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Immunomagnetic separation of Salmonella from foods
International Journal of Food Microbiolog% 14 (199 I) 11 - 18
11
¢' 1991 Elsevier Science Publishers B.V. All rights reserved 0168-1605/91/$03.50 FOOD 00,125
Immunomagnetic separation of Salmonella from foods Eystein Skjerve 1 and Orjan Olsvik
2
1Department of Food Hygiene and 2 Department of Microbiology and Immunology, Norwegian College of Veterinary Medicine, Oslo, Norway (Received 31 January 1991; accepted 22 May 1991)
Salmonella could be separated from different inoculated foods using antibody-coated immunomagnetic beads. When applied on suitable foods, the immunomagnetic separation technique showed a sensitivity of 10-20 Salmonella cells/g of the original sample. The technology appeared less useful for some food items. Key words: Salmonella; lmmunomagnetic separation; Food
Introduction
Methods for the detection and identification of Salmonella in foods have been of major interest for food microbiologists for many years. New media for enrichment and plating have been introduced (Beckers et al., 1987a,b), and various immunological methods, such as enzyme immunoassay (Minnisch et al., 1982; Prusak-Sochaczewski and Luong, 1989; Blais and Yamazaki, 1989; Lee et al., 1990) fluorescence labelling (Rodriguez and Kroll, 1990) and different agglutination methods (Metzler and Nachamkin, 1988; Benge, 1989) have been applied. More recently DNA hybridization methods have also been described (Fitts et al., 1983; Flowers et al., 1987). Preliminary selective a n d / o r non-selective enrichment of the sample before using the more recent methods results in rather long test times. I f a way could be found to circumvent the pre-enrichment procedure, this would reduce test time by at least 1 day. Immunomagnetic separation (IMS) has been shown to be a very functional tool for the isolation and extraction of bacteria from heterogeneous matrices. IMS has been used to extract Staphylococcus aureus from milk (Johne and Jarp, 1988; Johne et al., 1989), and specific Escherichia coli strains have been isolated from bacterial suspensions and faeces (Lund et al., 1988). Correspondence address." E. Skjerve, Department of Food Hygiene, Norwegian College of Veterinary Medicine, P.O. Box 8146 DEP, 0033 Oslo 1, Norway.
12 Immunomagnetic techniques for the detection of pathogens from foods have not been studied very much, though Skjerve et al. (1990) recently published results demonstrating the detection of Listeria monocytogenes by IMS. Some reports have also indicated the use of immunomagnetic separation to isolate Salmonella (Blackburn and Patel, 1989). The present paper describes the use of IMS for the direct detection of Salmonella in different food items.
Materials and Methods
Bacterial strain One strain each of Salmonella St. Paul and E. coli, both from the strain collection at the Department of Food Hygiene, Norwegian College of Veterinary Medicine, were used in the experiments. Fresh cultures of the bacteria were inoculated into Brain Heart Infusion Broth (BHIB; Oxoid, Basingstoke, U.K.) and incubated at 37 °C for 18 h. The broth was then diluted in phosphate-buffered saline pH 7.4 (PBS) to appropriate concentrations. lmmunomagnetic beads Immunomagnetic beads (IMB), size 2.8 /~m (Dynabeads R M-280, tosyl activated, Dynal A / S , Oslo, Norway) were used. Antibodies Affinity-purified antibodies against Salmonella common structural antigen-l, produced in goats (BacTrace R Salmonella CSA-1, Kirkegaard & Perry, Gaithersburg, MD), were used. Coating of immunomagnetic beads The beads were coated according to the manufacturer's instructions, using covalent coupling in 50 mM borate buffer at pH 9.5, followed by washing in PBS pH 7.4, and finally blocking with 0.1% bovine serum albumin (BSA; Sigma Chemical Company, St. Louis, MO). After preparation, the coated beads were stored at 4 ° C in PBS/0.1% BSA with 0.02% sodium azide. Before use, the beads were washed in PBS pH 7.4 with 0.1% Tween 20. Each mg of beads (6-7 × 107 IMB) was coated with 20 ~g antibodies. Washing and other separation of the beads were carried out using a magnetic particle concentrator (MPC-6, Dynal). Immunomagnetic separation BHIB dilutions of bacteria in PBS pH 7.4, were added to food samples diluted 1 : 10 in phosphate-buffered peptone water, (PBPW; Difco, Detroit, MI) in 10-ml polystyrene tubes. The tubes were washed with PBS with 0.05% Tween 20 before use. IMB were added, and the samples were rotated on a Luckham cell mixer (Luckham, Sussex, U.K.) for 10 min at room temperature in a near horizontal position. After rotation, the IMB were allowed to settle for 5 min before washing.
13 The magnetic particle concentrator (MPC-6) was employed to trap IMB against the wall of the tube, and residual liquid was removed by aspiration. IMB were then washed with 8 ml P B S / Tween 20, resuspended in 100 ~1 PBS, and spread on Brilliant Green Agar (BGA; Oxoid) or MacConkey agar (Oxoid) with a sterile glass rod. Agar plates were incubated for 18 h at 37 ° C, after which the number of colonies were counted. Uninoculated samples were used as controls. All samples were run in duplicate, and results were recorded as the mean of the two samples. Dubious colonies were agglutinated with a suspension of the described antibodycoated IMB using a slide agglutination technique for verification.
Specificity of immunomagnetic separation Cultures of S. St. Paul and E. coli were diluted in PBS to different concentrations. Immunomagnetic separation was applied on pure cultures and on mixtures of the two bacteria present in different ratios. 5 x 106 IMB were added to 1 ml of sample. Before transfer to MacConkey agar, the IMB were washed three times with P B S / T w e e n 20. Influence of food matrices on immunomagnetic separation Food items bought from a local retail grocery shop - - soft cheese (Brie), pasteurized whole milk, whole milk powder, yoghurt, chicken liver, vacuum-packed sliced ham and raw vegetable blend - - were mixed 1:10 in PBPW using a stomacher (BA 6021, Seward Laboratories, Suffolk, U.K.). BHIB dilutions of S. St. Paul were added, and immunomagnetic separation using 5 × 106 beads in 1 ml was accomplished as previously described. The beads were washed only once before being plated on BGA. Uninoculated PBPW was used as control. Catching efficiency and sensitit'ity in bigger t:olumes Dilutions of S. St. Paul grown in BHIB were added to whole milk powder diluted 1 : 10 in PBPW. IMB in amounts of either 0.5 X 107, 1 X 107, 1.5 × 107 or 2 x 107 were added to 5-ml volumes of sample containing 1250 S. St. Paul cells/ml. Before adding the IMB, a volume of 0.1 ml from each tube was plated on BGA. This was also done from the supernatant after IMS to estimate the proportion of bound bacteria. The experiment was continued, using 2 × 107 IMB in 5 ml, adding 60, 300 or 3 × 103 Salmonella cells/mi. The practical sensitivity of the system was tested by repeat experiments in which milk powder was diluted 1 : 10 in PBPW, and different numbers of S. St. Paul and 2 x 107 IMB were added to 10 ml of sample.
Results Immunomagnetic separation resulted in effective isolation of S. St. Paul from pure cultures and from mixtures with E. coli in PBS, as shown in Table I. Overall, a range of 4 - 8 cells of S. St. Paul resulted in one colony on the agar plate when cells were added in numbers ranging from below 10/ml up to 2 x 103/ml.
14 TABLE I l m m u n o m a g n e t i c separation of Salmonella St. Paul and E. coli from PBS Colonies on MacConkey agar
Bacteria added, c e l l s / m l
Salmonefla
E. coli
Salmonel~
20000 1600 200 160 16
E. coli
> 300 > 300 30 35 2 900000 22000 9000 900
160
9 3 1 0
220000 22 000 2 200
160 160
45 34 37
3
0 0
Recovery in pure cultures and in mixtures, recorded as n u m b e r of typical colonies on MacConkey agar.
Repeating the same experiment with E. coli, between 104 and 10 s ceils/mi in the test system resulted in more than one colony on MacConkey agar after IMS. Mixing 2 × 103 to 2 )< 105 E. coli cells with 160 cells/ml of S. St. Paul did not interfere with the recovery of S. St. Paul by IMS. Immunomagnetic separation of inoculated S. St. Paul cells from milk, milk powder, ham, and vegetable blend was successful. A distinct pellet of beads was observed on the sides of the tubes held in the magnetic particle concentration after separation, and few beads were lost. Though soft cheese gave visually a rather impure pellet, most of the IMB were still recovered. For yoghurt and chicken liver, a substantial loss of beads was observed during initial separation and washing.
>30O J [ ] PBPW '~
<
80 82 --48 52l i
I [ ] Cheese [] Milk-~d~r
~
a o
o
o
I
t,
L/
0
i::
60 Salmonella. cells/ml
Fig. 1. l m m u n o m a g n e t i c separation of Salmonella St. Paul from phosphate-buffered peptone water (PBPW). and milk powder, ham and soft cheese diluted 1:10 in PBPW. Different numbers of Salmonella added to 1 ml of sample. Results recorded as n u m b e r s of typical colonies of S. St. Paul on Brilliant G r e e n Agar (BGA).
15 Furthermore, the residual IMB did not form a distinct pellet, and recovery after IMS was poor. Fig. 1 shows results for the recovery of S. St. Paul from PBPW. vacuum-packed ham, soft cheese, and whole milk powder. In all these cases there was good separation of the IMB from the food matrix. Catching efficiency was slightly lower than in PBS. Increasing the number of IMB in milk powder resuspended in PBPW with 1250 Salmonella cells/ml, increased the binding ratio from 18% to 28% (data not shown). The use of 2 × 107 IMB in 5-ml samples of PBPW-suspended milk powder gave recoveries ranging from 50% with 60 cells/ml, down to 30% (300 cells/ml) and 16% for 3 × 103 cells/ml. Using 10 ml of the sample and 2 x 107 IMB, S. St. Paul could be detected at a sensitivity down to 1-2 cells/ml using the described test system. No experiment (n --- 15) failed to demonstrate Salmonella if at least 2-10 cells/ml were present (data not included).
Discussion
Immunomagnetic separation enabled us to distinguish between S. St. Paul and E. coli in mixtures of these bacteria in PBS, even when the proportion of E. coli present was very large (E. c o l i / S . St. Paul > 1000: 1). By increasing the sample volume to 5 or 10 ml, the sensitivity could be increased down to as few as 1-2 cells/ml, corresponding to 10-20 cells/g of the original sample. These results demonstrate a concentration benefit when compared to direct plating (max. sensitivity 10 cells/ml) of 5-10-fold. Another important advantage is the possibility of obtaining pure cultures directly, due to the specificity of the method. The antibodies used in the experiments were affinity-purified. The question then arises as to whether these antibodies offer bonds of sufficient strength. Skjerve (1990) reported a primary binding of up to 90% using a monoclonal antibody against Listeria monocytogenes. The results indicated that IMS can be utilized for separation of Salmonella directly from foods. When a detection sensitivity of 10-20 cells/g of the original sample is acceptable. Under normal food testing conditions Salmonella should be absent in 25 g of product. Using the IMS technique at least 500 salmonellae should be present in 25 g to obtain a positive result by examining the pre-enrichment medium directly. IMS did not prove suitable for all food items. This was shown by the size and appearance of the pellet of the IMB after initial separation in the MPC. Some of the difficulties might be overcome by changing the broth or buffer used for catching IMB, although preliminary tests using different proteins, with or without different detergents, has so far not solved the problem. Samples with large numbers of competing flora ( > 107/ml), as found in yoghurt were least suited for IMS, though chicken liver also gave problems. High-fat samples such as soft cheese gave an altered pellet, but still allowed separation. Similar problems were encountered in our preliminary experiments with IMS of
16
Salmonella from faeces (not published). IMS of Listeria from non-selective pre-enriched food samples was also reported to be difficult by Skjerve et al. (1990), while Cudjoe et al. (1991) reported a reduced sensitivity of E L I S A systems using IMS in faeces as compared to meats. Larger beads with a diameter of 450 /zm were employed in previous successful attempts to use IMS on faeces (Lund et al., 1988). The smaller beads might be more susceptible to aggregation and subsequent washing off, even though our results have so far revealed no such differences. The washing technique is important. Especially after the primary separation, care should be taken to avoid loss of IMB. The difference observed between the duplicate samples also indicates a need for careful handling of the IMB during separation and washing. Using the described method, preliminary results for the detection of Salmonella in foods could be available within 18-24 h. If other detection methods are employed, such as E L I S A or immunofluorescence, the result may be available within less than 2 h, depending on the number of Salmonella present. The much lower sensitivity inherent in immunodetection methods compared to plating would, however, reduce the applicability of a direct detection step for Salmonella. For other pathogens, such as Listeria monocytogenes and Yersinia enterocolitica, when the demand for sensitivity is not that strict, immunological detection could be a sensible approach. Of more interest for Salmonella detection could be the use of sensitive detection systems such as polymerase chain reaction (PCR; Mullis and Faioona, 1987; Mullis et al., 1986) in combination with IMS. Immunomagnetic separation would reduce the problem of background proteins and foreign DNA, and therefore allow the use of the extremely sensitive P C R detection method. Further work should be instituted in this field to obtain truly rapid methods for the detection of Salmonella.
References Beckers, H.J., Roberts, D., Pietzsch, O., van Schothorst, M., Vassiliadis, P. and Kampelmacher, E.H. (1987a) Replacement of Miiller Kaufmann's tetrathionate brilliant green bile broth by Rappaport Vasiliadis magnesium chloride malachite green broth in the standard method for the detection of salmonellae. Int. J. Food Microbiol. 4; 59-64. Beckers, H.J.. Peters, R. and Pateer, P.M. (1987b) Collaborative study on the isolation of Salmonella from reference material using selective enrichment media, prepared from individual ingredients or commercial dehydrated products. Int. J. Food Microbiol. 4, l-I 1. Benge, G.R. (1989) Detection of Salmonella species in faeces by latexagglutination in enrichment broth. Eur. J. Clin. Microbiol. Infect. Dis. 8. 294-298. Blackburn. C. de W. and Patel, P.D. (1989) Interaction of Salmonella with immunomagneticparticles. Proc. Soc. Appl. Bacteriol. Conf. ii, 50. Blais, B.W. and Yamazaki, H. (1989) Extraction of Salmonella antigens in non-sedimentable forms for enzyme immunoassay. Int. J. Food Microbiol. 9, 63-72. Cudjoe, K.S., Thorsen, L.I.. Sorensen, T., Reseland, J., Olsvik, O. and Granum, P.E. (1991) Detection of Clostridium perfringens type A enterotoxin in fecal and food samples using immunomagnetic separation (IMS)-ELISA. Int. J. Food Microbiol. 12, 313-222. Fitts, R.. Diamond, M,, Hamilton, C. and Neri, M. (1983) DNA-DNA hybridization assay for detection of Salmonella spp. in foods. Appl. Environ. Microbiol. 43, 1146-1151.
17 Flowers, R.S., Klatt, M.J., Mozola, M.A.. Curciale, M.S.. Gabis, D.A. and Sillikker. J.H. (1987) DNA hybridization assays for detection of Salmonella in foods: A corroberative study. J. Assoc. Off. Anal. Chem. 70, 521-535. Johne, B. and Jarp, J. (1988) A rapid assay for protein-A in Staphylococcus aureus strains using immunomagnetic monosized polymer particles. APMiS 96, 43-49. Johne, B., Jarp, J. and Haaheim, L.R. (1989) Staphylococcus aureus exopolysaccharide in vivo demonstrated by immunomagnetic separation and electron microscopy. J. Clin. Microbiol. 27, 1631-1635. Lee, H.A., Wyatt, G.M., Brahmam, S.M. and Morgan. M.R.A. (1990) Enzyme-linked immunosorbent assay for Salmonella typhymurium in food: Feasibility of 1-day Salmonella detection. Appl. Environ. Microbiol. 56: 1541-1546. Lund, A., Hellemann, A.L. and Vartdal, F. (1988) Rapid isolation of K88+ Escherichia coil by using immunomagnetic particles. J. Clin. Microbiol. 26, 2572-2575. Metzler, J. and Nachamkin, I. (1988) Evaluation of a latex agglutination test for the detection of Salmonella and Shigella spp. by using broth enrichment. J. Clin. Microbiol. 26, 2501-2504. Minnisch, S.A., Hartmann, P.A. and Heimsch, R.C. (1982) Enzyme immunoassay for detection of salmonellae in foods. Appl. Environ. Microbiol. 43, 877-883. Mullis, K.B. and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335-350. Mullis, K.B., Faloona, F.A., Scharf, S., Saiki, R., Horn, G. and Ehrlich, H. (1986) Enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harbour Symp. Ouant. Biol. 51,263-273. Prusak-Sochaczewski, E. and Luong, J.H.T. (1989) Utilization of two improved immunoassays based on avidin-biotin interaction for the detection of Salmonella. Int. J, Food Microbiol. 8, 321-333. Rodriguez, U.M. and Kroll, R.G. (1990) Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique. J. Appl. Microbiol. 68. 213-223. Skjerve, E., Rorvik, L.M. and Olsvik, ~. (1990) Detection of Listeria monocytogenes in foods using immunomagnetic separation. Appl. Environ. Microbiol. 56, 3478-3481.
11
¢' 1991 Elsevier Science Publishers B.V. All rights reserved 0168-1605/91/$03.50 FOOD 00,125
Immunomagnetic separation of Salmonella from foods Eystein Skjerve 1 and Orjan Olsvik
2
1Department of Food Hygiene and 2 Department of Microbiology and Immunology, Norwegian College of Veterinary Medicine, Oslo, Norway (Received 31 January 1991; accepted 22 May 1991)
Salmonella could be separated from different inoculated foods using antibody-coated immunomagnetic beads. When applied on suitable foods, the immunomagnetic separation technique showed a sensitivity of 10-20 Salmonella cells/g of the original sample. The technology appeared less useful for some food items. Key words: Salmonella; lmmunomagnetic separation; Food
Introduction
Methods for the detection and identification of Salmonella in foods have been of major interest for food microbiologists for many years. New media for enrichment and plating have been introduced (Beckers et al., 1987a,b), and various immunological methods, such as enzyme immunoassay (Minnisch et al., 1982; Prusak-Sochaczewski and Luong, 1989; Blais and Yamazaki, 1989; Lee et al., 1990) fluorescence labelling (Rodriguez and Kroll, 1990) and different agglutination methods (Metzler and Nachamkin, 1988; Benge, 1989) have been applied. More recently DNA hybridization methods have also been described (Fitts et al., 1983; Flowers et al., 1987). Preliminary selective a n d / o r non-selective enrichment of the sample before using the more recent methods results in rather long test times. I f a way could be found to circumvent the pre-enrichment procedure, this would reduce test time by at least 1 day. Immunomagnetic separation (IMS) has been shown to be a very functional tool for the isolation and extraction of bacteria from heterogeneous matrices. IMS has been used to extract Staphylococcus aureus from milk (Johne and Jarp, 1988; Johne et al., 1989), and specific Escherichia coli strains have been isolated from bacterial suspensions and faeces (Lund et al., 1988). Correspondence address." E. Skjerve, Department of Food Hygiene, Norwegian College of Veterinary Medicine, P.O. Box 8146 DEP, 0033 Oslo 1, Norway.
12 Immunomagnetic techniques for the detection of pathogens from foods have not been studied very much, though Skjerve et al. (1990) recently published results demonstrating the detection of Listeria monocytogenes by IMS. Some reports have also indicated the use of immunomagnetic separation to isolate Salmonella (Blackburn and Patel, 1989). The present paper describes the use of IMS for the direct detection of Salmonella in different food items.
Materials and Methods
Bacterial strain One strain each of Salmonella St. Paul and E. coli, both from the strain collection at the Department of Food Hygiene, Norwegian College of Veterinary Medicine, were used in the experiments. Fresh cultures of the bacteria were inoculated into Brain Heart Infusion Broth (BHIB; Oxoid, Basingstoke, U.K.) and incubated at 37 °C for 18 h. The broth was then diluted in phosphate-buffered saline pH 7.4 (PBS) to appropriate concentrations. lmmunomagnetic beads Immunomagnetic beads (IMB), size 2.8 /~m (Dynabeads R M-280, tosyl activated, Dynal A / S , Oslo, Norway) were used. Antibodies Affinity-purified antibodies against Salmonella common structural antigen-l, produced in goats (BacTrace R Salmonella CSA-1, Kirkegaard & Perry, Gaithersburg, MD), were used. Coating of immunomagnetic beads The beads were coated according to the manufacturer's instructions, using covalent coupling in 50 mM borate buffer at pH 9.5, followed by washing in PBS pH 7.4, and finally blocking with 0.1% bovine serum albumin (BSA; Sigma Chemical Company, St. Louis, MO). After preparation, the coated beads were stored at 4 ° C in PBS/0.1% BSA with 0.02% sodium azide. Before use, the beads were washed in PBS pH 7.4 with 0.1% Tween 20. Each mg of beads (6-7 × 107 IMB) was coated with 20 ~g antibodies. Washing and other separation of the beads were carried out using a magnetic particle concentrator (MPC-6, Dynal). Immunomagnetic separation BHIB dilutions of bacteria in PBS pH 7.4, were added to food samples diluted 1 : 10 in phosphate-buffered peptone water, (PBPW; Difco, Detroit, MI) in 10-ml polystyrene tubes. The tubes were washed with PBS with 0.05% Tween 20 before use. IMB were added, and the samples were rotated on a Luckham cell mixer (Luckham, Sussex, U.K.) for 10 min at room temperature in a near horizontal position. After rotation, the IMB were allowed to settle for 5 min before washing.
13 The magnetic particle concentrator (MPC-6) was employed to trap IMB against the wall of the tube, and residual liquid was removed by aspiration. IMB were then washed with 8 ml P B S / Tween 20, resuspended in 100 ~1 PBS, and spread on Brilliant Green Agar (BGA; Oxoid) or MacConkey agar (Oxoid) with a sterile glass rod. Agar plates were incubated for 18 h at 37 ° C, after which the number of colonies were counted. Uninoculated samples were used as controls. All samples were run in duplicate, and results were recorded as the mean of the two samples. Dubious colonies were agglutinated with a suspension of the described antibodycoated IMB using a slide agglutination technique for verification.
Specificity of immunomagnetic separation Cultures of S. St. Paul and E. coli were diluted in PBS to different concentrations. Immunomagnetic separation was applied on pure cultures and on mixtures of the two bacteria present in different ratios. 5 x 106 IMB were added to 1 ml of sample. Before transfer to MacConkey agar, the IMB were washed three times with P B S / T w e e n 20. Influence of food matrices on immunomagnetic separation Food items bought from a local retail grocery shop - - soft cheese (Brie), pasteurized whole milk, whole milk powder, yoghurt, chicken liver, vacuum-packed sliced ham and raw vegetable blend - - were mixed 1:10 in PBPW using a stomacher (BA 6021, Seward Laboratories, Suffolk, U.K.). BHIB dilutions of S. St. Paul were added, and immunomagnetic separation using 5 × 106 beads in 1 ml was accomplished as previously described. The beads were washed only once before being plated on BGA. Uninoculated PBPW was used as control. Catching efficiency and sensitit'ity in bigger t:olumes Dilutions of S. St. Paul grown in BHIB were added to whole milk powder diluted 1 : 10 in PBPW. IMB in amounts of either 0.5 X 107, 1 X 107, 1.5 × 107 or 2 x 107 were added to 5-ml volumes of sample containing 1250 S. St. Paul cells/ml. Before adding the IMB, a volume of 0.1 ml from each tube was plated on BGA. This was also done from the supernatant after IMS to estimate the proportion of bound bacteria. The experiment was continued, using 2 × 107 IMB in 5 ml, adding 60, 300 or 3 × 103 Salmonella cells/mi. The practical sensitivity of the system was tested by repeat experiments in which milk powder was diluted 1 : 10 in PBPW, and different numbers of S. St. Paul and 2 x 107 IMB were added to 10 ml of sample.
Results Immunomagnetic separation resulted in effective isolation of S. St. Paul from pure cultures and from mixtures with E. coli in PBS, as shown in Table I. Overall, a range of 4 - 8 cells of S. St. Paul resulted in one colony on the agar plate when cells were added in numbers ranging from below 10/ml up to 2 x 103/ml.
14 TABLE I l m m u n o m a g n e t i c separation of Salmonella St. Paul and E. coli from PBS Colonies on MacConkey agar
Bacteria added, c e l l s / m l
Salmonefla
E. coli
Salmonel~
20000 1600 200 160 16
E. coli
> 300 > 300 30 35 2 900000 22000 9000 900
160
9 3 1 0
220000 22 000 2 200
160 160
45 34 37
3
0 0
Recovery in pure cultures and in mixtures, recorded as n u m b e r of typical colonies on MacConkey agar.
Repeating the same experiment with E. coli, between 104 and 10 s ceils/mi in the test system resulted in more than one colony on MacConkey agar after IMS. Mixing 2 × 103 to 2 )< 105 E. coli cells with 160 cells/ml of S. St. Paul did not interfere with the recovery of S. St. Paul by IMS. Immunomagnetic separation of inoculated S. St. Paul cells from milk, milk powder, ham, and vegetable blend was successful. A distinct pellet of beads was observed on the sides of the tubes held in the magnetic particle concentration after separation, and few beads were lost. Though soft cheese gave visually a rather impure pellet, most of the IMB were still recovered. For yoghurt and chicken liver, a substantial loss of beads was observed during initial separation and washing.
>30O J [ ] PBPW '~
<
80 82 --48 52l i
I [ ] Cheese [] Milk-~d~r
~
a o
o
o
I
t,
L/
0
i::
60 Salmonella. cells/ml
Fig. 1. l m m u n o m a g n e t i c separation of Salmonella St. Paul from phosphate-buffered peptone water (PBPW). and milk powder, ham and soft cheese diluted 1:10 in PBPW. Different numbers of Salmonella added to 1 ml of sample. Results recorded as n u m b e r s of typical colonies of S. St. Paul on Brilliant G r e e n Agar (BGA).
15 Furthermore, the residual IMB did not form a distinct pellet, and recovery after IMS was poor. Fig. 1 shows results for the recovery of S. St. Paul from PBPW. vacuum-packed ham, soft cheese, and whole milk powder. In all these cases there was good separation of the IMB from the food matrix. Catching efficiency was slightly lower than in PBS. Increasing the number of IMB in milk powder resuspended in PBPW with 1250 Salmonella cells/ml, increased the binding ratio from 18% to 28% (data not shown). The use of 2 × 107 IMB in 5-ml samples of PBPW-suspended milk powder gave recoveries ranging from 50% with 60 cells/ml, down to 30% (300 cells/ml) and 16% for 3 × 103 cells/ml. Using 10 ml of the sample and 2 x 107 IMB, S. St. Paul could be detected at a sensitivity down to 1-2 cells/ml using the described test system. No experiment (n --- 15) failed to demonstrate Salmonella if at least 2-10 cells/ml were present (data not included).
Discussion
Immunomagnetic separation enabled us to distinguish between S. St. Paul and E. coli in mixtures of these bacteria in PBS, even when the proportion of E. coli present was very large (E. c o l i / S . St. Paul > 1000: 1). By increasing the sample volume to 5 or 10 ml, the sensitivity could be increased down to as few as 1-2 cells/ml, corresponding to 10-20 cells/g of the original sample. These results demonstrate a concentration benefit when compared to direct plating (max. sensitivity 10 cells/ml) of 5-10-fold. Another important advantage is the possibility of obtaining pure cultures directly, due to the specificity of the method. The antibodies used in the experiments were affinity-purified. The question then arises as to whether these antibodies offer bonds of sufficient strength. Skjerve (1990) reported a primary binding of up to 90% using a monoclonal antibody against Listeria monocytogenes. The results indicated that IMS can be utilized for separation of Salmonella directly from foods. When a detection sensitivity of 10-20 cells/g of the original sample is acceptable. Under normal food testing conditions Salmonella should be absent in 25 g of product. Using the IMS technique at least 500 salmonellae should be present in 25 g to obtain a positive result by examining the pre-enrichment medium directly. IMS did not prove suitable for all food items. This was shown by the size and appearance of the pellet of the IMB after initial separation in the MPC. Some of the difficulties might be overcome by changing the broth or buffer used for catching IMB, although preliminary tests using different proteins, with or without different detergents, has so far not solved the problem. Samples with large numbers of competing flora ( > 107/ml), as found in yoghurt were least suited for IMS, though chicken liver also gave problems. High-fat samples such as soft cheese gave an altered pellet, but still allowed separation. Similar problems were encountered in our preliminary experiments with IMS of
16
Salmonella from faeces (not published). IMS of Listeria from non-selective pre-enriched food samples was also reported to be difficult by Skjerve et al. (1990), while Cudjoe et al. (1991) reported a reduced sensitivity of E L I S A systems using IMS in faeces as compared to meats. Larger beads with a diameter of 450 /zm were employed in previous successful attempts to use IMS on faeces (Lund et al., 1988). The smaller beads might be more susceptible to aggregation and subsequent washing off, even though our results have so far revealed no such differences. The washing technique is important. Especially after the primary separation, care should be taken to avoid loss of IMB. The difference observed between the duplicate samples also indicates a need for careful handling of the IMB during separation and washing. Using the described method, preliminary results for the detection of Salmonella in foods could be available within 18-24 h. If other detection methods are employed, such as E L I S A or immunofluorescence, the result may be available within less than 2 h, depending on the number of Salmonella present. The much lower sensitivity inherent in immunodetection methods compared to plating would, however, reduce the applicability of a direct detection step for Salmonella. For other pathogens, such as Listeria monocytogenes and Yersinia enterocolitica, when the demand for sensitivity is not that strict, immunological detection could be a sensible approach. Of more interest for Salmonella detection could be the use of sensitive detection systems such as polymerase chain reaction (PCR; Mullis and Faioona, 1987; Mullis et al., 1986) in combination with IMS. Immunomagnetic separation would reduce the problem of background proteins and foreign DNA, and therefore allow the use of the extremely sensitive P C R detection method. Further work should be instituted in this field to obtain truly rapid methods for the detection of Salmonella.
References Beckers, H.J., Roberts, D., Pietzsch, O., van Schothorst, M., Vassiliadis, P. and Kampelmacher, E.H. (1987a) Replacement of Miiller Kaufmann's tetrathionate brilliant green bile broth by Rappaport Vasiliadis magnesium chloride malachite green broth in the standard method for the detection of salmonellae. Int. J. Food Microbiol. 4; 59-64. Beckers, H.J.. Peters, R. and Pateer, P.M. (1987b) Collaborative study on the isolation of Salmonella from reference material using selective enrichment media, prepared from individual ingredients or commercial dehydrated products. Int. J. Food Microbiol. 4, l-I 1. Benge, G.R. (1989) Detection of Salmonella species in faeces by latexagglutination in enrichment broth. Eur. J. Clin. Microbiol. Infect. Dis. 8. 294-298. Blackburn. C. de W. and Patel, P.D. (1989) Interaction of Salmonella with immunomagneticparticles. Proc. Soc. Appl. Bacteriol. Conf. ii, 50. Blais, B.W. and Yamazaki, H. (1989) Extraction of Salmonella antigens in non-sedimentable forms for enzyme immunoassay. Int. J. Food Microbiol. 9, 63-72. Cudjoe, K.S., Thorsen, L.I.. Sorensen, T., Reseland, J., Olsvik, O. and Granum, P.E. (1991) Detection of Clostridium perfringens type A enterotoxin in fecal and food samples using immunomagnetic separation (IMS)-ELISA. Int. J. Food Microbiol. 12, 313-222. Fitts, R.. Diamond, M,, Hamilton, C. and Neri, M. (1983) DNA-DNA hybridization assay for detection of Salmonella spp. in foods. Appl. Environ. Microbiol. 43, 1146-1151.
17 Flowers, R.S., Klatt, M.J., Mozola, M.A.. Curciale, M.S.. Gabis, D.A. and Sillikker. J.H. (1987) DNA hybridization assays for detection of Salmonella in foods: A corroberative study. J. Assoc. Off. Anal. Chem. 70, 521-535. Johne, B. and Jarp, J. (1988) A rapid assay for protein-A in Staphylococcus aureus strains using immunomagnetic monosized polymer particles. APMiS 96, 43-49. Johne, B., Jarp, J. and Haaheim, L.R. (1989) Staphylococcus aureus exopolysaccharide in vivo demonstrated by immunomagnetic separation and electron microscopy. J. Clin. Microbiol. 27, 1631-1635. Lee, H.A., Wyatt, G.M., Brahmam, S.M. and Morgan. M.R.A. (1990) Enzyme-linked immunosorbent assay for Salmonella typhymurium in food: Feasibility of 1-day Salmonella detection. Appl. Environ. Microbiol. 56: 1541-1546. Lund, A., Hellemann, A.L. and Vartdal, F. (1988) Rapid isolation of K88+ Escherichia coil by using immunomagnetic particles. J. Clin. Microbiol. 26, 2572-2575. Metzler, J. and Nachamkin, I. (1988) Evaluation of a latex agglutination test for the detection of Salmonella and Shigella spp. by using broth enrichment. J. Clin. Microbiol. 26, 2501-2504. Minnisch, S.A., Hartmann, P.A. and Heimsch, R.C. (1982) Enzyme immunoassay for detection of salmonellae in foods. Appl. Environ. Microbiol. 43, 877-883. Mullis, K.B. and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335-350. Mullis, K.B., Faloona, F.A., Scharf, S., Saiki, R., Horn, G. and Ehrlich, H. (1986) Enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harbour Symp. Ouant. Biol. 51,263-273. Prusak-Sochaczewski, E. and Luong, J.H.T. (1989) Utilization of two improved immunoassays based on avidin-biotin interaction for the detection of Salmonella. Int. J, Food Microbiol. 8, 321-333. Rodriguez, U.M. and Kroll, R.G. (1990) Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique. J. Appl. Microbiol. 68. 213-223. Skjerve, E., Rorvik, L.M. and Olsvik, ~. (1990) Detection of Listeria monocytogenes in foods using immunomagnetic separation. Appl. Environ. Microbiol. 56, 3478-3481.