Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe

Summary

Background

Toxins A and B are the primary virulence factors of Clostridium difficile. Since 2002, an epidemic of C difficile-associated disease with increased morbidity and mortality has been present in Quebec province, Canada. We characterised the dominant strain of this epidemic to determine whether it produces higher amounts of toxins A and B than those produced by non-epidemic strains.

Methods

We obtained isolates from 124 patients from Centre Hospitalier Universitaire de Sherbrooke in Quebec. Additional isolates from the USA, Canada, and the UK were included to increase the genetic diversity of the toxinotypes tested. Isolate characterisation included toxinotyping, pulsed-field gel electrophoresis (PFGE), PCR ribotyping, detection of a binary toxin gene, and detection of deletions in a putative negative regulator for toxins A and B (tcdC). By use of an enzyme-linked immunoassay, we measured the in-vitro production of toxins A and B by epidemic strain and non-dominant strain isolates.

Findings

The epidemic strain was characterised as toxinotype III, North American PFGE type 1, and PCR-ribotype 027 (NAP1/027). This strain carried the binary toxin gene cdtB and an 18-bp deletion in tcdC. We isolated this strain from 72 patients with C difficile-associated disease (58 [67%] of 86 with health-care-associated disease; 14 [37%] of 38 with community-acquired disease). Peak median (IQR) toxin A and toxin B concentrations produced in vitro by NAP1/027 were 16 and 23 times higher, respectively, than those measured in isolates representing 12 different PFGE types, known as toxinotype 0 (toxin A, median 848 μg/L [IQR 504–1022] vs 54 μg/L [23–203]; toxin B, 180 μg/L [137–210] vs 8 μg/L [5–25]; p<0·0001 for both toxins).

Interpretation

The severity of C difficile-associated disease caused by NAP1/027 could result from hyperproduction of toxins A and B. Dissemination of this strain in North America and Europe could lead to important changes in the epidemiology of C difficile-associated disease.

Introduction

Clostridium difficile infection results in a broad spectrum of disease ranging from mild diarrhoea to severe life-threatening conditions. Colonic injury and inflammation results from the production of two protein toxins: toxin A and toxin B. Isolates that produce neither toxin are non-pathogenic.¹ Moreover, some isolates produce toxin B only and can cause pseudomembranous colitis.² A small group of isolates produce a separate binary toxin in addition to toxins A and B; however, the role of this binary toxin in C difficile-associated disease is not known. Whether variations in disease severity can result from differences in toxin A and B production is unknown.

The genes encoding toxin A and toxin B are part of the pathogenicity locus (PaLoc), which is a short chromosomal segment carried by pathogenic strains of C difficile. Transcription analysis studies in reference strain C difficile VPI 10463 (toxinotype 0) have shown that production of toxin A and toxin B is coregulated and growth-dependent.³ The logarithmic phase is associated with strong expression of the tcdC gene and weak transcription of the genes encoding toxin A (tcdA), toxin B (tcdB), a positive regulator (tcdD), and a holin-like protein (tcdE).³ The inverse is seen during the stationary phase, suggesting that tcdC negatively regulates toxin expression.³ Variations in the PaLoc sequence can be detected by toxinotyping;⁴ 22 toxinotype variants (I–XXII), in addition to the reference toxinotype 0, have been characterised. Analysis of two large isolate collections showed that 78–88% isolates were toxinotype 0 and 2–3% were toxinotype III.4, 5, 6 Whether some toxinotypes are more virulent than others is not known.

In 2002, hospitals in Montreal and southern Quebec, Canada, began experiencing an epidemic of C difficile-associated disease. Between 2003 and 2004, about 14 000 nosocomial cases of the disease were reported.⁷ In January, 2005, 30 hospitals in Quebec reported rates of nosocomial disease higher than 15 per 10 000 patient-days, at least five times greater than the historical average.7, 8 At the Centre Hospitalier Universitaire de Sherbrooke in Quebec, the proportion of patients with C difficile-associated disease who died within 30 days after diagnosis rose from 4·7% in 1991–92 to 13·8% in 2003, suggesting increased virulence of C difficile.⁹ The incidence of C difficile-associated disease per 100 000 individuals aged 65 years or more in Sherbrooke increased from 102 in 1991–92 to 210 in 2002 and 866 in 2003.⁹

Because toxins A and B are the primary virulence factors of C difficile, we postulated that increased virulence could be due to increased toxin production. Thus, we attempted to identify the epidemic strain at Sherbrooke and we compared toxin A and B production in this strain with that in other contemporary isolates.