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Letters in Applied Microbiology 2002, 34, 238–243 Ribotyping of Clostridium perfringens from industrially produced ground meat Ü. Kilic, B. Schalch and A. Stolle Institute for Hygiene and Technology of Food of Animal Origin, Veterinary Faculty, Ludwig-MaximiliansUniversity Munich., Veterinärstr. 13, D-80539 Munich, Germany 2001/151: received 16 May 2001 and accepted 18 December 2001 Ü . K I L I C , B . S C H A L C H A N D A . S T O L L E . 2002. Aims: Clostridium (Cl.) perfringens is a common cause of food poisoning outbreaks. Ribosomal DNA analysis (ribotyping), a method which analyses restriction fragment length polymorphisms in the chromosomal genes that encode rRNA, has been shown to be useful for microbial species identification and subtyping. Methods and Results: The current study has used ribotyping to examine 111 Cl. perfringens isolates from industrially produced ground meat in order to collect a basis for a contamination survey. Among the 111 isolates 107 distinctly different ribopatterns were detected. In only four cases two Cl. perfringens isolates showed an identical ribopattern. The isolates gave identical ribotype patterns in three different runs, carried out 3–4 months apart from each other. Conclusions, Significance and Impact of the Study: The discriminatory index for EcoRI ribotyping of the Cl. perfringens isolates was 0Æ99. Results showed that ribotyping is suitable for subtyping Cl. perfringens isolates from raw meat. Ribotyping appeared to be a useful tool for profound epidemiologic studies of Cl. perfringens-contamination in food production and processing. INTRODUCTION Clostridium (Cl.) perfringens is a Gram-positive, sporeforming, anaerobic bacillus which is one of the most widely distributed pathogenic bacteria in the environment. It is found in the intestinal tract as part of the normal flora. Cl. perfringens spores are present in small numbers in human faecal specimens and raw food samples. Human food poisoning caused by Cl. perfringens is due to an enterotoxin which is produced during sporulation of the bacteria in the intestinal tract following ingestion of vegetative cells in large numbers (Labbe 1989). In 1986, Grimont and Grimont proposed ribotyping as a general method for molecular bacterial identification. This technique can discriminate between isolates of the same species (Stull et al. 1988). It has proven to be a useful epidemiological tool in the study of various bacterial 1 pathogens (Altwegg et al. 1989; Blumberg et al. 1991; Sarafian et al. 1991; Esteban et al. 1993; Sexton et al. 1993). Correspondence to: A. Stolle, Institute for Hygiene and Technology of Food of Animal Origin, Veterinary Faculty, Ludwig-Maximilians-University Munich., Veterina¨rstr. 13, D-80539 Munich, Germany (e-mail: Sekretariat@lmhyg. vetmed.uni-muenchen.de). Ribosomal DNA analysis (ribotyping) is a method for analysing restriction fragment length polymorphisms in the chromosomal genes that encode rRNA. Ribotyping is based on the visualization of segments of these ribosomal genes by a labelled probe. Cl. perfringens contains 10 highly conserved operons for 16S and 23S rRNA genes distributed throughout the 2 chromosome (Garnier et al. 1991). 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. perfringens is necessary to gain further information about the origin, the contamination routes of these microorganisms in food producing plants, for investigating and controlling food poisoning sources (McDonel 1986). MATERIALS AND METHODS Strains In this study, 111 Cl. perfringens isolates from raw meat were investigated. The isolates examined were taken from the 4 Institute’s collection of sulphate-reducing anaerobes which originated from industrially produced ground meat. These isolates had been collected from April 1994 to June 1996 ª 2002 The Society for Applied Microbiology RIBOTYPING OF CLOSTRIDIUM PERFRINGENS using the procedure described in the official methods’ 5 collection according to § 35 of the German Law on Food and Commodities. During cultivation and differentiation of the anaerobic isolates, Reverse CAMP Test and Acid Phosphatase reactions were used for the confirmation of Cl. perfringens. The isolates were stored in a Microbankä storage system (Mast Diagnostica, Reinfeld, Germany) at ) 18°C. Prior to ribotyping, the purity of the isolates was re-ensured by subculturing twice on Columbia sheep blood agar (Oxoid CM. 331, UK). DNA preparation Cl. perfringens isolates were cultured on Columbia sheep blood agar. The isolates were then incubated under anaerobic conditions at + 37°C for 16 h in Brain Heart Infusion Broth (Oxoid CM. 225, UK) and then centrifuged (13 000 g, 10 min). DNA was isolated from the cell pellet using the guanidium thiocyanate method according to Pitcher et al. (1989). Ribotyping Ribotyping was carried out according to the method described by Grimont and Grimont (1986). Following DNA isolation, the DNA concentrations were measured with a UV spectrophotometer (UV/VIS spectrometer Lambda 2; Perkin-Elmer, Norwalk, CT, USA). Five micrograms of DNA were then digested with EcoRI at + 37°C for a maximum of 2 h. Digoxigenin (DIG)labelled phage lambda DNA cleaved with HindIII (Boehringer Mannheim GmbH) was used as a molecular weight marker. The restriction fragments were separated by agarose gel electrophoresis at 25 V for 16 h, 19 h, 22 h and 24 h. The fragments were then transferred onto a nylon membrane via Southern Blot. After drying at room temperature, the DNA was fixed at + 120°C for 0Æ5 h. The membrane was welded in a plastic bag and prehybridized for 3 h in a + 58°C water bath. A cDNA probe was prepared from Escherichia coli 16S and 23S rRNA (Boehringer Mannheim GmbH) and labelled by incorporation of DIG-dUTP using Avian myeloblastosis virus reverse transcriptase. The solutions used for hybridization, washes and detection of the DIG label were prepared according to the DIG DNA labelling and detection kit (Boehringer Mannheim GmbH). Finally, the membrane was stained with nitroblue tetrazolium chloride and 5-Bromo-4-chloro-3-indolyl-phosphate, 4-toluidine-salt (Boehringer Mannheim GmbH) overnight and washed with TE solution (10:1). Ribotyping membranes were photographed and documented using the Gel Doc 1000 imaging system (Bio-Rad, Hercules, CA, USA) and Multi-AnalystÒ/PC Software (Bio-Rad, Hercules, CA, USA). 239 Ribotyping pattern analysis Cl. perfringens isolates were first examined visually. The band profiles were then arranged into a dendrogram using the unweighted pair group method with arithmetic averages (UPGMA). The ribotype patterns of Cl. perfringens were analysed via computer cluster analysis with Molecular AnalystÒ Software (Fingerprinting Version 1Æ12 Applied Maths 1992–94, Bio-Rad Laboratories). The numerical index of the discriminatory power of each typing assay was calculated based on the probability of two unrelated strains being characterized as the same type (Hunter and Gaston 1988). In addition, the reproducibility of ribotyping for the characterization of Cl. perfringens was determined in this study. 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). RESULTS Among 111 Cl. perfringens isolates from ground meat 107 distinctly different ribotype patterns were detected. In only four cases two Cl. perfringens isolates showed an identical ribopattern. Figure 1 shows an example of the variability of the ribotype patterns. The number of DIG labelled bands of Cl. perfringens varied between 12 and 17. Typical ribotype patterns of Cl. perfringens were as follows: 7–11 bands between 23 kb and 4Æ3 kb, 0–4 bands between 4Æ3 kb and 2Æ0 kb and 2–5 bands below 2Æ3 kb. The latter showed typical band patterns which could be differentiated easily. Based on these patterns of bands smaller than 2Æ3 kb, all 111 Cl. perfringens isolates were divided into five groups, named A to E. Seventy-three percent of all Cl. perfringens isolates examined belonged to profile type A, 12Æ6% to profile B, 9Æ9% to profile C, 2Æ7% to profile D and 1Æ8% to profile E (Fig. 1). 6 All Cl. perfringens isolates were typeable with EcoRI ribotyping and the rDNA patterns were clearly reproducible. 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. perfringens isolates was above 0Æ99. During the experiments for this study, it was observed that the following points have to be kept to closely when ribotyping Cl. perfringens: The DNA from Cl. perfringens isolates may be stored at + 4°C for a maximum of four weeks in TE buffer. The incubation time for restriction with EcoRI at + 37°C must not exceed two hours. In this study, an electrophoresis running time of 24 h gave the best results as compared to 16 h, 19 h, and 22 h (Fig. 2). Figure 3 shows the dendrogram of 74 Cl. perfringens isolates, which was determined by comparing the complete ª 2002 The Society for Applied Microbiology, Letters in Applied Microbiology, 34, 238–243 240 Ü . K I L I C ET AL. kb M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M kb 23 23 9.4 9.4 6.5 6.5 4.3 4.3 2.3 2.0 2.3 2.0 (a) kb M 1 1 2 2 3 3 4 4 M kb 23 23 9.4 9.4 6.5 6.5 4.3 4.3 2.3 2.3 2.0 2.0 A B C D E (c) (b) Fig. 1 Ribotyping of genomic DNA from Cl. perfringens isolates digested with EcoRI and probed for 16S and 23S rRNA genes. Lanes 1 through 18 show the different ribotype patterns of Cl. perfringens isolates. (a). Lanes 1 through 4 show the four identical ribotype patterns within 111 Cl. perfringens isolates (b). Typical patterns of the bands beneath 2,3 kb of Cl. perfringens isolates (c). The digoxigenin-labelled molecular weight marker II (M) was used a size marker. Band sizes in kb are indicated band patterns over the whole length of the electrophoretic run (24 h). DISCUSSION Ribotyping has been useful for subtyping diverse bacteria including species of Listeria monocytogenes (Grimont and Grimont 1986), Escherichia coli (Lipuma et al. 1989), Campylobacter spp. (Kiehlbauch et al. 1991), Yersinia enterocolitica (Lobato et al. 1998), and Cl. perfringens (Forsblom et al. 1995; Schalch et al. 1997). Ribotyping has several advantages over other typing methods. As the ribosomal genes are located on the chromosome, they are not easily lost like plasmids. Most bacteria contain multiple copies of the ribosomal operons; therefore, an acceptable number of fragments (3–15) that hybridize with the probe are generated. In addition, the rDNA patterns are not sensitive to antibiotic pressure, and they are not dependent on susceptibility to phages or production of bacteriocins. For molecular subtyping methods, the following criteria are important in comparative evaluation. First, ª 2002 The Society for Applied Microbiology, Letters in Applied Microbiology, 34, 238–243 RIBOTYPING OF CLOSTRIDIUM PERFRINGENS M 241 kb 23 9.4 6.5 M 23 4.3 9.4 6.5 4.3 2.3 2.3 2.0 Fig. 2 Ribopatterns of Cl. perfringens with electrophoresis running times of 16 h, 19 h, 22 h, and 24 h at 25 V the reproducibility of a typing method, which is the percentage of strains with the same subtype on repeated testing, preferably after a period of a few months. Second, the discriminatory power of a method is an estimate of its ability to differentiate between two unrelated strains (Hunter 1990; Swaminathan and Matar 1993). The acceptable level of discrimination depends on a number of factors, but an index of greater than 0Æ90 is considered necessary if the typing results are to be interpreted with confidence (Hunter and Gaston 1988). In the current study, the discriminatory index for ribotyping of the Cl. perfringens isolates studied was above 0Æ99. This indicates that ribotyping is very well suited to subtype Cl. perfringens isolates from raw meat. All isolates included in this study were typeable with EcoRI ribotyping. Within 111 Cl. perfringens isolates from ground meat, ribotyping distinguished 107 distinctly different ribotype patterns. These results clearly show the great genetic variety of Cl. perfringens contaminating ground meat. Several methods are available for the subtyping of Cl. perfringens isolates; these include bacteriocin typing, phage typing, antimicrobial susceptibility testing, and plasmid profile analysis (Traub et al. 1986; Yan 1989; Mahony et al. 1992). These methods are based on metabolic features or on the detection of extra-hromosomal genetic information which are prone to rapid changes. In addition, plasmid profile and antibiotic susceptibility results are particularly sensitive to environmental or antibiotic 2.0 16h 19h 22h 24h pressures. As the genes encoding rRNA are located on the chromosome and are not readily lost, ribotyping provides a more reliable discrimination especially for characterizing strains collected over extended periods of time (Grattard et al. 1993; Mulligan and Arbeit 1991; Schmitz et al. 1995). Manual ribotyping is time and cost intensive and requires profoundly trained personnel for carrying out the tests and evaluating the results. Nevertheless, this study underlines the discriminatory power and reproducibility of ribotyping of Cl. perfringens isolates from ground meat. Therefore, ribotyping has to be considered suitable for establishing a data bank as a basis for comprehensive contamination studies and seems to be very promising for the epidemiological investigation of contamination studies in the field of food hygiene and food industry. A recently developed automated RiboPrinterä system (Qualicon, Wilmington, Del, USA) allows acquisition and normalization of ribotype patterns. The system may be used for identification of bacteria at genus and species levels and to determine similarity of strain patterns. The RiboPrinterä system was designed specifically to satisfy the needs of the food industry. All the process steps required to characterize a bacterium to the strain level, from cell lysis to image 9 analysis, are performed in 8 h. RiboPrinterä may be an attractive alternative but, at the present time, the equipment and supplies are prohibitively expensive for most 10 laboratories. ª 2002 The Society for Applied Microbiology, Letters in Applied Microbiology, 34, 238–243 242 Ü . K I L I C ET AL. Fig. 3 Dendrogram obtained from cluster analysis of EcoRI ribotypes of Cl. perfringens isolates by using the unweighted pair group method with arithmetic averages (UPGMA) ACKNOWLEDGEMENTS We are grateful to Prof. Dr. H. Eisgruber for advice and helpful comments, and to H. Dietz and A. Fendel for technical assistance. 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