Journal of Food Protection, Vol. 59, No.1, Pages 24-27 Copyright©, International Association of Milk, Food and Environmental Sanitarians

Virulence of monocytogenes, , and Assayed with In Vitro Murine Macrophagocytosis

HUGH L. DALLAS,l DELMA P. THOMAS,2 and ANTHONY D. HlTCHINS1*

IDivision of Microbiological Studies, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 200 C Street, S.w., Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/1/24/1661793/0362-028x-59_1_24.pdf by guest on 26 September 2021 Washington, D.C. 20204; and 'Center for Devices and Radiological Health, U.S. Food and Drug Administration, Rockville, Maryland 20857, USA

(MS# 95-104: Received 8 May 1995/Accepted 15 August 1995)

ABSTRACT quantitative comparison was made of uptake, survival, and growth of Listeria spp. in a murine macrophage-like cell The survival of virulent and avirulent Listeria species internal- line, RAW264.7 (21). The Listeria species compared were ized in cells of a murine macrophage-like cell line, RAW264. 7, was L. monocytogenes, which is hemolytic and virulent for monitored. Mouse macrophage cells (ca. 5 X 105/ml) suspended in humans and animals; L. innocua, a nonhemolytic avirulent fresh RPMI medium 1640 containing fetal bovine serum were species; and L. seeligeri, a weakly hemolytic avirulent mixed with 5 X 107 to 5 X 108 Listeria cells per ml and incubated I species (14). The hemolysin of L. monocytogenes, listerioly- h at 37°C with COTenriched air. Gentamicin (10 flg/ml) was added to kill not internalized by the cells. At 2, 4, and 6 h sin 0, is a 60-kilodalton cytolysin which contains a sulfhy- postinfection, IO-fl! amounts of the suspensions were lysed in dryl group that is essential for activity; the L. seeligeri microtiter plate wells during serial decimal dilution in water. hemolysin is similar but not fully characterized (7). Al- Triplicate dilutions (10 fl! each) were plated on trypticase soy agar, though generally regarded as avirulent, L. seeligeri has and colonies were counted after 48 h incubation at 35°C. About 0.1 caused illness in humans (22). to I % of the added hemolytic pathogen L. monocytogenes Scott A and the avirulent nonhemolytic L. innocua were internalized at 2 h. MATERIALS AND METHODS The number of internal L. monocytogenes cells increased signifi- cantly by 6 h, but L. innocua cells showed no significant change. A Bacterial strains and growth media strain of the hemolytic species L. seeligeri behaved like the The bacterial strains used were L. monocytogenes Scott A var. nonhemolytic L. innocua. This distinction between the intracellular Lm82, serovar 4b; L. monocytogenes KC1703, serovar la; L. behavior of pathogenic and nonpathogenic species, if a general monocytogenes KC1708, serovar 3a; L. innocua ATCC 33090, phenomenon, may be useful as an in vitro virulence assessment serovar 6a; and L. seeligeri ATCC 35967 (CIP 100100), serovar parameter. 1/2b. Bacteria for infection studies were grown in trypticase soy broth (Becton Dickinson Co., Cockeysville, MD) with 0.6% Key words: Listeria spp., macrophagocytosis assay, virulence, (wt/vol) yeast extract (TSYE) at 35°C. Cultures were maintained tissue culture, microscopy on slants of TS YE agar and stored at 4°C. Duplicate cultures were stored at -75°C in TSYE broth with 10% (vol!vol) glycerol.

Listeria monocytogenes is the gram-positive facultative Macrophage cell line intracellular pathogen which causes the disease listeriosis. The murine macrophage cell line was RAW264.7, which The disease is transmitted through ingestion of contami- originated from the ascites of a tumor induced in a male mouse by nated food, which leads to infection of gastrointestinal the intraperitoneal injection of Abelson leukemia virus (16). Stock epithelial and other cells. In macrophage cells, Listeria cell suspensions (I ml) were stored at -70°C in 10% (vol/vol) monocytogenes can survive, multiply, and spread intracellu- glycerol or dimethyl sulfoxide (DMSO) in RPMI 1640 growth larly, thereby avoiding effective humoral response by the medium (Gibco Laboratories, Grand Island, NY). host (24). The hemolysin of L. monocytogenes is a virulence factor that is necessary for the escape of phagocytosed L. Macrophage cell cultures monocytogenes from the inhospitable phagolysosome envi- The RAW264.7 cell line (American Type Culture Collection, ronment into the more amenable cytoplasmic environment. Rockville, MD) was maintained in 75 cm2 tissue-culture flasks with The objective of this study was to develop an in vitro test for RPMI 1640 medium plus 10% (vol/vol) fetal bovine serum (FBS; distinguishing the virulence of Listeria species and strains. A Hyclone Labs, Inc., Logan UT) at 37°C in a CO2 incubator (Lab-Line Instruments, Inc., Melrose, IL) containing a humidified atmosphere of 5% (vol/vol) CO2 and 95% (vol/vol) air. Monolay- ered cells were detached and split I in 4 into a new flask with 20 ml * Author for correspondence. Tel: 202-260-0874; Fax: 202-401-7740. of prewarmed RPMI 1640 every 3 to 5 days. MACROPHAGOCYTOSIS OF LISTERIA SPP. 25

Preparation of macrophage suspensions for phagocytosis 30 min. FBS complement was heat inactivated at 56°C for 30 min. Macrophage mono layers for the experiments were grown for Triton X-lOO was used as a diluent additive at a concentration of 72 h in RPMI 1640 with 2 mM L-glutamine (Gibco) supplemented 1% (voUvol). with FBS (10%, voUvol) in polystyrene flasks (Coming Glass- works, Coming, NY) at 37°C in an atmosphere of 5% (voUvol) RESULTS CO2 and 95% (vol/vol) air. Spent medium was decanted and discarded; monolayers were washed twice with 10 ml of pre- warmed (37°C) Dulbecco's phosphate-buffered saline (PBS; Gibco) In five of six trials, intracellular levels of L. monocyto- without Ca ++ and Mg++. Monolayers were detached by using 5-ml genes Scott A were significantly higher than those of L. amounts of prewarmed Hank's balanced salt solution (Gibco) seeligeri at 2 h postinfection (Table 1). The intracellular without Ca++ and Mg++ for 6 min at 37°C. After detachment, 0.4 presence of Listeria was qualitatively confirmed by light ml of cell" suspension was used for phagocytosis experiments. microscopy of stained preparations and by electron micros- Suspended macrophage concentrations were estimated hemocyto- copy of thin sections of macrophages 2 h after infection. metrically, assuming 100% viability. At 4 h after infection, the mean log CFU values were

4.48 (3.94 to 5.45) for L. monocytogenes and <3.1 «3.0 to Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/1/24/1661793/0362-028x-59_1_24.pdf by guest on 26 September 2021 Macrophage infection 3.7) for L. seeligeri. Although the levels of L. monocyto- The macrophage cell line was grown for 72 h at 37°C, genes declined at 4 and 6 h, they were still significantly harvested, and suspended in Hank's balanced salt solution. Listeria higher than those of L. seeligeri. Population levels of L. species were grown overnight at 35°C in trypticase soy broth with monocytogenes at 24 h were generally higher than those at 6 0.6% (wt/vol) yeast extract to about 2 X 109 colony-forming units (CFU)/ml. The cells in 1 ml of culture were sedimented in a h. The levels of L. seeligeri were lower than the sensitivity microfuge tube at 14,000 X g for 1 min and resuspended in RPMI of the counting system «3.0 log CFU) after 2 h of growth medium. For infection, 2.5 ml of RPMI 1640 medium plus incubation. Levels of internalized L. monocytogenes were 0.4 ml of macrophage suspension and 0.1 ml of Listeria suspension significantly higher than those of L. innocua at 6 h in three were placed in a 35-mm diameter well of a 6-well tissue-culture trials, one of which (trial 5) is shown in Table 2. In the plate. Control wells contained medium and bacteria only. At 1 h absence of macrophages, all Listeria cells were killed by the after the initial infection, gentamicin (8) (Sigma Chemical Co., St. antibiotic. Louis, MO) was added (final concentration, 10 I1g/ml). Bacteria Experiments were performed to improve the measur- were enumerated at 2, 4, 6, and 24 h postinfection. All culture able internalization, e.g., Triton X-lOO diluent for lysis, manipulations were performed in a biological safety cabinet with variations in serum lots, serum types, environmental condi- laminar airflow (Forma Scientific, Inc., Marietta, OH). tions of the experiment, and effect of serotype differences among strains. None of these factors significantly improved Enumeration of intracellular bacteria internalization of Listeria (Table 2). In fact, use of Triton Quadruplicate samples (10 Ill) of infected cultures were X-IOO solution as the diluent for release and enumeration of pipetted into the round-bottomed wells of a 96-well microtiter plate viable phagocytosed Listeria had an adverse effect on the containing 90 111of distilled water and immediately diluted decimally. From each well, 10 111was removed with a motorized levels recovered. multichanneled pipette and placed on a plate of brain heart in- fusion agar (Difco Laboratories, Detroit, MI). The plates were incubated at 35°C for 48 h. Listeria colonies were counted using a TABLE 1. Infection (log CFU/ml) of macro phages by L. monocyto- dissecting microscope modified for Henry oblique transmitted genes Scott A and L. seeligeri ATCC 35967 illumination. log CFU/ml postinfection time (h)a Microscopy Intracellular Listeria were observed in infected macrophages Trialb Strain 0 2 4 6 24 by light microscopy. Infected washed macrophage monolayers on NDc l2-mm diameter covers lips in 60-mm plastic petri dishes were 1 Scott A 8.59 5.67 4.51 4.34 fixed and differentially stained by using a Hematology Three-Step 35967 8.58 3.36 <3.00 <3.60 ND Stain Set (AccraLab, Bridgeport, NJ) or a Diff-Quik Stain Set 2 Scott A 7.44 4.00 3.94 3.36 6.51 (American Scientific Products, McGaw Park, IL) according to the 35967 7.73 4.04 <3.30 <3.24 <3.00 manufacturer's instructions. 3 Scott A 7.17 6.47 4.32 3.98 4.25 For electron microscopy, the method of Hayat (10) was used. 35967 7.68 3.95 <3.78 <3.30 <3.00 Infected and uninfected macrophages were washed, fixed with 4 Scott A 8.80 3.98 3.73 3.56 5.15 glutaraldehyde, and postfixed with OS04. The cells were embedded 35967 8.71 3.43 <3.00 <3.00 <3.00 in Poly/Bed 812 plastic (Polysciences, Inc., Warrington PA), and 5 Scott A 7.62 4.85 4.71 4.12 4.66 sectioned. Sections were stained with lead citrate and uranyl 35967 5.39 <3.00 <3.00 <3.00 <3.00 acetate and examined in a JEM -lOOC electron microscope (Jeol, 6 Scott A 7.41 3.77 4.06 4.20 5.58 Ltd, Tokyo, Japan). 35967 7.27 3.36 3.10 3.00 3.78

a Gentamicin added at 1 h. Other treatments b Within trials, differences of ~0.30 between log CFU/ml values L. monocytogenes strain Lm82 cells were opsonized by are statistically significant. incubation in 5% (voUvol) mouse serum in RPMI 1640 at 37°C for eND, not done. 26 DALLAS, THOMAS, AND HITCHINS

TABLE 2. Infection of macrophages by L. monocytogenes Scott A Based on chemotactility data (1), this cell resembles a strains resident macrophage more than a monocyte. Like 1774.Al, log CFU/ml at postinfection the RAW264.7 cell line produces modest basal (unstimu- time (h)a lated) levels of H202, although only 1774.Al H202 levels can be dramatically stimulated by gamma-interferon (17). Trialb Strain Variable 0 2 4 6 24 At baseline levels, the nonoxidative properties ofRAW264.7 and 1774.1 seem to be similar (12), i.e., gamma-interferon- Scott A Control 7.62 4.85 4.71 4.12 4.66 Triton 7.45 4.03 3.39 <3.0 4.25 activated RAW264.7 cells kill bacteria (17). We felt that use 2 Scott A 4b 7.61 4.38 5.45 5.64 6.27 of inactive RAW264.7 cells would minimize killing of L. KC1703 la 7.27 4.11 4.57 4.55 6.69 monocytogenes since in primary macrophage cultures, kill- KC1708 3a 7.33 4.66 4.02 4.15 6.28 ing and multiplication of the ingested cells can proceed 3 Scott A FBSI 7.37 4.21 4.03 4.58 4.51 simultaneously (5). FBS2 7.44 4.37 4.29 4.03 4.51 The macrophage cultures used in this study were naive FBS2 heated 7.40 4.35 4.44 3.73 4.80

with respect to Listeria exposure. It was hoped that survival Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/1/24/1661793/0362-028x-59_1_24.pdf by guest on 26 September 2021 4 Scott A Control 8.01 4.27 5.37 4.25 6.28 differences between species and strains would be more Serum 7.65 4.02 4.41 3.84 6.29 apparent and reproducible if naive cells were used. 5 Scott A Control 8.50 6.59 4.52 5.60 6.63 Most experiments were done with L. monocytogenes 33090c Species 8.42 5.42 4.38 4.13 3.62 and L. seeligeri, which are both hemolytic and would be a Gentamicin added at 1 h. expected to escape from macrophage phagolysosomes. Thus b Within trials, differences of 2:0.26 between log CFU/ml values the difference in their behavior is somewhat surprising. are statistically significant. Although hemolysin is known to promote intracellular C L. innocua ATCC 33090. survival of L. monocytogenes, it is not essential for entry into macrophages or nonprofessional phagocytic cells (16). The DISCUSSION fact that L. seeligeri is generally less hemolytic than L. monocytogenes could account for the observed difference. In vitro methods are needed to determine the virulence Opsonization of L. monocytogenes appears to be impor- of Listeria strains (23). Nonmacrophage cell lines (nonpro- tant in their killing in adults as opposed to newborns (3). fessional phagocytes) have been used successfully to distin- Pretreatment of the Scott A strain with nonspecific mouse guish between pathogenic and nonpathogenic Listeria spp. serum did not enhance its level in mouse macrophages. by testing for the cytolytic activity of their culture filtrates Likewise, heating the FBS to inactivate complement did not (6). However, that method was not developed further and did decrease L. monocytogenes uptake. These observations not test L. seeligeri. Bhunia et al. (2) used myeloma and suggest that uptake was due to a nonopsonic mechanism hybridoma cells to distinguish pathogenic from nonpatho- (19). The number of serogroups examined in this study was genic Listeria by their killing (trypan blue uptake) ability in limited; therefore, no final conclusions can be made about 6 h. In 1992, Ho et al. (13) also used a cell lysis assay based the possible role of serotype determinants in uptake of on plaque formation in infected macrophage monolayers to Listeria by macrophages. However, serotypes la and 3a of distinguish pathogenic and nonpathogenic Listeria. Bunduki L. monocytogenes behaved like Scott A (serotype 4b) in et al. (4) used uptake by peripheral blood macrophages to macrophages (Table 2). The serotype of the L. seeligeri distinguish L. monocytogenes from L. innocua by their strain was 112b and that of the L. innocua strain was 6a. phagocytic indices. The result of the present study, using Phagocytosis by this macrophagic cell line seems to be internalized Listeria survival as the parameter, is similar to promising as an in vitro method for distinguishing between those of previous studies using different parameters or cell pathogenic and nonpathogenic Listeria species. The ultimate systems (2, 4, 6, 8), in that it distinguished pathogenic and goal is to distinguish virulent strains of L. monocytogenes nonpathogenic Listeria. speCIes. L. monocytogenes can survive and multiply in macro- phages (18). To measure differences of phagocytic suscepti- ACKNOWLEDGMENTS bility of L. monocytogenes and other Listeria spp., it is convenient to use macrophage-like cell lines rather than The authors thank V. M. Hitchins, Center for Devices and Radiologi- natural macrophages. The former, e.g., P388D1 (15), mimic cal Health, U.S. Food and Drug Administration (FDA), for providing the natural macrophages quite well; however, depending on macrophage cell line, and R. W. Bennett, FDA, for providing Listeria their progression in the monocyte-macrophage lineage at the strains KC1703 and KC1708. time of derivation, they may only partially reflect the total spectrum of baseline and inducible capabilities of mature REFERENCES macrophages (17). Most studies of Listeria phagocytosis have used the cell 1. Aksamit, R. R., W. Falk, and E. J. Leonard. 1981. Chemotaxis by line 1774.Al, following the initial example of Portnoy et al. mouse macrophage cell lines. J. Immunol. 126:2194-2199. 2. Bhunia, A. K., P.J. Steele, D. G. Westbrook, L. A. Bly, T. P.Maloney, (20). However, other macrophage cell lines have been and M. G. Johnson. 1994. A six-hour in vitro virulence assay for independently derived (9) and we chose to study one of using myeloma and hybridoma cells from these, RAW264.7 (21) because of its convenient availability. murine and human sources. Microbial Pathogen. 16:99-110. MACROPHAGOCYTOSIS OF LISTERIA SPP. 27

3. Bortolussi, R., A. Issekutz, and G. Faulkner. 1986. Opsonization of tal samples for virulent Listeria. Abstr. Annu. Meet. Am. Soc. Listeria monocytogenes Type 4b by human adult and newborn sera. Microbiol. 92:328. Infect. Immun. 52:493--498. 14. Hof, H., and P. Hefner. 1988. Pathogenicity of Listeria monocyto- 4. Bunduki, M. C., C M. Beliveau, and C W. Donnelly. 1993. genes in comparison to other Listeria species. Infection 16 (suppl. Examination of attachment and phagocytic uptake of Listeria species 2):SI41-S144. by mammalian intestinal cells. Food Microbiol. 10:507-516. 15. Kuhn, M., and W. Goebel. 1994. Induction of cytokines in phagocytic 5. de Chastellier, C., and P. Berche. 1994. Fate of Listeria monocyto- mammalian cells infected with virulent and avirulent Listeria strains. genes in murine macrophages: evidence for simultaneous killing and Infect. Immun. 62:348-356. survival of intracellular bacteria. Infect. Immun. 62:543-553. 16. Kuhn, M., S. Kathariou, and W. Goebel. 1988. Hemolysin supports 6. Farber, J. M., and J. I. Speirs. 1987. Potential use of continuous cell survival but not entry of the intracellular bacterium Listeria monocy- lines to distinguish between pathogenic and apathogenic Listeria spp. togenes. Infect. Immun. 56:79-82. J. Clin. Microbiol. 25:1463-1466. 17. Lambert, L. E., and D. M. Paulnock. 1989. Differential induction of 7. Geoffroy, C, J. L. Gaillard, J. E. Alouf, and P. Berche. 1989. activation markers in macrophage cell lines by interferon-gamma. Production of thiol hemolysins by Listeria monocytogenes and related Cell. Immunol. 120:401--418. species. J. Gen. Microbiol. 135:481--487. 18. Mackaness, G. B. 1962. Cellular resistance to infection. J. Exp. Med. 8. Havell, E. A. 1986. Synthesis and secretion of interferon by murine 116:381--406. fibroblasts in response to intracellular Listeria monocytogenes. Infect. 19. Ofek, I., R. E Rest, and N. Sharon. 1992. Nonopsonic phagocytosis of

Immun.54:787-792. microorganisms. ASM News 58:429--435. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/1/24/1661793/0362-028x-59_1_24.pdf by guest on 26 September 2021 9. Hay, R., J. Caputo, T. R. Chen, M. Macy, P. McClintock, and Y. Reid. 20. Portnoy, D. A., P. S. Jacks, and D. J. Hinrichs. 1988. Role of 1992. Tumor immunology bank, p. 285-305. In Catalogue of cell hemolysin for the intracellular growth of Listeria monocytogenes. lines and hybridomas. American type culture collection, 7th ed. J. Exp. Med. 167:1459-1471. ATCC, Rockville, MD. 21. Raschke, W. C, S. Baird, P. Ralph, and I. Nakoinz. 1978. Fuctional 10. Hayat, M. A. 1986. Rinsing, dehydration and embedding, p. 56-125. macrophage cell lines transformed by Abelson leukemia virus. Cell In Basic techniques for transmission electron microscopy. Academic 15:261-267. Press, San Diego, CA. 22. Rocourt, J., H. Hof, A. Schrettenbrunner, R. Maluvemi and J. Bille. 1985. II. Hether, N. W., and L. L. Jackson. 1983. Lipoteichoic acid from Acute purulent meningitis due to L seeligeri. 9th International Symposium Listeria monocytogenes. J. Bacteriol. 156:809-817. on Problems of Listeriosis. Nantes, France. September 16-20. 12. Hiemstra, P. S., P. B. Eisenhauer, S. S. L. Harwig, M. T. van den 23. Schlech, W. E, III. 1989. Methods to determine the virulence of Barselaar, R. van Furth, and R. I. Lehrer. 1993. Antimicrobial proteins Listeria strains. Int. J. Food Microbiol. 8:273-276. of murine macrophages. Infect. Immun. 61:3038-3046. 24. Tilney, L. G., and D. A. Portnoy. 1989. Actin filaments and the growth, 13. Ho, Y. W., B. Jackson, T. J. Hansen, and J. L. Chen-Wu. 1992. One day movement and spread of the intracellular bacterial parasite, Listeria quantitative cytopathogeneicity test to assay food and environmen- monocytogenes. J. Cell BioI. 109:1597-1608.