- Email: [email protected]
Characteristics of induction of virulence factor expression by activated charcoal in Listeria monocytogenes
FEMS Microbiology Letters 174 (1999) 137^141
Characteristics of induction of virulence factor expression by activated charcoal in Listeria monocytogenes Svetlana Ermolaeva *, Yurii Belyi, Igor Tartakovskii Gamaleya Research Institute of Epidemiology and Microbiology, Gamaleya st. 18, Moscow 123098, Russia Received 15 February 1999; accepted 3 March 1999
Abstract Activated charcoal has been previously shown to induce in vitro expression of virulence factors by Listeria monocytogenes. In trying to elucidate the nature of the charcoal action, we found that the treatment of brain heart infusion medium with activated charcoal followed by charcoal removal does not result in an increase of virulence factor expression. At the same time, the addition of fresh charcoal to the charcoal-treated medium induces expression, suggesting that the effect of activated charcoal cannot be explained only by changes in medium composition. In addition, we observed that activated charcoal induced expression of virulence factors even when L. monocytogenes was physically separated from charcoal particles by either a nitrocellulose membrane or a thin layer of agar. We propose that the interaction of charcoal with some listerial product(s) might be responsible for the effect observed. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Listeria monocytogenes ; Listeriolysin ; Lecithinase; Activated charcoal
1. Introduction Listeria monocytogenes is a Gram-positive facultative intracellular pathogen. Its infection cycle includes invasion of mammalian cells, escaping from phagocytic vacuoles and multiplication in the cytoplasm of the host, through which bacteria can rapidly move (see [1]). Many genes whose products are needed for successful intracellular survival have been identi¢ed [2]. A pore-forming hemolysin, listeriolysin O (LLO), and a broad-range phospholipase C, leci-
* Corresponding author. Tel.: +7 (095) 193 6371; E-mail: [email protected]
thinase, has been shown to play important roles in the egress of bacteria from the vacuole, a critical step in infection [1,2]. Expression of LLO and lecithinase as well as almost all other known listerial virulence factors is under the control of the transcription regulator PrfA [1,2]. Levels of their expression strictly depend upon environmental conditions [3,4]. In part, activated charcoal, when added to the cultivation medium, results in the induction of virulence factor expression [5,6]. This level is almost as high as that shown for mutant strains with a constitutive virulence factor expression [6,7]. It has been suggested that in the presence of activated charcoal, the levels of expression reached those needed for in vivo host infection [6]. Thus, activated charcoal might mimic a
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 3 2 - 9
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signal that L. monocytogenes understands as a command to induce the synthesis of virulence factors. We are interested in the nature of this signal. In this work we demonstrate that the e¡ect of activated charcoal could not be explained by the exhaustion of the cultivation medium.
1.5% agar layer supplemented with charcoal was overlaid by the 1.5% charcoal-free agar layer with a thickness of about 1 mm. In other experiments supplemented with egg yolk, agar was overlaid by a sterile membrane (pore size 0.1 Wm, Millipore) and L. monocytogenes was grown on the membrane. 2.2. Activity assays
2. Materials and methods 2.1. Bacterial strain and culture conditions L. monocytogenes strain NCTC 10527 (serogroup 4b), kindly provided by Dr. W. Goebel, University of Wurzburg, was used. Bacteria were cultivated in brain heart infusion medium (BHI; Sigma). Activated charcoal (CECA S, Hauts-de-Seine, France) was added to the BHI medium after autoclaving from the sterilized 6% (w/v) water suspension to a ¢nal concentration speci¢ed in the text. To treat BHI with activated charcoal concentrations from 0.2 to 2% were used, and after autoclaving incubated on the shaker at 37³C for 1 h. Charcoal was removed by sterilization through a membrane (pore size 0.22 Wm, Millipore). L. monocytogenes was grown overnight in BHI broth, harvested by centrifugation, suspended in PBS bu¡er (pH 7.2) and diluted 1:10 into fresh broth up to OD600 about 0.2. Cultures were shaken at 37³C for 8 h. Then bacteria were harvested by centrifugation and culture supernatants were assayed. To assay plated bacteria L. monocytogenes was plated by glass rod and cultivated for 48 h. To separate charcoal particles from bacteria a bottom
To assay hemolytic and lecithinase activities of plated bacteria, BHI, BHI supplemented with 0.2% activated charcoal (BHI-AC) or BHI treated with 0.2% charcoal, agar was supplemented with 5% rabbit blood or 5% egg yolk emulsion [8], respectively. Lecithinase activity of plated bacteria was determined as a ratio of the area of the halos surrounding the colony to the area of the colony itself. Lecithinase activity in culture supernatants was assayed by fermentation for 1 h of egg yolk prepared as follows: an egg yolk was diluted 1:10 with 0.05 M phosphate bu¡er, pH 7.2, containing 0.15 M NaCl and 1 mM CaCl2 , then coaline (Russia) was added up to 4% (w/ v) and the mixture was vigorously shaken, centrifuged at 300Ug and sterilized through a membrane. Results were scanned by Multiscan at 450 nm and arbitrary units were used: OD450 /culture OD600 . OD600 of the culture grown in the presence of 1% activated charcoal was extrapolated as equal to OD600 of a charcoal-free culture with the same CFU. LLO activity in culture supernatants was assayed by quanti¢cation of cell-lysis activity in serial dilutions of the supernatant, using rabbit blood. Arbitrary units were used: the last dilution number OD600 .
Fig. 1. E¡ect of preliminary BHI treatment with activated charcoal on LLO and lecithinase production. LLO and lecithinase production were measured by assaying culture supernatants for hemolytic or lecithinase activities, respectively, (see Section 2). Data were average from at least three experiments. BHI, brain heart infusion broth ; BHI-AC, BHI broth supplemented with 0.2% activated charcoal ; 0.2%, 1%, 2%, BHI broth was treated before bacteria inoculation with 0.2%,1% or 2% activated charcoal, respectively, followed by charcoal removal (see Section 2).
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139
3. Results 3.1. The presence of activated charcoal during cultivation is needed for increased expression of hemolysin and lecithinase BHI broth was treated with increasing concentrations from 0.2% to 2% of activated charcoal as described in Section 2. Before the inoculation of L. monocytogenes, charcoal was eliminated by ¢ltration through a sterile nitrocellulose membrane. Culture supernatants were assayed on lecithinase and hemolytic activity after 8 h growth. As a positive control we used 0.2% charcoal-containing BHI broth (BHIAC on Fig. 1). Very weak if any lecithinase activity was detected in supernatants of cultures grown in treated BHI, while in the presence of 0.2% activated charcoal this activity was readily detectable and was well determined (Fig. 1). No signi¢cant increase in hemolytic activity was detected in the culture supernatant even when BHI broth was treated with 2% AC, a concentration that is 10 times higher than normally used (Fig. 1). The principal e¡ect of BHI treatment with activated charcoal was a substantial decrease of the growth rate (Fig. 2). Treatment of BHI with activated charcoal did not increase lecithinase expression by plated bacteria (Table 1). 3.2. Smaller activated charcoal concentrations are needed in treated medium We compared the e¡ects of di¡erent activated charcoal concentrations on LLO and lecithinase ex-
Fig. 2. E¡ect of BHI treatment with activated charcoal on L. monocytogenes growth. L. monocytogenes was grown overnight in BHI broth, then harvested by centrifugation, suspended in PBS and diluted 1:10 at time 0 into BHI or BHI treated with indicated concentrations of activated charcoal followed by charcoal removal. Bacterial growth was determined by measuring cell density at 600 nm.
pression. Fresh activated charcoal was added to BHI or BHI treated previously with 1% charcoal, in concentrations from 0.002% to 1% (Fig. 3). In the presence of 0.2% activated charcoal in BHI broth a signi¢cant expression of lecithinase was observed, a result that is consistent with previous reports [6,7], hemolytic activity was increased by a factor of 10. Reduction of charcoal concentration by 10 times, i.e. up to 0.02%, resulted in an intermediate level of LLO and lecithinase production. Reduction of the concentration down to 0.002% eliminated the e¡ect.
Table 1 Lecithinase activitya of plated L. monocytogenes Nothingb Membranec
BHI
BHI-ACd
2% treatede
6.6 þ 2.0f 0
33.4 þ 11.3 3.3 þ 1.3
3.4 þ 0.21 ndg
a Lecithinase activity was determined as the ratio of the area of the halo surrounding the colony plated by the glass rod to the area of the colony. b L. monocytogenes was plated directly on the agar layer. c L. monocytogenes was plated on membrane (pore size 0.1 Wm), covering the agar layer. d BHI agar supplemented with 0.2% activated charcoal. e BHI broth was treated with 2% activated charcoal followed by charcoal removal as indicated in Section 2, then Bacto agar (Difco) was added up to 1.5%. f Data were average from at least three experiments, and standard deviation was calculated. g Not determined.
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Fig. 3. E¡ect of di¡erent charcoal concentrations on LLO and lecithinase production. L. monocytogenes was grown overnight in BHI broth, then harvested by centrifugation, suspended in PBS, diluted 1:10 into (a) BHI or (b) BHI treated with 1% activated charcoal, in the presence of indicated (x axis) concentrations of freshly added charcoal. Bacteria were grown for 8 h. Culture supernatants were assayed for hemolytic and lecithinase activity. Data were average from at least three experiments.
On the other hand, the increase of charcoal concentration up to 1% resulted in decreased LLO and lecithinase activities. For BHI medium treated with 1% charcoal, the maximum of hemolytic and lecithinase activities was observed when up to 0.02% fresh charcoal was added. Concentrations as low as 0.002% succeeded in increasing hemolytic activity by a factor of 2 and in bringing about a distinct appearance of lecithinase. In the presence of 0.2% charcoal hemolytic activity was only slightly more than in control charcoal treated BHI broth without fresh charcoal, and lecithinase was about one half of the maximum. Bacterial growth was not observed in treated BHI when 1% fresh charcoal was added. 3.3. A direct contact with activated charcoal particles is not necessary for induction of virulence factor expression No hemolytic and weak lecithinase activities were
observed for the L. monocytogenes strain NCTC 10527 plated on BHI agar (Table 1, Fig. 4). In accord with the previous report [6], the adding of activated charcoal up to 0.2% resulted in the appearance of considerable hemolytic activity. Lecithinase activity was increased by a factor of 5 (Table 1). Halos around colonies plated on charcoal-containing agar (BHI-AC) were substantially more distinct than on BHI agar. If L. monocytogenes was physically separated from the charcoal containing medium by a nitrocellulose membrane with pore size 0.1 Wm, lecithinase activity was still clearly visible on BHI-AC agar while it was not observed on BHI plates (Table 1). We next used a thin substrate-containing agar layer, to separate the bacteria from charcoal particles. The induction of hemolytic activity was observed only when BHIAC was used as a bottom layer but not with BHI alone (Fig. 4). Hemolysis was not as clearly visible as when bacteria were directly plated onto BHI-AC agar. This e¡ect was lost if the agar plate thickness was more than 2 mm.
4. Discussion
Fig. 4. E¡ect of separating of plated L. monocytogenes from activated charcoal on LLO expression. L. monocytogenes was plated by a glass rod and cultivated at 37³C for 48 h on rabbit blood containing agar. A: BHI agar; B: BHI-AC agar; C : double layer agar, the bottom is BHI, the upper is BHI; D: double layer agar, the bottom is BHI-AC, the upper is BHI, bacteria were plated on the upper layer.
Activated charcoal promotes the growth of some bacteria on nutritional media due to the removal of toxic compounds. For example, the growth-promoting e¡ect of activated charcoal on Legionella was believed to be due to the decomposition of active oxygen [9]. A similar mechanism can be assumed for L. monocytogenes, namely, a medium component
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acting as a repressor on virulence factor expression, was removed by charcoal resulting in an induction of expression of virulence factors. The data presented here suggest that this was not the case. Conditioning of BHI with 2% activated charcoal followed by its removal did not change LLO and lecithinase expression, while subsequent supplementing of treated medium with only 0.02% charcoal resulted in induction. It is clear that activated charcoal absorbs certain medium components and, possibly, listerial secreted products. Treatment of BHI with charcoal clearly resulted in the exhaustion of essential nutrients in the medium, thus reducing L. monocytogenes growth. We believed, that the reduction of hemolytic and lecithinase activities in culture supernatants, when charcoal concentrations were more than optimal, could be due to absorption of secreted LLO and lecithinase themselves to this substrate. One can envisage another mechanism of charcoal action, i.e. induction of immobilization of L. monocytogenes on charcoal particles is the signal for gene expression. We observed the induction of virulence factor expression when plated L. monocytogenes was separated from a charcoal-containing medium with a membrane or thin layer of agar. Thus, the direct contact with charcoal was not obligatory, although the increase of virulence factor expression by L. monocytogenes was observed only in the presence of activated charcoal. Taken together, these data suggested that absorption of some listerial product(s) by activated charcoal might be responsible for the switching of virulence factor production. If this is true, the absorptive capacity of activated charcoal is important for the e¡ect. In BHI, exhausted by charcoal, the capacity of freshly added charcoal should be increased for bacterial products, and indeed, smaller charcoal concentrations resulted in induction. On the other hand, a necessity of product di¡usion to the charcoal layer could be responsible for reducing of lecithinase and hemolytic activities when L. monocytogenes was physically separated from charcoal. Activated charcoal has been recently shown to be able to promote the growth of certain bacteria over a distance in certain conditions, thus excluding the
141
possibility of the di¡usion of some chemical substance [10]. The emission of a physical signal by charcoal itself was proposed as being responsible for the growth-promoting e¡ect. Although our present data suggest that this mechanism is not responsible for the induction of L. monocytogenes gene expression, mechanisms of the action of activated charcoal on L. monocytogenes other than those discussed above can not be excluded.
References [1] Sheehan, B., Kocks, C., Dramsi, S., Goiuin, F., Klars¢eld, A.D., Mengaud, J. and Cossart, P. (1994) Molecular and genetic determinants of the Listeria monocytogenes infectious process. Curr. Top. Microbiol. Immunol. 192, 187^216. [2] Portnoy, D.A., Chakraborty, T., Goebel, W. and Cossart, P. (1992) Molecular determinants of Listeria monocytogenes pathogenesis. Infect. Immun. 60, 1263^1267. [3] Leimeister-Wachter, M., Domann, E. and Chakraborty, T. (1992) The expression of virulence genes in Listeria monocytogenes is thermoregulated. J. Bacteriol, 174, 947^952. [4] Milenbachs, A., Brown, D., Moors, M. and Youngman, P. (1997) Carbon-source regulation of virulence gene expression in Listeria monocytogenes. Mol. Microbiol. 23, 1075^1085. [5] Ge¡roy, C., Gaillard, J.-L., Alouf, J. and Berche, P. (1987) Puri¢cation, characterization and toxicity of the sulfhydrylactivated hemolysin listeriolysin O from Listeria monocytogenes. Infect. Immun. 55, 1641-1646. [6] Ripio, M.-T., Dominguez-Bernal, G., Suarez, M., Brehm, K., Berche, P., Vazquez-Boland and J.-A. (1996) Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition. Res. Microbiol. 147, 371^384. [7] Ermolaeva, S., Varfolomeeva, N., Belyi, Yu. and Tartakovskii, I. (1997) Isolation and characterization of a Listeria monocytogenes mutant strain hyperproducing virulence factors. FEMS Microbiol. Lett. 150, 89^195. [8] O'Leary, W.M. and Weld, J.T. (1964) Lypolytic activities of Staphylococcus aureus. J. Bacteriol. 88, 1356^1364. [9] Ho¡man, P.S., Pine, L. and Bell, S. (1983) Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila, catalytic decomposition by charcoal. Appl. Environ. Microbiol. 45, 784^791. [10] Matsuhashi, M., Endoh, K., Pankrushina, A.N., Watanabe, H., Yamamura, H., Komiyama, M., Endo, S., Tobi, M., Ohshima, H., Mano, Y., Hyodo, M., Kaneko, T. and Otani, S. (1997) Growth-promoting e¡ect of carbon material upon bacterial cells propagating through a distance. J. Gen. Appl. Microbiol. 43, 225^230.
FEMSLE 8729 15-4-99
Characteristics of induction of virulence factor expression by activated charcoal in Listeria monocytogenes Svetlana Ermolaeva *, Yurii Belyi, Igor Tartakovskii Gamaleya Research Institute of Epidemiology and Microbiology, Gamaleya st. 18, Moscow 123098, Russia Received 15 February 1999; accepted 3 March 1999
Abstract Activated charcoal has been previously shown to induce in vitro expression of virulence factors by Listeria monocytogenes. In trying to elucidate the nature of the charcoal action, we found that the treatment of brain heart infusion medium with activated charcoal followed by charcoal removal does not result in an increase of virulence factor expression. At the same time, the addition of fresh charcoal to the charcoal-treated medium induces expression, suggesting that the effect of activated charcoal cannot be explained only by changes in medium composition. In addition, we observed that activated charcoal induced expression of virulence factors even when L. monocytogenes was physically separated from charcoal particles by either a nitrocellulose membrane or a thin layer of agar. We propose that the interaction of charcoal with some listerial product(s) might be responsible for the effect observed. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Listeria monocytogenes ; Listeriolysin ; Lecithinase; Activated charcoal
1. Introduction Listeria monocytogenes is a Gram-positive facultative intracellular pathogen. Its infection cycle includes invasion of mammalian cells, escaping from phagocytic vacuoles and multiplication in the cytoplasm of the host, through which bacteria can rapidly move (see [1]). Many genes whose products are needed for successful intracellular survival have been identi¢ed [2]. A pore-forming hemolysin, listeriolysin O (LLO), and a broad-range phospholipase C, leci-
* Corresponding author. Tel.: +7 (095) 193 6371; E-mail: [email protected]
thinase, has been shown to play important roles in the egress of bacteria from the vacuole, a critical step in infection [1,2]. Expression of LLO and lecithinase as well as almost all other known listerial virulence factors is under the control of the transcription regulator PrfA [1,2]. Levels of their expression strictly depend upon environmental conditions [3,4]. In part, activated charcoal, when added to the cultivation medium, results in the induction of virulence factor expression [5,6]. This level is almost as high as that shown for mutant strains with a constitutive virulence factor expression [6,7]. It has been suggested that in the presence of activated charcoal, the levels of expression reached those needed for in vivo host infection [6]. Thus, activated charcoal might mimic a
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 3 2 - 9
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signal that L. monocytogenes understands as a command to induce the synthesis of virulence factors. We are interested in the nature of this signal. In this work we demonstrate that the e¡ect of activated charcoal could not be explained by the exhaustion of the cultivation medium.
1.5% agar layer supplemented with charcoal was overlaid by the 1.5% charcoal-free agar layer with a thickness of about 1 mm. In other experiments supplemented with egg yolk, agar was overlaid by a sterile membrane (pore size 0.1 Wm, Millipore) and L. monocytogenes was grown on the membrane. 2.2. Activity assays
2. Materials and methods 2.1. Bacterial strain and culture conditions L. monocytogenes strain NCTC 10527 (serogroup 4b), kindly provided by Dr. W. Goebel, University of Wurzburg, was used. Bacteria were cultivated in brain heart infusion medium (BHI; Sigma). Activated charcoal (CECA S, Hauts-de-Seine, France) was added to the BHI medium after autoclaving from the sterilized 6% (w/v) water suspension to a ¢nal concentration speci¢ed in the text. To treat BHI with activated charcoal concentrations from 0.2 to 2% were used, and after autoclaving incubated on the shaker at 37³C for 1 h. Charcoal was removed by sterilization through a membrane (pore size 0.22 Wm, Millipore). L. monocytogenes was grown overnight in BHI broth, harvested by centrifugation, suspended in PBS bu¡er (pH 7.2) and diluted 1:10 into fresh broth up to OD600 about 0.2. Cultures were shaken at 37³C for 8 h. Then bacteria were harvested by centrifugation and culture supernatants were assayed. To assay plated bacteria L. monocytogenes was plated by glass rod and cultivated for 48 h. To separate charcoal particles from bacteria a bottom
To assay hemolytic and lecithinase activities of plated bacteria, BHI, BHI supplemented with 0.2% activated charcoal (BHI-AC) or BHI treated with 0.2% charcoal, agar was supplemented with 5% rabbit blood or 5% egg yolk emulsion [8], respectively. Lecithinase activity of plated bacteria was determined as a ratio of the area of the halos surrounding the colony to the area of the colony itself. Lecithinase activity in culture supernatants was assayed by fermentation for 1 h of egg yolk prepared as follows: an egg yolk was diluted 1:10 with 0.05 M phosphate bu¡er, pH 7.2, containing 0.15 M NaCl and 1 mM CaCl2 , then coaline (Russia) was added up to 4% (w/ v) and the mixture was vigorously shaken, centrifuged at 300Ug and sterilized through a membrane. Results were scanned by Multiscan at 450 nm and arbitrary units were used: OD450 /culture OD600 . OD600 of the culture grown in the presence of 1% activated charcoal was extrapolated as equal to OD600 of a charcoal-free culture with the same CFU. LLO activity in culture supernatants was assayed by quanti¢cation of cell-lysis activity in serial dilutions of the supernatant, using rabbit blood. Arbitrary units were used: the last dilution number OD600 .
Fig. 1. E¡ect of preliminary BHI treatment with activated charcoal on LLO and lecithinase production. LLO and lecithinase production were measured by assaying culture supernatants for hemolytic or lecithinase activities, respectively, (see Section 2). Data were average from at least three experiments. BHI, brain heart infusion broth ; BHI-AC, BHI broth supplemented with 0.2% activated charcoal ; 0.2%, 1%, 2%, BHI broth was treated before bacteria inoculation with 0.2%,1% or 2% activated charcoal, respectively, followed by charcoal removal (see Section 2).
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139
3. Results 3.1. The presence of activated charcoal during cultivation is needed for increased expression of hemolysin and lecithinase BHI broth was treated with increasing concentrations from 0.2% to 2% of activated charcoal as described in Section 2. Before the inoculation of L. monocytogenes, charcoal was eliminated by ¢ltration through a sterile nitrocellulose membrane. Culture supernatants were assayed on lecithinase and hemolytic activity after 8 h growth. As a positive control we used 0.2% charcoal-containing BHI broth (BHIAC on Fig. 1). Very weak if any lecithinase activity was detected in supernatants of cultures grown in treated BHI, while in the presence of 0.2% activated charcoal this activity was readily detectable and was well determined (Fig. 1). No signi¢cant increase in hemolytic activity was detected in the culture supernatant even when BHI broth was treated with 2% AC, a concentration that is 10 times higher than normally used (Fig. 1). The principal e¡ect of BHI treatment with activated charcoal was a substantial decrease of the growth rate (Fig. 2). Treatment of BHI with activated charcoal did not increase lecithinase expression by plated bacteria (Table 1). 3.2. Smaller activated charcoal concentrations are needed in treated medium We compared the e¡ects of di¡erent activated charcoal concentrations on LLO and lecithinase ex-
Fig. 2. E¡ect of BHI treatment with activated charcoal on L. monocytogenes growth. L. monocytogenes was grown overnight in BHI broth, then harvested by centrifugation, suspended in PBS and diluted 1:10 at time 0 into BHI or BHI treated with indicated concentrations of activated charcoal followed by charcoal removal. Bacterial growth was determined by measuring cell density at 600 nm.
pression. Fresh activated charcoal was added to BHI or BHI treated previously with 1% charcoal, in concentrations from 0.002% to 1% (Fig. 3). In the presence of 0.2% activated charcoal in BHI broth a signi¢cant expression of lecithinase was observed, a result that is consistent with previous reports [6,7], hemolytic activity was increased by a factor of 10. Reduction of charcoal concentration by 10 times, i.e. up to 0.02%, resulted in an intermediate level of LLO and lecithinase production. Reduction of the concentration down to 0.002% eliminated the e¡ect.
Table 1 Lecithinase activitya of plated L. monocytogenes Nothingb Membranec
BHI
BHI-ACd
2% treatede
6.6 þ 2.0f 0
33.4 þ 11.3 3.3 þ 1.3
3.4 þ 0.21 ndg
a Lecithinase activity was determined as the ratio of the area of the halo surrounding the colony plated by the glass rod to the area of the colony. b L. monocytogenes was plated directly on the agar layer. c L. monocytogenes was plated on membrane (pore size 0.1 Wm), covering the agar layer. d BHI agar supplemented with 0.2% activated charcoal. e BHI broth was treated with 2% activated charcoal followed by charcoal removal as indicated in Section 2, then Bacto agar (Difco) was added up to 1.5%. f Data were average from at least three experiments, and standard deviation was calculated. g Not determined.
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Fig. 3. E¡ect of di¡erent charcoal concentrations on LLO and lecithinase production. L. monocytogenes was grown overnight in BHI broth, then harvested by centrifugation, suspended in PBS, diluted 1:10 into (a) BHI or (b) BHI treated with 1% activated charcoal, in the presence of indicated (x axis) concentrations of freshly added charcoal. Bacteria were grown for 8 h. Culture supernatants were assayed for hemolytic and lecithinase activity. Data were average from at least three experiments.
On the other hand, the increase of charcoal concentration up to 1% resulted in decreased LLO and lecithinase activities. For BHI medium treated with 1% charcoal, the maximum of hemolytic and lecithinase activities was observed when up to 0.02% fresh charcoal was added. Concentrations as low as 0.002% succeeded in increasing hemolytic activity by a factor of 2 and in bringing about a distinct appearance of lecithinase. In the presence of 0.2% charcoal hemolytic activity was only slightly more than in control charcoal treated BHI broth without fresh charcoal, and lecithinase was about one half of the maximum. Bacterial growth was not observed in treated BHI when 1% fresh charcoal was added. 3.3. A direct contact with activated charcoal particles is not necessary for induction of virulence factor expression No hemolytic and weak lecithinase activities were
observed for the L. monocytogenes strain NCTC 10527 plated on BHI agar (Table 1, Fig. 4). In accord with the previous report [6], the adding of activated charcoal up to 0.2% resulted in the appearance of considerable hemolytic activity. Lecithinase activity was increased by a factor of 5 (Table 1). Halos around colonies plated on charcoal-containing agar (BHI-AC) were substantially more distinct than on BHI agar. If L. monocytogenes was physically separated from the charcoal containing medium by a nitrocellulose membrane with pore size 0.1 Wm, lecithinase activity was still clearly visible on BHI-AC agar while it was not observed on BHI plates (Table 1). We next used a thin substrate-containing agar layer, to separate the bacteria from charcoal particles. The induction of hemolytic activity was observed only when BHIAC was used as a bottom layer but not with BHI alone (Fig. 4). Hemolysis was not as clearly visible as when bacteria were directly plated onto BHI-AC agar. This e¡ect was lost if the agar plate thickness was more than 2 mm.
4. Discussion
Fig. 4. E¡ect of separating of plated L. monocytogenes from activated charcoal on LLO expression. L. monocytogenes was plated by a glass rod and cultivated at 37³C for 48 h on rabbit blood containing agar. A: BHI agar; B: BHI-AC agar; C : double layer agar, the bottom is BHI, the upper is BHI; D: double layer agar, the bottom is BHI-AC, the upper is BHI, bacteria were plated on the upper layer.
Activated charcoal promotes the growth of some bacteria on nutritional media due to the removal of toxic compounds. For example, the growth-promoting e¡ect of activated charcoal on Legionella was believed to be due to the decomposition of active oxygen [9]. A similar mechanism can be assumed for L. monocytogenes, namely, a medium component
FEMSLE 8729 15-4-99
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acting as a repressor on virulence factor expression, was removed by charcoal resulting in an induction of expression of virulence factors. The data presented here suggest that this was not the case. Conditioning of BHI with 2% activated charcoal followed by its removal did not change LLO and lecithinase expression, while subsequent supplementing of treated medium with only 0.02% charcoal resulted in induction. It is clear that activated charcoal absorbs certain medium components and, possibly, listerial secreted products. Treatment of BHI with charcoal clearly resulted in the exhaustion of essential nutrients in the medium, thus reducing L. monocytogenes growth. We believed, that the reduction of hemolytic and lecithinase activities in culture supernatants, when charcoal concentrations were more than optimal, could be due to absorption of secreted LLO and lecithinase themselves to this substrate. One can envisage another mechanism of charcoal action, i.e. induction of immobilization of L. monocytogenes on charcoal particles is the signal for gene expression. We observed the induction of virulence factor expression when plated L. monocytogenes was separated from a charcoal-containing medium with a membrane or thin layer of agar. Thus, the direct contact with charcoal was not obligatory, although the increase of virulence factor expression by L. monocytogenes was observed only in the presence of activated charcoal. Taken together, these data suggested that absorption of some listerial product(s) by activated charcoal might be responsible for the switching of virulence factor production. If this is true, the absorptive capacity of activated charcoal is important for the e¡ect. In BHI, exhausted by charcoal, the capacity of freshly added charcoal should be increased for bacterial products, and indeed, smaller charcoal concentrations resulted in induction. On the other hand, a necessity of product di¡usion to the charcoal layer could be responsible for reducing of lecithinase and hemolytic activities when L. monocytogenes was physically separated from charcoal. Activated charcoal has been recently shown to be able to promote the growth of certain bacteria over a distance in certain conditions, thus excluding the
141
possibility of the di¡usion of some chemical substance [10]. The emission of a physical signal by charcoal itself was proposed as being responsible for the growth-promoting e¡ect. Although our present data suggest that this mechanism is not responsible for the induction of L. monocytogenes gene expression, mechanisms of the action of activated charcoal on L. monocytogenes other than those discussed above can not be excluded.
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