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Applied and Environmental Microbiology, April 2008, p. 2391-2397, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02587-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Effects of Carbon Dioxide on Neurotoxin Gene Expression in Nonproteolytic Clostridium botulinum Type E

Ingrid Artin,^1,2,3 Andrew T. Carter,² Elisabet Holst,³ Maria Lövenklev,^1, David R. Mason,² Michael W. Peck,² and Peter Rådström^1*

Applied Microbiology, Lund Institute of Technology, Lund University, SE-221 00 Lund, Sweden,¹ Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom,² Division of Medical Microbiology, Department of Laboratory Medicine, Lund University, SE-223 62 Lund, Sweden³

Received 16 November 2007/ Accepted 14 February 2008

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Carbon dioxide is an antimicrobial gas commonly used in modified atmosphere packaging. In the present study, the effects of carbon dioxide on the growth of and neurotoxin production by nonproteolytic Clostridium botulinum type E were studied during the growth cycle. Quantitative reverse transcription-PCR and an enzyme-linked immunosorbent assay were used to quantify expression of the type E botulinum neurotoxin gene (cntE) and the formation of type E neurotoxin. The expression levels of cntE were similar in two strains, with relative expression peaking in the transition between exponential phase and stationary phase. In stationary phase, cntE mRNA expression declined rapidly. The cntE mRNA half-life was calculated to be approximately 9 minutes. Neurotoxin formation occurred in late exponential phase and stationary phase. High carbon dioxide concentrations delayed growth by increasing the lag time and decreasing the maximum growth rate. The effects of carbon dioxide concentration on relative neurotoxin gene expression and neurotoxin formation were significant. Expression of cntE mRNA and the formation of extracellular neurotoxin were twofold higher with a headspace carbon dioxide concentration of 70% (vol/vol) compared to 10% (vol/vol). This finding sheds a new, cautionary light on the potential risks of botulism associated with the use of modified atmosphere packaging.

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Clostridium botulinum is a group of four distinct spore-forming, obligately anaerobic bacteria that share the common feature of forming a botulinum neurotoxin (BoNT). There are seven different PNT serotypes ([BoNT/A to BoNT/G), and these are the most potent toxins known. Food-borne botulism is caused by consuming preformed neurotoxin in foods, with as little as 30 ng of neurotoxin being potentially lethal (32). Proteolytic C. botulinum (group I C. botulinum) and nonproteolytic C. botulinum (group II C. botulinum) are the two groups of C. botulinum most frequently associated with food-borne botulism. BoNT/E produced by nonproteolytic C. botulinum type E is the most frequent cause of food-borne botulism in the colder regions of the northern hemisphere. Foods associated with these outbreaks include vacuum-packed smoked fish, salted fish, and fermented marine mammals (7, 8, 11, 22, 25, 31, 34). Nonproteolytic C. botulinum derives energy by degrading sugars and can grow and produce toxin at temperatures as low as 3°C (19, 31).

Recent consumer demand for mildly processed foods that are convenient, easy to use, and low in preservatives (e.g., salt and nitrite) has led to the development of new processes and packaging techniques. The safety and quality of minimally heated chilled foods commonly relies on a combination of a mild heat treatment, refrigerated storage, and low-oxygen modified-atmosphere packaging (MAP). Carbon dioxide is frequently included in a modified atmosphere because of its antimicrobial activity (13). While the mild heat treatments applied to these foods are sufficient to kill vegetative cells, they will not eliminate all bacterial spores; in fact, they may even induce their germination. The anaerobic atmosphere used in MAP is strongly inhibitory to aerobic organisms but has a lesser effect on anaerobic bacteria such as nonproteolytic C. botulinum (16, 18). Nonproteolytic C. botulinum is the principal microbiological safety hazard in these new minimally heated chilled foods (31). Fish packed in modified atmosphere is one of the greatest hazards, as fish may be heavily contaminated with spores of nonproteolytic C. botulinum (26), and toxin production may precede microbial spoilage and sensory rejection (1, 17).

Formations of botulinum neurotoxin are reported to vary with serotype, strain, medium, and culture condition (21). However, little is known about the direct regulation of the neurotoxin gene (cnt) or of neurotoxin formation by nonproteolytic C. botulinum type E. While a positive regulator, cntR, has been found in other types of C. botulinum, it appears to be absent in C. botulinum type E (9, 24, 36). Several in vitro methods have been developed for monitoring cnt expression in proteolytic C. botulinum and nonproteolytic C. botulinum, including a gene reporter system (12), competitive reverse transcription (RT)-PCR (29, 39, 40), and quantitative RT-PCR (qRT-PCR) (10, 23, 27, 28, 42). Experiments using these methods found a peak in neurotoxin gene expression in late exponential or early stationary phase. However, many of these studies examined only one or two points during growth, making it uncertain if the full cnt expression profile had been found. To establish the position for C. botulinum type E, a qRT-PCR method was developed to quantify the relative expression of cntE mRNA during all stages of growth, using two different strains. An enzyme-linked immunosorbent assay (ELISA) was used to correlate the levels of cntE mRNA with the amount of extracellular neurotoxin formed.

A recent study of nonproteolytic C. botulinum type B (27) showed that the expression of cntB and the formation of BoNT/B in a 70% carbon dioxide atmosphere were fivefold greater than those in a 10% carbon dioxide atmosphere. Intermediate gene expression and neurotoxin formation occurred in a 35% carbon dioxide atmosphere (27). In order to determine if this response to carbon dioxide is also shown by nonproteolytic C. botulinum type E, the effects of three concentrations of carbon dioxide on the growth of cntE expression and BoNT/E production by C. botulinum type E strain ATCC 9564 were investigated. Finally, in order to establish that there is a close relationship between cntE mRNA accumulation (as measured by qRT-PCR) and its rate of gene expression, the rate of cntE mRNA breakdown was determined.

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Bacterial strains and culture conditions.
Two strains of nonproteolytic C. botulinum type E, ATCC 9564 and BL 1177, were used. Strain BL 1177 was isolated from salmon implicated in a case of food-borne botulism in Sweden. Overnight cultures of each strain were prepared in tryptone-peptone-yeast extract (TPY) broth and incubated anaerobically for 18 to 20 h at 30°C. The TPY broth contained tryptone (50 g/liter; Oxoid, Basingstoke, United Kingdom), proteose peptone (5 g/liter; Oxoid), yeast extract (20 g/liter; Oxoid), and sodium thioglycolate (1 g/liter; Merck, Darmstadt, Germany). After sterilization, the medium was incubated for 24 to 36 h in an anaerobic workstation (Electrotek AW 800 TG; addVise, Bromma, Sweden) to allow for temperature and gas atmosphere equilibration. The standard atmosphere inside the workstation contained nitrogen, carbon dioxide, and hydrogen (80:10:10 [per vol]). Flasks containing 237.5 ml TPY-C broth, i.e., TPY supplemented with 0.4% (wt/vol) glucose (BDH Laboratory Supplies, Poole, United Kingdom), 0.1% (wt/vol) each of the disaccharides maltose (ICN Biochemicals, Aurora, OH) and cellobiose (Sigma-Aldrich, St. Louis, MO), and 0.1% (wt/vol) soluble starch (Merck), were each inoculated with 12.5 ml of an overnight single-strain culture and incubated under the same conditions described above. Growth was followed using measurements of optical density at 620 nm (OD₆₂₀) with a Hitachi UV-1500 spectrophotometer (Hitachi Instruments). Samples for total RNA extraction were withdrawn at different sampling times throughout growth (after 4.75 h, 5.75 h, 6.5 h, 7.25 h, 8.75 h, 9.25 h, 10.25 h, 12.5 h, 29 h 55 min, and 75.5 h). At the same times, separate samples were removed and frozen immediately for subsequent ELISA analysis for neurotoxin.

qRT-PCR primer and probe design.
Two assays were used, one for cntE and one for the housekeeping gene encoding the 16S rRNA (rrn). This gene has previously been used as a reference gene for quantification of both the toxin gene in nonproteolytic C. botulinum type B (27, 28) and the germination gene (gerA) in proteolytic C. botulinum type B (5). The primers and probes used in this study are listed in Table 1. Primers and probes previously designed for the detection of the cntE gene were used (2). The primers of Lövenklev et al. (28) for the rrn gene were compared to the sequence of the rrn gene for nonproteolytic C. botulinum type E and found to be compatible, as this is a highly conserved region. Several probes were designed and compared. The hybridization probes consist of two parts: a donor probe labeled with fluorescein at the 3' end and an acceptor probe labeled with LC Red640 (LC) at the 5' end with its 3' end blocked with a phosphate moiety. Agarose gel electrophoresis showed that the amplified products were of the expected sizes.

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TABLE 1. Sequences and fluorescent dye of primers and hybridization probes used for qRT-PCR

Analysis of neurotoxin gene expression.
Total RNA was extracted using a modified method for extraction of total RNA from Bacillus spp. with acidic phenol (35). Cells from a 10-ml suspension were harvested by centrifugation (10,400 x g at 4°C) for 10 min and resuspended in 500 µl ice-cold TES buffer, pH 7.5 (50 mM Tris [ICN Biochemicals], 5 mM EDTA [sigma], and 50 mM NaCl [sigma]). Total RNA was recovered using the bead-beating method described by Lövenklev et al. (28). Before RT, contaminating DNA was degraded by treatment with RNase-free DNase (Promega, Madison, WI). Total RNA concentrations were determined using a RiboGreen RNA quantification kit (Molecular Probes, Leiden, The Netherlands) and by measuring fluorescence at 525 nm with a TD-700 fluorometer (Turner Designs, Sunnyvale, CA). The RNA was stored at –80°C before analysis.

First-strand cDNA was synthesized in two separate RT assays using the reverse primers for the cntE gene and the rrn gene (Table 1). Synthesis of cDNA was performed in a Gene Amp 9700 thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). The total volume of the reaction mixture was 20 µl and contained 0.1 µg total RNA, 0.5 µM of each primer (Sigma-Genosys), 0.5 mM each of nucleotides dATP, dTTP, dCTP, and dGTP (Roche Diagnostics GmbH, Mannheim, Germany), 20 U RNasin RNase inhibitor (Promega), 5 mM DTT (Life Technologies, Gaithersburg, MD), 1x first-strand buffer, and 200 U Superscript II RNase H^– reverse transecriptase (Life Technologies). Before RT enzymes were added, the reaction mixture was heated to 65°C for 5 min and then chilled on ice. After a brief centrifugation and the addition of RT enzymes, the reaction mixture was incubated at 42°C for 50 min and the reaction was terminated by incubation at 70°C for 15 min.

PCR amplification was carried out in a LightCycler instrument (Roche Diagnostics). The PCR mixture specific for the cntE gene contained 1x PCR buffer (Roche Diagnostics), 4 mM MgCl₂ (Roche Diagnostics), 0.2 mM of each dNTP (Roche Diagnostics), 0.3 µM of each primer, 0.3 µM of each probe (TIB Molbiol, Berlin, Germany), 1.25 U Tth DNA polymerase (Roche Diagnostics), and 4 µl of template solution. For the rrn gene, the mixture was the same except for the primer concentration (0.5 µM). The cDNA solution for the rrn gene was diluted 100-fold in water treated with diethyl pyrocarbonate before PCR amplification. The total volume added to each capillary was 20 µl. The LightCycler amplification protocol that was used started with an initial denaturation at 95°C for 60 s, followed by 45 cycles of 95°C for 0 s (i.e., no hold at 95°C), annealing and fluorescence acquisition at 56°C for 5 s, and elongation at 72°C for 25 s. The temperature transition rate was 20°C/s. The specificity was confirmed using melting curve analysis. It consisted of 95°C for 0 s, 50°C for 15 s, and then an increase in temperature of 0.2°C/s up to 90°C. Each sample was analyzed three times for each gene. The crossing point was determined using the second derivative maximum mathematical model in the LightCycler software (version 3.5; Roche Molecular Biochemicals).

A dilution series of total RNA was reverse transcribed and amplified with the LightCycler to determine the amplification efficiency (AE) and log-linear range of amplification for each real-time assay. The AE in the exponential phase was calculated as 10^(–1/s) – 1, where s is the slope of the crossing point value (CP) plotted against the logarithm of the amount of RNA transcribed. The cntE assay had a linear range of amplification between 50 pg and 5 µg of added total RNA, and the AE was determined to be E_cntE = 0.87. The rrn assay had a linear range of amplification between 5 pg and 0.5 µg of added total RNA, and the AE was E_rrn = 1.3.

To confirm the effectiveness of the DNase treatment, DNase-treated RNA was amplified using real-time PCR assays. None of the controls gave a fluorescent signal with real-time PCR, confirming that the DNase treatment removed all genomic DNA (data not shown).

The relative expression (RE) of the cntE gene compared to that of the rrn gene was calculated using these E values and the crossing point deviation (CP) of the unknown sample compared to a calibrator sample using the equation (33).

Measurement of neurotoxin concentration.
The concentrations of type E neurotoxin in culture supernatant samples were determined using a commercial ELISA procedure according to the manufacturer's instructions (Metabiologics, Madison, WI). The ELISA procedure incorporates polyclonal antibodies from rabbits specific for type E neurotoxin. Culture samples were centrifuged (4°C, 10,000 x g, 10 min) to separate the bacteria from the supernatant fluid. The supernatant samples were then diluted in casein buffer so that the concentration was within the linear range of the calibration curve (seven standards: 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ng/ml). A 100-µl sample of culture supernatant was placed in each well and the absorbance measured at 450 nm using an ELISA plate reader. Each sample was measured in duplicate, and the mean and standard deviation were calculated.

Assessment of mRNA degradation.
An aliquot (12.5 ml) of overnight culture of exponentially growing strain ATCC 9564 was inoculated into 237.5 ml of TPY-C broth and incubated in the standard anaerobic atmosphere at 37°C. Growth was monitored by determining the OD₆₂₀. The result was a typical sigmoid curve. When the cells were in mid-exponential phase (OD₆₂₀ = 2.3), rifampin was added at a final concentration of 100 mg/liter. Samples (10 ml) were removed at the time of the rifampin addition and thereafter at appropriate time intervals for RNA extraction, in order to quantify mRNA degradation. The RNA extraction was performed as stated earlier, with the exception that cells were harvested by centrifugation for 4 min at 12,500 x g at 4°C. The shorter centrifugation time enabled more-frequent sampling. RT-PCR for cntE mRNA was performed on 1 µg of total RNA, as described above. A standard curve was made by serial dilution of RNA from the reference sample, the sample taken at the time of the rifampin addition. The percentage of cntE mRNA remaining was calculated using the reference sample as 100%.

The effects of carbon dioxide concentration on growth, neurotoxin gene expression, and neurotoxin formation.
Strain ATCC 9564 was used. Three different modified atmospheres containing 10%, 35%, and 70% (per vol) CO₂ were tested. The concentration of H₂ was 10%, and the balance was N₂. Three flasks, each containing 237.5 ml TPY-C, were boiled to create anaerobic conditions and then flushed with the appropriate gas mixture, before autoclaving. The flasks were equilibrated in a Macs MG 1000 anaerobic workstation (Don Whitley, Shipley, United Kingdom) with the same atmosphere. Each flask was inoculated with 12.5 ml of an overnight culture and incubated at 30°C. Growth was followed by measuring the OD₆₂₀. Samples for total RNA extraction and ELISA were withdrawn in early exponential phase (OD₆₂₀ = 0.5 to 1), in late exponential phase (OD₆₂₀ 3), and in late stationary phase (after ca. 30 h). The growth experiment was carried out in triplicate for each CO₂ concentration. Maximum specific growth rates [µ (OD)] and lag times [ (OD)] of the change in OD₆₂₀ with time data were estimated by fitting the equation of Baranyi and Roberts (3). It is noted that (OD), the period during which the cell population reaches a detectable turbidity, is longer than the lag time () of the cell population estimated using viable count data. Regression analysis was carried out to describe the relationship between the percentage of CO₂ in the atmosphere and the growth parameters.

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Expression of the type E neurotoxin gene by C. botulinum.
A qRT-PCR method was developed to quantify the relative expression of cntE. Two assays were designed, one specific for cntE and one for rrn. The linear range of amplification and the AE for each qRT-PCR assay were determined by serial dilution of the total RNA before the RT. The AE was 0.87 for the cntE gene and 1.3 for the rrn gene.

Relative quantification of cntE expression in strain ATCC 9564 showed that the transcript level of cntE mRNA increased sharply during growth and reached a maximum in the transition between exponential phase and stationary phase (Fig. 1). It then decreased rapidly and did not rise again. Similar results were also obtained with strain BL 1177 (data not shown).

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FIG. 1. Effect of growth stage on relative expression of cntE and neurotoxin (BoNT/E) production by nonproteolytic C. botulinum strain ATCC 9564. Growth in TPY-C medium at 30°C with 10% H2, 10% CO2, and 80% N2. Diamonds, OD620; squares, RE of cntE; triangles, BoNT/E. Data are mean values of three independent experiments. Error bars show standard deviations.

The rate of cntE mRNA breakdown was measured to confirm that there is a close relationship between its mRNA accumulation (as measured by qRT-PCR) and its rate of gene expression (Fig. 2). Breakdown of mRNA showed a log-linear relationship, with less than 1 percent remaining after 1 hour. The cntE mRNA half-life was calculated to be approximately 9 minutes.

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FIG. 2. Breakdown of cntE mRNA in nonproteolytic C. botulinum strain ATCC 9564. The amount of cntE mRNA remaining is expressed as a percentage of the starting concentration (at the time of the rifampin addition). Data are from two independent experiments. The means and standard deviations are based on three technical replicates.

The effects of carbon dioxide on growth and neurotoxin expression.
A higher concentration of carbon dioxide resulted in slower growth of C. botulinum strain ATCC 9564 (Fig. 3). The effect of carbon dioxide on the µ (OD) was significant (P value of <0.05). Regression analysis was carried out to quantify the relationship between the concentration of carbon dioxide and the maximum specific growth rate (assuming that this relationship is linear). The resulting model can be written as follows: µ (OD) = 0.3949 – (0.00221 x [% CO₂]). The adjusted R² was 0.90, and the standard error of the fit was 0.0194. The (OD) increased with carbon dioxide concentration (Table 2).

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FIG. 3. Effect of carbon dioxide concentration on the growth of nonproteolytic C. botulinum strain ATCC 9564. Diamonds, 10% CO2; squares, 35% CO2; triangles, 70% CO2. The mean values and standard deviations are based on three independent growth experiments for each gas atmosphere.

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TABLE 2. Effect of carbon dioxide concentration on the growth parameters of nonproteolytic C. botulinum type E strain ATCC 9564 in TPY-C medium at 30°C in an atmosphere of 10% H2, 10 to 70% CO2, and 20 to 80% N2a

Relative expression of cntE was affected by both the stage of growth and the concentration of carbon dioxide (Fig. 4). A two-way factorial analysis was used to evaluate the effect of the stage of growth and the concentration of carbon dioxide on the relative expression of cntE (Table 3). The effect of the stage of growth and the carbon dioxide concentration on cntE expression were both highly significant (P value of <0.0001). A multiple-means comparison test was carried out to determine the differences between the three growth stages and between the three carbon dioxide concentrations. Expression of cntE was significantly higher in late exponential phase than in early exponential phase, and expression in stationary phase was significantly lower than in exponential phase (Table 3). Furthermore, cntE expression was significantly higher with 70% CO₂ than with lower concentrations of CO₂. The concentration of BoNT/E in late-stationary-phase cultures was consistent with the observations of relative cntE expression. The concentrations of BoNT/E after 30 h were 265 ± 78 ng/ml at 10% CO₂, 374 ± 80 ng/ml at 35% CO₂, and 570 ± 43 ng/ml at 70% CO₂ (Fig. 4).

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FIG. 4. Effect of carbon dioxide concentration and growth phase on the RE of cntE (bars) and the formation of neurotoxin (diamonds, BoNT/E) by nonproteolytic C. botulinum strain ATCC 9564. Light gray, 10% CO2; dark gray, 35% CO2; black, 70% CO2. The mean values and standard deviations are based on three independent growth experiments for each treatment.

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TABLE 3. Effect of carbon dioxide concentration and stage of growth on expression of the neurotoxin gene (cntE) by nonproteolytic C. botulinum type E strain ATCC 9564

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Expression of cntE by nonproteolytic C. botulinum type E strains ATCC 9564 and BL1177 was found to be related to growth phase. The relative expression of cntE mRNA peaked sharply, reached a maximum in the transition between late exponential phase and early stationary phase, and then fell rapidly to a low level during stationary phase. This observation is in agreement with previous reports on nonproteolytic C. botulinum type E strains HV and EVH (10, 39), although McGrath et al. (29), working with strain E VH Dolman, reported a greater expression of cntE in stationary phase than in mid-exponential phase. In the present study, while the decrease in cntE expression was rapid, it was not completely arrested in stationary phase. It was found that the concentration of cntE mRNA fell only 10-fold in the hour immediately after peaking, not 100-fold as would be expected based on the degradation rate of cntE mRNA if transcription of cntE had ceased immediately. This may be due to the continued transcription of cntE, albeit at a lower level, and/or the cells in the population not all entering stationary phase at exactly the same time. In addition, the degradation rate of cntE mRNA may be different in stationary phase than in mid-exponential phase.

The expression profile of cntE mRNA has both similarities and differences to those reported previously for other neurotoxin genes. The general trends for nonproteolytic C. botulinum type B at 30°C seem very similar, with an increase in cntB mRNA expression during growth, followed by a peak in early stationary phase, although the decrease in mRNA expression in stationary phase is neither as rapid nor as full as that in nonproteolytic C. botulinum type E (28). In proteolytic C. botulinum types A and B, a second rise in cnt mRNA expression has been observed in late stationary phase (10, 28). Furthermore, the decrease in mRNA expression in early stationary phase is not as rapid as that in cntE mRNA (4, 10, 28, 37, 42). The choice of media has also been reported to affect the expression profile (4). The differences between different types and strains may be due to differences in regulation, since nonproteolytic C. botulinum type E does not appear to have the positive regulator cntR, which has been found in the other botulinum toxin gene clusters (9, 24, 36). The tight regulation of expression of cntE in nonproteolytic C. botulinum type E does, however, suggest the presence of a positive regulator that remains to be identified.

Previous studies have reported that growth of nonproteolytic C. botulinum is slowed by high concentrations of carbon dioxide (16, 18, 27). The present study has extended these studies by demonstrating that this delay in turbidity is due to both an increased lag time and a decreased growth rate. However, from a food safety perspective, it is the production of neurotoxin, not cell growth, that is the important parameter. The present study has established that neurotoxin formation by nonproteolytic C. botulinum type E is greater at a higher concentration of carbon dioxide, confirming similar observations made previously by Lövenklev et al. (27) with nonproteolytic C. botulinum type B. In the present study, both relative gene expression and the amount of neurotoxin produced after 30 h were twofold higher when the headspace CO₂ concentration was 70% than when the CO₂ was 10%. It is possible that this is a growth rate phenomenon, where toxin production is enhanced due to a lowered growth rate, as reported for Clostridium difficile under stress conditions (30). Sharkey et al. (39), however, indicated that for nonproteolytic C. botulinum type E, an increased growth rate was associated with an increased expression of cntE, while the presence of sorbic acid and sodium nitrite reduced both the growth rate and expression of cntE (40). Lövenklev et al. (27, 28) also reported an inhibitory effect of nitrite on cntB expression. These reports, taken together, imply that the increase in neurotoxin gene expression and neurotoxin formation in our experiments is more likely to be related to the increased carbon dioxide concentration than to the decrease in growth rate. Similar trends have been found in other species. In Streptococcus pyogenes, it was found that expression of emm, the gene encoding M protein, and scpA, encoding the C5a endopeptidase Scp, which are major virulence factors, were stimulated by an increased concentration of carbon dioxide (6, 43). Carbon dioxide has also been found to stimulate toxin production in Staphylococcus aureus, Vibrio cholerae, and Bacillus anthracis (14, 15, 20, 38, 41, 43).

In conclusion, the finding that carbon dioxide, frequently used as an antimicrobial in MAP, actually stimulates neurotoxin production in nonproteolytic C. botulinum types B and E sheds a new, cautionary light on the risk of botulism and the use of MAP. Thus, in order to ensure the continued production of microbiologically safe food that also shows a high quality for human consumption, data about microbial virulence (including the formation of toxins) is required to complement existing knowledge on the growth and survival of pathogenic microbes. Further investigations are therefore required to identify the effect of factors such as food composition, gas atmosphere, preservatives, and temperature on the down- and upregulation of virulence factors (including toxin formation) in food-borne pathogens. This information will enable the development of new knowledge-led molecule-based strategies for food formulation and food preservation and will also inform quantitative risk assessments.

 
This work was supported by the Swedish Governmental Agency for Innovation Systems; the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning; the Competitive Strategic Grant of the BBSRC; and a Marie Curie training site fellowship.

We are very grateful to Carmen Pin for statistical advice.

 
* Corresponding author. Mailing address: Applied Microbiology, Lund Institute of Technology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Phone: 46 46-222 3412. Fax: 46 46-222 42 03. E-mail: Peter.Radstrom{at}tmb.lth.se

Published ahead of print on 29 February 2008.

Present address: SIK—The Swedish Institute for Food and Biotechnology, SE-402 29 Gothenburg, Sweden.

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Applied and Environmental Microbiology, April 2008, p. 2391-2397, Vol. 74, No. 8
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