Ribotyping for strain characterization of Clostridium perfringens isolates from food poisoning cases and outbreaks

Abstract

Ribotyping was used to characterize 34 Clostridium perfringens strains isolated from 10 food poisoning cases and outbreaks over a 7-year period. Twelve different ribopatterns were generated by EcoRI digestion. In eight food poisoning cases and outbreaks, all of the ribotypes of each food and stool isolate were found to be identical. Two C. perfringens isolates showed unique patterns. Ribotyping was found to be a useful tool for determining the genetic relationship of C. perfringens isolates in the context of foodborne poisoning cases. Clostridium perfringens is a gram-positive, spore-forming, anaerobic rod. This bacterium can be responsible for food spoilage and can produce an enterotoxin (CPE) which is released upon lysis of the vegetative cell during sporulation in the intestinal tract. CPE causes food poisoning in humans and some animals (2, 8, 10). C. perfringens is also a part of the normal intestinal flora. Therefore, typing of C. perfringens is of great importance for investigating food poisoning sources and for studying the epidemiology of this microorganism. Phenotypical differentiation procedures as well as DNAbased typing methods have been used successfully for strain differentiation of C. perfringens. Among them were plasmid isolation (3, 7), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6), and ribotyping of patients' and hospital environment isolates (4). This report describes the examination of 34 food poisoning-related C. perfringens isolates by ribotyping (5). MATERIALS AND METHODS Strains. Table 1 shows the 34 C. perfringens isolates investigated from 10 foodborne outbreaks and cases which occurred between 1984 and 1991 in Eastern Germany. Two to six isolates from foods and patient stool samples were available for each outbreak and case. Isolates were cultured, purified, identified, and kept in a Microbank storage system (Mast Diagnostica, Reinfeld, Germany) at Ϫ18°C as described by Eisgruber et al. (3). Before the ribotyping, the purity of the isolates was reensured by culture of the isolates twice on Columbia sheep blood agar (Unipath, Ltd., Basingstoke, Hampshire, United Kingdom). Ribotyping. Ribotyping was carried out according to the method described by Grimont and Grimont (5). Clostridia were grown anaerobically overnight in brain heart infusion broth (Unipath Ltd.). An aliquot of 1.5 ml of that suspension was centrifuged (13,000 ϫ g, 10 min). DNA was isolated by the guanidium thiocyanate method of Pitcher et al. (9) with the modifications described by Björkroth and Korkeala (1). Five micrograms of DNA was cleaved with EcoRI (Boehringer Mannheim GmbH, Mannheim, Germany) according to the manufacturer's instructions. The DNA concentrations were determined with a UV spectrophotometer (UV/VIS spectrometer Lambda 2; Perkin-Elmer, Norwalk, Conn.). DNA fragments were separated in 0.8% agarose gels (16 h, 25 V). Digoxigenin (DIG)-labeled phage lambda DNA (Boehringer Mannheim GmbH) was used as a molecular size marker. After gel electrophoresis, DNA fragments were transferred by the met...

Molecular methods for the analysis of Clostridium perfringens relevant to food hygiene

“…DNA was isolated using the guanidinium thiocyanate method of Pitcher et al [22] with modi¢cations [12]. Ensuing procedures were carried out as described previously [23]. The DNA probe was prepared from Escherichia coli 16S and 23S rRNA (Boehringer, Mannheim, Germany) according to the manufacturer's instructions.…”

Ribotyping of Clostridium perfringens from industrially produced ground meat

“…In this study, a convincing reproducibility was observed, as reported before for Cl. perfringens (Schalch et al 1997). The discriminatory index for ribotyping of Cl.…”

“…, Yersinia enterocolitica (Lobato et al 1998), and Cl. perfringens (Forsblom et al 1995;Schalch et al 1997).…”

“…10 of all 111 Cl. perfringens isolates were purely coincidental selected and ribotyped in three different runs, carried out 3-4 months apart from each other (Schalch et al 1997).…”

“…Ribotyping was reported to be an excellent technique for the typing of Cl. perfringens isolates (Forsblom et al 1995;Schalch et al 1997). Typing of Cl.…”