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A simple method for the differentiation of Listeria monocytogenes based on induction of lecithinase activity by charcoal
International Journal of Food Microbiology 82 (2003) 87 – 94 www.elsevier.com/locate/ijfoodmicro
Research note
A simple method for the differentiation of Listeria monocytogenes based on induction of lecithinase activity by charcoal Svetlana Ermolaeva a,b, Tatyana Karpova b, Susana Novella a, Martin Wagner a,c, Mariela Scortti a,d, Igor Tartakovskii b, Jose A. Vazquez-Boland a,d,* a
Molecular Bacterial Pathogenesis Group, Animal Health Department, Veterinary Faculty, University of Leon, Leon, Spain b Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia c Institute for Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, Austria d Veterinary Molecular Microbiology Section, Department of Clinical Veterinary Science, University of Bristol, Longford, BS40 5DU, UK Received 4 April 2002; received in revised form 29 July 2002; accepted 26 August 2002
Abstract The PlcB phospholipase C, or lecithinase, is an important listerial virulence factor of potential use as a pathogenicity marker for Listeria spp. food isolates. However, wild-type strains of Listeria monocytogenes express virulence factors very poorly in vitro and their lecithinase activity is normally difficult to detect on agar plates. We recently reported that the production of listerial virulence factors is strongly induced if L. monocytogenes is grown in the presence of activated charcoal. We report here a simple method for the rapid differentiation of L. monocytogenes from other Listeria spp. based on a comparison of lecithinase reactions in egg yolk agar plates with and without charcoal supplementation. All L. monocytogenes wild-type isolates tested showed a clear induction of lecithinase activity in charcoal-supplemented medium (CEYM), while nonpathogenic Listeria spp. remained negative in CEYM. The animal pathogen L. ivanovii was easily differentiated from L. monocytogenes because it showed a strong lecithinase reaction independently of the presence or absence of charcoal in the medium. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Listeria identification; Listeria monocytogenes; Lecithinase induction; Activated charcoal; Food products
1. Introduction Listeria spp. are ubiquitous contaminants of food products. Of the six species currently included in this bacterial genus, only Listeria monocytogenes, the
* Corresponding author. Veterinary Molecular Microbiology Section, Department of Clinical Veterinary Science, University of Bristol, Longford, BS40 5DU, UK. Tel.: +44(0)117-928-9615. E-mail address: [email protected] (J.A. Vasquez-Boland).
causative agent of listeriosis, represents a hazard for humans. The increasing occurrence in the last 20 years of listeriosis outbreaks associated with the consumption of contaminated food has led to L. monocytogenes being considered one of the most important foodborne pathogens today (Farber and Peterkin, 1991; Vazquez-Boland et al., 2001). The enrichment and plating media currently used in the monitoring of Listeria contamination in food are not selective for L. monocytogenes, making it necessary to carry out an individual differentiation of the isolated
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 2 ) 0 0 3 9 9 - 9
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Listeria spp. colonies (Donnelly, 1999). A number of conventional tests have been recommended to this end, including investigation of carbohydrate utilization, h-haemolysis and virulence for mice (Hitchins, 1995; Johnson, 1998; Rocourt, 1999). However, identification based on carbohydrate fermentation tests is not sufficient to differentiate L. monocytogenes from its closely related, nonpathogenic species L. innocua (Hitchins, 1995). Discrimination between these two species requires the demonstration of haemolytic activity, but interpretation of this assay is complicated by the weak haemolysis manifested by most L. monocytogenes isolates. Even the synergistic haemolysis (CAMP) tests, proposed as a means of discriminating between Listeria spp. (Rocourt et al., 1983), are difficult to interpret and may give false-positive or negative results (Ravlovich, 1984; Vazquez-Boland et al., 1990, 1992a). Clearly, the mouse virulence test is not suitable for the routine identification of Listeria spp. Therefore, there is a need for a simple method for the specific discrimination of L. monocytogenes isolates for routine use in food microbiology laboratories. The listerial phospholipases C, PlcA and PlcB, present in all isolates of L. monocytogenes, are potential virulence markers that can be used in the differentiation of pathogenic and nonpathogenic Listeria spp. (Notermans et al., 1991; Vazquez-Boland et al., 1992b; Coffey et al., 1996). Of these, the PlcB phospholipase, or lecithinase, is particularly suitable because its activity can be easily visualized on agar plates supplemented with egg yolk, an inexpensive substrate (Fuzi and Pillis, 1962). The problem is that, in in vitro conditions such as in bacteriological culture media, wild-type strains of L. monocytogenes normally produce low levels of virulence-associated factors, making it difficult to identify their corresponding activities on agar plates (Ripio et al., 1996; Gianfranceschi et al., 1998). Thus, the PlcB phospholipase is normally so weakly expressed that L. monocytogenes has even been reported to give negative results in the lecithinase test (Rocourt et al., 1983). We recently reported that the addition of 0.2% charcoal to broth medium strongly increases the production of virulence factors in L. monocytogenes (Geoffroy et al., 1991; Ripio et al., 1996, 1997; Ermolaeva et al., 1999). The precise mechanism underlying this phenomenon remains unknown, but it relates to the adsorption capacity of activated
charcoal (Ermolaeva et al., 1999, 2000), and involves induction of the L. monocytogenes virulence genes activated by the PrfA regulatory protein (Ripio et al., 1996). Thus, in the presence of charcoal, all PrfAregulated virulence genes are highly expressed, including that encoding lecithinase, leading to a dramatic increase in the cognate enzymatic activity. We used this property of L. monocytogenes to design a simple protocol for the reliable differentiation of this species on egg yolk agar plates.
2. Materials and methods 2.1. Strains The strains used in this study are listed in Table 1. They include strains from bacterial culture collections (SLCC, Special Listeria Culture Collection, Institute of Hygiene and Microbiology of the University of Wu¨rzburg, Germany; NCTC, National Collection of Type Cultures, United Kingdom; ATCC, American Type Culture Collection, USA; CLIP, Collection de Listeria de l’Institut Pasteur; PAM, collection of the Molecular Bacterial Pathogenesis Group, University of Leo´n, Spain) and strains freshly isolated from food products. Listeria were routinely cultured at 37 jC on Columbia sheep blood agar (CSBA) (bioMe´rieux, Marcy-l’Etoile, France). 2.2. Isolation and identification of Listeria spp. from food products Listeria spp. were isolated from food according to the USDA protocol (Johnson, 1998). Briefly, 25 g of the food sample was pre-enriched in 225 ml Fraser selective broth at 37 jC for 48 h, followed by plating on PALCAM and Oxford agar. Listeria-selective culture media were purchased from HiMedia Laboratories, Bombay, India. Approximately 10 esculinpositive colonies were subcultured on nonselective medium, tested for catalase activity and subjected to microscopy after Gram staining. Gram- and catalasepositive rods were presumed to be members of the genus Listeria and were characterised further using the API Listeria system (bioMe´rieux). Motility was determined at 22 and 37 jC in 0.35% agar (Listeria Motility Medium, HiMedia Laboratories). Haemolytic
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Table 1 Listeria spp. strains used in this study and their lecithinase reaction in BHI (Difco) egg-yolk medium with and without 0.5% charcoal at 37 jC Strain
Source/description
Lecithinase reaction charcoal Without
With
L. monocytogenes wild-type strains CLIP 75936 SLCC 2755 NCTC 5348 SLCC 2479 NCTC 5214 NCTC 10527 ATCC 19116 ATCC 19118 NCTC 10888 PAM 350 PAM 58 PAM 63 PAM 348 PAM 363 PAM 68 PAM 67 PAM 368 PAM 71 PAM 16 PAM 78 PAM 80 PAM 14 PAM 366 PAM 11 PAM 15 PAM 73 PAM 346 PAM 345 GIM 5 GIM 20 GIM 22 GIM 23 GIM 24 GIM 25 GIM 27 GIM 34 GIM 41 GIM 42 GIM 50 GIM 51
Collection; sva 1/2a Collection; sv 1/2b Collection; sv 1/2c Collection; sv 3c Collection; sv 4a Collection; sv 4b Collection; sv 4c Collection, sv 4c Collection; sv 4d Collection; sv 1/2a Collection; sv 1/2a Collection; sv 1/2a Collection; sv 1/2b Collection; sv 1/2c Collection; sv 1/2c Collection; sv 1/2c Collection; sv 1/2c Collection; sv 1/2c Collection; sv 3b Collection; sv 3b Collection; sv 3c Collection; sv 4b Collection; sv 4b Collection; sv 4b Collection; sv 4b Collection; sv 4b Collection; sv 4b Collection; sv 4b Fresh isolate; mixed minced meat Fresh isolate; sausage Fresh isolate; sausage Fresh isolate; sausage Fresh isolate; sausage Fresh isolate; sausage Fresh isolate; mixed minced meat Fresh isolate; cooked beef Fresh isolate; mixed minced meat Fresh isolate; minced turkey meat Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat
L. monocytogenes mutant strains SLCC 53 PAM 274 NCTC 7973
Collection; prfA deletion mutant sv 1/2a Collection; prfA deletion mutant sv 1/2a Collection; prfA* mutantb; sv 1/2a
+
+
L. ivanovii NCTC 11846 ATCC 19119 PAM 55
Collection; sv 5 Collection; sv 5 Collection; sv 5
+ + +
+ + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
(continued on next page)
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Table 1 (continued ) Strain
Source/description
Lecithinase reaction charcoal Without
L. seeligeri SLCC 3954 SLCC 5921
Collection; sv 1/2b Collection; sv 1/2b
L. innocua ATCC 33090 ATCC 33091 GIM 1 GIM 3 GIM 4 GIM 6 GIM 7 GIM 9 GIM 10 GIM 11 GIM 12 GIM 13 GIM 14 GIM 15 GIM 16 GIM 17 GIM 18 GIM 19 GIM 28 GIM 29 GIM 30 GIM 31 GIM 32 GIM 33 GIM 35 GIM 36 GIM 37 GIM 38 GIM 39 GIM 42 GIM 45 GIM 46 GIM 48 GIM 49 GIM 52 GIM 53
Collection; sv 6a Collection; sv 6b Fresh isolate; sliced chicken Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; sliced beef Fresh isolate; mixed minced meat Fresh isolate; minced pork Fresh isolate; cooked pork Fresh isolate; breaded chicken fillet Fresh isolate; breaded chicken fillet Fresh isolate; salami Fresh isolate; mixed minced meat Fresh isolate; salami Fresh isolate; salted lard Fresh isolate; mixed minced meat Fresh isolate; sliced chicken Fresh isolate; chicken grill Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; minced beef Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; minced turkey meat Fresh isolate; minced chicken meat Fresh isolate; sliced chicken Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; minced turkey meat Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; beef meat Fresh isolate; mixed minced meat Fresh isolate; sliced chicken Fresh isolate; meat pie
L. welshimeri SLCC 5334 GIM 2 GIM 8 GIM 44
Collection; sv 6a Fresh isolate; deboned beef Fresh isolate; salted lard Fresh isolate; mixed minced meat
L. grayi PAM 450
Collection
a
With
Serovar. b PrfA* phenotype (overproduction of all virulence determinants, including PlcB/lecithinase, due a Gly145Ser mutation in the transcriptional regulator PrfA (Ripio et al., 1997).
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activity was determined on CSBA. For confirmation of L. monocytogenes, all food isolates were tested with a L. monocytogenes-specific PCR method (Ermolaeva, 1995) using oligonucleotide primers PlcA1 (5VAGGGGGCCATTTTGTTATAAG) and PlcA2 (5VATCGTTGCTGTTTTGCTCGGT), which amplify a 476-bp DNA fragment internal to the plcA gene (Mengaud et al., 1991). PCR was performed on listerial cell lysates obtained as follows: 100 Al of an overnight culture was treated with 2 mg/ml lysozyme (Serva) in 0.5 ml 20% sucrose Tris –HCl buffer (10 mM Tris – HCl pH 8.0, 1 mM EDTA) at 37 jC; bacterial cells were then harvested by centrifugation and resuspended in 0.5 ml 3% Triton X-100 in Tris – HCl buffer containing 125 Ag/ml Proteinase K (Sigma); the mixture was incubated for 1 h at 56 jC, and the resulting lysate was boiled for 5 min to inactivate Proteinase K. PCR was performed in 35 cycles of 94 jC for 1 min, 55 jC for 1 min, 72 jC for 1 min, followed by a final elongation at 72 jC for 10 min. 2.3. Preparation of charcoal-supplemented egg yolk medium (CEYM) Activated charcoal powder (Merck) was added to the base agar medium (Table 2) before autoclaving. A 5% (v/v) sample of an egg yolk suspension, prepared by adding one fresh egg yolk to 100 ml of sterile saline, was added aseptically to the melted base agar
Table 2 Induction of L. monocytogenes lecithinase activity by 0.5% activated charcoal in various base media at 37 jC Base medium
Induction of lecithinase activity a
Brain – Heart Infusion (Difco Laboratories, USA) GRM 1 (NNPGIP, Obolensk, Russia) Brain – Heart Infusion (bioMe´rieux, France) Brain – Heart Infusion (HiMedia Laboratories, India) Listeria-selective broth (HiMedia) MYP (HiMedia)
+
a
+ F F F –
+, induction is observed in all strains tested; F induction in only a few strains, as a weak halo; – , no induction.
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medium stabilised at 45 jC. Inoculated egg yolk media were incubated at 37 jC for 48 h.
3. Results and discussion We previously determined that 0.2% (w/v) activated charcoal was the optimal concentration to fully induce virulence-associated factors of L. monocytogenes in liquid Brain –Heart Infusion (BHI) medium (Difco Laboratories, Detroit, MI, USA) (Ripio et al., 1996; Ermolaeva et al., 1999). To establish the optimal concentration of charcoal in solid BHI medium, we assessed the induction of lecithinase activity in the presence of various concentrations of the adsorbent, from 0.2% to 1%, using a set of wild-type collection strains of L. monocytogenes. All these strains were lecithinase-negative in egg yolk medium without charcoal. With 0.2% charcoal, only 6 of the 28 reference strains tested gave a clear positive reaction. With a concentration of 0.5% charcoal, all strains gave a positive reaction manifested as a dense halo of precipitation, with a radius c 3– 8 mm, around the bacterial streak (Table 1, Fig. 1). With concentrations higher than 0.8%, growth was impaired and the opacity halos were narrower. We therefore adopted 0.5% as the optimal concentration of charcoal for CEYM (see Fig. 2). We also investigated the effect of the base growth medium on lecithinase induction by 0.5% charcoal on agar plates, using the same set of reference strains of L. monocytogenes (Table 2). The medium routinely used in our studies on the charcoal effect is the abovementioned BHI from Difco. Two other BHI media, from bioMe´rieux and HiMedia, were tested. In contrast to Difco BHI, these media led to induction of the lecithinase in only a few strains, with weak opacity reactions. Similar results were obtained with solidified Listeria-selective broth (HiMedia). On MYP medium (HiMedia), specifically designed for assays of the lecithinase activity of Bacillus cereus, opacity reactions were not observed in any of the L. monocytogenes strains tested. Finally, a clear induction of lecithinase activity, similar to that in Difco BHI, was observed with GRM1 agar medium, derived from fish meal (NNPGIP media, Obolensk, Russia). Thus, base medium composition is an important factor to be
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Fig. 1. Behaviour of Listeria spp. in CEYM (L. monocytogenes, L. mon; L. ivanovii, L. iva; L. seeligeri, L. see; L. innocua, L. inn; L. welshimeri, L. wel.). The pathogenic species L. monocytogenes and L. ivanovii give a positive lecithinase reaction in CEYM but only L. monocytogenes shows induction of lecithinase activity by charcoal. The minimum concentration of charcoal producing optimal induction is 0.5%.
considered in the standardisation of CEYM for the correct detection of charcoal-mediated induction of L. monocytogenes lecithinase activity. We next assessed the feasibility of discriminating L. monocytogenes from other Listeria spp. using CEYM. In the genus Listeria, two other species, L. ivanovii and L. seeligeri, possess the plcB gene encoding the lecithinase (Vazquez-Boland et al., 2001). L. ivanovii is specifically pathogenic for ruminants, whereas L. seeligeri is nonpathogenic. L. ivanovii strains, which constitutively overexpress PrfAdependent virulence genes (Gonzalez-Zorn et al.,
2000), gave a strong positive reaction regardless of whether or not charcoal was present in the medium. L. seeligeri strains, which expresses virulence genes very weakly (if at all) due to a natural mutation in the PrfA regulatory system (Kreft and Vazquez-Boland, 2001), showed no induction of lecithinase activity in CEYM. As expected, all the other nonpathogenic Listeria spp., which lack the plcB gene (Vazquez-Boland et al., 2001), were lecithinase negative in CEYM (Table 1 and Fig. 1). The usefulness of the CEYM test was further assessed by applying it to the study of Listeria spp.
Fig. 2. Identification of L. monocytogenes by the induction of lecithinase activity in CEYM. The reaction of 14 freshly isolated Listeria spp. strains (see Table 1), including 12 L. monocytogenes and two L. innocua strains [inside the rectangle] are shown in the upper division of the plates; from left to right beginning at top row: GIM51, 50, 42, 41, 49, 48, 34, 27, 25, 24, 23, 22, 20, 5. In the lower division, control strains of L. monocytogenes including four wild-type collection strains, which are PAM 14 and CLIP 75936 in the upper row, and NCTC 5214 and NCTC 10527 below, and two mutant strains serving as negative ( ) and positive (+) lecithinase reaction controls (the strains PAM 274 and NCTC 7973, which are DprfA and prfA* mutants, respectively; see text for details). Note that, at 0.2% charcoal, only part of the L. monocytogenes strains show induction of lecithinase activity (marked with an asterisk), with reactions that are weaker than with the optimal (0.5%) concentration. Note also that L. innocua strains remain lecithinase negative in CEYM.
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isolated during a microbiological survey of marketed meat products conducted in 2001 in Moscow (Russia). A total of 12 of the 49 Listeria spp. isolates obtained from the 183 samples analysed were identified as L. monocytogenes, 34 as L. innocua and three as L. welshimeri (Table 1). One sample was contaminated with strains of all three species, three samples with L. monocytogenes and L. innocua, and one sample with L. innocua and L. welshimeri. All isolates identified as L. monocytogenes gave a positive result in CEYM test, whereas isolates identified as L. innocua and L. welshimeri displayed no lecithinase activity, either in the absence or presence of charcoal (Table 1, Fig. 2). Experimental evidence accumulated in our laboratories over several years of investigations on virulence factor regulation in Listeria indicates that the induction of lecithinase activity by charcoal is, within the genus Listeria, a unique and universal property of wild-type isolates of L. monocytogenes. So far, deviation from this behaviour has been observed only for strains of L. monocytogenes carrying mutations in the prfA gene encoding the listerial virulence gene activator, PrfA. These variant strains include SLCC 53, the type strain of L. monocytogenes (Kathariou and Pine, 1991; Mengaud et al., 1991). This strain, which is nonpathogenic, has a naturally occurring deletion in its prfA gene and displays no induction of lecithinase activity in charcoal-containing medium. PAM 274, a site-directed prfA-deletion mutant, had the same phenotype as SLCC 53 (Table 1, Fig. 2). Another type of variant with abnormal behaviour is represented by NCTC 7973, which produces a mutant PrfA* protein, leading to the constitutive overproduction of virulence factors (Ripio et al., 1997). PrfA* strains, like L. ivanovii, display a strong lecithinase activity in the presence and absence of charcoal (Fig. 2). Although both types of mutations may arise spontaneously in L. monocytogenes, they are extremely rare and have only been described in strains that have been maintained in culture collections for a long time, or subjected to repeated subculturing in vitro (Kathariou and Pine, 1991; Ripio et al., 1996). The remote possibility of finding such mutants does not invalidate the CEYM test, because this method will nonetheless distinguish between potentially pathogenic, (i.e. lecithi-
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nase-positive) and nonpathogenic, (i.e. lecithinasenegative) Listeria spp.
4. Conclusions We report here a simple, inexpensive and reliable method for rapid differentiation of the foodborne pathogen L. monocytogenes from other Listeria spp. This test involves comparing the lecithinase reactions of Listeria spp. in an appropriate egg yolk medium with and without charcoal supplementation. L. monocytogenes isolates typically display a negative lecithinase reaction in medium without charcoal and a positive lecithinase reaction in charcoal-supplemented egg yolk medium (CEYM). The second pathogenic species of the genus, L. ivanovii, is distinguished from L. monocytogenes because it gives a strong positive reaction not only in CEYM, but also in the absence of charcoal. Isolates of the nonpathogenic species L. innocua, L. welshimeri and L. seeligeri remain lecithinase-negative in CEYM. The simplicity and reliability of the CEYM test described here makes this method suitable for routine screening for L. monocytogenes in food Listeria spp. isolates.
Acknowledgements This work was supported by INTAS (grant 20000471). S.E. wishes to thank EMBO and NATO for the fellowships awarded.
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Research note
A simple method for the differentiation of Listeria monocytogenes based on induction of lecithinase activity by charcoal Svetlana Ermolaeva a,b, Tatyana Karpova b, Susana Novella a, Martin Wagner a,c, Mariela Scortti a,d, Igor Tartakovskii b, Jose A. Vazquez-Boland a,d,* a
Molecular Bacterial Pathogenesis Group, Animal Health Department, Veterinary Faculty, University of Leon, Leon, Spain b Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia c Institute for Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, Austria d Veterinary Molecular Microbiology Section, Department of Clinical Veterinary Science, University of Bristol, Longford, BS40 5DU, UK Received 4 April 2002; received in revised form 29 July 2002; accepted 26 August 2002
Abstract The PlcB phospholipase C, or lecithinase, is an important listerial virulence factor of potential use as a pathogenicity marker for Listeria spp. food isolates. However, wild-type strains of Listeria monocytogenes express virulence factors very poorly in vitro and their lecithinase activity is normally difficult to detect on agar plates. We recently reported that the production of listerial virulence factors is strongly induced if L. monocytogenes is grown in the presence of activated charcoal. We report here a simple method for the rapid differentiation of L. monocytogenes from other Listeria spp. based on a comparison of lecithinase reactions in egg yolk agar plates with and without charcoal supplementation. All L. monocytogenes wild-type isolates tested showed a clear induction of lecithinase activity in charcoal-supplemented medium (CEYM), while nonpathogenic Listeria spp. remained negative in CEYM. The animal pathogen L. ivanovii was easily differentiated from L. monocytogenes because it showed a strong lecithinase reaction independently of the presence or absence of charcoal in the medium. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Listeria identification; Listeria monocytogenes; Lecithinase induction; Activated charcoal; Food products
1. Introduction Listeria spp. are ubiquitous contaminants of food products. Of the six species currently included in this bacterial genus, only Listeria monocytogenes, the
* Corresponding author. Veterinary Molecular Microbiology Section, Department of Clinical Veterinary Science, University of Bristol, Longford, BS40 5DU, UK. Tel.: +44(0)117-928-9615. E-mail address: [email protected] (J.A. Vasquez-Boland).
causative agent of listeriosis, represents a hazard for humans. The increasing occurrence in the last 20 years of listeriosis outbreaks associated with the consumption of contaminated food has led to L. monocytogenes being considered one of the most important foodborne pathogens today (Farber and Peterkin, 1991; Vazquez-Boland et al., 2001). The enrichment and plating media currently used in the monitoring of Listeria contamination in food are not selective for L. monocytogenes, making it necessary to carry out an individual differentiation of the isolated
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 2 ) 0 0 3 9 9 - 9
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Listeria spp. colonies (Donnelly, 1999). A number of conventional tests have been recommended to this end, including investigation of carbohydrate utilization, h-haemolysis and virulence for mice (Hitchins, 1995; Johnson, 1998; Rocourt, 1999). However, identification based on carbohydrate fermentation tests is not sufficient to differentiate L. monocytogenes from its closely related, nonpathogenic species L. innocua (Hitchins, 1995). Discrimination between these two species requires the demonstration of haemolytic activity, but interpretation of this assay is complicated by the weak haemolysis manifested by most L. monocytogenes isolates. Even the synergistic haemolysis (CAMP) tests, proposed as a means of discriminating between Listeria spp. (Rocourt et al., 1983), are difficult to interpret and may give false-positive or negative results (Ravlovich, 1984; Vazquez-Boland et al., 1990, 1992a). Clearly, the mouse virulence test is not suitable for the routine identification of Listeria spp. Therefore, there is a need for a simple method for the specific discrimination of L. monocytogenes isolates for routine use in food microbiology laboratories. The listerial phospholipases C, PlcA and PlcB, present in all isolates of L. monocytogenes, are potential virulence markers that can be used in the differentiation of pathogenic and nonpathogenic Listeria spp. (Notermans et al., 1991; Vazquez-Boland et al., 1992b; Coffey et al., 1996). Of these, the PlcB phospholipase, or lecithinase, is particularly suitable because its activity can be easily visualized on agar plates supplemented with egg yolk, an inexpensive substrate (Fuzi and Pillis, 1962). The problem is that, in in vitro conditions such as in bacteriological culture media, wild-type strains of L. monocytogenes normally produce low levels of virulence-associated factors, making it difficult to identify their corresponding activities on agar plates (Ripio et al., 1996; Gianfranceschi et al., 1998). Thus, the PlcB phospholipase is normally so weakly expressed that L. monocytogenes has even been reported to give negative results in the lecithinase test (Rocourt et al., 1983). We recently reported that the addition of 0.2% charcoal to broth medium strongly increases the production of virulence factors in L. monocytogenes (Geoffroy et al., 1991; Ripio et al., 1996, 1997; Ermolaeva et al., 1999). The precise mechanism underlying this phenomenon remains unknown, but it relates to the adsorption capacity of activated
charcoal (Ermolaeva et al., 1999, 2000), and involves induction of the L. monocytogenes virulence genes activated by the PrfA regulatory protein (Ripio et al., 1996). Thus, in the presence of charcoal, all PrfAregulated virulence genes are highly expressed, including that encoding lecithinase, leading to a dramatic increase in the cognate enzymatic activity. We used this property of L. monocytogenes to design a simple protocol for the reliable differentiation of this species on egg yolk agar plates.
2. Materials and methods 2.1. Strains The strains used in this study are listed in Table 1. They include strains from bacterial culture collections (SLCC, Special Listeria Culture Collection, Institute of Hygiene and Microbiology of the University of Wu¨rzburg, Germany; NCTC, National Collection of Type Cultures, United Kingdom; ATCC, American Type Culture Collection, USA; CLIP, Collection de Listeria de l’Institut Pasteur; PAM, collection of the Molecular Bacterial Pathogenesis Group, University of Leo´n, Spain) and strains freshly isolated from food products. Listeria were routinely cultured at 37 jC on Columbia sheep blood agar (CSBA) (bioMe´rieux, Marcy-l’Etoile, France). 2.2. Isolation and identification of Listeria spp. from food products Listeria spp. were isolated from food according to the USDA protocol (Johnson, 1998). Briefly, 25 g of the food sample was pre-enriched in 225 ml Fraser selective broth at 37 jC for 48 h, followed by plating on PALCAM and Oxford agar. Listeria-selective culture media were purchased from HiMedia Laboratories, Bombay, India. Approximately 10 esculinpositive colonies were subcultured on nonselective medium, tested for catalase activity and subjected to microscopy after Gram staining. Gram- and catalasepositive rods were presumed to be members of the genus Listeria and were characterised further using the API Listeria system (bioMe´rieux). Motility was determined at 22 and 37 jC in 0.35% agar (Listeria Motility Medium, HiMedia Laboratories). Haemolytic
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Table 1 Listeria spp. strains used in this study and their lecithinase reaction in BHI (Difco) egg-yolk medium with and without 0.5% charcoal at 37 jC Strain
Source/description
Lecithinase reaction charcoal Without
With
L. monocytogenes wild-type strains CLIP 75936 SLCC 2755 NCTC 5348 SLCC 2479 NCTC 5214 NCTC 10527 ATCC 19116 ATCC 19118 NCTC 10888 PAM 350 PAM 58 PAM 63 PAM 348 PAM 363 PAM 68 PAM 67 PAM 368 PAM 71 PAM 16 PAM 78 PAM 80 PAM 14 PAM 366 PAM 11 PAM 15 PAM 73 PAM 346 PAM 345 GIM 5 GIM 20 GIM 22 GIM 23 GIM 24 GIM 25 GIM 27 GIM 34 GIM 41 GIM 42 GIM 50 GIM 51
Collection; sva 1/2a Collection; sv 1/2b Collection; sv 1/2c Collection; sv 3c Collection; sv 4a Collection; sv 4b Collection; sv 4c Collection, sv 4c Collection; sv 4d Collection; sv 1/2a Collection; sv 1/2a Collection; sv 1/2a Collection; sv 1/2b Collection; sv 1/2c Collection; sv 1/2c Collection; sv 1/2c Collection; sv 1/2c Collection; sv 1/2c Collection; sv 3b Collection; sv 3b Collection; sv 3c Collection; sv 4b Collection; sv 4b Collection; sv 4b Collection; sv 4b Collection; sv 4b Collection; sv 4b Collection; sv 4b Fresh isolate; mixed minced meat Fresh isolate; sausage Fresh isolate; sausage Fresh isolate; sausage Fresh isolate; sausage Fresh isolate; sausage Fresh isolate; mixed minced meat Fresh isolate; cooked beef Fresh isolate; mixed minced meat Fresh isolate; minced turkey meat Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat
L. monocytogenes mutant strains SLCC 53 PAM 274 NCTC 7973
Collection; prfA deletion mutant sv 1/2a Collection; prfA deletion mutant sv 1/2a Collection; prfA* mutantb; sv 1/2a
+
+
L. ivanovii NCTC 11846 ATCC 19119 PAM 55
Collection; sv 5 Collection; sv 5 Collection; sv 5
+ + +
+ + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
(continued on next page)
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Table 1 (continued ) Strain
Source/description
Lecithinase reaction charcoal Without
L. seeligeri SLCC 3954 SLCC 5921
Collection; sv 1/2b Collection; sv 1/2b
L. innocua ATCC 33090 ATCC 33091 GIM 1 GIM 3 GIM 4 GIM 6 GIM 7 GIM 9 GIM 10 GIM 11 GIM 12 GIM 13 GIM 14 GIM 15 GIM 16 GIM 17 GIM 18 GIM 19 GIM 28 GIM 29 GIM 30 GIM 31 GIM 32 GIM 33 GIM 35 GIM 36 GIM 37 GIM 38 GIM 39 GIM 42 GIM 45 GIM 46 GIM 48 GIM 49 GIM 52 GIM 53
Collection; sv 6a Collection; sv 6b Fresh isolate; sliced chicken Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; sliced beef Fresh isolate; mixed minced meat Fresh isolate; minced pork Fresh isolate; cooked pork Fresh isolate; breaded chicken fillet Fresh isolate; breaded chicken fillet Fresh isolate; salami Fresh isolate; mixed minced meat Fresh isolate; salami Fresh isolate; salted lard Fresh isolate; mixed minced meat Fresh isolate; sliced chicken Fresh isolate; chicken grill Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; minced beef Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; minced turkey meat Fresh isolate; minced chicken meat Fresh isolate; sliced chicken Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; minced turkey meat Fresh isolate; mixed minced meat Fresh isolate; mixed minced meat Fresh isolate; beef meat Fresh isolate; mixed minced meat Fresh isolate; sliced chicken Fresh isolate; meat pie
L. welshimeri SLCC 5334 GIM 2 GIM 8 GIM 44
Collection; sv 6a Fresh isolate; deboned beef Fresh isolate; salted lard Fresh isolate; mixed minced meat
L. grayi PAM 450
Collection
a
With
Serovar. b PrfA* phenotype (overproduction of all virulence determinants, including PlcB/lecithinase, due a Gly145Ser mutation in the transcriptional regulator PrfA (Ripio et al., 1997).
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activity was determined on CSBA. For confirmation of L. monocytogenes, all food isolates were tested with a L. monocytogenes-specific PCR method (Ermolaeva, 1995) using oligonucleotide primers PlcA1 (5VAGGGGGCCATTTTGTTATAAG) and PlcA2 (5VATCGTTGCTGTTTTGCTCGGT), which amplify a 476-bp DNA fragment internal to the plcA gene (Mengaud et al., 1991). PCR was performed on listerial cell lysates obtained as follows: 100 Al of an overnight culture was treated with 2 mg/ml lysozyme (Serva) in 0.5 ml 20% sucrose Tris –HCl buffer (10 mM Tris – HCl pH 8.0, 1 mM EDTA) at 37 jC; bacterial cells were then harvested by centrifugation and resuspended in 0.5 ml 3% Triton X-100 in Tris – HCl buffer containing 125 Ag/ml Proteinase K (Sigma); the mixture was incubated for 1 h at 56 jC, and the resulting lysate was boiled for 5 min to inactivate Proteinase K. PCR was performed in 35 cycles of 94 jC for 1 min, 55 jC for 1 min, 72 jC for 1 min, followed by a final elongation at 72 jC for 10 min. 2.3. Preparation of charcoal-supplemented egg yolk medium (CEYM) Activated charcoal powder (Merck) was added to the base agar medium (Table 2) before autoclaving. A 5% (v/v) sample of an egg yolk suspension, prepared by adding one fresh egg yolk to 100 ml of sterile saline, was added aseptically to the melted base agar
Table 2 Induction of L. monocytogenes lecithinase activity by 0.5% activated charcoal in various base media at 37 jC Base medium
Induction of lecithinase activity a
Brain – Heart Infusion (Difco Laboratories, USA) GRM 1 (NNPGIP, Obolensk, Russia) Brain – Heart Infusion (bioMe´rieux, France) Brain – Heart Infusion (HiMedia Laboratories, India) Listeria-selective broth (HiMedia) MYP (HiMedia)
+
a
+ F F F –
+, induction is observed in all strains tested; F induction in only a few strains, as a weak halo; – , no induction.
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medium stabilised at 45 jC. Inoculated egg yolk media were incubated at 37 jC for 48 h.
3. Results and discussion We previously determined that 0.2% (w/v) activated charcoal was the optimal concentration to fully induce virulence-associated factors of L. monocytogenes in liquid Brain –Heart Infusion (BHI) medium (Difco Laboratories, Detroit, MI, USA) (Ripio et al., 1996; Ermolaeva et al., 1999). To establish the optimal concentration of charcoal in solid BHI medium, we assessed the induction of lecithinase activity in the presence of various concentrations of the adsorbent, from 0.2% to 1%, using a set of wild-type collection strains of L. monocytogenes. All these strains were lecithinase-negative in egg yolk medium without charcoal. With 0.2% charcoal, only 6 of the 28 reference strains tested gave a clear positive reaction. With a concentration of 0.5% charcoal, all strains gave a positive reaction manifested as a dense halo of precipitation, with a radius c 3– 8 mm, around the bacterial streak (Table 1, Fig. 1). With concentrations higher than 0.8%, growth was impaired and the opacity halos were narrower. We therefore adopted 0.5% as the optimal concentration of charcoal for CEYM (see Fig. 2). We also investigated the effect of the base growth medium on lecithinase induction by 0.5% charcoal on agar plates, using the same set of reference strains of L. monocytogenes (Table 2). The medium routinely used in our studies on the charcoal effect is the abovementioned BHI from Difco. Two other BHI media, from bioMe´rieux and HiMedia, were tested. In contrast to Difco BHI, these media led to induction of the lecithinase in only a few strains, with weak opacity reactions. Similar results were obtained with solidified Listeria-selective broth (HiMedia). On MYP medium (HiMedia), specifically designed for assays of the lecithinase activity of Bacillus cereus, opacity reactions were not observed in any of the L. monocytogenes strains tested. Finally, a clear induction of lecithinase activity, similar to that in Difco BHI, was observed with GRM1 agar medium, derived from fish meal (NNPGIP media, Obolensk, Russia). Thus, base medium composition is an important factor to be
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Fig. 1. Behaviour of Listeria spp. in CEYM (L. monocytogenes, L. mon; L. ivanovii, L. iva; L. seeligeri, L. see; L. innocua, L. inn; L. welshimeri, L. wel.). The pathogenic species L. monocytogenes and L. ivanovii give a positive lecithinase reaction in CEYM but only L. monocytogenes shows induction of lecithinase activity by charcoal. The minimum concentration of charcoal producing optimal induction is 0.5%.
considered in the standardisation of CEYM for the correct detection of charcoal-mediated induction of L. monocytogenes lecithinase activity. We next assessed the feasibility of discriminating L. monocytogenes from other Listeria spp. using CEYM. In the genus Listeria, two other species, L. ivanovii and L. seeligeri, possess the plcB gene encoding the lecithinase (Vazquez-Boland et al., 2001). L. ivanovii is specifically pathogenic for ruminants, whereas L. seeligeri is nonpathogenic. L. ivanovii strains, which constitutively overexpress PrfAdependent virulence genes (Gonzalez-Zorn et al.,
2000), gave a strong positive reaction regardless of whether or not charcoal was present in the medium. L. seeligeri strains, which expresses virulence genes very weakly (if at all) due to a natural mutation in the PrfA regulatory system (Kreft and Vazquez-Boland, 2001), showed no induction of lecithinase activity in CEYM. As expected, all the other nonpathogenic Listeria spp., which lack the plcB gene (Vazquez-Boland et al., 2001), were lecithinase negative in CEYM (Table 1 and Fig. 1). The usefulness of the CEYM test was further assessed by applying it to the study of Listeria spp.
Fig. 2. Identification of L. monocytogenes by the induction of lecithinase activity in CEYM. The reaction of 14 freshly isolated Listeria spp. strains (see Table 1), including 12 L. monocytogenes and two L. innocua strains [inside the rectangle] are shown in the upper division of the plates; from left to right beginning at top row: GIM51, 50, 42, 41, 49, 48, 34, 27, 25, 24, 23, 22, 20, 5. In the lower division, control strains of L. monocytogenes including four wild-type collection strains, which are PAM 14 and CLIP 75936 in the upper row, and NCTC 5214 and NCTC 10527 below, and two mutant strains serving as negative ( ) and positive (+) lecithinase reaction controls (the strains PAM 274 and NCTC 7973, which are DprfA and prfA* mutants, respectively; see text for details). Note that, at 0.2% charcoal, only part of the L. monocytogenes strains show induction of lecithinase activity (marked with an asterisk), with reactions that are weaker than with the optimal (0.5%) concentration. Note also that L. innocua strains remain lecithinase negative in CEYM.
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isolated during a microbiological survey of marketed meat products conducted in 2001 in Moscow (Russia). A total of 12 of the 49 Listeria spp. isolates obtained from the 183 samples analysed were identified as L. monocytogenes, 34 as L. innocua and three as L. welshimeri (Table 1). One sample was contaminated with strains of all three species, three samples with L. monocytogenes and L. innocua, and one sample with L. innocua and L. welshimeri. All isolates identified as L. monocytogenes gave a positive result in CEYM test, whereas isolates identified as L. innocua and L. welshimeri displayed no lecithinase activity, either in the absence or presence of charcoal (Table 1, Fig. 2). Experimental evidence accumulated in our laboratories over several years of investigations on virulence factor regulation in Listeria indicates that the induction of lecithinase activity by charcoal is, within the genus Listeria, a unique and universal property of wild-type isolates of L. monocytogenes. So far, deviation from this behaviour has been observed only for strains of L. monocytogenes carrying mutations in the prfA gene encoding the listerial virulence gene activator, PrfA. These variant strains include SLCC 53, the type strain of L. monocytogenes (Kathariou and Pine, 1991; Mengaud et al., 1991). This strain, which is nonpathogenic, has a naturally occurring deletion in its prfA gene and displays no induction of lecithinase activity in charcoal-containing medium. PAM 274, a site-directed prfA-deletion mutant, had the same phenotype as SLCC 53 (Table 1, Fig. 2). Another type of variant with abnormal behaviour is represented by NCTC 7973, which produces a mutant PrfA* protein, leading to the constitutive overproduction of virulence factors (Ripio et al., 1997). PrfA* strains, like L. ivanovii, display a strong lecithinase activity in the presence and absence of charcoal (Fig. 2). Although both types of mutations may arise spontaneously in L. monocytogenes, they are extremely rare and have only been described in strains that have been maintained in culture collections for a long time, or subjected to repeated subculturing in vitro (Kathariou and Pine, 1991; Ripio et al., 1996). The remote possibility of finding such mutants does not invalidate the CEYM test, because this method will nonetheless distinguish between potentially pathogenic, (i.e. lecithi-
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nase-positive) and nonpathogenic, (i.e. lecithinasenegative) Listeria spp.
4. Conclusions We report here a simple, inexpensive and reliable method for rapid differentiation of the foodborne pathogen L. monocytogenes from other Listeria spp. This test involves comparing the lecithinase reactions of Listeria spp. in an appropriate egg yolk medium with and without charcoal supplementation. L. monocytogenes isolates typically display a negative lecithinase reaction in medium without charcoal and a positive lecithinase reaction in charcoal-supplemented egg yolk medium (CEYM). The second pathogenic species of the genus, L. ivanovii, is distinguished from L. monocytogenes because it gives a strong positive reaction not only in CEYM, but also in the absence of charcoal. Isolates of the nonpathogenic species L. innocua, L. welshimeri and L. seeligeri remain lecithinase-negative in CEYM. The simplicity and reliability of the CEYM test described here makes this method suitable for routine screening for L. monocytogenes in food Listeria spp. isolates.
Acknowledgements This work was supported by INTAS (grant 20000471). S.E. wishes to thank EMBO and NATO for the fellowships awarded.
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