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The influence of commonly used selective agents on the growth of Listeria monocytogenes
International Journal of Food Microbiology 50 (1999) 221–226 www.elsevier.nl / locate / ijfoodmicro
Short communication
The influence of commonly used selective agents on the growth of Listeria monocytogenes Charlotte Nexmann Jacobsen* Royal Veterinary and Agricultural University, Deptartment of Dairy and Food Science, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Received 25 January 1999; received in revised form 6 May 1999; accepted 22 June 1999
Abstract The effects, upon addition of various selective agents to a rich growth medium (BHIEM) on the growth of Listeria monocytogenes and non-Listeria were studied in microtitre wells. A combination of nalidixic acid (0.1 g / l), acriflavine (0.02 g / l) and fosfomycin (0.1 g / l) added to the medium (BHIEM NAF ) was found to be the least inhibitory to Listeria while at the same time, effectively inhibitory to non-Listeria organisms. Recovery of heat injured Listeria monocytogenes in BHIEM, BHIEM NAF and the two traditional Listeria-media, UVM I and II, was compared. Recovery in BHIEM was obtained within 24 h of incubation at 308C, whereas recovery in BHIEM NAF took up to 48 h. Growth and recovery in UVM I and II never reached the same level as in BHIEM or BHIEM NAF . 1999 Elsevier Science B.V. All rights reserved.
1. Introduction It is readily known, that the present standard Listeria-media do not allow recovery of sublethally injured bacteria (Busch and Donnelly, 1992). The injured bacteria might recover and contribute to the development of listeriosis (Sheridan et al., 1994). This way there is a need for an enrichment medium which allows maximum growth of Listeria with simultaneous suppression of background flora. The selective agents previously utilized for Listeriaselective media are numerous. Beerens and TahonCastel (1966) were the first to report the advantage *Tel.: 1 45-35-283-284; fax: 1 45-35-283-214. E-mail address: [email protected] (C. Nexmann Jacobsen)
of using nalidixic acid for isolating Listeria. Since then nalidixic acid has been recognized as one of the most important selective agents for Listeria isolation and is now used alone, or more commonly in combination with other selective agents, such as acriflavine, lithium chloride, colistin, pheny ethanol, moxalactam, ceftazidime, fosfomycin, polymyxin B and cycloheximide, for isolation of L. monocytogenes (Lee and McClain, 1986; Curtis et al., 1989a; van Netten et al., 1989; Cox et al., 1990, Ryser and Marth, 1991; Curtis and Lee, 1995). It is a well known fact, that the selective agents not only inhibit non-Listeria organisms, but are also inhibitory to some degree to Listeria spp. (Curtis et al., 1989b). Various treatments such as heating and freezing can injure Listeria sublethally (Golden et al., 1988a,b;
0168-1605 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00101-4
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C. Nexmann Jacobsen / International Journal of Food Microbiology 50 (1999) 221 – 226
Budu-Amoako et al., 1992; Busch and Donnelly, 1992, Mackey et al., 1994). The injured cells have a lower resistance towards selective agents, thereby delaying recovery and detection. In order to avoid a false negative test, it is important to have a method that supports resuscitation. The objective of this study is to evaluate the influence of various selective agents on the growth of L. monocytogenes as well as non-listeria, when added to a nutritionally rich medium (Brain Heart Infusion broth supplemented with egg yolk and magnesium sulphate) that has been shown to support the growth of Listeria (Jacobsen, 1998).
2. Materials and methods
2.1. Cultures All strains used in this study were supplied by the Department of Veterinary Microbiology at the Royal Veterinary and Agricultural University, Copenhagen, Denmark (Table 1). Lactococcuslactis was grown in LM 17 where as Lactobacillusbrevis and Lactobacillus salivarius were grown anaerobically in De Man, Rogosa, Sharpe broth (MRS, Oxoid) for 20–24 h at 308C. The remaining strains were incubated in Brain Heart Infusion broth (BHI, Difco) for 20–24 h at 378C prior to testing. Stock cultures were maintained at 2 408C in 15% v / v glycerol.
2.2. Growth studies Listeria monocytogenes and various non-Listeria organisms, grown in in the presence of selective agents in an enriched growth medium composed of Brain Heart Infusion broth supplemented with 5 g / l magnesium sulphate and 25 ml / l egg yolk emulsion (Merck) (Jacobsen et al., 1997; Jacobsen, 1998) was compared to growth in the selective media University of Vermont (UVM) I and II (McClain and Lee, 1988). The studies were performed in microtitre plates (Corning, New York, USA). Each medium was inoculated at a level of 10 5 cells / ml and the wells of microtitre plates filled with 200 ml of each inoculated medium. Each medium was tested in 20 wells and the average of the results was calculated. Each measurement was blanked with the particular medium without inoculation. Measurements were
performed with a spectrophotometer (Multiscan MCC 340, Labsystem, Finland) at 600 nm. The microtitre plates were shaken for 5 s before optical density was measured. The following selective additives to BHIEM were tested: Lithium chloride (LiCl, Sigma) 0.5 g / l, 2 g / l, and 10 g / l, acriflavine (Chroma-Gesellschaft, ¨ Schmid GmbH, Kongen, Germany) 5 g / l, 10 g / l and 20 mg / l, nalidixic acid (Sigma) 0.01 g / l, 0.02 g / l, 0.05 g / l and 0.1 g / l, colistin (Sigma) 0.02 g / l, 0.05 g / l, phenyl ethanol (Sigma) 1.0 g / l and 2.5 g / l, moxalactam (Eli Lilly) 0.01 g / l and 0.02 g / l, ceftazidime (Glaxo) 0.02 g / l, fosfomycin (Boehringer) 0.04 g / l, and 0.1 g / l Polymyxin B (Sigma) 1 000 000 iu as well as cycloheximide (Sigma) 0.05 g / l and 0.2 g / l). Optical density was measured following 24 h of incubation at 308C. Not all L. monocytogenes strains were tested with LiCl, phenyl ethanol, moxalactam, polymyxin B and cycloheximide.
2.3. Heat injury Overnight cultures (2 ml) of Listeria monocytogenes strain 11139 were added to 200 ml preheated BHI and immersed in a waterbath for 20 min at 558C for heat treatment as described by Meyer and Donnelly (1992). An aliquot of 0.5 ml of unheated and heated cells was then transferred into 100 ml of BHIEM, BHIEM containing 0.1 g / l nalidixic acid, 0.02 g / l acriflavine and 0.1 g / l fosfomycin (BHIEM NAF ) as well as 100 ml of UVM I and II respectively. Optical density was measured after 0 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h and 48 h of incubation at 308C.
3. Results and discussion The results from the growth studies of grampositive and gram-negative bacteria in selective media are listed in Table 1. Addition of lithium chloride (LiCl) to BHIEM inhibits gram-positive bacteria with the exception of Bacillus licheniformis, while gram-negative bacteria are only slightly reduced. Unfortunately in this study, LiCl inhibited the growth of Listeria monocytogenes more than 50% in the concentrations tested compared with the growth in BHIEM. The inhibitory effect of LiCl on L.
Table 1 The influence of selective agents on the growth of Listeria monocytogenes and various non-Listeria following incubation for 24 h at 308C a Strain
Inhibition % 5 100 2 ((growth in BHIEM added the selective agent compared to growth in BHIEM) 3 100) Acriflavine (mg/l)
0.5
2
10
5
10
L. monocytogenes 11139 11578 14729 11137 T15 85 5101 3967.6
63 61 ND b ND ND ND ND ND
58 66 ND ND ND ND ND ND
62 65 ND ND ND ND ND ND
0 14 9 15 0 7 7 9
11 7 18 29 3 46 56 20
Staph. aureus Ent. faecium M. luteus Lact. lactis Lb. brevis Lb. salivarius B. cereus B. licheniformis
0 41 0 60 46 33 33 0
0 46 59 85 45 81 35 0
38 100 87 98 100 100 75 0
93 100 93 99 24 56 10 46
E. coli Ps. fluorescens Acine. anitratus Enterob. cloacae Prot. mirabilis Citrob. freundii Kl. aeroginosa
0 0 0 0 12 0 11
10 0 30 0 13 0 14
65 54 99 43 23 35 45
0 0 47 0 15 0 17
Nalidixic acid (g/l) 20
Colistin (mg/l)
Ph.eth. (g/l)
Moxal. (mg/l)
Ceftaz. (g/l)
Fosfom. (g/l)
Pol.B. (iu)
0.01
0.02
0.05
0.1
20
50
1
2.5
10
20
0.02
0.04
0.1
1 mill.
6 1 23 23 0 52 60 11
7 8 0 0 0 0 0 0
0 7 0 ND 0 ND 0 0
10 9 0 0 2 0 0 0
65 47 5 13 3 30 30 10
43 16 0 0 0 0 0 0
47 16 ND 0 ND ND ND 2
53 54 ND ND ND ND ND ND
53 54 ND ND ND ND ND ND
5 9 ND ND ND ND ND ND
3 5 ND ND ND ND ND ND
5 7 13 24 11 11 11 22
9 6 ND ND ND ND ND ND
2 0 17 24 9 ND ND 30
0 0 ND ND ND ND ND ND
99 100 88 98 52 74 0 96
100 100 86 97 41 64 96 91
20 0 0 0 26 12 97 64
70 0 33 0 19 9 98 86
90 0 70 0 23 8 98 86
88 0 77 0 17 13 96 86
0 29 92 40 30 76 26 5
0 20 90 33 35 87 34 4
15 34 85 33 52 65 53 17
48 37 75 54 13 62 53 6
100 0 34 0 0 0 0 69
100 0 60 100 8 28 0 61
100 7 53 100 44 7 0 69
52 10 77 9 0 0 52 0
97 5 88 3 0 0 64 0
12 0 40 78 36 0 25 6
0 30 55 0 17 5 15
22 40 74 0 27 20 39
98 41 98 99 62 96 99
94 52 95 88 97 86 97
97 90 98 99 100 95 100
98 98 98 100 100 94 100
98 75 71 82 17 98 89
98 98 57 79 24 98 97
34 79 54 54 44 42 56
94 95 91 99 76 94 94
89 0 24 81 68 70 81
91 0 42 74 79 80 81
87 53 74 78 69 69 86
5 0 0 0 16 11 0
1 0 15 0 13 5 0
80 40 76 67 35 28 70
C. Nexmann Jacobsen / International Journal of Food Microbiology 50 (1999) 221 – 226
LiCl g/l
a
Abbreviations: Staph.: Staphylococcus; Ent.: Enterococcus; M. luteus: Micrococcus luteus; Lact.: Lactococcus; Lb.: Lactobacillus; B.: Bacillus; E.: Escherichia; Ps.: Pseudomonas: Acine.: Acinetobacter; Enterob.: Enterobacter; Prot.: Proteus; Citrob.: Citrobacter; Kl.: Klebsiella; LiCl: Lithium chloride; Ph. eth.: Phenyl ethanol; Moxal.: Moxalactam; Ceftaz.: Ceftazidime; Fosfom.: Fosfomycin; Pol.B.:Polymyxin B; Cycloh.:Cycloheximide. BHIEM: Brain Heart Infusion broth containing 0.1 g / l nalidixic acid, 0.02 g / l acriflavine and 0.1 g / l fosfomycin. b ND.: Not determined. 223
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monocytogenes observed here confirms the observations of another study (Cox et al., 1990), where LiCl, at a level of 20 g / l in Tryptone soy broth, was found to inhibit and delay growth of L. monocytogenes. Other authors have observed unaffected growth of L. monocytogenes in the presence of 15 g / l LiCl (van Netten et al., 1989). When adding acriflavine, half of the eight strains of L. monocytogenes were slightly inhibited and the remaining four were unaffected by the presence of acriflavine, whereas strong inhibition of the cocci was observed. Other studies (Ralovich et al., 1971) report that the organism multiplies well when acriflavine is present with simultaneous suppression of gram-positive cocci. Bacilluscereus was inhibited almost completely by the highest concentration of acriflavine, whereas Lactobacillusbrevis and Lactobacillus salivarius were inhibited 52% and 64% respectively, compared to growth in the nonselective medium. The growth of the gram-negative bacteria was not inhibited to any noticeable degree by acriflavine. Another study, however, reports a negative effect from acriflavine on both lag time and generation time of L. monocytogenes (Beumer et al., 1996). Almost complete inhibition of gram-negative bacteria was observed when grown in the presence of nalidixic acid compared to growth in non-selective enrichment media. The efficiency of nalidixic acid towards gram-negative bacteria is a well known characteristic (Beerens and Tahon-Castel, 1966). Beerens and Tahon-Castel (1966) also observed partial inhibition of Staphylococcus aureus, Bacillus cereus and Lactobacillus acidophilus. Inhibition of Staph. aureus and B. cereus by nalidixic acid was also observed in this study, as well as inhibition of Micrococcus luteus. No other inhibition of grampositive bacteria was observed in this study. L. monocytogenes strain 11139 and to some degree 11578 were inhibited slightly at the highest concentration of nalidixic acid, whereas growth of the other L. monocytogenes strains was unaffected. Colistin, phenyl ethanol, moxalactam and ceftazidime inhibited mainly the gram-negative bacteria. Unfortunately, phenyl ethanol also inhibited the growth of L. monocytogenes as observed previously by van Netten et al. (1988). Polymyxin B inhibited only a few of the strains tested. At a concentration of 0.1 g / l, fosfomycin was very inhibitory to Staph. aureus and M. luteus strongly and to a lesser degree B. cereus. Fosfomycin has been reported to selective-
ly inhibit Listeria species, L. ivanovii, L. seeligeri and L. welshimeri (van Netten et al., 1989), resulting in more selective isolation of L. monocytogenes. This inhibition is reported to be enhanced by incubation at high temperature whereas L. monocytogenes seems to be unaffected by this (Curtis et al., 1989b). Cycloheximide did not seem to inhibit either L. monocytogenes or the other bacteria tested (results not shown). Since it is mainly used to avoid growth of yeast and fungi in media by inhibition of protein synthesis (Obrig, 1971), and is reported to be toxic to humans (Sax and Lewis, 1988; Basic-Zaninovic et al., 1991), addition of this agent seems irrelevant in a day to day enrichment method. The strongest inhibition of gram-positive bacteria was observed when adding LiCl. Unfortunately, L. monocytogenes was also strongly inhibited. This demonstrates how critical the use of LiCl in a selective enrichment medium is for the resuscitation of L. monocytogenes. Acriflavine demonstrated broad inhibition of gram-positive bacteria, and was only slightly inhibitory towards L. monocytogenes. As a consequence of these results, it was decided to add nalidixic acid in a concentration of 0.1 g / l to BHIEM for inhibition of gram-negative bacteria and acriflavine at a concentration of 0.02 g / l for inhibition of gram-positive bacteria in combination with fosfomycin at a concentration of 0.1 g / l. The medium was denoted BHIEM NAF . For heat injured cells of L. monocytogenes, the fastest recovery was observed in BHIEM, whereas recovery of injured cells, when grown in the medium BHIEM NAF , was delayed, requiring up to 48 h of incubation at 308C to reach the same level as the untreated bacteria (Table 2). This delayed recovery in selective media has also been reported by Wang and Hitchins (1994). Listeria monocytogenes never reached the same growth level, when grown in UVM I and II compared to BHIEM or BHIEM NAF during 48 h of incubation. Formulation of a selective medium, without any inhibitory effects towards stressed L. monocytogenes seems impossible. However BHIEM NAF seems to be less inhibitory than other Listeria media e.g. those recommended by the Nordic Committee on Food ¨ ¨ and Peterz, 1992), Analysis (UVM I and II) (Westoo although recovery in BHIEM NAF was delayed compared to recovery in BHIEM. The less inhibitory effect of the medium could be caused by the addition of egg yolk emulsion (van Netten et al., 1988; In’t
C. Nexmann Jacobsen / International Journal of Food Microbiology 50 (1999) 221 – 226
225
Table 2 The ability of heat stressed L. monocytogenes (strain 11139) to recover in BHIEM, BHIEM NAF , UVM I and UVM II, following 20 min of heat treatment at 558C ha
Untreated controls (OD600)b
0 18 19 20 21 22 23 24 48 a b
Heat treated bacteria (OD600)b
BHIEM
BHIEM NAF
UVM I
UVM II
BHIEM
BHIEM NAF
UVM I
UVM II
0.054 0.845 0.837 0.872 0.854 0.882 0.882 0.880 0.850
0.055 0.147 0.161 0.224 0.263 0.386 0.578 0.663 0.721
0.010 0.248 0.259 0.262 0.271 0.272 0.271 0.275 0.255
0.008 0.151 0.188 0.273 0.243 0.248 0.218 0.260 0.270
0.062 0.854 0.854 0.891 0.911 0.922 0.952 0.947 0.870
0.048 0.150 0.174 0.157 0.152 0.191 0.166 0.159 0.774
0.009 0.017 0.035 0.055 0.08 0.122 0.184 0.215 0.267
0.009 0.003 0.002 0.004 0.005 0.015 0.077 0.048 0.286
h: Hours of incubation at 308C. OD600: Optical density at 600 nm.
Veld and de Boer, 1991) and magnesium sulphate (Busch and Donnelly, 1992) that previously has been shown to protect and support growth of L. monocytogenes. To further support the growth of stressed bacteria, a two-stage enrichment procedure has proven beneficial (Lovett, 1988). Therefore incubation of L. monocytogenes in the rich, non-selective enrichment broth BHIEM for 6–8 h, is recommended to resuscitate sublethally damaged L. monocytogenes (Busch and Donnelly, 1992; Curtis and Lee, 1995), before the addition to the medium of the selective agents acriflavine, nalidixic acid and fosfomycin in the above mentioned concentrations. Further studies should be performed to establish the optimal duration of the resuscitation period before addition of selective agents.
Acknowledgements This work was financially supported by the Danish Research and Development Programme for Food Technology and the Danish Dairy Research Foundation. The technical assistance of Anette Ravn is highly appreciated.
References Basic-Zaninovic, T., Papes, D., Franekic, J., 1991. Cycloheximide in ‘In Vitro’ and ‘In Vivo’ test systems. Mutat. Res. 263, 203–210. Beerens, H., Tahon-Castel, M.M., 1966. Millieu a l’acide nalidixique pour l’isolement des streptocoques, D. pneumoniae,
Listeria, Erysipelothrix. Ann. Inst. Pasteur. 111, 90–93. Beumer, R.R., te Giffel, M.C., Anthonie, S.V.R., Cox, L.J., 1996. The effect of acriflavine and nalidixic acid on the growth of Listeria spp. in enrichment media. Food Microbiol. 13, 137– 148. Budu-Amoako, E., Toora, S., Ablett, R.F., Smith, J., 1992. Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells. Appl. Environ. Microbiol. 58, 3177–3179. Busch, S.V., Donnelly, C.W., 1992. Development of a repairenrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua. Appl. Environ. Microbiol. 58, 14–20. Cox, L.J., Dooley, D., Beumer, R., 1990. Effect of Lithium chloride and other inhibitors on the growth of Listeria spp. Food Microbiol. 7, 311–325. Curtis, G.D.W., Lee, W.H., 1995. Culture media and methods for the isolation of Listeria monocytogenes. Int. J. Food Microbiol. 26, 1–13. Curtis, G.D.W., Mitchell, R.G., King, R.G., Griffin, E.J., 1989a. A selective differential medium for the isolation of Listeria monocytogenes. Lett. Appl. Microbiol. 8, 95–98. Curtis, G.D.W., Nichols, W.W., Falla, T.J., 1989b. Selective agents for Listeria can inhibit their growth. Lett. Appl. Microbiol. 8, 169–172. Golden, D.A., Beuchat, L.R., Brackett, R.E., 1988a. Evaluation of selective direct-plating media for their suitability to recover uninjured, heat-injured and freeze-injured Listeria monocytogenes from foods. Appl. Environ. Microbiol. 54, 1451–1456. Golden, D.A., Beuchat, L.R., Brackett, R.E., 1988b. Inactivation and injury of Listeria monocytogenes as affected by heating and freezing. Food Microbiol. 5, 17–23. In’t Veld, P.H., de Boer, E., 1991. Recovery of Listeria monocytogenes on selective media in a collaborative study using reference samples. Int. J. Food Microbiol. 13, 295–300. Jacobsen, C.N., 1998. Flow cytometric detection of Listeria monocytogenes. Ph.D. Thesis. Royal Veterinary and Agricultural University, Denmark, pp 79–92. Jacobsen, C.N., Rasmussen, J., Jakobsen, M., 1997. Viability
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staining and flow cytometric detection of Listeria monocytogenes. J. Microbiol. Meth. 28, 35–43. Lee, W.H., McClain, D., 1986. Improved Listeria selective agar. Appl. Environ. Microbiol. 52, 1215–1217. Lovett, J., 1988. Isolation and enumeration of Listeria monocytogenes. Food Technol. 42, 172–175. Mackey, B.M., Boogard, E., Hayes, C.M., Baranyi, J., 1994. Recovery of heat-injured Listeria monocytogenes. Int. J. Food Microbiol. 22, 227–237. McClain, D., Lee, W.H., 1988. Development of the USDA-FSIS method for the isolation of Listeria monocytogenes. J. Assoc. Off. Anal. Chem. 71, 660–664. Meyer, D.H., Donnelly, C.W., 1992. Effect of incubation temperature on repair of heat-injured Listeria in milk. J. Food Prot. 55, 579–582. Obrig, T.G., 1971. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes – The effect of actidione on eukaryotic cells. J. Biol. Chem. 246, 174–181. ´ ¨ E.M., Malovich, ´ ´ Ralovich, B., Forray, A., Mero, H., Szazados, I., 1971. New selective medium for isolation of L. monocytogenes. Zbl. Bakt. I. Abt. Orig. 216, 88–91.
Ryser, E.T., Marth, E.H., 1991. Listeria, Listeriosis and Food Safety. Marcel Dekker, New York. Sax, N.I., Lewis, R.J., 1988. Dangerous Properties of Industrial Materials, 7th ed, Van Nostrand Reinhold, New York. Sheridan, J.J., Duffy, G., Buchanan, R.L., McDowell, D.A., Blair, I.S., 1994. The use of selective and non-selective enrichment broths for the isolation of Listeria species from meat. Food Microbiol. 11, 439–446. van Netten, P., van de Ven, A., Perales, I., Mossel, D.A.A., 1988. A selective and diagnostic medium for use in the enumeration of Listeria spp. in foods. Int. J. Food. Microbiol. 6, 187–198. van Netten, P., Perales, A., van de Moosdijk, A., Curtis, G.D.W., Mossel, D.A.A., 1989. Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp. Int. J. Food Microbiol. 8, 299–316. Wang, S.Y., Hitchins, A.D., 1994. Enrichment of severely and moderately heat-injured Listeria monocytogenes cells. J. Food Safety 14, 259–271. ¨ ¨ A., Peterz, M., 1992. Evaluation of methods for detection Westoo, of Listeria monocytogenes in foods: NMKL collaborative study. J. AOAC Int. 75, 46–52.
Short communication
The influence of commonly used selective agents on the growth of Listeria monocytogenes Charlotte Nexmann Jacobsen* Royal Veterinary and Agricultural University, Deptartment of Dairy and Food Science, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Received 25 January 1999; received in revised form 6 May 1999; accepted 22 June 1999
Abstract The effects, upon addition of various selective agents to a rich growth medium (BHIEM) on the growth of Listeria monocytogenes and non-Listeria were studied in microtitre wells. A combination of nalidixic acid (0.1 g / l), acriflavine (0.02 g / l) and fosfomycin (0.1 g / l) added to the medium (BHIEM NAF ) was found to be the least inhibitory to Listeria while at the same time, effectively inhibitory to non-Listeria organisms. Recovery of heat injured Listeria monocytogenes in BHIEM, BHIEM NAF and the two traditional Listeria-media, UVM I and II, was compared. Recovery in BHIEM was obtained within 24 h of incubation at 308C, whereas recovery in BHIEM NAF took up to 48 h. Growth and recovery in UVM I and II never reached the same level as in BHIEM or BHIEM NAF . 1999 Elsevier Science B.V. All rights reserved.
1. Introduction It is readily known, that the present standard Listeria-media do not allow recovery of sublethally injured bacteria (Busch and Donnelly, 1992). The injured bacteria might recover and contribute to the development of listeriosis (Sheridan et al., 1994). This way there is a need for an enrichment medium which allows maximum growth of Listeria with simultaneous suppression of background flora. The selective agents previously utilized for Listeriaselective media are numerous. Beerens and TahonCastel (1966) were the first to report the advantage *Tel.: 1 45-35-283-284; fax: 1 45-35-283-214. E-mail address: [email protected] (C. Nexmann Jacobsen)
of using nalidixic acid for isolating Listeria. Since then nalidixic acid has been recognized as one of the most important selective agents for Listeria isolation and is now used alone, or more commonly in combination with other selective agents, such as acriflavine, lithium chloride, colistin, pheny ethanol, moxalactam, ceftazidime, fosfomycin, polymyxin B and cycloheximide, for isolation of L. monocytogenes (Lee and McClain, 1986; Curtis et al., 1989a; van Netten et al., 1989; Cox et al., 1990, Ryser and Marth, 1991; Curtis and Lee, 1995). It is a well known fact, that the selective agents not only inhibit non-Listeria organisms, but are also inhibitory to some degree to Listeria spp. (Curtis et al., 1989b). Various treatments such as heating and freezing can injure Listeria sublethally (Golden et al., 1988a,b;
0168-1605 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00101-4
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C. Nexmann Jacobsen / International Journal of Food Microbiology 50 (1999) 221 – 226
Budu-Amoako et al., 1992; Busch and Donnelly, 1992, Mackey et al., 1994). The injured cells have a lower resistance towards selective agents, thereby delaying recovery and detection. In order to avoid a false negative test, it is important to have a method that supports resuscitation. The objective of this study is to evaluate the influence of various selective agents on the growth of L. monocytogenes as well as non-listeria, when added to a nutritionally rich medium (Brain Heart Infusion broth supplemented with egg yolk and magnesium sulphate) that has been shown to support the growth of Listeria (Jacobsen, 1998).
2. Materials and methods
2.1. Cultures All strains used in this study were supplied by the Department of Veterinary Microbiology at the Royal Veterinary and Agricultural University, Copenhagen, Denmark (Table 1). Lactococcuslactis was grown in LM 17 where as Lactobacillusbrevis and Lactobacillus salivarius were grown anaerobically in De Man, Rogosa, Sharpe broth (MRS, Oxoid) for 20–24 h at 308C. The remaining strains were incubated in Brain Heart Infusion broth (BHI, Difco) for 20–24 h at 378C prior to testing. Stock cultures were maintained at 2 408C in 15% v / v glycerol.
2.2. Growth studies Listeria monocytogenes and various non-Listeria organisms, grown in in the presence of selective agents in an enriched growth medium composed of Brain Heart Infusion broth supplemented with 5 g / l magnesium sulphate and 25 ml / l egg yolk emulsion (Merck) (Jacobsen et al., 1997; Jacobsen, 1998) was compared to growth in the selective media University of Vermont (UVM) I and II (McClain and Lee, 1988). The studies were performed in microtitre plates (Corning, New York, USA). Each medium was inoculated at a level of 10 5 cells / ml and the wells of microtitre plates filled with 200 ml of each inoculated medium. Each medium was tested in 20 wells and the average of the results was calculated. Each measurement was blanked with the particular medium without inoculation. Measurements were
performed with a spectrophotometer (Multiscan MCC 340, Labsystem, Finland) at 600 nm. The microtitre plates were shaken for 5 s before optical density was measured. The following selective additives to BHIEM were tested: Lithium chloride (LiCl, Sigma) 0.5 g / l, 2 g / l, and 10 g / l, acriflavine (Chroma-Gesellschaft, ¨ Schmid GmbH, Kongen, Germany) 5 g / l, 10 g / l and 20 mg / l, nalidixic acid (Sigma) 0.01 g / l, 0.02 g / l, 0.05 g / l and 0.1 g / l, colistin (Sigma) 0.02 g / l, 0.05 g / l, phenyl ethanol (Sigma) 1.0 g / l and 2.5 g / l, moxalactam (Eli Lilly) 0.01 g / l and 0.02 g / l, ceftazidime (Glaxo) 0.02 g / l, fosfomycin (Boehringer) 0.04 g / l, and 0.1 g / l Polymyxin B (Sigma) 1 000 000 iu as well as cycloheximide (Sigma) 0.05 g / l and 0.2 g / l). Optical density was measured following 24 h of incubation at 308C. Not all L. monocytogenes strains were tested with LiCl, phenyl ethanol, moxalactam, polymyxin B and cycloheximide.
2.3. Heat injury Overnight cultures (2 ml) of Listeria monocytogenes strain 11139 were added to 200 ml preheated BHI and immersed in a waterbath for 20 min at 558C for heat treatment as described by Meyer and Donnelly (1992). An aliquot of 0.5 ml of unheated and heated cells was then transferred into 100 ml of BHIEM, BHIEM containing 0.1 g / l nalidixic acid, 0.02 g / l acriflavine and 0.1 g / l fosfomycin (BHIEM NAF ) as well as 100 ml of UVM I and II respectively. Optical density was measured after 0 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h and 48 h of incubation at 308C.
3. Results and discussion The results from the growth studies of grampositive and gram-negative bacteria in selective media are listed in Table 1. Addition of lithium chloride (LiCl) to BHIEM inhibits gram-positive bacteria with the exception of Bacillus licheniformis, while gram-negative bacteria are only slightly reduced. Unfortunately in this study, LiCl inhibited the growth of Listeria monocytogenes more than 50% in the concentrations tested compared with the growth in BHIEM. The inhibitory effect of LiCl on L.
Table 1 The influence of selective agents on the growth of Listeria monocytogenes and various non-Listeria following incubation for 24 h at 308C a Strain
Inhibition % 5 100 2 ((growth in BHIEM added the selective agent compared to growth in BHIEM) 3 100) Acriflavine (mg/l)
0.5
2
10
5
10
L. monocytogenes 11139 11578 14729 11137 T15 85 5101 3967.6
63 61 ND b ND ND ND ND ND
58 66 ND ND ND ND ND ND
62 65 ND ND ND ND ND ND
0 14 9 15 0 7 7 9
11 7 18 29 3 46 56 20
Staph. aureus Ent. faecium M. luteus Lact. lactis Lb. brevis Lb. salivarius B. cereus B. licheniformis
0 41 0 60 46 33 33 0
0 46 59 85 45 81 35 0
38 100 87 98 100 100 75 0
93 100 93 99 24 56 10 46
E. coli Ps. fluorescens Acine. anitratus Enterob. cloacae Prot. mirabilis Citrob. freundii Kl. aeroginosa
0 0 0 0 12 0 11
10 0 30 0 13 0 14
65 54 99 43 23 35 45
0 0 47 0 15 0 17
Nalidixic acid (g/l) 20
Colistin (mg/l)
Ph.eth. (g/l)
Moxal. (mg/l)
Ceftaz. (g/l)
Fosfom. (g/l)
Pol.B. (iu)
0.01
0.02
0.05
0.1
20
50
1
2.5
10
20
0.02
0.04
0.1
1 mill.
6 1 23 23 0 52 60 11
7 8 0 0 0 0 0 0
0 7 0 ND 0 ND 0 0
10 9 0 0 2 0 0 0
65 47 5 13 3 30 30 10
43 16 0 0 0 0 0 0
47 16 ND 0 ND ND ND 2
53 54 ND ND ND ND ND ND
53 54 ND ND ND ND ND ND
5 9 ND ND ND ND ND ND
3 5 ND ND ND ND ND ND
5 7 13 24 11 11 11 22
9 6 ND ND ND ND ND ND
2 0 17 24 9 ND ND 30
0 0 ND ND ND ND ND ND
99 100 88 98 52 74 0 96
100 100 86 97 41 64 96 91
20 0 0 0 26 12 97 64
70 0 33 0 19 9 98 86
90 0 70 0 23 8 98 86
88 0 77 0 17 13 96 86
0 29 92 40 30 76 26 5
0 20 90 33 35 87 34 4
15 34 85 33 52 65 53 17
48 37 75 54 13 62 53 6
100 0 34 0 0 0 0 69
100 0 60 100 8 28 0 61
100 7 53 100 44 7 0 69
52 10 77 9 0 0 52 0
97 5 88 3 0 0 64 0
12 0 40 78 36 0 25 6
0 30 55 0 17 5 15
22 40 74 0 27 20 39
98 41 98 99 62 96 99
94 52 95 88 97 86 97
97 90 98 99 100 95 100
98 98 98 100 100 94 100
98 75 71 82 17 98 89
98 98 57 79 24 98 97
34 79 54 54 44 42 56
94 95 91 99 76 94 94
89 0 24 81 68 70 81
91 0 42 74 79 80 81
87 53 74 78 69 69 86
5 0 0 0 16 11 0
1 0 15 0 13 5 0
80 40 76 67 35 28 70
C. Nexmann Jacobsen / International Journal of Food Microbiology 50 (1999) 221 – 226
LiCl g/l
a
Abbreviations: Staph.: Staphylococcus; Ent.: Enterococcus; M. luteus: Micrococcus luteus; Lact.: Lactococcus; Lb.: Lactobacillus; B.: Bacillus; E.: Escherichia; Ps.: Pseudomonas: Acine.: Acinetobacter; Enterob.: Enterobacter; Prot.: Proteus; Citrob.: Citrobacter; Kl.: Klebsiella; LiCl: Lithium chloride; Ph. eth.: Phenyl ethanol; Moxal.: Moxalactam; Ceftaz.: Ceftazidime; Fosfom.: Fosfomycin; Pol.B.:Polymyxin B; Cycloh.:Cycloheximide. BHIEM: Brain Heart Infusion broth containing 0.1 g / l nalidixic acid, 0.02 g / l acriflavine and 0.1 g / l fosfomycin. b ND.: Not determined. 223
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C. Nexmann Jacobsen / International Journal of Food Microbiology 50 (1999) 221 – 226
monocytogenes observed here confirms the observations of another study (Cox et al., 1990), where LiCl, at a level of 20 g / l in Tryptone soy broth, was found to inhibit and delay growth of L. monocytogenes. Other authors have observed unaffected growth of L. monocytogenes in the presence of 15 g / l LiCl (van Netten et al., 1989). When adding acriflavine, half of the eight strains of L. monocytogenes were slightly inhibited and the remaining four were unaffected by the presence of acriflavine, whereas strong inhibition of the cocci was observed. Other studies (Ralovich et al., 1971) report that the organism multiplies well when acriflavine is present with simultaneous suppression of gram-positive cocci. Bacilluscereus was inhibited almost completely by the highest concentration of acriflavine, whereas Lactobacillusbrevis and Lactobacillus salivarius were inhibited 52% and 64% respectively, compared to growth in the nonselective medium. The growth of the gram-negative bacteria was not inhibited to any noticeable degree by acriflavine. Another study, however, reports a negative effect from acriflavine on both lag time and generation time of L. monocytogenes (Beumer et al., 1996). Almost complete inhibition of gram-negative bacteria was observed when grown in the presence of nalidixic acid compared to growth in non-selective enrichment media. The efficiency of nalidixic acid towards gram-negative bacteria is a well known characteristic (Beerens and Tahon-Castel, 1966). Beerens and Tahon-Castel (1966) also observed partial inhibition of Staphylococcus aureus, Bacillus cereus and Lactobacillus acidophilus. Inhibition of Staph. aureus and B. cereus by nalidixic acid was also observed in this study, as well as inhibition of Micrococcus luteus. No other inhibition of grampositive bacteria was observed in this study. L. monocytogenes strain 11139 and to some degree 11578 were inhibited slightly at the highest concentration of nalidixic acid, whereas growth of the other L. monocytogenes strains was unaffected. Colistin, phenyl ethanol, moxalactam and ceftazidime inhibited mainly the gram-negative bacteria. Unfortunately, phenyl ethanol also inhibited the growth of L. monocytogenes as observed previously by van Netten et al. (1988). Polymyxin B inhibited only a few of the strains tested. At a concentration of 0.1 g / l, fosfomycin was very inhibitory to Staph. aureus and M. luteus strongly and to a lesser degree B. cereus. Fosfomycin has been reported to selective-
ly inhibit Listeria species, L. ivanovii, L. seeligeri and L. welshimeri (van Netten et al., 1989), resulting in more selective isolation of L. monocytogenes. This inhibition is reported to be enhanced by incubation at high temperature whereas L. monocytogenes seems to be unaffected by this (Curtis et al., 1989b). Cycloheximide did not seem to inhibit either L. monocytogenes or the other bacteria tested (results not shown). Since it is mainly used to avoid growth of yeast and fungi in media by inhibition of protein synthesis (Obrig, 1971), and is reported to be toxic to humans (Sax and Lewis, 1988; Basic-Zaninovic et al., 1991), addition of this agent seems irrelevant in a day to day enrichment method. The strongest inhibition of gram-positive bacteria was observed when adding LiCl. Unfortunately, L. monocytogenes was also strongly inhibited. This demonstrates how critical the use of LiCl in a selective enrichment medium is for the resuscitation of L. monocytogenes. Acriflavine demonstrated broad inhibition of gram-positive bacteria, and was only slightly inhibitory towards L. monocytogenes. As a consequence of these results, it was decided to add nalidixic acid in a concentration of 0.1 g / l to BHIEM for inhibition of gram-negative bacteria and acriflavine at a concentration of 0.02 g / l for inhibition of gram-positive bacteria in combination with fosfomycin at a concentration of 0.1 g / l. The medium was denoted BHIEM NAF . For heat injured cells of L. monocytogenes, the fastest recovery was observed in BHIEM, whereas recovery of injured cells, when grown in the medium BHIEM NAF , was delayed, requiring up to 48 h of incubation at 308C to reach the same level as the untreated bacteria (Table 2). This delayed recovery in selective media has also been reported by Wang and Hitchins (1994). Listeria monocytogenes never reached the same growth level, when grown in UVM I and II compared to BHIEM or BHIEM NAF during 48 h of incubation. Formulation of a selective medium, without any inhibitory effects towards stressed L. monocytogenes seems impossible. However BHIEM NAF seems to be less inhibitory than other Listeria media e.g. those recommended by the Nordic Committee on Food ¨ ¨ and Peterz, 1992), Analysis (UVM I and II) (Westoo although recovery in BHIEM NAF was delayed compared to recovery in BHIEM. The less inhibitory effect of the medium could be caused by the addition of egg yolk emulsion (van Netten et al., 1988; In’t
C. Nexmann Jacobsen / International Journal of Food Microbiology 50 (1999) 221 – 226
225
Table 2 The ability of heat stressed L. monocytogenes (strain 11139) to recover in BHIEM, BHIEM NAF , UVM I and UVM II, following 20 min of heat treatment at 558C ha
Untreated controls (OD600)b
0 18 19 20 21 22 23 24 48 a b
Heat treated bacteria (OD600)b
BHIEM
BHIEM NAF
UVM I
UVM II
BHIEM
BHIEM NAF
UVM I
UVM II
0.054 0.845 0.837 0.872 0.854 0.882 0.882 0.880 0.850
0.055 0.147 0.161 0.224 0.263 0.386 0.578 0.663 0.721
0.010 0.248 0.259 0.262 0.271 0.272 0.271 0.275 0.255
0.008 0.151 0.188 0.273 0.243 0.248 0.218 0.260 0.270
0.062 0.854 0.854 0.891 0.911 0.922 0.952 0.947 0.870
0.048 0.150 0.174 0.157 0.152 0.191 0.166 0.159 0.774
0.009 0.017 0.035 0.055 0.08 0.122 0.184 0.215 0.267
0.009 0.003 0.002 0.004 0.005 0.015 0.077 0.048 0.286
h: Hours of incubation at 308C. OD600: Optical density at 600 nm.
Veld and de Boer, 1991) and magnesium sulphate (Busch and Donnelly, 1992) that previously has been shown to protect and support growth of L. monocytogenes. To further support the growth of stressed bacteria, a two-stage enrichment procedure has proven beneficial (Lovett, 1988). Therefore incubation of L. monocytogenes in the rich, non-selective enrichment broth BHIEM for 6–8 h, is recommended to resuscitate sublethally damaged L. monocytogenes (Busch and Donnelly, 1992; Curtis and Lee, 1995), before the addition to the medium of the selective agents acriflavine, nalidixic acid and fosfomycin in the above mentioned concentrations. Further studies should be performed to establish the optimal duration of the resuscitation period before addition of selective agents.
Acknowledgements This work was financially supported by the Danish Research and Development Programme for Food Technology and the Danish Dairy Research Foundation. The technical assistance of Anette Ravn is highly appreciated.
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