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Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
Correspondence
Brendan W. Wren
brendan.wren{at}lshtm.ac.uk
Received 2 November 2007
Accepted 29 December 2007
Abbreviations: CDAD, Clostridium difficile-associated disease; pHPA, p-hydroxyphenylacetate.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Disturbingly, the reported incidence of CDAD has increased significantly in the last decade, with highly virulent strains causing outbreaks of increased severity in North America and Europe (Loo et al., 2005; McDonald et al., 2005; Pépin et al., 2005; Warny et al., 2005). The origin of these strains (called BI/NAP1/027 hypervirulent strains) is uncertain, although it has been proposed that increased use of fluoroquinolone antibiotics may give the strains a selective advantage (Pépin et al., 2005).
CDAD characteristically occurs after treatment with broad-spectrum antibiotics, which disrupt the normal gut microflora, allowing C. difficile to become established, although the mechanism(s) by which C. difficile establishes itself in the gut under these conditions is unclear. CDAD covers a wide range of diseases from asymptomatic or mild diarrhoea to moderately severe diarrhoea, and even pseudomembranous colitis, which can be fatal (Johnson & Gerding, 1998). Host factors and genetic differences in C. difficile strains account for some of the differences in disease severity, but the status of the human gut microflora prior to and after infection is also likely to be important, particularly in the case of relapse.
C. difficile produces a number of factors that contribute to its virulence, including two related toxins, called toxin A and toxin B (Lyerly et al., 1986). However, production of these toxins cannot completely explain C. difficile pathogenesis. In recent years, increasing numbers of pathogenic strains have been reported that have truncated versions of toxin A and/or B (Borriello et al., 1992; Depitre et al., 1993; Pituch et al., 2003; Toyokawa et al., 2003; van den Berg et al., 2004). Our understanding of other factors involved in C. difficile pathogenesis and survival in the gut is poor.
It has been demonstrated that C. difficile produces a phenolic compound, p-cresol, via the degradation of tyrosine (Scheline, 1968), and can withstand up to 0.5 % p-cresol, a concentration that inhibits the growth of other anaerobic bacteria (Hafiz & Oakley, 1976). To date, the mechanism and relevance of p-cresol production and tolerance have not been demonstrated. Our hypothesis is that it may provide the bacterium with a competitive advantage over other intestinal microflora and consequently contribute to the progression of CDAD. When the C. difficile strain 630 genome sequence was scrutinized (Sebaihia et al., 2006), an intriguing genetic pathway was discovered that allows C. difficile to produce the bacteriostatic compound p-cresol (4-methylphenol) from tyrosine via the intermediate p-hydroxyphenylacetate (pHPA). The genes involved include those encoding components of pHPA decarboxylate (CD0153–CD0155, hpdBCA) (Sebaihia et al., 2006), which catalyse the decarboxylation of pHPA to yield p-cresol (Selmer & Andrei, 2001).
Given the undoubted and unusual ability of C. difficile to produce and tolerate p-cresol, the aim of this study was to ascertain whether different strains of C. difficile, including the hypervirulent 027 strains, were able to tolerate different concentrations of p-cresol, and to compare the relative p-cresol tolerance with other clostridial species.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The strains covered a range of clostridial species, including C. difficile R20291 (Stoke Mandeville), a hypervirulent ribotype 027 strain, isolated from a recent outbreak in Stoke Mandeville (provided by Jon Brazier, Anaerobe Reference Laboratory, Cardiff, UK), which caused infection of over 300 people, along with an epidemic C. difficile 027 strain B1-16 (provided by Dale Gerding, Edward Hines Jr Veterans Affairs Hospital, Hines, IL, USA), which are both fluoroquinolone-resistant. C. difficile CD196 was the primary 027 ribotype (provided by Michel Popoff, Institut Pasteur, Paris, France), an historical strain, which is fluoroquinolone-sensitive. C. difficile CF4 (provided by Dale Gerding) was chosen because it falls in the A–B+ clade (Fig. 1), which lacks toxin A, thought to be an important virulence factor, whilst C. difficile strain 630 (Wüst & Hardegger, 1983) has been fully sequenced by the Wellcome Trust Sanger Institute (Sebaihia et al., 2006), thus providing useful additional information. C. difficile 630 was isolated in Switzerland from a patient with severe pseudomembranous colitis (provided by Dr Peter Mullany, Eastman Dental Institute, London, UK). Clostridium perfringens NCTC 8237 and Clostridium sordellii NCTC 6929 were provided by Peter Donachie (London School of Hygiene and Tropical Medicine).
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Viability assays. Viability assays were performed using the growth conditions outlined above. After 4 h incubation in the p-cresol (0–0.2 %), serial dilutions were performed in pre-equilibrated PBS and plated in triplicate onto BHI plates, which were incubated for 24 h under the anaerobic conditions described above. Colony counts were determined for all of the test conditions and calculated as c.f.u. (ml culture)–1. The p-cresol stress c.f.u. data were normalized to the untreated control and expressed as a percentage of the untreated control. Data were presented in graph form using GraphPad Prism version 4.02, and analysed using a two-tailed Student's t-test with a cut-off value of P<0.01 in Microsoft Excel to determine whether any of the strains showed a significant difference in level of tolerance to p-cresol compared with C. difficile 630.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). It appeared that the C. difficile 027 strains R20291 and BI-16 were significantly more tolerant to p-cresol than the historical C. difficile 027 strain CD196, and were more tolerant than C. difficile strains 630 and CF4 (Fig. 1a–e). This indicates that the newly emerging hypervirulent strains of C. difficile may tolerate a higher level of p-cresol, which, in turn, may be linked to the level of p-cresol production by these strains. Growth curves were also undertaken for C. perfringens (NCTC 8237) and C. sordellii (NCTC 6929) over a similar time course with the same concentrations of p-cresol (0–0.2 %). Both strains appeared to tolerate similarly high concentrations of p-cresol, in line with the hypervirulent C. difficile 027 clinical isolates R20291 and BI-16.
Viability assays were then performed with a selection of the strains to determine the number of c.f.u. after exposure to various concentrations of p-cresol (0–0.2 %) (data not shown, except for 0.1 %; Fig. 2). The viability assays clearly showed that there was a significant increase in viable c.f.u. for C. difficile R20291 compared with C. difficile 630 (Fig. 2), with a value of P=4.29x10–9, when treated with 0.1 % p-cresol, thus demonstrating that C. difficile R20291 tolerates a higher level of p-cresol than C. difficile 630, as also indicated by the growth curves (Fig. 1). Also, in line with the growth-curve data, C. difficile CD196 and CF4 were not significantly different from C. difficile 630 in terms of p-cresol tolerance at a 99 % confidence interval (P=0.062) (Fig. 2). The hypervirulent C. difficile strains R20291 and BI-16 were more tolerant to p-cresol than the more historical C. difficile strains 630 and CD196. This evolutionary difference between the historic and current epidemic strains may be a contributory virulence factor of the C. difficile 027 strains, which have spread rapidly throughout North America and Europe, causing widespread disease.
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Viability assays for the other clostridial species indicated that C. perfringens had a similar level of tolerance for p-cresol to C. difficile 630 (Fig. 2), whereas, interestingly, C. sordellii was able to tolerate a similar level of p-cresol to the hypervirulent C. difficile R20291 strain (Fig. 2). This is of particular interest as it may indicate an overlap in tolerance pathways, or may suggest that the mechanism for p-cresol tolerance has been transferred horizontally or may have evolved via the acquisition of point mutations in similar pathways. However, Sivsammye & Sims (1990) revealed using GLC that neither C. perfringens nor C. sordellii produce p-cresol. To address this issue further, BLASTN analysis was performed with the current published genome sequences available for C. perfringens strains ATCC 13124 and SM101 (Myers et al., 2006), with the C. difficile 630 genes hpdA, hpbB and hpdC, which encode the decarboxylase responsible for converting pHPA to p-cresol. No homologues were identified in C. perfringens. Currently, there is no genome sequence available for C. sordellii; however, there may be an overlapping pathway that has been adapted by C. sordellii to enable tolerance of p-cresol. It has been shown that several clostridial species produce pHPA from tyrosine, but do not decarboxylate it to p-cresol (Elsden et al., 1976). Interestingly, in Lactobacillus strains, p-cresol, 5-hydroxyskatol and methylcatechol are all produced using the same method of decarboxylation (Yokoyama & Carlson, 1981), adding further support to the possibility of overlapping degradation pathways.
The current data from viability assays, along with the GLC data (Sivsammye & Sims, 1990), indicate that the mechanism of p-cresol tolerance may not be linked to the production of p-cresol, as C. sordellii tolerates p-cresol, yet does not produce it. This hypothesis is supported by preliminary data from a hpdB mutant constructed in an erythromycin-sensitive C. difficile 630 strain (
630erm), which indicate that, although the strain lacks the decarboxylase (via mutation) that enables the conversion of pHPA to p-cresol, it is able to tolerate the same level of p-cresol as the wild type (L. F. Dawson, unpublished data).
Future experiments will measure p-cresol tolerance from a larger selection of C. difficile strains, and will also measure the extent of p-cresol production from various strains using a combination of GLC, NMR and MS.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  I. R. Poxton Proceedings from the 2nd International Clostridium difficile Symposium, Maribor, Slovenia, June 2007. J. Med. Microbiol., June 1, 2008; 57(Pt 6): 683 - 794. [Full Text] [PDF]
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, Untreated bacteria; 
, 0.025 % p-cresol; 
, 0.05 % p-cresol; 
, 0.1 % p-cresol; 
, 0.2 % p-cresol.