Impedance microbiology is a rapid method that enables qualitative and quantitative tracing of microorganisms by measuring the change in the electrical conductivity. With direct impedance technology, the change in the conductivity of a liquid culture medium serves as a measuring parameter, whereas with indirect impediometry, the change in the electrical conductivity of a reaction solution, which occurs through the absorption of gases from the inoculated bacterial culture, is measured. Most investigations concerning the applicability of impediometry in food microbiology deal with the impedimetric detection or enumeration of Enterobacteriaceae, especially the detection of Salmonella. However, impediometry has been applied to other bacterial groups or species as well. Furthermore, a great number of published findings concern the impedimetric determination of the total bacterial count. The successful application of this fast method on further areas of food hygiene, such as tracing antibiotics and testing additives for their antimicrobiological effect, has also been described. In general the use of impediometry for the application areas stated has been judged positively. However, the time and expense required by the user to optimize the method, the deficits when testing slightly contaminated sample material or determining the bacterial count in those cases in which the microorganisms are sublethally damaged, and the necessity of performing individual calibration for each food category limit the applicability of impediometry.
The prevalence of the enterotoxin gene in a well-characterized collection of 71 Clostridium perfringens strains from 36 separate food-poisoning cases or outbreaks was analyzed with the polymerase chain reaction (PCR). The clonality of 39 strains originating from 14 outbreaks where at least two isolates were available was studied with pulsed-field gel electrophoresis (PFGE) using SmaI and ApaI restriction endonucleases. The cpe gene PCR assay was found to correlate well with Clostridium perfringens enterotoxin (CPE) production in vitro with reverse passive latex agglutination. Of the C. perfringens food and clinical food-poisoning isolates 24 (86%) and 38 (88%) were cpe-positive, respectively. Different PFGE patterns indicated that multiple cpe-positive clones are frequently present within one outbreak. The existence of cpe-positive and negative isolates with identical or nearly identical PFGE patterns in a single outbreak suggests that the cpe gene may be in a movable genetic element.
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...
In 1998, 21 inhabitants of a German nursing home fell ill with acute gastroenteritis after consumption of minced beef heart (P. Graf and L. Bader, Epidemiol. Bull. 41:327-329, 2000). Two residents died during hospital treatment. Seventeen Clostridium perfringens strains were collected from two different dishes and from patients' stool samples and autopsy materials. A majority of these isolates was not typeable by restriction fragment length polymorphism-pulsed-field gel electrophoresis (PFGE). Subsequent ribotyping of C. perfringens distinguished four different groups. The same ribopattern was detected in a minced beef heart dish, in autopsy material from the two deceased patients, and additionally in stool samples from six further residents who had fallen ill with diarrhea. Three further ribopatterns from food and autopsy materials could be differentiated. As chromosomal macrorestriction with subsequent PFGE is generally regarded more useful than ribotyping for molecular strain analysis, four selected isolates were lysed in parallel with a standard protocol and two nucleases inhibiting modifications. Neither of these methods could differentiate all of the isolates. These results suggest that PFGE with the current standard protocols is not able to characterize all C. perfringens isolates from food-borne disease investigations and that ribotyping is still a helpful method for molecular identification of clonal relationships.
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