agar and incubated for 7 h at 37°C anaerobically . The broth cultures were centrifuged at 10178xg for 40 min . The resulting pellets were washed twice in phosphate buffered saline (PBSA), resuspended in 50 ml of 0 .4% formol saline and stored at 4°C . Each of the formalised antigens was injected subcutaneously into the scruff of the neck of 3-3 .5 kg New Zealand white rabbits at 3-4 day intervals with increasing doses of antigen (0 .5, 0 .75 and 1 .0 ml, and then 0 .5-ml increments up to 3 .0 ml) . Rabbits were bled by cardiac puncture 1 week after the final injection . The separated sera were deprived of complement by heating at 60°C for 30 min to avoid cell lysis during the reaction with the antibody . Antibody stabilisation of capsule . All the bacterial growth from a plate was suspended in 200 PI of PBSA and an equal volume of neat complement-deprived homologous or heterologous rabbit antiserum was added . After gentle vortexing, the suspension was incubated for 1 h at room temperature and washed twice in PBSA by microcentrifugation . The pellet was suspended in 3% glutaraldehyde in 0 .1 M sodium cacodylate buffer, pH 7 .2, containing 0 .1% ruthenium red for 2 h at room temperature and washed three times in cacodylate buffer containing ruthenium red ." Fifteen strains were reacted with the same antiserum, this being heterologous in 14 of the cases ; four of these strains, M-1, B-1, DD and Bat, were reacted with homologous antisera and M-1 with antisera against the other three strains . Glutaraldehyde/diamine fixation. Bacterial growth was suspended in 3% glutaraldehyde in 0 .1 M sodium cacodylate buffer, pH 7 .2 with 10 MM L-lysine (L-5501 Sigma) containing 0 .1% ruthenium red as above for 10 min at room temperature ." The fixative was then replaced with fresh fixative, excluding L-lysine, for a further 2 h at room temperature, and the samples were washed as above . Electron microscopy . The samples were post-fixed in 1 % osmium tetroxide in cacodylate buffer containing ruthenium red for 2 h at room temperature . In some experiments, the cells were enrobed in 1 .5% low-gelling-temperature agarose Type VII (A-4018, Sigma) in distilled water . In others, the cells were processed in microcentrifuge tubes . All samples were dehydrated through a graded series of ethanol-water mixtures : 30, 50, 70, 80 and 90% ethanol for 5 min each, then 100% ethanol for 60 min with three changes . The blocks were held overnight in a 50 : 50 mixture of hard mix LR White resin (London Resin Company) and ethanol . The samples were subjected to four changes of LR resin before final anoxic polymerisation at 50°C for 24 h . Ultra-thin sections were cut on a LKBV ultramicrotome, stabilised with carbon and examined without post-staining in a Philips EM 300 electron microscope .

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