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Effect of Different Carbon and Nitrogen Sources on Clostridium argentinense Toxigenicity in Coculture withPseudomonas mendocina
Anaerobe (2001) 7, 323–328 doi:10.1006/anae.2001.0406
PHYSIOLOGY/STRUCTURAL BIOLOGY/BIOCHEMISTRY
E¡ect of Di¡erent Carbon and Nitrogen Sources on Clostridium argentinense Toxigenicity in Coculture with Pseudomonas mendocina H.J. Centorbi and H.J. Silva* Area de Microbiologı´a, Facultad de Quı´mica Bioquı´mica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina (Received 22 May 2001; accepted in revised form 4 December 2001) Key Words: carbon and nitrogen sources, cocultures, Clostridium argentinense, Pseudomonas mendocina
The effect of different carbon and nitrogen sources on the production of toxin by Clostridium argentinense was examined. The toxin production by C. argentinense in coculture with Pseudomonas mendocina increased in all the cases in relation to that produced by monocultures independent of the nature of the source. Using dextrin as carbon source C. argentinense produced the highest levels of toxin both in monocultures (300 LD50/mL) and in cocultures with P. mendocina (5000 LD50/mL). Experiments run in a microfermenter showed that the slow growth of cocultures associated with the assimilation of dextrin and the pH and Eh profiles favoured the production of toxin. Of the nitrogen sources assayed, corn steep liquor sustained the highest levels of toxin in both monocultures and cocultures with 3 and 2.8 fold increases with respect to that obtained using proteose peptone. The toxin production by C. argentinense cultures and C. argentinense–P. mendocina cocultures was highly dependent on the nature of the carbon and nitrogen sources used in the culture media. Growth of C. argentinense on substrates slowly assimilated stimulated the production of toxin. # 2001 Academic Press
Introduction Clostridium botulinum produces a potent neurotoxin responsible for botulism, a severe neuroparalytic illness which affects man and animals [1]. Based on their serological specificity, neurotoxins are divided into seven toxigenic types from A to G. The organisms
*Address correspondence to: H.J. Silva, Area de Microbiologı´a, Facultad de Quı´mica Bioquı´mica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina. Tel.: 54-02652-423789 (Ext 127), E-mail: [email protected]
1075–9964/01/060323 + 06 $35.00/0
belonging to type G differ phenotypically from other members and have been assigned to a new species, Clostridium argentinense [2]. Clostridia producing botulinum toxin 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. C. argentinense was first isolated by Gimenez and Cicarrelli from soils in the province of Mendoza, Argentina [3]. Several factors have been reported to influence growth and toxin production by C. botulinum. In addition to environmental and chemical factors, biological interactions between # 2001 Academic Press
324
H.J. Centorbi and H.J. Silva
C. botulinum and other bacteria can exert profound effects on its behaviour [4,5]. 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 such as Acinetobacter lwoffi and Pseudomonas sp. CH79 [6]. Apart from the effect on growth the toxin production is also influenced, cocultures of C. botulinum with these aerobes resulted in an increase in the amount of botulinum neurotoxin. Similarly, the production of toxin by C. argentinense was neatly enhanced by coculturing with Pseudomonas mendocina [7]. In the present work, the effect of different carbon and nitrogen sources on the toxigenicity of C. argentinense was evaluated in individual cultures as well as in cocultures with P. mendocina. In some cases a more detailed study was undertaken in a microfermenter to analyse the growth kinetics and toxin production by C. argentinense.
Materials and Methods Micro-organisms and culture conditions Clostridium argentinense strain G 89 [3] and Pseudomonas mendocina ATCC 25411 [8], originally isolated from soils in Mendoza, were kindly provided by the Institute of Microbiology, Faculty of Medicine, National University of Cuyo. The micro-organisms were maintained at 41C in cooked meat medium and nutrient agar (Difco), respectively, and prior to nutrient assays grown in the same media at 371C for 24 h. The evaluation of the carbon and nitrogen sources was performed in 10 mL test tubes using a base medium of the following composition (g/L): proteose peptone (Difco), 30; yeast extract (Merck), 5; Trypticase (Difco), 5; glucose (Merk), 11; L-cysteine hydrochloride (Mallinckrodt), 0.5. In this medium, glucose was replaced by the following carbon sources (g/L): molasses (Calsa, Argentine Company of Yeast), 30; saccharose (Merk), 15; lactose (Merk), 15; dextrine (Merk), 15. Similarly, proteose peptone was replaced by the following nitrogen sources in the amount required to maintain the total nitrogen content constant: soybean peptone (Merck), 42.9 g/L; casamino acids (Britania), 53.7 g/L; corn steep liquor (Sigma) 10.75 mL/L. The pH of media was adjusted to 7.6 and the media were sterilized at 1211C for 15 min. After cooling, media were immediately inoculated to prevent reoxygenation. The inoculum size for the coculture experiments consisted of a mixture of 0.1 mL of C. argentinense containing 6.2 107 CFU/mL and 0.1 mL of P. mendocina of
3.0 10ll CFU/mL obtained in the base medium. The experiments also included monocultures of both micro-organisms which represented positive (C. argentinense) and negative (P. mendocina) toxicity controls. After inoculation the cultures were incubated at 371C for 72 h in an anaerobic jar (Oxoid).
Microfermenter experiments Monocultures and cocultures were grown in an 800 mL fermenter without aeration and agitation [9] containing 700 mL of the base medium using either glucose (11 g/L) or dextrine (15 g/L) as carbon sources. The pH of media was adjusted to 7.6 and then sterilized at 1211C for 15 min. The fermenter was equipped with pH and Eh sensors to monitor their evolutions. Measurements were made with a digital pH/ORP controller (Cole Parmer). To prevent reoxygenation the fermentor was inoculated immediately after autoclaving with 8 mL (1% fermenter culture volume) of a growing culture of C. argentinense in base medium for monocultures and with 16 mL (2% fermenter culture volume) of 50% C. argentinense and 50% P. mendocina prepared in base medium for coculture experiments.
Analytical techniques Viable counts by the dilution pour plate method were used to enumerate micro-organisms in inocula for test tube experiments. Serial dilutions of stock cultures were made and used to inoculate plates in triplicate containing the basal medium for C. argentinense and nutrient broth for P. mendocina. Plates of C. argentinense were inoculated in an anaerobic glove box and incubated in an anaerobic jar for 72 h at 371C. In the fermenter experiments, samples were taken by duplicate at 10 h intervals for optical density (OD) and botulinum neurotoxin levels. Measurements of OD were made in a Baush and Lomb spectrophotometer at 600 nm. Biomass was estimated by dry weight determinations of samples centrifuged at 10 000 g for 20 min at 41C, washed twice with distilled water and dried at 1001C for 16 h. The specific growth rate (m) was calculated by the expression ln x/x0 tÿ1, where x0 was the initial biomass and x was the biomass at time t [10]. To estimate the production of toxin, the mouse bioassay was conducted in separate culture and in coculture supernatants obtained by centrifugation at 10 000 g for 20 min at 41C. Mice of the Rockland strain weighing between 18 and 21 g were injected intraperitoneally with 0.5 mL and for those with typical symptoms of botulinum intoxication the
E¡ect of C and N Sources on Cocultures Toxicity
325
cultures; however, with this sugar the level of toxin was lower than with glucose. Using dextrine as a carbon source, C. argentinense produced the highest levels of toxin both in monocultures (300 LD50/mL) and in cocultures with P. mendocina (5000 LD50 /mL). Low synergistic effect was observed in cocultures using saccharose and lactose, with a five- and sevenfold increase, respectively, but the level of toxin obtained with these sugars was significantly lower than with glucose. The evolution in the production of biomass and toxin in C. argentinense cultures (Figure 1) and C. argentinense–P. mendocina cocultures (Figure 2) was analysed in a microfermenter using glucose and dextrin as carbon sources. C. argentinense grown on glucose showed a high specific growth rate (m = 0.21/h), the maximum level of biomass being obtained after 10 h of evolution of the culture without the existence of a lag period (Figure 1A). Using
LD50/mL was calculated by the method of Reed and Muench [11].
Results The effects of the carbon sources on the productı´on of botulinum toxin of C. argentinense and C. argentinense– P. mendocina cocultures are presented in Table 1. P. mendocina alone was negative in the toxin assay by intraperitoneal inoculation in mice. The production of toxin by C. argentinense in coculture with P. mendocina increased in all the cases in relation to that produced by C. argentinense independent of the nature of the carbon source. The levels of toxicity were 17.5 times higher using glucose. A high effect on the level of botulinum toxin by cocultures was produced using sugar cane molasses, with a 40-fold increase in relation to the level of toxin produced by mono-
Table 1. Effect of the carbon source on the production of botulinum toxin in cocultures of C. argentinense–P. mendocina Carbon source (CS)
C. argentinense (LD50/mL)
C. argentinense–P. mendocina (LD50/mL)
LD50/mL coculture/ LD50/mL C. argentinense
LD50/mL CS/LD50/mL glucose1
100 30 50 300 100
1750 1200 350 5000 500
17.5 40 7 16.6 5
1 0.69 0.2 2.86 0.29
Glucose Molasses Lactose Dextrine Saccharose 1
Ratio for cocultures. Values are means of two separate experiments.
1.0
(A)
1.6 1.2 OD 600 nm
OD 600 nm
0.8 0.6 0.4 0.2
0
10
20
30
40 50 60 Time (h)
70
80
90 100
(B)
0
10 20 30 40 50 60 70 80 90 100 Time (h)
0
10 20 30 40 50 60 70 80 90 100 110 Time (h)
300 250 LD50 /ml
LD50 /ml
0.4 0
0
100 90 80 70 60 50 40 30 20 10 0
0.8
200 150 100 50 0
0 10 20 30 40 50 60 70 80 90 100 110 120 Time (h)
Figure 1. Growth (A) and toxin production (B) of C. argentinense in base medium containing glucose (&), left and dextrine (~), right.
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H.J. Centorbi and H.J. Silva 3.5
(A)
2.5
OD 600 nm
OD 600 nm
3.0 2.0 1.5 1.0 0.5
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
0 0
40
60 80 Time (h)
100
0
120
(B)
5000
1500
4000
1000
0
20
40
60
80 100 120 140 160 Time (h)
6000
2000 LD50 /ml
LD50 /ml
2500
20
3000 2000
500
1000 0
0 0 10 20 30 40 50 60 70 80 90 100 110 Time (h)
0
20 40 60
80 100 120 140 160 180 Time (h)
0 −50 −100 −150 −200 −250 −300 −350 −400 −450 −500
8.0 7.5 7.0 pH ( )
dextrin as carbon source, after a l0 h lag period, the highest productivity of biomass was generated in 30 h of culture, and was found to be higher than that obtained with glucose and growing at a lower specific growth rate. The highest toxin level occurred at the end of the cultures (100 h) for both sources of carbon (Figure 1B). The toxin levels obtained with dextrin were three times higher than with glucose. Comparing the evolution of biomass in C. argentinense culture (Figure 1A) with the toxin production (Figure 1B) it was observed that with both carbon sources toxin liberation took place as a consequence of cellular lysis. The behaviour of cocultures of C. argentinense– P. mendocina in the microfermenter for the same carbon sources is presented in Figure 2A. The higher toxin levels were again reached for the two carbon sources when cultures entered the stationary phase with clear cellular lysis when using dextrine (Figure 2B). However, this process was less intense than that in C. argentinense culture due to the contribution of P. mendocina biomass. With dextrine, the pH dropped to acid values in the logarithmic growth phase, remaining fairly constant until the end of the culture (Figure 3). The levels of redox potential were of the order of ÿ300 to ÿ450 mV up to 120 h of culture, indicating the existence of a strongly reducing atmosphere, appropriate for growth and toxin production by C. argentinense (Figure 3). The effects of the nitrogen sources in cultures of C. argentinense and cocultures of C. argentinense– P. mendocina are presented in Table 2. The levels of
6.5 6.0 5.5 5.0 0
20
40
60 80 Time (h)
100
120
Eh (mV) ( )
Figure 2. Growth (A) and toxin production (B) of C. argentinense–P. mendocina cocultures in base medium containing glucose (&), left and dextrine, (~), right.
140
Figure 3. Eh (&) and pH (&) profiles of C. argentinense–P. mendocina cocultures in base medium with dextrine as carbon source.
toxin were higher in cocultures independent of the nitrogen source. The cocultures showed similar increases in the level of toxin with respect to C. argentinense cultures using proteose peptone, soybean peptone and corn steep liquor (17.5, 16 and 16.6 times increase, respectively). Of these three nitrogen sources, both in C. argentinense cultures and in cocultures, corn steep liquor sustained the highest levels of toxin, with 3- and 2.86-fold higher levels than that obtained with proteose peptone. Casamino acids proved to be a poor nitrogen source, showing a small increase in toxin production in cocultures and a level of toxin significantly lower than that obtained with proteose peptone (LD50/mL casamino acids/LD50/ mL proteose peptone = 0.07).
E¡ect of C and N Sources on Cocultures Toxicity
327
Table 2. Effect of the nitrogen source on the production of botulinum toxin in cocultures of C. argentinense–P. mendocina Nitrogen source (NS) Proteose peptone (Pp) Soybean peptone Corn-steep liquor Casamino acid
C. argentinense (LD50/mL)
C. argentinense–P. mendocina (LD50/mL)
LD50/mL coculture/ LD50/mL C. argentinense
LD50/mL NS/ LD50/mL Pp1
100 50 300 20
1750 800 5000 125
17.5 16 16.6 6.25
1 0.457 2.86 0.07
1
Ratio for cocultures. Values are means of two separate experiments.
Discussion and Conclusions The optimal and limiting conditions for the growth and toxin production of Clostridium strains, mainly those related with substrates, pH, Eh, aw, and temperature have been well established in the literature [12–14]. However, this is not the case when Clostridium shares the same habitat with other bacteria. This knowledge is of special interest due to its theoretical and practical implications for food and environmental microbiology, and indeed for human and animal pathology. It is in this sense that we studied the effect of carbon and nitrogen nutrition, as an environmental factor, on the growth and toxin production of Clostridium argentinense sharing the same culture medium with P. mendocina. The results obtained provide useful information mainly because both microorganisms were originally isolated from soils in the Province of Mendoza, Argentina [3,8], and may be directly or indirectly involved as potential agents of contamination of some foods, as saprophytic or pathogenic inhabitants of the human or animal intestine and other environments. Gimenez et al. [6] observed a metabiotic effect in cocultures of some aerobic bacteria including Acinetobacter and Pseudomonas strains and C. botulinum type A. Among the aerobic bacteria, P. mendocina increased growth and the level of botulinum toxin. Previous studies, also indicated the existence of an important interaction between P. mendocina and C. argentinense [7]. The production of toxin by coculture of these micro-organisms was clearly enhanced, which correlated with an increased level of biomass. Nutritional factors, among others, may influence their interaction. The results obtained in this work show that the toxin levels attained in cocultures depend on the nature of the carbon sources (Table 1) and nitrogen sources (Table 2) used in the culture media. Variations in the levels of toxin were observed both in C. argentinense cultures and in cocultures with P. mendocina. Of all the carbon and nitrogen sources employed, the highest levels of toxin were obtained with dextrine and corn steep liquor with a 2.8-fold increase in relation to that obtained using glucose and proteose peptone.
Corn steep liquor, a complex organic nitrogen source commonly used for the production of bioactive drugs [15], provides a non-homogeneous and readily available source of amino acids, which best suited the nitrogen requirements of C. argentinense for growth and toxin production. While soybean peptone in cocultures produced increments in the toxin level in relation to C. argentinense cultures, it was more than half less effective than proteose peptone. Casamino acid was not an appropriate nitrogen source for growth and toxigenicity of C. argentinense in individual cultures and cocultures, producing the lowest toxin levels of all the nitrogen sources assayed. Apart from its poor nitrogen nutritional value, the content of iron of casamino acids of 15 mg% (p/p) was probably limiting. It has been observed that other Clostridium strains such as C. botulinum strain Hall type A is highly demanding of iron, requiring concentrations higher than 10 mg/L in the culture medium for toxin production [16]. A more detailed study in a microfermenter, using glucose and dextrine as carbon sources, on growth evolution and toxin production by C. argentinense cultures (Figure 1) and in cocultures with P. mendocina (Figure 2), showed that in cocultures the levels of biomass and toxin were higher. In previous studies it was found that with glucose the quality of the coculture biomass producing toxin, expressed as specific toxicity (LD50/g ), was four times higher than that of C. argentinense [7]. The increased level of biomass observed in the cocultures with glucose with respect to monocultures (Figure 2A) could be explained on the grounds of changes in metabolic fluxes in P. mendocina under anaerobic conditions. P. mendocina has been observed to display a remarkable metabolic versatility in reduced environments, being able to redirect its anabolic flux towards the synthesis of uronic acids or alginate which are released in the extracellular medium [17]. These compounds may have been utilized as a non-readily available carbon source by C. argentinense when glucose was exhausted improving its growth at low specific growth rates in later stages of cocultures. The culture evolution of C. argentinense–P. mendocina coculture with dextrine showed two distinct phases,
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a growth phase followed by a lytic phase with the production of toxin. The Eh values observed in the growth phase were representative of highly reducing conditions (Figure 3), which resulted similar to that displayed by cocultures grown on glucose [9]. In this atmosphere the synthesis of microbial exopolysaccharides or growth factors by the aerobic micro-organism may have also been an influential factor for growth stimulation and toxin production of C. argentinense. With either glucose or dextrine the cocultures grew at lower specific growth rates than that of C. argentinense. Particularly remarkable was the growth evolution with dextrine, showing a very low specific growth rate (m = 0.021/h) and similar to what was observed in cultures of C. argentinense, the final level of biomass was higher than those with glucose. Dextrine being a polymer of glucose with bonds a (1-4) and a (1-6) at the ramification points, a controlled liberation of glucose takes place during its dissimilation resulting in a limitation of the carbon source when using this carbohydrate. The results obtained in the study indicate that the production of toxin by C. argentinense cultures and C. argentinense–P. mendocina cocultures is highly dependent on the nature of the carbon and nitrogen sources used in the culture media. For glucose and dextrine, the slow use of the substrate in the growth phase of cocultures allowed the production of more toxin levels. However, additional studies on growth and toxin production kinetics with other carbohydrates and complex nitrogenous nutrients of slow assimilation, either naturally occurring or by slow feeding will confirm whether this observation is a general fact for C. argentinense cultures.
Acknowledgements This work was supported by the Secretariat of Science and Technology, UNSL, and the National Council of Scientific and Technical Research (CONICET) Argentine.
References 1. Smith L.D. and Williams B.L. (1984) The Clostridia. In Balows A. (ed.), The Pathogenic Anaerobic Bacteria, 3rd ed., pp 94–100. Thomas CC, Springfield, IL, U.S.A. 2. Suen J.C., Hatheway C.L., Steingerwalt A.G. and Brenner D.J. (1988) 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. Gime´nez D.F. and Ciccarelli A.S. (1970) Another type of Clostridium botulinum. Zbl Backt Hyg I Abr Orig 215: 221–224 4. Okereke A. and Montville T.J. (1991) Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperature. Appl Environ Microbiol 57: 3423–3428 5. Crandall A.D., Winkowski K. and Montville T.J. (1994) Inability of Pediococcus pentosaceous to inhibit Clostridium botulinum in sous vide beef with gravy at 4 and 101C. J Food Prot 57: 104–107 6. Gime´nez M.A., Solanes R.E. and Gime´nez D.F. (1988) Efectos metabio´tico y sine´rgico de algunas bacterias aerobias estrictas sobre el crecimiento y toxinoge´nesis de Clostridium botulinum tipo A. Rev Fao Ciencias Agrerias 24: 59–68 7. Centorbi H.J. and Silva H.J. (2000) Enhancement of growth and toxin production of Clostridium argentinense by co-culture with Pseudomonas mendocina. Microbios 101: 15–22 8. Palleroni N.J., Doudoroff M., Stainer R.Y., Solanes R.E. and Mandel M. (1970) Taxonomy of the Pseudomonas stutzeri group. J Gen Microbiol 60: 215–231 9. Centorbi H.J. and Silva H.J. (1999) Effect of the culture system on Clostridium argentinense toxigenicity in coculture with Pseudomonas mendocina. Anaerobe 5: 491–493 10. Pirt S.J. (1975) Parameters of growth and analysis of growth data. In Pirt S.J. (ed.) Principles of Microbe and Cell Cultivation, 1st ed., pp 4–14. Blackwell Scientific Publications, Oxford 11. Reed L.J. and Muench M. (1938) A simple method of estimating fifty percent end points. Am J Hyg 27: 493–497 12. Baird-Parker A.C. and Freams B. (1967) Combined effect of water activity, pH and temperature on the growth of Clostridium botulinum from spore and vegetative cell inocula. J Appl Bacteriol 30: 420–429 13. Lund B.M., Knox M.R. and Sims A.P. (1984) The effect of oxygen and redox potential on growth of Clostridium botulinum type E from a spore inoculum. Food Microbiol 1: 277–287 14. Dodds K.L. (1989) Combined effect of water activity and pH on inhibition of toxin production by Clostridium botulinum in cooked vacuum-packed potatoes. Appl Environ Microbiol 55(3): 656–660 15. Pfefferle C., Theobald U., Gu¨rtler H. and Fiedler H.P. (2000) Improved secondary metabolite production in the genus Streptosporangium by optimization of the fermentation conditions. J Biotechnol 80: 135–142 16. Sieguel L.S. (1981) Effect of iron on growth and toxin production by Clostridium botulinum toxin type A. Curr Microbiol 6: 127–130 17. Verdoni N., Aon M.A. and Lebeault J.M. (1992) Metabiotic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxygen-limited conditions in chemostat cultures. Appl Environ Microbiol 58: 3150–3156
PHYSIOLOGY/STRUCTURAL BIOLOGY/BIOCHEMISTRY
E¡ect of Di¡erent Carbon and Nitrogen Sources on Clostridium argentinense Toxigenicity in Coculture with Pseudomonas mendocina H.J. Centorbi and H.J. Silva* Area de Microbiologı´a, Facultad de Quı´mica Bioquı´mica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina (Received 22 May 2001; accepted in revised form 4 December 2001) Key Words: carbon and nitrogen sources, cocultures, Clostridium argentinense, Pseudomonas mendocina
The effect of different carbon and nitrogen sources on the production of toxin by Clostridium argentinense was examined. The toxin production by C. argentinense in coculture with Pseudomonas mendocina increased in all the cases in relation to that produced by monocultures independent of the nature of the source. Using dextrin as carbon source C. argentinense produced the highest levels of toxin both in monocultures (300 LD50/mL) and in cocultures with P. mendocina (5000 LD50/mL). Experiments run in a microfermenter showed that the slow growth of cocultures associated with the assimilation of dextrin and the pH and Eh profiles favoured the production of toxin. Of the nitrogen sources assayed, corn steep liquor sustained the highest levels of toxin in both monocultures and cocultures with 3 and 2.8 fold increases with respect to that obtained using proteose peptone. The toxin production by C. argentinense cultures and C. argentinense–P. mendocina cocultures was highly dependent on the nature of the carbon and nitrogen sources used in the culture media. Growth of C. argentinense on substrates slowly assimilated stimulated the production of toxin. # 2001 Academic Press
Introduction Clostridium botulinum produces a potent neurotoxin responsible for botulism, a severe neuroparalytic illness which affects man and animals [1]. Based on their serological specificity, neurotoxins are divided into seven toxigenic types from A to G. The organisms
*Address correspondence to: H.J. Silva, Area de Microbiologı´a, Facultad de Quı´mica Bioquı´mica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina. Tel.: 54-02652-423789 (Ext 127), E-mail: [email protected]
1075–9964/01/060323 + 06 $35.00/0
belonging to type G differ phenotypically from other members and have been assigned to a new species, Clostridium argentinense [2]. Clostridia producing botulinum toxin 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. C. argentinense was first isolated by Gimenez and Cicarrelli from soils in the province of Mendoza, Argentina [3]. Several factors have been reported to influence growth and toxin production by C. botulinum. In addition to environmental and chemical factors, biological interactions between # 2001 Academic Press
324
H.J. Centorbi and H.J. Silva
C. botulinum and other bacteria can exert profound effects on its behaviour [4,5]. 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 such as Acinetobacter lwoffi and Pseudomonas sp. CH79 [6]. Apart from the effect on growth the toxin production is also influenced, cocultures of C. botulinum with these aerobes resulted in an increase in the amount of botulinum neurotoxin. Similarly, the production of toxin by C. argentinense was neatly enhanced by coculturing with Pseudomonas mendocina [7]. In the present work, the effect of different carbon and nitrogen sources on the toxigenicity of C. argentinense was evaluated in individual cultures as well as in cocultures with P. mendocina. In some cases a more detailed study was undertaken in a microfermenter to analyse the growth kinetics and toxin production by C. argentinense.
Materials and Methods Micro-organisms and culture conditions Clostridium argentinense strain G 89 [3] and Pseudomonas mendocina ATCC 25411 [8], originally isolated from soils in Mendoza, were kindly provided by the Institute of Microbiology, Faculty of Medicine, National University of Cuyo. The micro-organisms were maintained at 41C in cooked meat medium and nutrient agar (Difco), respectively, and prior to nutrient assays grown in the same media at 371C for 24 h. The evaluation of the carbon and nitrogen sources was performed in 10 mL test tubes using a base medium of the following composition (g/L): proteose peptone (Difco), 30; yeast extract (Merck), 5; Trypticase (Difco), 5; glucose (Merk), 11; L-cysteine hydrochloride (Mallinckrodt), 0.5. In this medium, glucose was replaced by the following carbon sources (g/L): molasses (Calsa, Argentine Company of Yeast), 30; saccharose (Merk), 15; lactose (Merk), 15; dextrine (Merk), 15. Similarly, proteose peptone was replaced by the following nitrogen sources in the amount required to maintain the total nitrogen content constant: soybean peptone (Merck), 42.9 g/L; casamino acids (Britania), 53.7 g/L; corn steep liquor (Sigma) 10.75 mL/L. The pH of media was adjusted to 7.6 and the media were sterilized at 1211C for 15 min. After cooling, media were immediately inoculated to prevent reoxygenation. The inoculum size for the coculture experiments consisted of a mixture of 0.1 mL of C. argentinense containing 6.2 107 CFU/mL and 0.1 mL of P. mendocina of
3.0 10ll CFU/mL obtained in the base medium. The experiments also included monocultures of both micro-organisms which represented positive (C. argentinense) and negative (P. mendocina) toxicity controls. After inoculation the cultures were incubated at 371C for 72 h in an anaerobic jar (Oxoid).
Microfermenter experiments Monocultures and cocultures were grown in an 800 mL fermenter without aeration and agitation [9] containing 700 mL of the base medium using either glucose (11 g/L) or dextrine (15 g/L) as carbon sources. The pH of media was adjusted to 7.6 and then sterilized at 1211C for 15 min. The fermenter was equipped with pH and Eh sensors to monitor their evolutions. Measurements were made with a digital pH/ORP controller (Cole Parmer). To prevent reoxygenation the fermentor was inoculated immediately after autoclaving with 8 mL (1% fermenter culture volume) of a growing culture of C. argentinense in base medium for monocultures and with 16 mL (2% fermenter culture volume) of 50% C. argentinense and 50% P. mendocina prepared in base medium for coculture experiments.
Analytical techniques Viable counts by the dilution pour plate method were used to enumerate micro-organisms in inocula for test tube experiments. Serial dilutions of stock cultures were made and used to inoculate plates in triplicate containing the basal medium for C. argentinense and nutrient broth for P. mendocina. Plates of C. argentinense were inoculated in an anaerobic glove box and incubated in an anaerobic jar for 72 h at 371C. In the fermenter experiments, samples were taken by duplicate at 10 h intervals for optical density (OD) and botulinum neurotoxin levels. Measurements of OD were made in a Baush and Lomb spectrophotometer at 600 nm. Biomass was estimated by dry weight determinations of samples centrifuged at 10 000 g for 20 min at 41C, washed twice with distilled water and dried at 1001C for 16 h. The specific growth rate (m) was calculated by the expression ln x/x0 tÿ1, where x0 was the initial biomass and x was the biomass at time t [10]. To estimate the production of toxin, the mouse bioassay was conducted in separate culture and in coculture supernatants obtained by centrifugation at 10 000 g for 20 min at 41C. Mice of the Rockland strain weighing between 18 and 21 g were injected intraperitoneally with 0.5 mL and for those with typical symptoms of botulinum intoxication the
E¡ect of C and N Sources on Cocultures Toxicity
325
cultures; however, with this sugar the level of toxin was lower than with glucose. Using dextrine as a carbon source, C. argentinense produced the highest levels of toxin both in monocultures (300 LD50/mL) and in cocultures with P. mendocina (5000 LD50 /mL). Low synergistic effect was observed in cocultures using saccharose and lactose, with a five- and sevenfold increase, respectively, but the level of toxin obtained with these sugars was significantly lower than with glucose. The evolution in the production of biomass and toxin in C. argentinense cultures (Figure 1) and C. argentinense–P. mendocina cocultures (Figure 2) was analysed in a microfermenter using glucose and dextrin as carbon sources. C. argentinense grown on glucose showed a high specific growth rate (m = 0.21/h), the maximum level of biomass being obtained after 10 h of evolution of the culture without the existence of a lag period (Figure 1A). Using
LD50/mL was calculated by the method of Reed and Muench [11].
Results The effects of the carbon sources on the productı´on of botulinum toxin of C. argentinense and C. argentinense– P. mendocina cocultures are presented in Table 1. P. mendocina alone was negative in the toxin assay by intraperitoneal inoculation in mice. The production of toxin by C. argentinense in coculture with P. mendocina increased in all the cases in relation to that produced by C. argentinense independent of the nature of the carbon source. The levels of toxicity were 17.5 times higher using glucose. A high effect on the level of botulinum toxin by cocultures was produced using sugar cane molasses, with a 40-fold increase in relation to the level of toxin produced by mono-
Table 1. Effect of the carbon source on the production of botulinum toxin in cocultures of C. argentinense–P. mendocina Carbon source (CS)
C. argentinense (LD50/mL)
C. argentinense–P. mendocina (LD50/mL)
LD50/mL coculture/ LD50/mL C. argentinense
LD50/mL CS/LD50/mL glucose1
100 30 50 300 100
1750 1200 350 5000 500
17.5 40 7 16.6 5
1 0.69 0.2 2.86 0.29
Glucose Molasses Lactose Dextrine Saccharose 1
Ratio for cocultures. Values are means of two separate experiments.
1.0
(A)
1.6 1.2 OD 600 nm
OD 600 nm
0.8 0.6 0.4 0.2
0
10
20
30
40 50 60 Time (h)
70
80
90 100
(B)
0
10 20 30 40 50 60 70 80 90 100 Time (h)
0
10 20 30 40 50 60 70 80 90 100 110 Time (h)
300 250 LD50 /ml
LD50 /ml
0.4 0
0
100 90 80 70 60 50 40 30 20 10 0
0.8
200 150 100 50 0
0 10 20 30 40 50 60 70 80 90 100 110 120 Time (h)
Figure 1. Growth (A) and toxin production (B) of C. argentinense in base medium containing glucose (&), left and dextrine (~), right.
326
H.J. Centorbi and H.J. Silva 3.5
(A)
2.5
OD 600 nm
OD 600 nm
3.0 2.0 1.5 1.0 0.5
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
0 0
40
60 80 Time (h)
100
0
120
(B)
5000
1500
4000
1000
0
20
40
60
80 100 120 140 160 Time (h)
6000
2000 LD50 /ml
LD50 /ml
2500
20
3000 2000
500
1000 0
0 0 10 20 30 40 50 60 70 80 90 100 110 Time (h)
0
20 40 60
80 100 120 140 160 180 Time (h)
0 −50 −100 −150 −200 −250 −300 −350 −400 −450 −500
8.0 7.5 7.0 pH ( )
dextrin as carbon source, after a l0 h lag period, the highest productivity of biomass was generated in 30 h of culture, and was found to be higher than that obtained with glucose and growing at a lower specific growth rate. The highest toxin level occurred at the end of the cultures (100 h) for both sources of carbon (Figure 1B). The toxin levels obtained with dextrin were three times higher than with glucose. Comparing the evolution of biomass in C. argentinense culture (Figure 1A) with the toxin production (Figure 1B) it was observed that with both carbon sources toxin liberation took place as a consequence of cellular lysis. The behaviour of cocultures of C. argentinense– P. mendocina in the microfermenter for the same carbon sources is presented in Figure 2A. The higher toxin levels were again reached for the two carbon sources when cultures entered the stationary phase with clear cellular lysis when using dextrine (Figure 2B). However, this process was less intense than that in C. argentinense culture due to the contribution of P. mendocina biomass. With dextrine, the pH dropped to acid values in the logarithmic growth phase, remaining fairly constant until the end of the culture (Figure 3). The levels of redox potential were of the order of ÿ300 to ÿ450 mV up to 120 h of culture, indicating the existence of a strongly reducing atmosphere, appropriate for growth and toxin production by C. argentinense (Figure 3). The effects of the nitrogen sources in cultures of C. argentinense and cocultures of C. argentinense– P. mendocina are presented in Table 2. The levels of
6.5 6.0 5.5 5.0 0
20
40
60 80 Time (h)
100
120
Eh (mV) ( )
Figure 2. Growth (A) and toxin production (B) of C. argentinense–P. mendocina cocultures in base medium containing glucose (&), left and dextrine, (~), right.
140
Figure 3. Eh (&) and pH (&) profiles of C. argentinense–P. mendocina cocultures in base medium with dextrine as carbon source.
toxin were higher in cocultures independent of the nitrogen source. The cocultures showed similar increases in the level of toxin with respect to C. argentinense cultures using proteose peptone, soybean peptone and corn steep liquor (17.5, 16 and 16.6 times increase, respectively). Of these three nitrogen sources, both in C. argentinense cultures and in cocultures, corn steep liquor sustained the highest levels of toxin, with 3- and 2.86-fold higher levels than that obtained with proteose peptone. Casamino acids proved to be a poor nitrogen source, showing a small increase in toxin production in cocultures and a level of toxin significantly lower than that obtained with proteose peptone (LD50/mL casamino acids/LD50/ mL proteose peptone = 0.07).
E¡ect of C and N Sources on Cocultures Toxicity
327
Table 2. Effect of the nitrogen source on the production of botulinum toxin in cocultures of C. argentinense–P. mendocina Nitrogen source (NS) Proteose peptone (Pp) Soybean peptone Corn-steep liquor Casamino acid
C. argentinense (LD50/mL)
C. argentinense–P. mendocina (LD50/mL)
LD50/mL coculture/ LD50/mL C. argentinense
LD50/mL NS/ LD50/mL Pp1
100 50 300 20
1750 800 5000 125
17.5 16 16.6 6.25
1 0.457 2.86 0.07
1
Ratio for cocultures. Values are means of two separate experiments.
Discussion and Conclusions The optimal and limiting conditions for the growth and toxin production of Clostridium strains, mainly those related with substrates, pH, Eh, aw, and temperature have been well established in the literature [12–14]. However, this is not the case when Clostridium shares the same habitat with other bacteria. This knowledge is of special interest due to its theoretical and practical implications for food and environmental microbiology, and indeed for human and animal pathology. It is in this sense that we studied the effect of carbon and nitrogen nutrition, as an environmental factor, on the growth and toxin production of Clostridium argentinense sharing the same culture medium with P. mendocina. The results obtained provide useful information mainly because both microorganisms were originally isolated from soils in the Province of Mendoza, Argentina [3,8], and may be directly or indirectly involved as potential agents of contamination of some foods, as saprophytic or pathogenic inhabitants of the human or animal intestine and other environments. Gimenez et al. [6] observed a metabiotic effect in cocultures of some aerobic bacteria including Acinetobacter and Pseudomonas strains and C. botulinum type A. Among the aerobic bacteria, P. mendocina increased growth and the level of botulinum toxin. Previous studies, also indicated the existence of an important interaction between P. mendocina and C. argentinense [7]. The production of toxin by coculture of these micro-organisms was clearly enhanced, which correlated with an increased level of biomass. Nutritional factors, among others, may influence their interaction. The results obtained in this work show that the toxin levels attained in cocultures depend on the nature of the carbon sources (Table 1) and nitrogen sources (Table 2) used in the culture media. Variations in the levels of toxin were observed both in C. argentinense cultures and in cocultures with P. mendocina. Of all the carbon and nitrogen sources employed, the highest levels of toxin were obtained with dextrine and corn steep liquor with a 2.8-fold increase in relation to that obtained using glucose and proteose peptone.
Corn steep liquor, a complex organic nitrogen source commonly used for the production of bioactive drugs [15], provides a non-homogeneous and readily available source of amino acids, which best suited the nitrogen requirements of C. argentinense for growth and toxin production. While soybean peptone in cocultures produced increments in the toxin level in relation to C. argentinense cultures, it was more than half less effective than proteose peptone. Casamino acid was not an appropriate nitrogen source for growth and toxigenicity of C. argentinense in individual cultures and cocultures, producing the lowest toxin levels of all the nitrogen sources assayed. Apart from its poor nitrogen nutritional value, the content of iron of casamino acids of 15 mg% (p/p) was probably limiting. It has been observed that other Clostridium strains such as C. botulinum strain Hall type A is highly demanding of iron, requiring concentrations higher than 10 mg/L in the culture medium for toxin production [16]. A more detailed study in a microfermenter, using glucose and dextrine as carbon sources, on growth evolution and toxin production by C. argentinense cultures (Figure 1) and in cocultures with P. mendocina (Figure 2), showed that in cocultures the levels of biomass and toxin were higher. In previous studies it was found that with glucose the quality of the coculture biomass producing toxin, expressed as specific toxicity (LD50/g ), was four times higher than that of C. argentinense [7]. The increased level of biomass observed in the cocultures with glucose with respect to monocultures (Figure 2A) could be explained on the grounds of changes in metabolic fluxes in P. mendocina under anaerobic conditions. P. mendocina has been observed to display a remarkable metabolic versatility in reduced environments, being able to redirect its anabolic flux towards the synthesis of uronic acids or alginate which are released in the extracellular medium [17]. These compounds may have been utilized as a non-readily available carbon source by C. argentinense when glucose was exhausted improving its growth at low specific growth rates in later stages of cocultures. The culture evolution of C. argentinense–P. mendocina coculture with dextrine showed two distinct phases,
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a growth phase followed by a lytic phase with the production of toxin. The Eh values observed in the growth phase were representative of highly reducing conditions (Figure 3), which resulted similar to that displayed by cocultures grown on glucose [9]. In this atmosphere the synthesis of microbial exopolysaccharides or growth factors by the aerobic micro-organism may have also been an influential factor for growth stimulation and toxin production of C. argentinense. With either glucose or dextrine the cocultures grew at lower specific growth rates than that of C. argentinense. Particularly remarkable was the growth evolution with dextrine, showing a very low specific growth rate (m = 0.021/h) and similar to what was observed in cultures of C. argentinense, the final level of biomass was higher than those with glucose. Dextrine being a polymer of glucose with bonds a (1-4) and a (1-6) at the ramification points, a controlled liberation of glucose takes place during its dissimilation resulting in a limitation of the carbon source when using this carbohydrate. The results obtained in the study indicate that the production of toxin by C. argentinense cultures and C. argentinense–P. mendocina cocultures is highly dependent on the nature of the carbon and nitrogen sources used in the culture media. For glucose and dextrine, the slow use of the substrate in the growth phase of cocultures allowed the production of more toxin levels. However, additional studies on growth and toxin production kinetics with other carbohydrates and complex nitrogenous nutrients of slow assimilation, either naturally occurring or by slow feeding will confirm whether this observation is a general fact for C. argentinense cultures.
Acknowledgements This work was supported by the Secretariat of Science and Technology, UNSL, and the National Council of Scientific and Technical Research (CONICET) Argentine.
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