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Clinical features of Clostridium difficile-associated diarrhoea due to binary toxin (actin-specific ADP-ribosyltransferase)-producing strains

¹Service de Microbiologie, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75012 Paris cedex 12, France ²Hôpital Beaujon, 92118 Clichy, France ³Hôpital Cochin, 75679 Paris cedex 14, France ⁴Hôpital Ambroise Paré, 92104 Boulogne Billancourt cedex, France ⁵Hôpital Lariboisière, 75475 Paris cedex 10, France ⁶Hôpital Pitié-Salpétrière, 75651 Paris cedex 13, France ⁷Centre Hospitalier de Montfermeil, 93370 Montfermeil, France ⁸UPRES no. EA2392, Faculté de Médecine, UFR Saint-Antoine, Université Paris 6, France

Correspondence Frédéric Barbut frederic.barbut{at}sat.ap-hop-paris.fr

Received June 30, 2004
Accepted September 30, 2004

Toxins A and B are known to be the primary virulence factors of Clostridium difficile. Other potential virulence factors have been identified such as binary toxin (actin-specific ADP-ribosyltransferase toxin, or CDT). A retrospective case–control study was performed in order to identify clinical features and risk factors of C. difficile-associated diarrhoea due to binary toxin-producing strains. Each case (a patient with diarrhoea due to binary toxin-producing strain) was compared with two controls (patients with diarrhoea due to a C. difficile strain that did not produce binary toxin) matched for ward and date of hospitalization. cdtA and cdtB genes were screened by PCR. Production of CDT was studied by Western blotting using an antiserum against Ia and Ib from the Clostridium perfringens iota toxin, and the activity of the binary toxin was assessed using an ADP-ribosyltransferase assay. Twenty-six cases (14 males and 12 females) were identified in 1999 and 2000. Cases and controls did not differ significantly for sex, age, previous administration of antibiotics or frequency of endoscopic examination. Diarrhoea was community-acquired more often in cases than in controls (65.4 vs 35.7 %, P = 0.017) and more often represented the cause of hospitalization (61.5 vs 26.2 %, P = 0.003). Moreover, diarrhoea in cases was more frequently associated with abdominal pain (63.6 vs 39.4 %, P = 0.07) and with liquid stools (76.9 vs 59.5 %, P = 0.14) than in controls. These results suggest that there could be a correlation between the production of binary toxin and the severity of diarrhoea.

Abbreviations: AAD, antibiotic-associated diarrhoea; CDAD, C. difficile-associated diarrhoea; PMC, pseudomembranous colitis.

	Introduction

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Clostridium difficile is a spore-forming, anaerobic, Gram-positive bacillus, responsible for almost all cases of pseudomembranous colitis (PMC) and for 15–25 % of cases of antibiotic-associated diarrhoea (AAD) (Kelly et al., 1994; Bartlett, 2002). In the last 20 years, C. difficile has also emerged as a major cause of nosocomial diarrhoea in adult patients and has been implicated in large outbreaks in hospital settings (McFarland et al., 1989; Cartmill et al., 1992).

The main virulence factors are the two large clostridial cytotoxins: toxin A (TcdA) and toxin B (TcdB). TcdA and TcdB both disrupt the actin cytoskeleton of intestinal epithelial cells by the UDP–glucose-dependent glycosylation of proteins from the Rho and Ras subfamilies (Just et al., 1995a, b). A third toxin, named CDT (actin-specific ADP-ribosyltransferase), was described from the C. difficile strain CD196 in 1988 (Popoff et al., 1988). CDT belongs to the group of clostridial binary toxins, which include the iota toxin of Clostridium perfringens type E, the toxin of Clostridium spiroforme and the C2 toxin of Clostridium botulinum C and D (Hateway, 1990; Popoff & Boquet, 1988; Popoff et al., 1989). CDT is composed of two independent unlinked protein chains, CDTa (the enzymic component) and CDTb (the binding component) (Considine & Simpson, 1991; Gülke et al., 2001). The binding component recognizes a cell-surface receptor, resulting in internalization into the cytosol of the enzymic component, which catalyses the ADP-ribosylation of monomeric actin and leads to disorganization of the cytoskeleton (Aktories & Wegner, 1992). CDT is cytotoxic to Vero cells in culture (Perelle et al., 1997) and the cytotoxicity is neutralized by anti-Ib antibodies directed against the iota toxin of C. perfringens. CDT is encoded by two genes (cdtA and cdtB), which have been cloned and sequenced in C. difficile strain CD196 (Perelle et al., 1997). The prevalence of binary toxin genes in human C. difficile isolates varies from 1.6 to 20.8 % (Goncalves et al., 2004; Rupnik et al., 2003). To date, little is known about the clinical relevance and pathogenic role of the binary toxin CDT in C. difficile-associated human infections. The aims of this study were to characterize the clinical features and to identify risk factors of C. difficile-associated diarrhoea due to a binary toxin-producing strain.

	Methods

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Patients.

We retrospectively studied 26 patients (= cases) presenting with an AAD due to a binary toxin-producing strain of C. difficile. These patients were hospitalized in six different care facilities in Paris or the surrounding area during 1999 and 2000. Each case was compared with two controls. Controls were defined as patients with an AAD due to a TcdA- and TcdB-positive and a binary toxin-negative C. difficile strain. Controls were matched with cases on the ward and the date of hospitalization (± 3 months). Clinical charts of cases and controls were reviewed using a standardized questionnaire.

Strains were isolated from stools by culture on selective medium (TCCA: brain heart infusion agar supplemented with 5 % defibrinated horse blood, 0.1 % sodium taurocholate, 10 µg cefoxitin ml^–1 and 250 µg cycloserine ml^–1) in an anaerobic atmosphere at 37 °C for 48 h.

Detection of the cdtA and cdtB genes.

DNA was extracted with the Instagene Matrix kit (Bio-Rad). PCR primers designed to amplify regions of cdtA and cdtB and PCR reactions were as previously described by Stubbs et al. (2000). Detection of cdtA and cdtB was confirmed by Southern blot analysis. Genomic DNA was digested with HindIII (Ozyme) and the restriction fragments were separated by electrophoresis on a 0.8 % agarose gel. They were transferred to a nylon membrane (Pharmacia Biotech) and probed with internal fragments of the cdtA and cdtB genes (353 and 490 bp, respectively) from strain CD196. The probe fragments were obtained by PCR and labelled with [³²P]dCTP (Boehringer), according to the manufacturer's instructions.

Detection of the binary toxin CDT by Western blotting and ADP-ribosyltransferase assay.

Western blotting was performed as described by Popoff et al. (1988). All strains positive for cdtA and cdtB were cultured in Wilkins–Chalgren broth for 48 h. Proteins were then precipitated from the culture supernatant with 70 % ammonium sulfate. These supernatant proteins (30 µg) dissolved in 200 µl distilled water were dialysed overnight against PBS and concentrated with Aquacide II (Calbiochem Biosciences). They were subjected to SDS-PAGE in 10 % acrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked by incubation for 1 h in 5 % skimmed milk powder in PBS and incubated overnight at room temperature with rabbit polyclonal antisera (diluted 1 : 5000) specific for the enzymic (Ia) or binding (Ib) components of C. perfringens iota toxin. Bound antibody was detected with peroxidase-labelled protein A and the Signal Plus kit (Pierce Chemical).

The ADP-ribosyltransferase assay was performed according to the method described by Stubbs et al. (2000). Concentrated supernatant proteins (10 µg), prepared as described above, were incubated at 37 °C for 1 h with 50 µl 50 mM triethanolamine/HCl (pH 7.5), 5 mM MgCl₂, 10 mM dithiothreitol, 10 mM thymidine and [³²P]NAD (10⁶ c.p.m. per reaction). The reaction mix also contained brain extract as a source of actin (10 µg). Proteins were then precipitated by the addition of 20 µl of a solution containing 1 mg BSA ml^–1, 10 % (w/v) SDS and 0.5 ml trichloroacetic acid (10 %, w/v); the reaction mixture was incubated on ice for 1 h. The precipitate was dissolved in distilled water and subjected to 10 % SDS-PAGE. After radiography, qualitative differences in ADP-ribosyltransferase activity were assessed by eye.

Production of TcdA and TcdB in vitro.

Strains were incubated anaerobically in brain–heart infusion broth for 5–7 days as described by Barbut et al. (1993). TcdA production was detected with the Premier C. difficile Toxin A assay (Meridian Diagnostics) according to the manufacturer's instructions. TcdB was detected by a cytotoxicity assay using MRC-5 monolayers (bioMérieux): supernatants were filtered through a 0.45 µm pore-size filter and 10x dilutions were applied to cells grown in 96-well plates. The characteristic cytotoxic effect (cell rounding) was observed after 18 h at 37 °C (Barbut et al., 1993).

Statistical analysis.

All data were analysed with Epi Info software (6.04d; CDC, Atlanta, GA, USA). Qualitative variables were compared using the S² test or Fisher's exact test and quantitative variables with the Wilcoxon test. P < 0.05 was considered significant.

	Results and Discussion

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Detection of TcdA and TcdB, cdtA and cdtB and CDTa and CDTb

All strains from both cases and controls were TcdB-positive by the cytotoxicity assay and TcdA-positive by ELISA. All strains from the cases contained both cdtA and cdtB genes. The culture supernatant of strains from cases reacted with anti-Ia and anti-Ib antiserum from C. perfringens iota toxin and exhibited a positive actin-specific ADP-ribosyltransferase activity (data not shown).

Comparison of cases and controls

Twenty-six cases (14 males and 12 females) were compared with 42 controls (23 males and 19 females). All cases and controls were in-patients. Ten cases could be matched to only one control. The age of patients, previous administration of antibiotics, frequency of endoscopic examination and the presence of fever >38.5 °C and hyperleukocytosis were not statistically different between cases and controls (Table 1). The duration of hospitalization after the onset of diarrhoea was longer in cases and stools were more frequently liquid or loose than in controls, but the differences were not statistically significant. Using univariate analysis, diarrhoea due to a binary toxin-positive strain was more often community-acquired (65.4 vs 35.7 %, P = 0.017) and associated with abdominal pain (63.6 vs 39.4 %, P = 0.07) than diarrhoea due to binary toxin-negative strains. Diarrhoea was more often the cause of hospitalization in cases than in controls (61.5 vs 26.2 %, P = 0.003). Cases (88.5 %) and controls (82.9 %) were specifically treated with oral metronidazole or vancomycin, respectively. Improvements in diarrhoea and the rate of relapse during the following 2 months were similar in cases and controls (76 vs 73.7 %, P = 0.9, and 4 vs 10.5 %, P = 0.46, respectively).

Pathogenic role of binary toxins

Binary toxins produced by some clostridia could play a role in human and animal digestive diseases. For example, iota toxin from C. perfringens type E has been implicated in animal enterotoxaemia (Borriello & Carman, 1985), although the production of other toxins could explain the pathogenicity. In contrast, binary toxin is the only virulence factor identified to date in C. spiroforme, which is responsible for rabbit enteritis, and in one case of human colitis (Babudieri et al., 1986; Petit et al., 1999). In addition, C. difficile CD196, the strain in which CDT was first identified, was isolated from a woman with severe PMC (Popoff et al., 1988).

Little is known about the clinical relevance and pathogenic role of the ADP-ribosylating toxin CDT in C. difficile human infections. C. difficile induces diseases of varying severity, from mild diarrhoea to PMC colitis, toxic megacolon and even fulminant colitis. Differences in the levels of production of toxins TcdA and TcdB alone cannot account for the wide spectrum of clinical presentations. Georges et al. (1982) reported that there were no significant differences between clinical presentations relative to the titre of cytotoxin. Similarly, there was no correlation between virulence in a hamster model of AAD and production of TcdA and TcdB in vitro (Borriello et al., 1987). Therefore, C. difficile binary toxin CDT could be expected to be an additional virulence factor and may act in synergy with other toxins, depolymerizing the actin cytoskeleton by a complementary mechanism.

To our knowledge, the study we have performed is the first clinical study aimed at evaluating the role of binary toxins in human disease. Our results show that C. difficile-associated diarrhoea (CDAD) due to a binary toxin-producing strain was more often associated with community-acquired diarrhoea and with abdominal pain than CDAD due to a binary toxin-negative strain. Moreover, diarrhoea due to a binary toxin-positive strain was more often the cause of hospitalization of patients, indicating the severity of the diarrhoea. These results suggest that binary toxin could be an additional virulence factor and could be associated with a more severe form of disease. Nevertheless, we assume that the retrospective design of this study represents a major bias: the evaluation of the severity of diarrhoea is a difficult issue if the study is not specifically designed for it. Additional prospective clinical studies are needed to confirm these preliminary results before recommending routine testing for binary toxin.

The prevalence of binary toxin genes in human clinical isolates of C. difficile varies from 1.6 to 20.8 % (Rupnik et al., 2003; Goncalves et al., 2004; Terhes et al., 2004). Most binary toxin-positive strains are isolated from C. difficile strains that are also positive for TcdA and TcdB; however, an interesting feature of these strains is that they belong to specific toxinotypes that are different from toxinotype 0 (Rupnik, 2001). These toxinotypes are characterized by changes in tcdA and tcdB genes in comparison with the reference strain VPI 10463. However, binary toxin-positive strains have also been isolated from TcdA- and TcdB-negative strains. A recent study showed that the prevalence of binary genes in non-cytotoxic strains (TcdA- and TcdB-negative strains) is 1.9 % (Geric et al., 2003). These strains are of particular interest because they represent a suitable model for studying the role of binary toxins in pathogenesis. They have been used in the hamster model of ileocolitis and in the rabbit ileal ligated loop. The results have been recently presented and at first glance seem contradictory (S. Johnson, B. Geric, R. J. Carmen, M. Rupnik, S. P. Sambol, M. Grabnar, D. M. Lyerly & D. N. Gerding, unpublished data). Indeed, these strains produced no symptoms in the hamster model, despite colonization, but culture supernatants of the same strains were positive in the ileal loop model. These contradictory results raise the question of the suitability of the hamster model, which has previously shown limitations for studying the pathogenesis of some C. difficile strains, i.e. strains from toxinotype VIII (TcdA^– TcdB⁺).

In conclusion, preliminary results suggest that binary toxin in C. difficile strains could be associated with a more severe form of human disease, but these results must be confirmed by prospective studies before recommending testing for these toxins in clinical practice.

	ACKNOWLEDGEMENTS

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This study was supported by grants from the Unité Propre de Recherche de l'Enseignement Supérieur (UPRES) no. EA2392. We thank M. R. Popoff (Pasteur Institute, Paris) for providing strain CD196 and the antibodies against the iota toxin of C. perfringens.

	References

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