APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p. 3478-3481 0099-2240/90/113478-04$02.00/0 Copyright C) 1990, American Society for Microbiology

Vol. 56, No. 11

Detection of Listeria monocytogenes in Foods by Immunomagnetic Separation EYSTEIN SKJERVE,l* LIV MARIT R0RVIK,' AND 0RJAN OLSVIK2

Department of Food Hygiene' and Department of Microbiology and Immunology,2 Norwegian College of Veterinary Medicine, P.O. Box 8146 DEP, 0033 Oslo 1, Norway Received 29 May 1990/Accepted 22 August 1990

Immunomagnetic separation with immunomagnetic beads was used to isolate strains of Listeria monocytoboth from pure cultures and from heterogeneous suspensions. The monoclonal antibodies used recognized all six strains of serotype 4 but only one of three strains of serotype 1. Coating procedure, incubation time, and number of immunomagnetic beads influenced the sensitivity of the isolation method. Less than 1 x 102 bacteria per ml in pure cultures and less than 2 x 102 bacteria per ml in enriched foods could be detected. The method represents a new approach to extraction and isolation of pathogenic bacteria directly from foods, after resuscitation, or from enrichment broths.

genes

Listeria monocytogenes is a pathogen of growing concern in public health and food hygiene. As one of the classic pathogens, it has recently become the subject of renewed interest because of acknowledged epidemics caused by food contaminated with Listeria spp. (1, 2, 9, 13, 20, 29). Substantial interest has been shown in establishing new methods as well as improving classic methods for the detection and isolation of Listeria spp. in foods. Although traditional culturing methods have been improved upon, there is still no agreement among scientists on a superior standard method (3, 4, 20, 25-28). Immunological methods of detection based on enzyme-linked immunosorbent assay procedures have been developed and are now commercially available (5, 7, 18). These methods offer possibilities for screening a large number of samples but are, nevertheless, laborious and time consuming, as well as being dependent on preenrichment in selective media. Flow cytometry has been used with some success for detecting L. monocytogenes in milk and offers some possibilities for application as a rapid method (6). However, the equipment needed is very expensive and the method lacks the sensitivity needed to detect bacteria directly in foods. Methods employing DNA-DNA hybridization have also been tried with some success but are still dependent on an initial, selective preenrichment step (14-16). Immunomagnetic separation (IMS) has been shown to be a very effective tool for the separation and isolation of specific cells from heterogeneous cell suspensions, especially for the isolation of specific cells from blood (22). An increasing number of immunoassays that use IMS have been described (10, 19, 21-23). Of more interest in the context of this paper is the use of IMS for the isolation and extraction of bacteria from heterogeneous suspensions. IMS has been used to extract Staphylococcus aureus from milk (11, 12), and specific Escherichia coli strains have been isolated from bacterial suspensions and feces (17). IMS has not been used for the extraction and isolation of pathogens from foods. The present paper deals with the possibility of establishing more rapid methods for the detection of L. monocytogenes in foods, with adequate sensitivity and specificity, by using IMS. *

Corresponding author. 3478

MATERIALS AND METHODS Bacterial strains. L. monocytogenes strains isolated from various foods in our laboratory were used in the experiments. The criteria used to verify their status as L. monocytogenes were those of Skovgaard and Morgen (28). Serotyping was performed with Listeria 0 antisera 1 and 4 (2300-50-2 and 2301-50-1; Difco Laboratories, Detroit, Mich.) by using the procedure of the manufacturer. The strains and their origins and serotypes are shown in Table 1. Other bacterial strains, including Streptococcus faecalis, Streptococcus dysgalactiae, and Listeria strains (non L. monocytogenes) from the collection of our departments were also used to test the system. Isolation of flagella. A strain of L. monocytogenes (L 10, serotype 4) was cultured at 22°C for 4 days in tryptic soy broth (TSB) (Oxoid Ltd., Basingstoke, England) with 0.2% glucose and 0.6% yeast extract (Difco). Flagella were isolated by following the procedure of Peel et al. (24), using mechanical disruption with glass beads followed by centrifugation steps to remove debris before the final ultracentrifugation step. Isolated flagella were kept frozen at -20°C until use. Monoclonal antibodies. Monoclonal antibodies (immunoglobulin G2b [IgG2b]) produced against flagella from L. monocytogenes were kindly provided by Willie Donachie, Moredon Research Institute, Edinburgh, Scotland. The concentration of antibodies in the supernatant was 10 ,ug/ml. Dot blot assay. Nine L. monocytogenes strains were grown in TSB at 22°C (40 h) and 37°C (18 h). A 10-ml volume of the culture was centrifuged at 5,000 x g for 15 min, the supernatant was aspirated, and the bacteria were washed once in phosphate-buffered saline (pH 7.4) (PBS). The final pellet was suspended in 1 ml of PBS. A 5-,u volume of this suspension was spotted on a nitrocellulose sheet (approximately 5 x 107 bacteria per dot). Isolated flagella from L. monocytogenes were used as a positive control. Nitrocellulose membranes were incubated for 1 h at 37°C, washed twice with high-salt buffer (0.5 M NaCl-0.01 M Tris [pH 7.4]), and blocked with high-salt buffer with 0.5% Tween 20 for 18 h at room temperature. Monoclonal antibodies were diluted 1:40 in high-salt-Tween buffer and incubated with the nitrocellulose membrane for 30 min. The sheet was then washed for 5 min in high-salt-Tween buffer, followed by low-salt buffer (0.15 M NaCI-0.01 M Tris-0.5% Tween 20

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TABLE 1. L. monocytogenes strains used in the experiments and their origins and serotypes, typed by the Difco Listeria 0 antisera 1 and 4 Strain

Serotype

Origin

L 20 L 31 L 244 L1 L6 L 45 L 10 L 256 L 265

1 1 1 4 4 4 4 4 4

Cow milk (mastitis) Cheese (Touree de l'aubier) Minced meat Cheese (La folie de chef) Cheese (Brie Ker Noelle) Poultry carcass Cheese (Blue Castello) Shrimp Stomach contents of human infant

[pH 7.4]). Goat anti-mouse IgG conjugated with horseradish peroxidase (Tago Inc., Burlingame, Calif.) was diluted 1:1,000 in low-salt buffer and incubated with the membrane for 30 min. Sheets were then washed twice in low-salt buffer, and substrate (10 ,ul of 3% chloronaphthol in ethanol plus 0.2 ,ul of H202 per ml of Tris buffer [pH 7.6]) was then added. The color was allowed to develop for 15 min, and the reaction was then stopped by drying the nitrocellulose membrane between sheets of blotting paper. IMB. Immunomagnetic beads (IMB), 2.8 ,um in diameter, with covalently linked sheep anti-mouse IgG antibodies (Dynabeads M-280, sheep anti-mouse IgG; Dynal A/S, Oslo, Norway) were used in the experiments by following the recommendation of the manufacturer. Coating of IMB. IMB (sheep anti-mouse IgG) were incubated with the monoclonal antiserum for 3 h at room temperature with sufficient shaking to avoid settling of IMB. Different ratios of antibodies/IMB were tested. Isolation of live bacteria by using IMS. Strains of L. monocytogenes were grown in TSB under different conditions of temperature and time. The broth was diluted in PBS (pH 7.4)-0.1% bovine serum albumin (no. B 2518; Sigma Chemical Co., St. Louis, Mo.) in Eppendorf tubes, 1 ml in each tube. Various numbers of IMB were added, and the mixture was incubated at room temperature for periods ranging from 10 min to 1 h. The mixture was shaken to avoid sedimentation of beads. A magnetic particle concentrator for microtubes of the Eppendorf type (MPC-E; Dynal A/S) was used to trap IMB against the wall of the tube for removing residual liquid by aspiration. IMB were then washed in PBS with 0.1% bovine serum albumin before being transferred to Oxford agar (Oxoid CM 856, supplement SR 140; Oxoid) or 7% ox blood agar (Oxoid CM 271) and then incubated for 24 h at 30°C. Colonies on agar plates were counted. Agglutination reaction. IMB coated with monoclonal antibodies (approximately 106 IMB per sample) in 10 p.l of PBS were mixed on an ordinary glass slide with TSB cultures of different bacterial strains (L. monocytogenes grown at 25 and 37°C and different other strains). The slide was tilted, and agglutination was allowed to develop for 1 min. Visual agglutination was recorded by using IMB without secondary antibodies as negative control. Determination of optimal coating procedures. IMB were mixed with monoclonal antibodies at ratios ranging from 0.05 to 5 p.g/106 IMB. After coating, 2 x 106 IMB were incubated for 15 min in 1 ml of PBS with 0.1% bovine serum albumin and approximately 2 x 103 L. monocytogenes L 10. After incubation and three additional washings, IMB were spread onto Oxford agar and incubated for 24 h at 30°C, and the number of colonies was then counted. Evaluation of IMS. To determine the effect of the number

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TABLE 2. Dot blot assay and agglutination reactions with IMB

for different strains of L. monocytogenesa Reaction at:

Strain

L 20 L 31 L 244 L1 L6 L45 L 10 L 256 L 265

Serotype

1 1 1 4 4 4 4 4 4

25°C

37°C

Dot blot

Agglutination

Dot blot

Agglutination

+ + + + + + +

+ + + + + + +

-

-

-

-

-

-

" IMB were coated with monoclonal antibodies against flagella from L. monocytogenes

of IMB used, different numbers (105, 106, and 107) of IMB were incubated in PBS with 4 x 103 L. monocytogenes L 10 for 15 min. After magnetic separation, 0.1 ml of the supernatant was spread onto Oxford agar plates to determine the fraction of bacteria that bound to IMB. The effects of incubation time and washing procedures on the system were tested by incubating IMB (5 x 106) with 4 x 104 L. monocytogenes L 10 in 1 ml of PBS for 15, 30, and 60 min. IMB were washed six times before being spread inoculated onto agar plates. Samples were taken from the supernatant after each step. The sensitivity of the system was tested by incubating different numbers of L. monocytogenes L 10 with 107 IMB per sample in 1 ml of PBS. IMB were washed three times, and the supernatant was sampled after each step to determine the number of unbound bacteria in each step. The ability of the system to extract L. monocytogenes from heterogeneous suspensions was tested in samples of cheese (Brie) and vacuum-packed boiled ham that were obtained from a retail grocery shop. The samples were homogenized with 10 volumes of TSB and UVM (modified Listeria enrichment broth, Difco no. 0223-17-2) by use of a model BA 6021 stomacher (Seward Laboratories, London, England). The broths were incubated at 30°C for 24 h. L. monocytogenes L 10 was grown in TSB at 30°C for 18 h. Different numbers of L. monocytogenes were added to the incubated broths. Bacteria were caught by using IMB, washed three times, and spread onto Oxford agar. A similar experiment was undertaken by mixing 103 L. monocytogenes L 10 with 106 to 107 S. faecalis. An incubation time of 15 min was used in these experiments. RESULTS A close correlation between agglutination tests and dot blots was found (Table 2). The monoclonal antibodies recognized all six strains of serotype 4 but only one of the three serotype 1 strains grown at 25°C. All results were consistently negative when L. monocytogenes was grown at 37°C. One of the definitely positive strains (strain L 10, serotype 4) was used in the subsequent experiments. Strains of S. faecalis, S. dysgalactiae, and Listeria sp. (non L. monocytogenes) gave no positive reactions on either test. A marked reduction in the number of CFU of L. monocytogenes was found when the amount of monoclonal antibody relative to IMB was increased (Fig. 1). Microscopic exami-

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SKJERVE ET AL.

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150-

0

O

100

\ 0

0

1

2

3

4

5

microgram antibodies / 1 million beads FIG. 1. IMS from PBS containing 2 x 103 L. monocytogenes per ml by using IMB. The number of colonies on Oxford agar after separation by using IMB coated with different amounts of monoclonal antibodies is shown.

nation also showed that IMB with more linked monoclonal antibodies formed more aggregates. In subsequent experiments, a ratio of 0.5 ,ug of antibodies to 106 IMB was used, giving nearly maximum binding capacities without using too much monoclonal antibody. Increasing the number of IMB in each assay raised the bound fraction of bacteria from below 5% (105 IMB) to 12% (106 IMB3) and 33% (107 IMB) of the original 4 x 103 L. monocytogenes.

An increase of the incubation time from 15 to 60 min reduced the number of unbound bacteria from 50 to 5% (Fig. 2). With an incubation time of 60 min, an additional 30% of the initially bound bacteria was washed away, leaving an estimated 65% of the original bacteria present on the IMB after six washings. For shorter incubation times, 35% (30 min) and 15% (15 min) were present after six washing steps. The sensitivity of the system in PBS in different experiments varied by between 10 and 100 L. monocytogenes per ml. Increasing the number of bacteria also gave an increase in the fraction of bound bacteria, from less than 10% (102 L. monocytogenes per ml) up to 50% (1.5 x 104 L. monocytogenes per ml).

% of CFU 100 75

50 25 0

15 min. 30 min. 60 min. Incubation time

FIG. 2. IMS of L. monocytogenes by using 5 x 106 IMB in a suspension of 4 x 104 L. monocytogenes per ml in PBS. Fractions of bacteria bound after six washing steps after primary incubation for 15, 30, and 60 min (-), bound but washed away (O), and not bound (O) are indicated.

Vacuum-packed ham had a heterogeneous bacterial flora of 6 x 104 CFU/ml after growth for 24 h in UVM and 107 CFU/ml in TSB. Brie cheese grown in UVM showed a heterogeneous population of approximately 3.5 x 104 CFU/ml after 24 h at 30°C and 1 x 107 CFU/ml in TSB. L. monocytogenes L 10 could be recovered from all of these broths with IMS at different sensitivities, ranging from below 2 x 102 in ham grown in UVM-TSB and Brie cheese in UVM to not less than 2 x 104 L. monocytogenes per ml in Brie cheese grown in TSB. Considerable nonspecific binding was seen after IMS in broths containing 107 CFU/ml. Mixing L. monocytogenes L 10 with large numbers (106 to 107) of S. faecalis in PBS did not seem to interfere with the recovery of the target organism. DISCUSSION L. monocytogenes is motile and expresses flagella at room temperature but not at 37°C. Although this is classic knowledge, it has recently been studied in more detail by Peel et al. (24). Their findings were also verified by the present experiments which demonstrated no reaction on dot blots or agglutination from strains grown at 370C. The results indicate that a slide agglutination reaction is a simple and reliable way of assessing whether a system using IMB can extract specific strains of bacteria in suspension. All strains giving agglutination with IMB coated with specific antibodies were also recovered from dilutions in PBS. By optimizing the coating of IMB and by increasing the number of IMB used and the incubation time up to 60 min, more of the target bacteria could be recovered. Such a prolonged incubation time will, however, also increase the occurrence of nonspecific binding. With an achievable reacting surface on the beads of up to 50 to 100 cm2/ml of sample, it is preferable to reduce the incubation time. In spite of the optimization of the assay, a large proportion of the originally bound bacteria was flushed away by the washing steps. As demonstrated by the experiments, the monoclonal antibodies used here reacted with only some of the strains. IMS was also able to retrieve target bacteria from very heterogeneous suspensions. The high level of nonspecific binding is not surprising with such large numbers (up to 107) of other bacteria. The results indicate a possible practical sensitivity of 1 x 102 to 2 x 102 L. monocytogenes per ml, thus supporting the findings from experiments carried out with PBS. The sensitivities in pure cultures and in heterogeneous suspensions are thus not dramatically different. The use of improved antibodies, reduced incubation time, and possibly better blocking solutions could reduce the number of nonspecific interactions. The detection system of spreading the IMB onto agar described in this paper offers a theoretical sensitivity of less than 102 bacteria per ml; in principle, one bacterium would elicit a signal by the production of one colony the following day. Another approach would be to establish an immunoassay in which antibodies are trapped on the IMB and an enzyme-labeled secondary antibody is added after extraction from the sample. The use of large numbers of IMB might increase the sensitivity of these forms of enzyme-linked immunosorbent assay, as compared with previously described immunoassays. The system described in this paper cannot offer, at present, a new method for the detection of L. monocytogenes in foods. IMS could, however, represent an interesting approach in microbiology in general, provided that

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antibodies of acceptable quality are available. By using IMS directly on food samples or other heterogeneous suspensions such as feces, or more likely after some hours of resuscitation or preenrichment in nonselective or slightly selective media, substantia,l time can be saved. Furthermore, bacteria can be isolated without the stress most selective media put upon them, making it possible to obtain bacteria with the same biochemical and genetic properties as those originally present in the food (8). Further experiments should use either genus- or species-specific affinity-purified polyclonal antibodies, new monoclonal antibodies with specificity for more strains within the family, or pooled cocktails of monoclonal antibodies which will improve the sensitivity and the specificity of the system. LITERATURE CITED 1. Archer, D. L., and F. E. Young. 1988. Contemporary issues: diseases with a food vector. Clin. Microbiol. Rev. 1:377-398. 2. Beckers, H. J., P. S. S. Soentoro, and E. H. F. Delfgou van Asch. 1987. The occurrence of Listeria monocytogenes in soft cheeses and raw milk and its resistance to heat. Int. J. Food Microbiol. 4:249-256. 3. Brackett, R. E., and L. R. Beuchat. 1989. Methods and media for the isolation and cultivation of Listeria monocytogenes from various foods. Int. J. Food Microbiol. 8:219-223. 4. Buchanan, R. L., H. G. Stahl, M. M. Bencivengo, and F. del Corral. 1989. Comparison of lithium chloride-phenylethanolmoxalactam and modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats and poultry. Appl. Environ. Microbiol. 55:599-603. 5. Butman, B. T., M. C. Plank, R. J. Durham, and J. A. Mattingly. 1988. Monoclonal antibodies which identify a genus-specific Listeria antigen. Appl. Environ. Microbiol. 54:1564-1569. 6. Donnelly, C. W., G. J. Baigent, and E. H. Briggs. 1988. Flow cytometry for automated analysis of milk containing Listeria monocytogenes. J. Assoc. Off. Anal. Chem. 71:655-658. 7. Farber, J. M., and J. I. Speirs. 1987. Monoclonal antibodies directed against the flagellar antigens of Listeria species and their potential in EIA-based methods. J. Food Protect. 50:479484. 8. Hill, W. E., J. L. Ferreira, W. L. Payne, and V. M. Johns. 1985. Probability of recovering pathogenic Escherichia coli from foods. Appl. Environ. Microbiol. 49:1374-1378. 9. Hird, D. W. 1987. Review of evidence for zoonotic listeriosis. J. Food Protect. 50:429-433. 10. Johansen, L., K. Nustad, T. B. Oerstadvik, J. Ugelstad, A. Berge, and T. Ellingsen. 1983. Excess antibody immunoassays for rat glandular kallikrein. Monosized polymer particles as the preferred solid phase material. J. Immunol. Methods 59:255264. 11. Johne, B., and J. Jarp. 1988. A rapid assay for protein-A in Staphylococcus aureus strains using immunomagnetic monosized polymer particles. Acta Pathol. Microbiol. Immunol. Scand. 96:43-49. 12. Johne, B., J. Jarp, and L. R. Haaheim. 1989. Staphylococcus aureus exopolysaccharide in vivo demonstrated by immunomagnetic separation and electron microscopy. J. Clin. Microbiol. 27:1631-1635. 13. Kampelmacher, E. H., and D. A. A. Mossel. 1989. Evaluation

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