TY - JOUR
AU - Stolle, Andreas
AB - Abstract Clostridium perfringens continues to be a common cause of food-borne disease [1,2]. It produces an enterotoxin (CPE) which is released upon lysis of the vegetative cell during sporulation in the intestinal tract. Catering premises with insufficient cooling and reheating devices often seem to be the cause of outbreaks of C. perfringens food poisoning. Typing of C. perfringens is of great importance for investigating sources of food poisoning cases and for studying the epidemiology of this microorganism. This report describes the examination of 155 C. perfringens isolates by molecular methods. Isolates were taken from 10 food poisoning outbreaks and cases (n=34, food and fecal isolates) and from meat and fish pastes (n=121). Isolates were characterized by plasmid profiling, ribotyping, and/or macrorestriction analysis by pulsed-field gel electrophoresis (PFGE). Results show that all three methods are suitable for classifying C. perfringens isolates below the species level. Ribopatterns and PFGE patterns can be interpreted more easily than plasmid profiling results and can be recommended for contamination studies and epidemiologic investigation of food poisonings associated with C. perfringens. Clostridium perfringens, Food poisoning, Minced meat, Plasmid profiling, Ribotyping, Pulsed-field gel electrophoresis 1 Introduction A variety of methods have been described for the strain differentiation of bacteria, including biochemical characterization, bacteriocin typing, antibiotic resistance profiles, protein gel electrophoresis fingerprinting, SDS-PAGE, or HPLC profiling of bacterial fatty acids [3–6]. Furthermore, several DNA-based fingerprinting methods are being applied to an increasing extent in routine settings. For Clostridium perfringens, there are plasmid isolation [7], ribotyping of patients' and hospital environment isolates [8] and macrorestriction enzyme analysis by pulsed-field gel electrophoresis (PFGE) [4,9]. Further DNA-based methods are polymerase chain reaction (PCR)-related methods such as random amplified polymorphic DNA (RAPD). Bacterial strain typing methods are mainly used for epidemiological investigations of the sources and spread of the causative agent in food-borne disease outbreaks but also supply valuable information for contamination studies in the food industry [10–12]. This article describes and evaluates the use of plasmid profiling, ribotyping and PFGE for strain differentiation of C. perfringens from food poisonings and meat. Strains tested include patient stool and food isolates from 10 food-borne outbreaks and cases. Furthermore, as meat is described to be the main vehicle of infection in C. perfringens-associated food poisoning, 121 meat and food isolates were analyzed. 2 Materials and methods 2.1 Strains A total of 34 C. perfringens isolates associated with 10 food-borne outbreaks and cases (Table 1, designated 1–10) were analyzed by plasmid isolation, ribotyping and PFGE. Isolates from food and stool were available. The strains were identified with the RAPID ID 32A® identification kit (BioMerieux, Nürtingen, Germany) and additional confirmation procedures as described previously [13,14]. Table 1 Plasmid, ribotype and PFGE patterns for 34 C. perfringens isolates from food poisoning cases Outbreak/case  Isolate  Plasmids (molecular weight in MDa)  Ribotype [23]  PFGE [4, 9]  1  rabbit meat  721/84  7.1  1  1    feces  731/84  7.1  1  1    feces  732/84  7.1  1  1  2  rabbit meat  310/85  28.7  2  2    feces  313/85  28.7  2  2    feces  314/85  28.7  2  2  3  heart goulash  945/85  25.5, 22.5  3  3    cauliflower salad  948/85  27.1  3  3    cauliflower salad  949/85  27.1  3  3    feces  953/85  25.5, 22.5  3  3    feces  954/85  21.1  11  11    feces  955/85  22.5  3  3  4  pork  10/86  28.7, 8.4, 6.6  4  4    feces  18/86  >33.0, 8.4, 6.6  4  4    feces  26/86  28.7, 8.4, 6.6  4  4  5  beef  349/86  30.7  5  5    feces  344/86  30.7  5  5    feces  345/86  30.7  5  5  6  pea mash  834/87  5.6  6  6    feces  835/87  29.3, 5.6  6  6  7  poultry meat  216/88  0  7  7    feces  227/88  0  7  7    feces  231/88  0  7  7    feces  234/88  0  7  7  8  chicken fricassee  1291/88  29.3, 8.7, 2.4  8  8    feces  1295/88  29.3, 8.7, 2.4, 2.3  8  8  9  poultry fricassee  174/90  27.9, 3.0  12  12    feces  175/90  27.9  9  9    feces  176/90  0  9  9    feces  192/90  0  9  9    feces  195/90  0  9  9  10  beef  344/91  0  10  10    feces  346/91  8.8  10  10    feces  347/91  0  10  10  Outbreak/case  Isolate  Plasmids (molecular weight in MDa)  Ribotype [23]  PFGE [4, 9]  1  rabbit meat  721/84  7.1  1  1    feces  731/84  7.1  1  1    feces  732/84  7.1  1  1  2  rabbit meat  310/85  28.7  2  2    feces  313/85  28.7  2  2    feces  314/85  28.7  2  2  3  heart goulash  945/85  25.5, 22.5  3  3    cauliflower salad  948/85  27.1  3  3    cauliflower salad  949/85  27.1  3  3    feces  953/85  25.5, 22.5  3  3    feces  954/85  21.1  11  11    feces  955/85  22.5  3  3  4  pork  10/86  28.7, 8.4, 6.6  4  4    feces  18/86  >33.0, 8.4, 6.6  4  4    feces  26/86  28.7, 8.4, 6.6  4  4  5  beef  349/86  30.7  5  5    feces  344/86  30.7  5  5    feces  345/86  30.7  5  5  6  pea mash  834/87  5.6  6  6    feces  835/87  29.3, 5.6  6  6  7  poultry meat  216/88  0  7  7    feces  227/88  0  7  7    feces  231/88  0  7  7    feces  234/88  0  7  7  8  chicken fricassee  1291/88  29.3, 8.7, 2.4  8  8    feces  1295/88  29.3, 8.7, 2.4, 2.3  8  8  9  poultry fricassee  174/90  27.9, 3.0  12  12    feces  175/90  27.9  9  9    feces  176/90  0  9  9    feces  192/90  0  9  9    feces  195/90  0  9  9  10  beef  344/91  0  10  10    feces  346/91  8.8  10  10    feces  347/91  0  10  10  View Large Table 1 Plasmid, ribotype and PFGE patterns for 34 C. perfringens isolates from food poisoning cases Outbreak/case  Isolate  Plasmids (molecular weight in MDa)  Ribotype [23]  PFGE [4, 9]  1  rabbit meat  721/84  7.1  1  1    feces  731/84  7.1  1  1    feces  732/84  7.1  1  1  2  rabbit meat  310/85  28.7  2  2    feces  313/85  28.7  2  2    feces  314/85  28.7  2  2  3  heart goulash  945/85  25.5, 22.5  3  3    cauliflower salad  948/85  27.1  3  3    cauliflower salad  949/85  27.1  3  3    feces  953/85  25.5, 22.5  3  3    feces  954/85  21.1  11  11    feces  955/85  22.5  3  3  4  pork  10/86  28.7, 8.4, 6.6  4  4    feces  18/86  >33.0, 8.4, 6.6  4  4    feces  26/86  28.7, 8.4, 6.6  4  4  5  beef  349/86  30.7  5  5    feces  344/86  30.7  5  5    feces  345/86  30.7  5  5  6  pea mash  834/87  5.6  6  6    feces  835/87  29.3, 5.6  6  6  7  poultry meat  216/88  0  7  7    feces  227/88  0  7  7    feces  231/88  0  7  7    feces  234/88  0  7  7  8  chicken fricassee  1291/88  29.3, 8.7, 2.4  8  8    feces  1295/88  29.3, 8.7, 2.4, 2.3  8  8  9  poultry fricassee  174/90  27.9, 3.0  12  12    feces  175/90  27.9  9  9    feces  176/90  0  9  9    feces  192/90  0  9  9    feces  195/90  0  9  9  10  beef  344/91  0  10  10    feces  346/91  8.8  10  10    feces  347/91  0  10  10  Outbreak/case  Isolate  Plasmids (molecular weight in MDa)  Ribotype [23]  PFGE [4, 9]  1  rabbit meat  721/84  7.1  1  1    feces  731/84  7.1  1  1    feces  732/84  7.1  1  1  2  rabbit meat  310/85  28.7  2  2    feces  313/85  28.7  2  2    feces  314/85  28.7  2  2  3  heart goulash  945/85  25.5, 22.5  3  3    cauliflower salad  948/85  27.1  3  3    cauliflower salad  949/85  27.1  3  3    feces  953/85  25.5, 22.5  3  3    feces  954/85  21.1  11  11    feces  955/85  22.5  3  3  4  pork  10/86  28.7, 8.4, 6.6  4  4    feces  18/86  >33.0, 8.4, 6.6  4  4    feces  26/86  28.7, 8.4, 6.6  4  4  5  beef  349/86  30.7  5  5    feces  344/86  30.7  5  5    feces  345/86  30.7  5  5  6  pea mash  834/87  5.6  6  6    feces  835/87  29.3, 5.6  6  6  7  poultry meat  216/88  0  7  7    feces  227/88  0  7  7    feces  231/88  0  7  7    feces  234/88  0  7  7  8  chicken fricassee  1291/88  29.3, 8.7, 2.4  8  8    feces  1295/88  29.3, 8.7, 2.4, 2.3  8  8  9  poultry fricassee  174/90  27.9, 3.0  12  12    feces  175/90  27.9  9  9    feces  176/90  0  9  9    feces  192/90  0  9  9    feces  195/90  0  9  9  10  beef  344/91  0  10  10    feces  346/91  8.8  10  10    feces  347/91  0  10  10  View Large As C. perfringens was found in 20% of minced meat samples examined microbiologically according to the EU Directive 88/657/ECC [15], 111 C. perfringens isolates were analyzed by ribotyping in order to collect basic information for a contamination study. Isolates were obtained by the pour-plate technique with sulfite-cycloserine-azide medium [16] and confirmed by acid phosphatase and reverse-CAMP testing as described [17]. Ten C. perfringens food isolates from doner kebab, shrimp paste, crab paste, and fish sauce ‘tai pla’ were analyzed by ribotyping and PFGE (Table 2). Table 2 Ribotype and PFGE patterns for 10 C. perfringens food isolates Origin  Isolate  Ribotype  PFGE  Doner kebab  24019–1  A  NT    24019–2  B  B    24019–3  A  NT  Crab paste  22431–1  C  C    22431–3  D  NT  Shrimp paste  42–1  E  E 1    42–2  E  E 1    42–3  E  E 2  Fish sauce  264–1b  F  F    264–2  F  F  NT, not typeable.  Origin  Isolate  Ribotype  PFGE  Doner kebab  24019–1  A  NT    24019–2  B  B    24019–3  A  NT  Crab paste  22431–1  C  C    22431–3  D  NT  Shrimp paste  42–1  E  E 1    42–2  E  E 1    42–3  E  E 2  Fish sauce  264–1b  F  F    264–2  F  F  NT, not typeable.  View Large Table 2 Ribotype and PFGE patterns for 10 C. perfringens food isolates Origin  Isolate  Ribotype  PFGE  Doner kebab  24019–1  A  NT    24019–2  B  B    24019–3  A  NT  Crab paste  22431–1  C  C    22431–3  D  NT  Shrimp paste  42–1  E  E 1    42–2  E  E 1    42–3  E  E 2  Fish sauce  264–1b  F  F    264–2  F  F  NT, not typeable.  Origin  Isolate  Ribotype  PFGE  Doner kebab  24019–1  A  NT    24019–2  B  B    24019–3  A  NT  Crab paste  22431–1  C  C    22431–3  D  NT  Shrimp paste  42–1  E  E 1    42–2  E  E 1    42–3  E  E 2  Fish sauce  264–1b  F  F    264–2  F  F  NT, not typeable.  View Large 2.2 Plasmid analysis Plasmid analysis was performed according to Mahony et al. [18,19] and Eisgruber et al. [7,20] and was carried out twice per isolate. 2.3 Ribotyping Ribotyping was performed according to the method described by Grimont and Grimont [21]. DNA was isolated using the guanidinium thiocyanate method of Pitcher et al. [22] with modifications [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. Patterns were compared visually. All 34 C. perfringens isolates from food poisonings were analyzed three times. The 111 minced meat and 10 food isolates were ribotyped once, some isolates twice. 2.4 PFGE The 34 isolates from food poisonings and the 10 food isolates were analyzed once as described [24–26]. 3 Results 3.1 Food poisoning isolates A summary of the results for plasmid typing, ribotyping and PFGE of all 34 C. perfringens isolates is shown in Table 1. 3.2 Plasmid profiling For outbreaks 1, 2, and 5 (Table 1) all C. perfringens isolates, from foods as well as from fecal samples, showed identical plasmid profiles. One out of two available fecal isolates as well as the food isolates from outbreak 4 showed identical plasmid profiles. For outbreak 3, one out of three fecal isolates showed a plasmid profile identical to the isolate from one (heart goulash) of the three epidemiologically implicated foods. Considering that strains differing only by the presence/absence of a single plasmid might be clonally related, outbreaks 6 and 8 are also characterized by the isolation of clonally related strains from foods and fecal samples. For three outbreaks, the plasmid profiling results do not allow a clear interpretation. All of the five isolates from outbreak 7 lacked any detectable plasmids. For outbreak 9, one of the four fecal isolates appears to be clonally related to the food isolate as it differs only by the presence/absence of a single plasmid. The three other fecal isolates carried no detectable plasmids. 3.3 Ribotyping Altogether, 12 distinct ribotype patterns were found among the 34 food poisoning-associated C. perfringens isolates. Those 12 patterns were clearly reproducible in three different runs. Patterns differing by one or more bands were considered different. In eight of 10 food poisonings (Table 1: 1, 2, 4–8, 10) identical ribotype patterns for all isolates were detected. Two unique ribotypes were found in outbreaks 3 (954/85, fecal isolate) and 9 (174/90, poultry fricassee isolate). Outbreak 3 showed five identical ribotypes among six isolates derived from foods and feces. In outbreak 9 all four fecal isolates showed identical ribotypes. However, the C. perfringens strain isolated from the presumptive outbreak-related chicken fricassee (174/90) had a different and unique pattern. 3.4 PFGE Our own examinations and those of other authors showed that PFGE could distinguish C. perfringens isolates with identical ribopatterns. But PFGE patterns interpreted according to the criteria of Tenover et al. [27], i.e. patterns within an outbreak that differ by one to three fragments only are considered to be subtypes, categorized the isolates as closely related. 3.5 Meat and food isolates Results of ribotyping and PFGE of 10 C. perfringens isolates from doner kebab, shrimp paste, crab paste, and fish sauce ‘tai pla’ are shown in Table 2. As far as the isolates were typeable by PFGE, corresponding results were obtained by both methods. Three isolates from doner kebab and crab paste could not be typed by PFGE. Within 111 C. perfringens isolates from minced meat, 107 distinctly different ribopatterns were detected. Fig. 1 shows the ribopatterns of 15 isolates. Considering only the pattern of fragments smaller than 2.3 kb all 111 C. perfringens isolates could be divided into five groups. The profiles (A–E) are presented in Fig. 2. 70.3% of all C. perfringens isolated from minced meat belonged to profile type A (see also Fig. 1, lanes 2, 4, 10), 12.6% to profile B and 9.9% to profile C. Four C. perfringens strains showed different profile types (D, E). Figure 1 View largeDownload slide Ribotype patterns of 15 C. perfringens isolates of minced meat. Figure 1 View largeDownload slide Ribotype patterns of 15 C. perfringens isolates of minced meat. Figure 2 View largeDownload slide Typical fragment patterns under 2322 bp of C. perfringens isolates from minced meat. Figure 2 View largeDownload slide Typical fragment patterns under 2322 bp of C. perfringens isolates from minced meat. The discriminatory power of ribotyping for these minced meat isolates was above 0.99, estimated according to the recommendations of Hunter and Gaston [28]. 4 Discussion This study compared the utility of plasmid profiling, ribotyping and PFGE for the differentiation of C. perfringens isolates from food poisonings, minced meat and food. 4.1 Plasmid profiling Plasmid profiling has been used for strain differentiation in a variety of Gram-positive and Gram-negative bacteria [29]. The utility of plasmid profiling for the differentiation of isolates within a given species depends on multiple factors including (i) variability of plasmid patterns within a species, (ii) frequency of isolates without plasmids, (iii) stability of plasmids, and (iv) reproducibility of plasmid patterns. Our own investigations have shown that about 30% of all C. perfringens isolates carry no detectable plasmids. Among plasmid-carrying isolates a significant diversity of plasmid patterns has been found. This is consistent with results reported [19,30]. Mahony et al. [19] reported that 92% of the clinical C. perfringens isolates tested carried plasmids while only 77% of the isolates from other sources had detectable plasmids. Phillips Jones et al. [30] found detectable plasmids for 71% of their C. perfringens isolates from food poisonings. A lower frequency of plasmid carriage was found among isolates from other sources including pork, lamb and ground beef. Even the absence of plasmids can give a hint on epidemiological relationship, e.g. within outbreak 7 (Table 1) no plasmids were isolated from all three clinical isolates and the associated food isolate. The probability of such a finding for four entirely unrelated isolates can be considered very small. Similarly, Mahony et al. [31] reported two outbreaks for which C. perfringens isolates without plasmids were obtained. In general, the high diversity of plasmid patterns appears to make plasmid profiling a sensitive method for the discrimination of C. perfringens isolates. 4.2 Ribotyping and PFGE Ribotyping and PFGE both gave corresponding identical results for eight cases of food poisoning (Table 1). This indicates that the isolates within each of these food poisonings are genetically closely related. Since the food isolates tested were epidemiologically implicated as the source of the outbreak or case, it can be assumed that the C. perfringens strain found in food and stool caused the disease. In two cases different patterns were detectable within the isolates of one food poisoning. Among the six isolates of outbreak 3 (Table 1) one unique pattern was detected (954/85, feces isolate) by ribotyping and PFGE. The other five isolates from foods and stool were identical. In outbreak 9 the ribopattern and PFGE pattern of the food isolate (174/90) differed from those of the four feces isolates and therefore can be judged to be genetically not related to any of the patients' stool isolates. This conclusion could not be drawn from the plasmid profiling results. Within the 10 C. perfringens isolates from doner kebab, shrimp paste, crab paste, and fish sauce corresponding results were obtained by ribotyping and PFGE, as far as isolates were PFGE-typeable. The ribotyping results of 111 C. perfringens isolates from minced meat underline the high discriminatory power of the method. Furthermore, these results clearly show that meat is contaminated by C. perfringens isolates with a great genetic variety. The use of multiple strain typing methods is necessary to achieve reliable information regarding the connection between presumptive epidemiologically related isolates. Plasmid typing as outlined in this communication is a relatively simple typing method with low initial setup costs and represents a useful method for preliminary strain typing of C. perfringens isolates. Manual ribotyping and PFGE are more expensive and laborious, but results can be interpreted easily. Both methods seem to be very promising for the epidemiological investigation of food-borne diseases caused by C. perfringens and for contamination studies in the field of food hygiene and industry. Acknowledgements We thank Dr. habil. H.-P. 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TI - Molecular methods for the analysis of Clostridium perfringens relevant to food hygiene
JF - Journal of the Endocrine Society
DO - 10.1111/j.1574-695X.1999.tb01295.x
DA - 1999-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/molecular-methods-for-the-analysis-of-clostridium-perfringens-relevant-JITHxGbs4T
SP - 281
EP - 286
VL - 24
IS - 3
DP - DeepDyve
ER -