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Effect of the Culture System on Clostridium argentinense Toxigenicity in Coculture with Pseudomonas mendocina
Anaerobe (1999) 5, 491±493 Article No. anae.1999.0220
VIRULENCE FACTORS AND HOST DEFENSES (POSTER PRESENTATION)
E¡ect of the Culture System on Clostridium argentinenseToxigenicity in Coculture with Pseudomonas mendocina H.J. Centorbi and H.J. Silva Area de Microbiologia, Universidad Nacional de San Luis, Chacabuco y Pedernera, San Luis, Argentina
Key Words: culture system, coculture, Clostridium argentinense, toxigenicity
Introduction Clostridium botulinum is a Gram-positive sporeforming anaerobic bacterium which produces a potent neurotoxin causing botulism in man and other animals. Based on serological specificity, botulinum neurotoxin is classified into seven toxigenic types named A to G [1]. The organismproducing type G neurotoxin has recently been assigned to a new species Clostridium argentinense [2] based on its phenotypic and genetic differences from C. botulinum. Clostridia producing botulinum neurotoxin are widely distributed in the environment. They have been isolated from geographically diverse soils and marine sediments, where they share their ecological niche with other anaerobic and aerobic bacteria. Certain bacteria interact by metabiosis, in which one species promotes the growth of a second species. Metabiosis has been observed in culture media between C. botulinum type A and aerobic bacteria 1075-9964/99/030491 + 00 $30.00/0
such as Acinetobacter lwoffi and Pseudomonas sp. CH79. Cocultures of C. botulinum with these aerobes resulted in the potentiation of botulinum neurotoxin production through mechanisms not fully understood [3]. In the present study two methods for coculturing C. argentinense and P. mendocina were compared for the ability of C. argentinense to grow and produce neurotoxin. We examined several growth parameters and variables to generate hypotheses on the possible mechanisms involved in metabiosis.
Materials and Methods C. argentinense strain G 89 [4], and P. mendocina ATCC 25411 [5], both originally isolated from soils in Mendoza Province, Argentina, were cultivated in the following culture medium at pH 7.6 proteose peptone, 30 g/L, yeast extract, 5 g/L, trypticase, 5 g/L, glucose, 11 g/L, cysteine chlorohydrate, 0.5 g/L. The cocultures grew in an 800-mL fermentor equipped with pH # 1999 Academic Press
492
Centorbi and Silva
and Eh sensors. The fermentor was operated either under homogeneous conditions inoculating a mixture of 50% C. argentinense and 50% P. mendocina or under heterogeneous ones employing a dialysis membrane (Spectra/por, MWCO 12,000±14,000), which physically separated both micro-organisms in the fermentor. Both systems were operated under atmospheric conditions. Samples were taken by duplicate at 10-h intervals for OD, glucose and botulinum neurotoxin levels, and the runs were repeated twice.
homogeneous cocultures (Figure 2). Glucose was totally consumed after 30 h of incubation in the homogeneous system, and only after 90 h in the heterogeneous system (Figure 3). The pH values
Results and Discussion When C. argentinense was cocultured with P. mendocina under homogeneous conditions, the toxin level after 100 h of incubation was 21 times higher (1750 LD50/mL) than the level obtained in the heterogeneous system (80 LD50/mL) (Figure 1.) The specific growth rate and final biomass of C. argentinense was 3 and 3.5 times higher, respectively, in
Figure 3. Glucose consumption in C. argentinense±P. mendocina cocultures. ÐÐ, homogeneous system; ± ±, heterogeneous system.
Figure 1. Toxin levels in cocultures of C. argentinense±P. mendocina. &, homogeneous system; &, heterogeneous system. Figure 4. pH profiles of C. argentinense±P. mendocina cocultures. ÐÐ, homogeneous system; ± ±, heterogeneous system.
Figure 2. Growth of C. argentinense±P. Mendocina cocultures. ÐÐ, homogeneous system; ± ±, heterogeneous system; ÐÐÐÐ, C. argentinense monocultures.
Figure 5. Eh evolution of C. argentinense±P. mendocina cocultures. ÐÐ, homogeneous system; ± ±, heterogeneous system.
Clostridium argentinenseToxigenicity decreased in accordance with glucose consumption (Figure 4). The redox potential remained at high negative values (7400 mV) in both systems. After 50 h of cultivation, Eh increased slightly to 7300 mV in the homogeneous system when toxin was produced by the anaerobe (Figure 5). It has been observed that during transition from aerobic to oxygen-limited (microaerophilic) conditions, P. mendocina can redirect its metabolism leading to a mid-type fermentation pathway and the synthesis of extracellular uronic or alginic acids under severe electron acceptor limitations [6]. A possible explanation for the increased neurotoxin production of C. argentinense in homogeneous coculture with P. mendocina is that uronic acids are alginate produced by P. mendocina in a reduced environment are utilized by C. argentinense for neurotoxin production when glucose is depleted.
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References 1. Hatheway C.L. (1990) Toxigenic clostridia. Clin Microbiol Rev 3: 66±98 2. Suen J.C., Hatheway C.L., Steingerwalt A.G. and Brenner D.J. (1998) Clostridium argentinense, sp. nov. a genetically homogeneous group composed of all strains of Clostridium botulinum toxin type G and some nontoxigenic strain previously identified as Clostridium subterminale or Clostridium hastiforme. J Syst Bacteriol 38: 375±381 3. Gimenez M.A., Solanes R.E. and Gimenez D.F. (1988) Efectos metabioÂtico y sineÂrgico de algunas bacterias aerobias estrictas sobre el crecimiento y toxinogeÂnesis de Clostridium botulinum tipo A. Rev Cs Agr 24: 59±58 4. Gimenez D.F. and Cicarelli A.S. (1970) Another type of Clostridium botulinum. Zbl Backt Hyg I Abr Orig 215: 221±224 5. Palleroni N.J., Doudoroff M., Stanier R.Y., Solanes R.E. and Mandel M. (1970) Taxonomy of the Pseudomonas stutzeri group. J Gen Microbiol 60: 215±231 6. Verdoni N., Aon M.A. and Lebeault J.M. (1992) Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxigen-limited conditions in chemostat cultures. Appl Environ Microbiol 58: 3150±3156
VIRULENCE FACTORS AND HOST DEFENSES (POSTER PRESENTATION)
E¡ect of the Culture System on Clostridium argentinenseToxigenicity in Coculture with Pseudomonas mendocina H.J. Centorbi and H.J. Silva Area de Microbiologia, Universidad Nacional de San Luis, Chacabuco y Pedernera, San Luis, Argentina
Key Words: culture system, coculture, Clostridium argentinense, toxigenicity
Introduction Clostridium botulinum is a Gram-positive sporeforming anaerobic bacterium which produces a potent neurotoxin causing botulism in man and other animals. Based on serological specificity, botulinum neurotoxin is classified into seven toxigenic types named A to G [1]. The organismproducing type G neurotoxin has recently been assigned to a new species Clostridium argentinense [2] based on its phenotypic and genetic differences from C. botulinum. Clostridia producing botulinum neurotoxin are widely distributed in the environment. They have been isolated from geographically diverse soils and marine sediments, where they share their ecological niche with other anaerobic and aerobic bacteria. Certain bacteria interact by metabiosis, in which one species promotes the growth of a second species. Metabiosis has been observed in culture media between C. botulinum type A and aerobic bacteria 1075-9964/99/030491 + 00 $30.00/0
such as Acinetobacter lwoffi and Pseudomonas sp. CH79. Cocultures of C. botulinum with these aerobes resulted in the potentiation of botulinum neurotoxin production through mechanisms not fully understood [3]. In the present study two methods for coculturing C. argentinense and P. mendocina were compared for the ability of C. argentinense to grow and produce neurotoxin. We examined several growth parameters and variables to generate hypotheses on the possible mechanisms involved in metabiosis.
Materials and Methods C. argentinense strain G 89 [4], and P. mendocina ATCC 25411 [5], both originally isolated from soils in Mendoza Province, Argentina, were cultivated in the following culture medium at pH 7.6 proteose peptone, 30 g/L, yeast extract, 5 g/L, trypticase, 5 g/L, glucose, 11 g/L, cysteine chlorohydrate, 0.5 g/L. The cocultures grew in an 800-mL fermentor equipped with pH # 1999 Academic Press
492
Centorbi and Silva
and Eh sensors. The fermentor was operated either under homogeneous conditions inoculating a mixture of 50% C. argentinense and 50% P. mendocina or under heterogeneous ones employing a dialysis membrane (Spectra/por, MWCO 12,000±14,000), which physically separated both micro-organisms in the fermentor. Both systems were operated under atmospheric conditions. Samples were taken by duplicate at 10-h intervals for OD, glucose and botulinum neurotoxin levels, and the runs were repeated twice.
homogeneous cocultures (Figure 2). Glucose was totally consumed after 30 h of incubation in the homogeneous system, and only after 90 h in the heterogeneous system (Figure 3). The pH values
Results and Discussion When C. argentinense was cocultured with P. mendocina under homogeneous conditions, the toxin level after 100 h of incubation was 21 times higher (1750 LD50/mL) than the level obtained in the heterogeneous system (80 LD50/mL) (Figure 1.) The specific growth rate and final biomass of C. argentinense was 3 and 3.5 times higher, respectively, in
Figure 3. Glucose consumption in C. argentinense±P. mendocina cocultures. ÐÐ, homogeneous system; ± ±, heterogeneous system.
Figure 1. Toxin levels in cocultures of C. argentinense±P. mendocina. &, homogeneous system; &, heterogeneous system. Figure 4. pH profiles of C. argentinense±P. mendocina cocultures. ÐÐ, homogeneous system; ± ±, heterogeneous system.
Figure 2. Growth of C. argentinense±P. Mendocina cocultures. ÐÐ, homogeneous system; ± ±, heterogeneous system; ÐÐÐÐ, C. argentinense monocultures.
Figure 5. Eh evolution of C. argentinense±P. mendocina cocultures. ÐÐ, homogeneous system; ± ±, heterogeneous system.
Clostridium argentinenseToxigenicity decreased in accordance with glucose consumption (Figure 4). The redox potential remained at high negative values (7400 mV) in both systems. After 50 h of cultivation, Eh increased slightly to 7300 mV in the homogeneous system when toxin was produced by the anaerobe (Figure 5). It has been observed that during transition from aerobic to oxygen-limited (microaerophilic) conditions, P. mendocina can redirect its metabolism leading to a mid-type fermentation pathway and the synthesis of extracellular uronic or alginic acids under severe electron acceptor limitations [6]. A possible explanation for the increased neurotoxin production of C. argentinense in homogeneous coculture with P. mendocina is that uronic acids are alginate produced by P. mendocina in a reduced environment are utilized by C. argentinense for neurotoxin production when glucose is depleted.
493
References 1. Hatheway C.L. (1990) Toxigenic clostridia. Clin Microbiol Rev 3: 66±98 2. Suen J.C., Hatheway C.L., Steingerwalt A.G. and Brenner D.J. (1998) Clostridium argentinense, sp. nov. a genetically homogeneous group composed of all strains of Clostridium botulinum toxin type G and some nontoxigenic strain previously identified as Clostridium subterminale or Clostridium hastiforme. J Syst Bacteriol 38: 375±381 3. Gimenez M.A., Solanes R.E. and Gimenez D.F. (1988) Efectos metabioÂtico y sineÂrgico de algunas bacterias aerobias estrictas sobre el crecimiento y toxinogeÂnesis de Clostridium botulinum tipo A. Rev Cs Agr 24: 59±58 4. Gimenez D.F. and Cicarelli A.S. (1970) Another type of Clostridium botulinum. Zbl Backt Hyg I Abr Orig 215: 221±224 5. Palleroni N.J., Doudoroff M., Stanier R.Y., Solanes R.E. and Mandel M. (1970) Taxonomy of the Pseudomonas stutzeri group. J Gen Microbiol 60: 215±231 6. Verdoni N., Aon M.A. and Lebeault J.M. (1992) Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxigen-limited conditions in chemostat cultures. Appl Environ Microbiol 58: 3150±3156