Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Wed, 15 Jun 2016 14:23:23 Journal of Medical Microbiology (2005), 54, 159–162 DOI 10.1099/jmm.0.45809-0 45809 & 2005 SGM Printed in Great Britain 159 Correspondence R. Alonso ralonso.hgugm@salud. madrid.org Received 2 July 2004 Accepted 27 September 2004 Toxigenic status of Clostridium difficile in a large Spanish teaching hospital R. Alonso, A. Martı ´n, T. Pela ´ ez, M. Marı ´n, M. Rodrı ´guez-Creixe ´ ms and E. Bouza Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario ‘‘Gregorio Maran ˜o ´ n’’, C/Doctor Esquerdo, 46, 28007 Madrid, Spain The aim of this study was to evaluate the toxigenic status of circulating strains of Clostridium difficile in a large teaching hospital. Overall 220 isolates were studied of which 199 (90 . 5 %) produced both large clostridial toxins detected by conventional methods. Ten more strains (4 . 5 %) had toxin A and B genes detectable by PCR. Eleven (5 . 0 %) variant strains (A  B þ ) were detected among the isolates studied and 10 strains (4 . 5 %) had the binary toxin genes (cdtA and cdtB). INTRODUCTION Clostridium difficile is the main aetiologic agent of antibiotic- associated diarrhoea (CDAD) (Bartlett, 1992; Kelly et al., 1994) and is responsible for both sporadic cases and epidemic outbreaks (Johnson et al., 1990). Most cases are nosocomially acquired, but community-acquired CDAD is being increas- ingly recognized (Hirschhorn et al., 1994; Kyne et al., 1998). C. difficile can produce two large clostridial toxins (LCTs), toxin A (TcdA), which is mainly enterotoxic, and toxin B (TcdB), which is highly cytotoxic. Both toxins disrupt the cytoskeleton by acting on regulatory proteins involved in actin polymerization (Lyerly et al., 1988). Traditionally, it was considered that toxigenic strains produced both LCTs whereas non-toxigenic strains did not produce either. Some years ago, the isolation of toxigenic strains that produced toxin B only was reported (A  B þ ) (Lyerly et al., 1992) and it was soon demonstrated that this phenomenon was not as rare among clinical isolates as previously thought (Kato et al., 1998; Depitre et al., 1993; Rupnik et al., 2003). CDAD is usually caused by strains producing both LCTs although A  B þ isolates may also cause the disease (Alfa et al., 2000). The presence of an additional toxin in C. difficile has recently been detected. This actin-specific ADP-ribosyltransferase toxin has been designated binary toxin (CDT) due to its two independent proteins, CDTa, the catalytic component, and CDTb, the binding component. C. difficile binary toxin is related genetically, immunologically and functionally to the group of clostridial binary toxins, which includes the well known iota toxin of Clostridium perfringens (Popoff et al., 1988). It is not clear how the production of the binary toxin by strains of C. difficile can determine its virulence. It has been suggested that, although strains that produce CDT only have a relatively low virulence, the toxin could act synergis- tically in strains that produce both LCTs (Stubbs et al., 2000; Geric et al., 2003). Data regarding the prevalence of CDT in C. difficile are scarce, but figures range from 4 to 12 . 0% (Perelle et al., 1997; Gulke et al., 2001). The aim of this study was to evaluate the toxigenic status of circulating strains of C. difficile in a large Spanish hospital with a high prevalence of CDAD. METHODS Strains and identification. Two hundred and twenty isolates of C. difficile were collected prospectively over a 6-month period (January– June 2001) from 1154 diarrhoeic stool samples submitted for C. difficile investigation (hospitalized patients treated with antibiotics). Samples were cultured on CCFA (cycloserine–cefoxitin–fructose agar) plates which were incubated under anaerobic conditions at 37 8C for 48 h. C. difficile isolates were presumptively identified by their colony morph- ology, yellow colour, ground-glass texture, characteristic horse-dung smell and Gram-stain appearance. Additional biochemical tests were also used (ATB 32A; bioMe ´ rieux). Only one isolate was collected from each positive sample. Toxin detection. The presence of C. difficile toxin B was determined by demonstrating a specific cytopathic effect on MRC-5 cells, as described previously (Chang et al., 1979; Bowman & Riley, 1988; Bartlett, 1994), either directly from fecal samples or, if negative, from pure cultures of the micro-organism. An enzyme immunoassay (CdTOX A OIA; BioStar) was used to detect the presence of toxin A in fecal samples. The test was repeated from pure cultures when a negative result was observed in the direct clinical specimen. Molecular methods. LCTs and cdt genes were detected by PCR assays. DNA was extracted from pure C. difficile cultures using a Chelex resin- based commercial system (InstaGene Matrix; Bio-Rad) following the manufacturer’s recommendations. The tcdA gene was detected using a previously published PCR assay (Kato et al., 1991). Briefly, oligonucleo- tides 59-CCC AAT AGA AGA TTC AAT ATT AAG CTT-39 and 59-GGA AGA AAA GAA CTT CTG GCT CAC TCA GGT-39 were used to prime This paper was presented at the First International Clostridium difficile Symposium, Kranjska Gora, Slovenia, 5 – 7 May 2004. Abbreviation: LCTs, large clostridial toxins.