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Prevalence of Enterotoxigenic Clostridium perfringens in Meats in San Luis, Argentina
Anaerobe (2002) 8, 253–258 doi:10.1006/anae.2002.0433
FOOD MICROBIOLOGY
Prevalence of Enterotoxigenic Clostridium perfringens in Meats in San Luis, Argentina Patricia Virginia Stagnitta, Blas Micalizzi and Ana Mar|¤ a Stefanini de Guzma¤n* ´ rea Microbiologı´a, Departamento de A Bioquı´mica y Ciencias Biolo´gicas. Facultad de Quı´mica, Bioquı´mica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700- San Luis, Argentina (Received 29 November 2001; accepted 24 October 2002) Key Words: Enterotoxigenic, Clostridium perfringens, meats
Clostridium perfringens is an important pathogen agent causing, among other diseases, enteritis in humans and enterotoxemia in domestic animals. This bacterium can produce more than 15 toxins, one of which is its enterotoxin (CPE), that causes human food poisoning. The aim of this work was (i) to determine the prevalence of C. perfringens in some non-industrial meat foods in San Luis, Argentina, (ii) to characterize the C. perfringens enterotoxigenic strains by PCR, RPLA and the slide reverse passive latex agglutination test, (iii) to type the C. perfringens strains isolated and identification by PCR and (iv) to develop a slide RPLA test. A total of 515 samples of meat food (315 fresh sausages, 100 hamburgers and 100 samples of minced meat) were studied. A 126 C. perfringens strains (24.46%) were isolated and characterized. Of these C. perfringens-positive samples, 48 contained counts higher than 2 log/g. No significant differences were observed between counts performed in iron–milk medium and tryptose– sulfite–cycloserine agar (r= 0.99). Twelve samples (9.52%) exhibited counts with MPN 45 log bacteria/g. Modified To´rtora medium (Tm) with thiotone replaced by proteose peptone turned out to be the most useful medium for both sporulation and enterotoxin production. Of the 126 samples tested by PCR and RPLA, nine strains (7.14%) were enterotoxigenic. Similar results were obtained by Slide RPLA, which exhibited a sensitivity of 8 ng/mL. Of the 126 C. perfringens strains, 123 were of type A (97.20%), two were of type C (1.59%) and one of type E (0.79%). All enterotoxigenic strains were classified as type A. # 2003 Published by Elsevier Science Ltd.
Introduction * ´ rea Microbiologı´a, Departamento Address correspondence to: A de Bioquı´mica y Ciencias Biolo´gicas. Facultad de Quı´mica, Bioquı´mica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700- San Luis, Argentina. Tel: +54-2652423789 int. 127. Fax: +54-2652-431301. E-mail: [email protected]
Clostridium perfringens is an important pathogen agent causing, among other diseases, enteritis in humans and enterotoxemia in domestic animals [1–5]. This Gram-positive, anaerobic bacterium is widely distributed in the environmentFsoil and waterFand
1075–9964/03/$ - see front matter # 2003 Published by Elsevier Science Ltd.
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usually found in the intestinal tract of humans and animals [1,2,6]. It can produce more than 15 toxins, among these, C. perfringens enterotoxin (CPE) [7], which causes human food poisoning [7, 8]. The first studies on food poisoning by C. perfringens did not distinguish between positive and negative enterotoxigenic strains. C. perfringens enterotoxin has been implicated in sporadic diarrheas and antibiotic-associated diarrheas [7, 9]. Intoxication with C. perfringens can be caused by ingestion of food containing X5 log vegetative cells/g of food of an enterotoxigenic strain [8,10]. The in vivo production of the enterotoxin is associated with the sporulation in intestine [10] and the in vitro production of the enterotoxin is obtained in appropriate sporulation media [11,12]. Enterotoxigenic strains can be characterized by the presence of the cpe gene by means of molecular methods or CPE determination by immunological methodsFELISA, reverse passive latex agglutination (RPLA) and cytotoxicity assays [2,5,6,13–16]. Five types of C. perfringens have been classified based on the production of four major toxins: a, b, e and i, which can be determined by serologic methods or by molecular methods such as PCR [2,13,15,17–20]. Considering that there are no available reports on food poisoning outbreaks caused by C. perfringens enterotoxigenic strains in San Luis, Argentina, the aims of this work were: (i) to determine the prevalence of C. perfringens in some non-industrial meat foods in San Luis, Argentina, by the most probable number (MPN) and the count on plate methods, (ii) to characterize the C. perfringens enteroxigenic strains by molecular methods such as PCR and immunological methods such as RPLA and Slide RPLA in order to detect CPE production, (iii) to type, by PCR, C. perfringens strains isolated from food, and (iv) to develop a slide RPLA test.
Materials and Methods Strains C. perfringens isolates and two reference strains, kindly supplied by Dr Ronald G. Labbe´ of University of Massachusetts, U.S.A.: NCTC 8798 cpe+ and ATCC 3694 cpe- were used. Samples A total of 515 food samples consisting of 315 fresh sausages, 100 hamburgers and 100 samples of minced meat were obtained from local markets of San Luis city, Argentina. Samples were immediately processed
or stored in refrigerator (4–61C) until processed, no longer than 24 h. Then, 2.5 g of each sample was homogenized in a mortar with 2.5 g of fine sand, previously sterilized in oven for 1 h at 1801C. The sample was resuspended in 22.5 mL of sterile 0.1% peptone water (dilution 1:10). Serial ten-fold dilutions were made according to the amount of suspected bacteria adding 1 ml of original suspension to tubes containing 9 ml of sterile 0.1 % peptone water. Culture and count of C. perfringens Culture and counts were carried out by MPN using de Man’s table in iron–milk medium [21–23], with aliquots of 1:10 dilutions in triplicate. Cultures were incubated at 451C for 16–18 h. The plate count was carried out on tryptose–sulfite–cycloserine agar (TSCA) [24] according to the manufacturer’s instructions. Plates were inoculated with aliquots of 0.2 mL of each dilution in triplicate and incubated in jars under anaerobic conditions by the evacuation-replacement method with a mixture of propane–butane at 371C for 48 h. Isolation and identification Suspected black colonies of C. perfringens were purified on TSCA [24] and identified by Gram’s stain and by standard biochemical tests. Strains were kept in cooked meat medium and replicated in brain–heart infusion or thioglycolate broth according to the experiments. Enterotoxin production C. perfringens strains isolated from food and reference strains were grown in thioglycolate broth at 371C for 18–24 h. One milliliter of culture was grown in the following sporulation media: To´rtora medium (Tm) modified in our laboratory, replacing thiotone by proteose- peptone [12]; Duncan–Strong medium (D–S) [25] and reinforced Clostridium medium (RCM) [26]. Tm was chosen as the CPE production medium because of the elevated titres obtained. The Tm medium was incubated at 371C for 12 days and spores were counted on days 3, 6, 9 and 12. Counts were carried out in a Neubauer chamber by using a Carl Zeiss Microscope with a 100 objective for phase contrast. The enterotoxin was determined in the culture supernatants by the RPLA technique. CPE detection by RPLA To quantify CPE in culture supernatants, a PET-RPLA commercial kit (Oxoid) of 2 ng/mL sensitivity was
Prevalence of Clostridium perfringens in Meats used. The test was carried out according to the manufacturer’s instructions. As control, the culture filtrates of CPE-positive and negative reference strains were used. Slide RPLA test Immunosera obtainment in rabbits. Lots of three white rabbits of approximately 2.5 kg were inoculated subcutaneously with 0.5 mL of the supernatant containing the enterotoxin mixed with Freund’s complete adjuvant (Oxoid) (1:1) [27]. Two doses 2 weeks apart were applied. Before the antigen administration, bleeding was carried out to discard the presence of natural antibodies. Immunosera were obtained by bleeding white rabbits 2 weeks after the last dose. Previously, animals were anesthetized with Ketalars (8 mg/kg of weight). Blood was left to coagulate at 371C and serum was separated, 1/10.000 thimerosal (Sigma) was added and the serum was kept at 201C until use. Purification of immunoglobulin G (IgG). The IgG antibodies of rabbit sera were purified by saline precipitation [28,29] and by affinity chromatography using a column of Sepharose CL 4B-protein A (Sigma) [30]. Concentration of purified IgG was determined by an ELISA test developed in our laboratory. Slide RPLA test. Latex bead sensitization. Latex bead (Sigma) sensitization was carried out according to Fach and Popoff, and Tamiya et al. [10,14]. Slide RPLA test was performed by mixing 25 mL sensitized latex and 25 mL of each of the supernatants of sporulation medium containing the CPE on a glass slide. The mixture was constantly shaken and the reaction was observed for the first 3 min. The sensitivity of this test was 8 ng/mL. Molecular biology methods C. perfringens cpe gene determination by PCR. Each of the 126 isolated strains was cultured in tubes with 10 mL thioglycolate broth and incubated anaerobically overnight at 371C. For DNA isolation and purification, a combination of chemical and enzymatic treatments was used for lysis of C. perfringens cells according to the method of Kokai-Kun et al. [31]. PCR amplification was performed in a thermal cycler (Perkin Elmer PCR System 2000) incubating the obtained DNA with the following primers: 50 -TGTTAATACTTTAAGGATATGTATCC-30 and 50 -TCCATCACCTAAGGACTG30 (Promega, Madison, WI, USA) at a concentration of 0.4 mM; 0.4 mM deoxynucleoside; 2 mM Cl2Mg (Pro-
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mega) and 1.5 U of Taq polymerase (Promega) in a total reaction volume of 50 mL per reaction tube. The first PCR cycle lasted 300 s at 941C, followed by 34 cycles of 1.5 min at 941C, 1 min at 501C and 1 min at 721C, and finishing with a 600 s cycle at 721C. Products of amplification were determined by electrophoresis in 1% agarose gel (Promega) at 80 V for 90 min using BioRad Power PAC 1000 Midicell EC 3500. The gel was stained with ethidium bromide (0.5 mg/mL) (Promega), and bands were visualized by UV illumination (White Ultraviolet Transilluminator UVP (Upland Sigma). Gels were photographed by a Polaroid Gel Cam EPH % 0.85 Electrophoresis Hood. C. perfringens typing by PCR. From a culture obtained in solid medium, C. perfringens strains were typified by using the technique of Uzal et al. [32] with the following primers: Fa-Toxin:50 -TGCTAATGTTACTGCCGTTGATAG-30 and 50 -ATAATCCCAATCATCCCAACTATG-30 . Fb-Toxin: 50 -AGGAGGTTTTTTTATGAAG-30 and 50 TCTAAATAGCTGTTACTTTGT-30 . Fe-Toxin:50 -TACTCATACTGTGGGAACCTTCGATACAAGC-30 and 50 -CTCATCTCCCATAACTGCACTATAATTTCC-30 . Ft-Toxin: 50 -TTTTAACTAGTTCATTTCCTAGTTA-30 and 50 -TTTTTGTATTCTTTTTCTCTAGATT-30 . The cycling consisted of 28 1-min cycles at 481C, 2-min cycles at 721C, and 1-min cycles at 931C for 1 min; one 1-min cycle at 481C and one 5-min cycle at 721C. Once the cycling was finished, the products were determined by electrophoresis in a 2% agarose gel with ethidium bromide. Gel visualization and photographs were performed as above described. Statistical tests The statistical study of results was performed by Student’s t-test, sign test, correlation coefficient ‘r’ and statistical X2 as appropriate.
Results Count, isolation and identification of C. perfringens Of the 515 samples analysed, 126 strains of C. perfringens were isolated and characterized by standard biochemical tests (Table 1). Of the 126 isolates, 48 (38.1%) exhibited C. perfringens counts 42 log/g, thus exceeding the maximum allowed by the Co´digo Alimentario Argentino (Argentine Alimentary Code, CAA) of 2 log sporulated anaerobes/g [33]. There were no significant differences between counts performed in iron–milk medium and TSCA (r= 0.99)
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Table 1. Presence of C. perfringens in meat foods: number of samples and MPN (most probable number) in iron–milk medium
Table 3. C. perfringens: enterotoxigenic strains and enterotoxin production in Tm and DS
MPN* (decimal ratio)
Total of samples
o10 101 102 103 104 105
21 33 24 24 12 12
Culture media **
Strain
Decimal ratio and count of C. perfringens determined by MPN in iron–milk medium. * MPN/g of sample.
1 2 3 4 5 6 7 8 9
Modified Tm
Duncan–Strong [24]
8.192* 4.096 4.096 1.024 8.192 8.192 8.192 8. 192 4.096
64* 32 32 16 128 64 128 64 32
*
Concentration of enterotoxin expressed as ng/mL, determined by RPLA in supernatants of sporulation media on day 12 of incubation. Sensitivity of the test: 2 ng/mL. ** The other 117 strains were enterotoxin negative.
Table 2. C. perfringens strain NCTC 8798: spore count and enterotoxin production in different sporulation media Media Tm Day 3 6 9 12
DS *
Spores
CPE 7
1.37 10 1.9 107 1.84 107 1.93 107
16 384 16 384 16 384 16 384
Spores ND 1.2 106 1.28 106 1.12 106
RCM CPE 16 32 32 32
Spores ND ND ND ND
CPE ND ND ND ND
* Determination of enterotoxin concentration was carried out by RPLA expressed as ng/ml. Test sensitivity: 2 ng/mL. ND: not detected. Tm: modified To´rtora medium. DS: Duncan–Strong medium. RCM: reinforced Clostridium medium.
at MPN values above 200 bacteria/g. Twelve samples (9.52%) showed counts 45 log bacteria/g by the MPN method. There were significant differences with respect to other sample counts (Po0.000001). Sporulation, in vitro CPE production and RPLA Table 2 shows the results obtained by sporulation and enterotoxin production of the reference strain. When compared with other sporulation methods, such as Duncan–Strong [25] and reinforced Clostridium medium (26), it was noted that the modified To´rtora medium (Tm) is very useful for sporulation as well as enterotoxin production since on day 3, high counts of spores and high level of enterotoxin, determined by RPLA, were obtained. Of the 126 C. perfringens strains isolated and tested by the above methods, nine were enterotoxigenic while the other 117 were enterotoxin negative (Table 3). cpe-gene determination by PCR Of the 126 C. perfringens strains tested by PCR, nine strains (7.14%) turned out to be enterotoxigenic. Figure 1 shows the 935-bp PCR product corresponding to cpe-gene obtained with the primers used. Table 4 exhibits the origin of enterotoxigenic strains.
Figure 1. Agarose gel showing representative PCR results for enterotoxin gene detection with DNA. Lanes MM show molecular mass markers (from bottom to top: 564, 831, 947, 1375, 1584, 1904, 2027 4973 and 21226 bp); Lanes A–I: show amplification of cpe of the nine enterotoxigenic strains Clostridium perfringens isolated from meat foods (935 bp).
No significant differences were observed (Po 0.05) between fresh sausage, hamburger and minced meat samples when X2 frequency analysis was applied. Four out of the nine enterotoxigenic strains belonged to samples with 4 105 bacteria g –1. Typing of C. perfringens isolated strain by PCR In order to type C. perfringens strains based on their main toxins, the 126 strains were analysed by PCR. Of these, 123 (97.62%) belonged to Type A, 2 (1.59%) to Type C and 1 (0.79%) to Type E. All enterotoxigenic strains were classified as Type A.
Discussion As far as we know, this work constitutes the first study of the prevalence of C. perfringens in commercial meat samples in Argentina. An important number of
Prevalence of Clostridium perfringens in Meats Table 4. C. perfringens: origin of enterotoxigenic strains Origin Fresh sausages Hamburgers Minced meat Total
257
Conclusions
Number of samples
C. perfringens isolates
Enterotoxigenic strains
315 100 100 515
83 (26,35%) 19 (19%) 24 (24%) 126
8 0 1 9
Frequency of C. perfringens enterotoxigenic strains according to the origin of samples. No significant differences were observed (Po 0.05).
raw meat samples exhibited elevated C. perfringens counts, above the limits allowed by the CAA, which has established threshold levels not higher than 2 log CFU/g for sporulated anaerobes [33]. Our results also show the microbiological and epidemiological importance of C. perfringens as potentially responsible for food poisoning. The Tm medium was the most useful for sporulation [11] and enterotoxin production, since the highest spore number values as well as toxin concentration determined by RPLA were obtained. These results were observed when both the isolates and the reference strains were tested, and they were obtained in a shorter time period. These results are consistent with those obtained by Ando et al. [34] and To´rtora [12]. A comparison of CEP determinations by the RPLA commercial test and the plate test gave a 100% correlation, this is, all the enterotoxigenic strains were positive in both tests. This indicates that the plate test is a good alternative to commercial RPLA, considering that this is an expensive and not easily available reagent in Argentina. Although slide RPLA exhibits a slightly lower sensitivity (8 ng/mL) than commercial RPLA (2 ng/mL and), determinations with this test take only 3 min at a lower cost. The nine strains that were PCR-positive for cpe exhibited an amplification product of 935-bp [15] with the above-mentioned primers, while Saito et al. [35] obtained a 364-bp PCR product using other primers. PCR technique is a rapid method by which C. perfringens cpe-positive detection is possible in samples with bacterial loads as low as 10 bacteria/g and, unlike conventional methods which require previous enrichment, it can be directly applied when food poisoning is suspected. The fact that these nine cpe-positive strains were RPLA positive indicates that there were no ‘‘silent’’ strains, as reported by Billington et al. for C. perfringens type E [18]. The high percentage of Type A isolates and the fact that no Type B or D strains were detected in our samples might mainly be due to the origin of the samples used in this work. Similar results were obtained in 1996 by Songer et al., in a study with samples of different origins [16]. It should be noted that the nine enterotoxigenic strains were Type A.
1. According to our results, iron-milk medium is recommended because, besides being selective, results can be obtained in a shorter time (16–18 h). 2. The percentage of cpe-positive isolates obtained in this work, taking into account all the samples, was similar to those obtained in other geographical areas. 3. Of the three analyzed foods, the ones potentially most hazardous to health were the fresh sausages with 8 cpe-positive isolates, possibly because they are made with several ingredients and processed for longer times in deficient hygienic conditions. 4. The preparation of slide RPLA reagent for determining the enterotoxin will allow for lower costs and shorter times for the obtainment of results. 5. The studied foods may constitute a health hazard if they are not processed under adequate hygienic conditions or if the cold storage chain is not preserved.
Acknowledgements The authors thank Professor Isabel Teresa Gimenez for her assistance in statistical studies and Mr Alfredo R. Villegas for technical assistance. This work was supported by Secretarı´a de Ciencia y Te´cnica of Universidad Nacional de San Luis. Project 8803.
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FOOD MICROBIOLOGY
Prevalence of Enterotoxigenic Clostridium perfringens in Meats in San Luis, Argentina Patricia Virginia Stagnitta, Blas Micalizzi and Ana Mar|¤ a Stefanini de Guzma¤n* ´ rea Microbiologı´a, Departamento de A Bioquı´mica y Ciencias Biolo´gicas. Facultad de Quı´mica, Bioquı´mica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700- San Luis, Argentina (Received 29 November 2001; accepted 24 October 2002) Key Words: Enterotoxigenic, Clostridium perfringens, meats
Clostridium perfringens is an important pathogen agent causing, among other diseases, enteritis in humans and enterotoxemia in domestic animals. This bacterium can produce more than 15 toxins, one of which is its enterotoxin (CPE), that causes human food poisoning. The aim of this work was (i) to determine the prevalence of C. perfringens in some non-industrial meat foods in San Luis, Argentina, (ii) to characterize the C. perfringens enterotoxigenic strains by PCR, RPLA and the slide reverse passive latex agglutination test, (iii) to type the C. perfringens strains isolated and identification by PCR and (iv) to develop a slide RPLA test. A total of 515 samples of meat food (315 fresh sausages, 100 hamburgers and 100 samples of minced meat) were studied. A 126 C. perfringens strains (24.46%) were isolated and characterized. Of these C. perfringens-positive samples, 48 contained counts higher than 2 log/g. No significant differences were observed between counts performed in iron–milk medium and tryptose– sulfite–cycloserine agar (r= 0.99). Twelve samples (9.52%) exhibited counts with MPN 45 log bacteria/g. Modified To´rtora medium (Tm) with thiotone replaced by proteose peptone turned out to be the most useful medium for both sporulation and enterotoxin production. Of the 126 samples tested by PCR and RPLA, nine strains (7.14%) were enterotoxigenic. Similar results were obtained by Slide RPLA, which exhibited a sensitivity of 8 ng/mL. Of the 126 C. perfringens strains, 123 were of type A (97.20%), two were of type C (1.59%) and one of type E (0.79%). All enterotoxigenic strains were classified as type A. # 2003 Published by Elsevier Science Ltd.
Introduction * ´ rea Microbiologı´a, Departamento Address correspondence to: A de Bioquı´mica y Ciencias Biolo´gicas. Facultad de Quı´mica, Bioquı´mica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700- San Luis, Argentina. Tel: +54-2652423789 int. 127. Fax: +54-2652-431301. E-mail: [email protected]
Clostridium perfringens is an important pathogen agent causing, among other diseases, enteritis in humans and enterotoxemia in domestic animals [1–5]. This Gram-positive, anaerobic bacterium is widely distributed in the environmentFsoil and waterFand
1075–9964/03/$ - see front matter # 2003 Published by Elsevier Science Ltd.
254
P.V. Stagnitta et al.
usually found in the intestinal tract of humans and animals [1,2,6]. It can produce more than 15 toxins, among these, C. perfringens enterotoxin (CPE) [7], which causes human food poisoning [7, 8]. The first studies on food poisoning by C. perfringens did not distinguish between positive and negative enterotoxigenic strains. C. perfringens enterotoxin has been implicated in sporadic diarrheas and antibiotic-associated diarrheas [7, 9]. Intoxication with C. perfringens can be caused by ingestion of food containing X5 log vegetative cells/g of food of an enterotoxigenic strain [8,10]. The in vivo production of the enterotoxin is associated with the sporulation in intestine [10] and the in vitro production of the enterotoxin is obtained in appropriate sporulation media [11,12]. Enterotoxigenic strains can be characterized by the presence of the cpe gene by means of molecular methods or CPE determination by immunological methodsFELISA, reverse passive latex agglutination (RPLA) and cytotoxicity assays [2,5,6,13–16]. Five types of C. perfringens have been classified based on the production of four major toxins: a, b, e and i, which can be determined by serologic methods or by molecular methods such as PCR [2,13,15,17–20]. Considering that there are no available reports on food poisoning outbreaks caused by C. perfringens enterotoxigenic strains in San Luis, Argentina, the aims of this work were: (i) to determine the prevalence of C. perfringens in some non-industrial meat foods in San Luis, Argentina, by the most probable number (MPN) and the count on plate methods, (ii) to characterize the C. perfringens enteroxigenic strains by molecular methods such as PCR and immunological methods such as RPLA and Slide RPLA in order to detect CPE production, (iii) to type, by PCR, C. perfringens strains isolated from food, and (iv) to develop a slide RPLA test.
Materials and Methods Strains C. perfringens isolates and two reference strains, kindly supplied by Dr Ronald G. Labbe´ of University of Massachusetts, U.S.A.: NCTC 8798 cpe+ and ATCC 3694 cpe- were used. Samples A total of 515 food samples consisting of 315 fresh sausages, 100 hamburgers and 100 samples of minced meat were obtained from local markets of San Luis city, Argentina. Samples were immediately processed
or stored in refrigerator (4–61C) until processed, no longer than 24 h. Then, 2.5 g of each sample was homogenized in a mortar with 2.5 g of fine sand, previously sterilized in oven for 1 h at 1801C. The sample was resuspended in 22.5 mL of sterile 0.1% peptone water (dilution 1:10). Serial ten-fold dilutions were made according to the amount of suspected bacteria adding 1 ml of original suspension to tubes containing 9 ml of sterile 0.1 % peptone water. Culture and count of C. perfringens Culture and counts were carried out by MPN using de Man’s table in iron–milk medium [21–23], with aliquots of 1:10 dilutions in triplicate. Cultures were incubated at 451C for 16–18 h. The plate count was carried out on tryptose–sulfite–cycloserine agar (TSCA) [24] according to the manufacturer’s instructions. Plates were inoculated with aliquots of 0.2 mL of each dilution in triplicate and incubated in jars under anaerobic conditions by the evacuation-replacement method with a mixture of propane–butane at 371C for 48 h. Isolation and identification Suspected black colonies of C. perfringens were purified on TSCA [24] and identified by Gram’s stain and by standard biochemical tests. Strains were kept in cooked meat medium and replicated in brain–heart infusion or thioglycolate broth according to the experiments. Enterotoxin production C. perfringens strains isolated from food and reference strains were grown in thioglycolate broth at 371C for 18–24 h. One milliliter of culture was grown in the following sporulation media: To´rtora medium (Tm) modified in our laboratory, replacing thiotone by proteose- peptone [12]; Duncan–Strong medium (D–S) [25] and reinforced Clostridium medium (RCM) [26]. Tm was chosen as the CPE production medium because of the elevated titres obtained. The Tm medium was incubated at 371C for 12 days and spores were counted on days 3, 6, 9 and 12. Counts were carried out in a Neubauer chamber by using a Carl Zeiss Microscope with a 100 objective for phase contrast. The enterotoxin was determined in the culture supernatants by the RPLA technique. CPE detection by RPLA To quantify CPE in culture supernatants, a PET-RPLA commercial kit (Oxoid) of 2 ng/mL sensitivity was
Prevalence of Clostridium perfringens in Meats used. The test was carried out according to the manufacturer’s instructions. As control, the culture filtrates of CPE-positive and negative reference strains were used. Slide RPLA test Immunosera obtainment in rabbits. Lots of three white rabbits of approximately 2.5 kg were inoculated subcutaneously with 0.5 mL of the supernatant containing the enterotoxin mixed with Freund’s complete adjuvant (Oxoid) (1:1) [27]. Two doses 2 weeks apart were applied. Before the antigen administration, bleeding was carried out to discard the presence of natural antibodies. Immunosera were obtained by bleeding white rabbits 2 weeks after the last dose. Previously, animals were anesthetized with Ketalars (8 mg/kg of weight). Blood was left to coagulate at 371C and serum was separated, 1/10.000 thimerosal (Sigma) was added and the serum was kept at 201C until use. Purification of immunoglobulin G (IgG). The IgG antibodies of rabbit sera were purified by saline precipitation [28,29] and by affinity chromatography using a column of Sepharose CL 4B-protein A (Sigma) [30]. Concentration of purified IgG was determined by an ELISA test developed in our laboratory. Slide RPLA test. Latex bead sensitization. Latex bead (Sigma) sensitization was carried out according to Fach and Popoff, and Tamiya et al. [10,14]. Slide RPLA test was performed by mixing 25 mL sensitized latex and 25 mL of each of the supernatants of sporulation medium containing the CPE on a glass slide. The mixture was constantly shaken and the reaction was observed for the first 3 min. The sensitivity of this test was 8 ng/mL. Molecular biology methods C. perfringens cpe gene determination by PCR. Each of the 126 isolated strains was cultured in tubes with 10 mL thioglycolate broth and incubated anaerobically overnight at 371C. For DNA isolation and purification, a combination of chemical and enzymatic treatments was used for lysis of C. perfringens cells according to the method of Kokai-Kun et al. [31]. PCR amplification was performed in a thermal cycler (Perkin Elmer PCR System 2000) incubating the obtained DNA with the following primers: 50 -TGTTAATACTTTAAGGATATGTATCC-30 and 50 -TCCATCACCTAAGGACTG30 (Promega, Madison, WI, USA) at a concentration of 0.4 mM; 0.4 mM deoxynucleoside; 2 mM Cl2Mg (Pro-
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mega) and 1.5 U of Taq polymerase (Promega) in a total reaction volume of 50 mL per reaction tube. The first PCR cycle lasted 300 s at 941C, followed by 34 cycles of 1.5 min at 941C, 1 min at 501C and 1 min at 721C, and finishing with a 600 s cycle at 721C. Products of amplification were determined by electrophoresis in 1% agarose gel (Promega) at 80 V for 90 min using BioRad Power PAC 1000 Midicell EC 3500. The gel was stained with ethidium bromide (0.5 mg/mL) (Promega), and bands were visualized by UV illumination (White Ultraviolet Transilluminator UVP (Upland Sigma). Gels were photographed by a Polaroid Gel Cam EPH % 0.85 Electrophoresis Hood. C. perfringens typing by PCR. From a culture obtained in solid medium, C. perfringens strains were typified by using the technique of Uzal et al. [32] with the following primers: Fa-Toxin:50 -TGCTAATGTTACTGCCGTTGATAG-30 and 50 -ATAATCCCAATCATCCCAACTATG-30 . Fb-Toxin: 50 -AGGAGGTTTTTTTATGAAG-30 and 50 TCTAAATAGCTGTTACTTTGT-30 . Fe-Toxin:50 -TACTCATACTGTGGGAACCTTCGATACAAGC-30 and 50 -CTCATCTCCCATAACTGCACTATAATTTCC-30 . Ft-Toxin: 50 -TTTTAACTAGTTCATTTCCTAGTTA-30 and 50 -TTTTTGTATTCTTTTTCTCTAGATT-30 . The cycling consisted of 28 1-min cycles at 481C, 2-min cycles at 721C, and 1-min cycles at 931C for 1 min; one 1-min cycle at 481C and one 5-min cycle at 721C. Once the cycling was finished, the products were determined by electrophoresis in a 2% agarose gel with ethidium bromide. Gel visualization and photographs were performed as above described. Statistical tests The statistical study of results was performed by Student’s t-test, sign test, correlation coefficient ‘r’ and statistical X2 as appropriate.
Results Count, isolation and identification of C. perfringens Of the 515 samples analysed, 126 strains of C. perfringens were isolated and characterized by standard biochemical tests (Table 1). Of the 126 isolates, 48 (38.1%) exhibited C. perfringens counts 42 log/g, thus exceeding the maximum allowed by the Co´digo Alimentario Argentino (Argentine Alimentary Code, CAA) of 2 log sporulated anaerobes/g [33]. There were no significant differences between counts performed in iron–milk medium and TSCA (r= 0.99)
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Table 1. Presence of C. perfringens in meat foods: number of samples and MPN (most probable number) in iron–milk medium
Table 3. C. perfringens: enterotoxigenic strains and enterotoxin production in Tm and DS
MPN* (decimal ratio)
Total of samples
o10 101 102 103 104 105
21 33 24 24 12 12
Culture media **
Strain
Decimal ratio and count of C. perfringens determined by MPN in iron–milk medium. * MPN/g of sample.
1 2 3 4 5 6 7 8 9
Modified Tm
Duncan–Strong [24]
8.192* 4.096 4.096 1.024 8.192 8.192 8.192 8. 192 4.096
64* 32 32 16 128 64 128 64 32
*
Concentration of enterotoxin expressed as ng/mL, determined by RPLA in supernatants of sporulation media on day 12 of incubation. Sensitivity of the test: 2 ng/mL. ** The other 117 strains were enterotoxin negative.
Table 2. C. perfringens strain NCTC 8798: spore count and enterotoxin production in different sporulation media Media Tm Day 3 6 9 12
DS *
Spores
CPE 7
1.37 10 1.9 107 1.84 107 1.93 107
16 384 16 384 16 384 16 384
Spores ND 1.2 106 1.28 106 1.12 106
RCM CPE 16 32 32 32
Spores ND ND ND ND
CPE ND ND ND ND
* Determination of enterotoxin concentration was carried out by RPLA expressed as ng/ml. Test sensitivity: 2 ng/mL. ND: not detected. Tm: modified To´rtora medium. DS: Duncan–Strong medium. RCM: reinforced Clostridium medium.
at MPN values above 200 bacteria/g. Twelve samples (9.52%) showed counts 45 log bacteria/g by the MPN method. There were significant differences with respect to other sample counts (Po0.000001). Sporulation, in vitro CPE production and RPLA Table 2 shows the results obtained by sporulation and enterotoxin production of the reference strain. When compared with other sporulation methods, such as Duncan–Strong [25] and reinforced Clostridium medium (26), it was noted that the modified To´rtora medium (Tm) is very useful for sporulation as well as enterotoxin production since on day 3, high counts of spores and high level of enterotoxin, determined by RPLA, were obtained. Of the 126 C. perfringens strains isolated and tested by the above methods, nine were enterotoxigenic while the other 117 were enterotoxin negative (Table 3). cpe-gene determination by PCR Of the 126 C. perfringens strains tested by PCR, nine strains (7.14%) turned out to be enterotoxigenic. Figure 1 shows the 935-bp PCR product corresponding to cpe-gene obtained with the primers used. Table 4 exhibits the origin of enterotoxigenic strains.
Figure 1. Agarose gel showing representative PCR results for enterotoxin gene detection with DNA. Lanes MM show molecular mass markers (from bottom to top: 564, 831, 947, 1375, 1584, 1904, 2027 4973 and 21226 bp); Lanes A–I: show amplification of cpe of the nine enterotoxigenic strains Clostridium perfringens isolated from meat foods (935 bp).
No significant differences were observed (Po 0.05) between fresh sausage, hamburger and minced meat samples when X2 frequency analysis was applied. Four out of the nine enterotoxigenic strains belonged to samples with 4 105 bacteria g –1. Typing of C. perfringens isolated strain by PCR In order to type C. perfringens strains based on their main toxins, the 126 strains were analysed by PCR. Of these, 123 (97.62%) belonged to Type A, 2 (1.59%) to Type C and 1 (0.79%) to Type E. All enterotoxigenic strains were classified as Type A.
Discussion As far as we know, this work constitutes the first study of the prevalence of C. perfringens in commercial meat samples in Argentina. An important number of
Prevalence of Clostridium perfringens in Meats Table 4. C. perfringens: origin of enterotoxigenic strains Origin Fresh sausages Hamburgers Minced meat Total
257
Conclusions
Number of samples
C. perfringens isolates
Enterotoxigenic strains
315 100 100 515
83 (26,35%) 19 (19%) 24 (24%) 126
8 0 1 9
Frequency of C. perfringens enterotoxigenic strains according to the origin of samples. No significant differences were observed (Po 0.05).
raw meat samples exhibited elevated C. perfringens counts, above the limits allowed by the CAA, which has established threshold levels not higher than 2 log CFU/g for sporulated anaerobes [33]. Our results also show the microbiological and epidemiological importance of C. perfringens as potentially responsible for food poisoning. The Tm medium was the most useful for sporulation [11] and enterotoxin production, since the highest spore number values as well as toxin concentration determined by RPLA were obtained. These results were observed when both the isolates and the reference strains were tested, and they were obtained in a shorter time period. These results are consistent with those obtained by Ando et al. [34] and To´rtora [12]. A comparison of CEP determinations by the RPLA commercial test and the plate test gave a 100% correlation, this is, all the enterotoxigenic strains were positive in both tests. This indicates that the plate test is a good alternative to commercial RPLA, considering that this is an expensive and not easily available reagent in Argentina. Although slide RPLA exhibits a slightly lower sensitivity (8 ng/mL) than commercial RPLA (2 ng/mL and), determinations with this test take only 3 min at a lower cost. The nine strains that were PCR-positive for cpe exhibited an amplification product of 935-bp [15] with the above-mentioned primers, while Saito et al. [35] obtained a 364-bp PCR product using other primers. PCR technique is a rapid method by which C. perfringens cpe-positive detection is possible in samples with bacterial loads as low as 10 bacteria/g and, unlike conventional methods which require previous enrichment, it can be directly applied when food poisoning is suspected. The fact that these nine cpe-positive strains were RPLA positive indicates that there were no ‘‘silent’’ strains, as reported by Billington et al. for C. perfringens type E [18]. The high percentage of Type A isolates and the fact that no Type B or D strains were detected in our samples might mainly be due to the origin of the samples used in this work. Similar results were obtained in 1996 by Songer et al., in a study with samples of different origins [16]. It should be noted that the nine enterotoxigenic strains were Type A.
1. According to our results, iron-milk medium is recommended because, besides being selective, results can be obtained in a shorter time (16–18 h). 2. The percentage of cpe-positive isolates obtained in this work, taking into account all the samples, was similar to those obtained in other geographical areas. 3. Of the three analyzed foods, the ones potentially most hazardous to health were the fresh sausages with 8 cpe-positive isolates, possibly because they are made with several ingredients and processed for longer times in deficient hygienic conditions. 4. The preparation of slide RPLA reagent for determining the enterotoxin will allow for lower costs and shorter times for the obtainment of results. 5. The studied foods may constitute a health hazard if they are not processed under adequate hygienic conditions or if the cold storage chain is not preserved.
Acknowledgements The authors thank Professor Isabel Teresa Gimenez for her assistance in statistical studies and Mr Alfredo R. Villegas for technical assistance. This work was supported by Secretarı´a de Ciencia y Te´cnica of Universidad Nacional de San Luis. Project 8803.
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