DOI 10.1007/s10517-015-2859-z
772
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 IMMUNOLOGY AND MICROBIOLOGY
Effects of Temperature on Biological Activity of Permafrost Microorganisms L. F. Kalyonova, M. A. Novikova, A. M. Subbotin, and A. S. Bazhin Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 737-741, December, 2014 Original article submitted April 25, 2013 The number and viability of microorganism specimens Bacillus spp. isolated from permafrost soil remained unchanged after incubation at temperatures of -16-37ºC. Experiments on F1 CBA/Black-6 mice showed that incubation of bacteria at -5ºC for 72 h promotes a decrease in their toxicity and an increase in their immunostimulating effect. Key Words: permafrost bacteria; incubation temperature; immune system
Temperature is one of the most important environmental factors that strongly affects biological properties of microorganisms. Freeze–thaw cycles can be stressful for several types of bacteria and can modulate growth intensity, metabolism, and pathogenicity [2,4-6]. However, negative temperatures are a stressful factor not for all bacteria. For example, psychrophilic bacteria can growth at temperatures from 5 to -10ºC. These bacteria growth faster at higher temperatures, but do not growth at temperatures above 27ºC [4,6]. Permafrost soil is the unique ecosystem characterized by constant physical, chemical, and temperature parameters that did not change for a long time (from several thousands to millions years). Viable bacteria were found in samples of permafrost formations from Central Yakutia aging 3.5 million years [1]. Probably, bacteria are “conserved” in the formation, are the same age as the formation, and are evolutionary adapted to constant negative temperatures. Degradation of the permafrost zone due to natural (cyclic changes of climate on the Earth, water and wind erosion) and artificial (increased anthropogenic stress) reasons can be followed by the release of microorganisms from relict permafrost to the environment and enter biological circles. New life conditions (e.g. temperature) can be a stressful factor for released ancient microorganisms and can result in the changes of their functional activTyumen Research Center, Siberian Division of the Russian Academy of Sciences, Russia. Address for correspondence: lkalenova@mail. ru. L. F. Kalyonova
ity. It is practically impossible to estimate the potential role of relict microorganisms in human life, as little is known about their biological potential. Here we studied the effects of temperature on biological activity of Bacillus spp. from permafrost soil under in vitro and in vivo conditions.
MATERIALS AND METHODS Experiments were performed on MG8 strain of Bacillus spp. isolated from the samples of permafrost soil from Central Yakutia. Mean temperature of the soil is -5ºC. Bacillus spp. (PMO) was stored in polystyrene tubes in liquid nitrogen at -180ºC in doses of 106 microbial cells (m.b.) in 1 ml distilled water. PMO were defrosted for 30 min at 20-22ºC before culturing. Preliminary studies have demonstrated that optimal temperature for the growth of MG8 bacteria after inoculation to nutrient mediums is 20-26ºC; bacterial growth was decelerated with decreasing of culturing temperature. At -5ºC, signs of colony growth were found in 2-3 months and at -16ºC they were not observed for 6 months. In our experiment, we estimated the effects of preincubation temperature on biological activity of bacteria in vitro (number and viability) and in vivo (viability and reactivity of the immune system of warmblooded organism). Modern JP 5832 strain (MMO) of Bacillus spp. from medicinal substance Baktisubtil was used as the control. MMO in aliquots of 106 cells in 1 ml distilled water were kept in liquid nitrogen for
0007-4888/15/15860772 © 2015 Springer Science+Business Media New York
L. F. Kalyonova, M. A. Novikova, et al.
1 week before analysis. Further manipulations were simultaneously performed on both MMO and PMO. At the first stage, PMO were divided into aliquots of 2000×106, 1000×106, 500×106, and 250×106 m.b. in 2 ml of physiological saline (0.9% NaCl) and put into sterile polystyrene tubes. Bacterial aliquots were incubated at positive temperature (37ºC), temperatures
773 similar to permafrost temperature and freeze–thaw cycle (4ºC and -5ºC), and negative temperature (-16ºC) for 72 h. Then they were incubated at room temperature (20-22ºC) for 30 min, inoculated to solid mediums (5 Petri dishes per dose), and cultured at 26ºC (optimal temperature for PMO growth). The number of growth colonies was calculated after 18 h. The effects of PMO on animal viability was studied on F1 CBA/Black-6 mice (n=264) weighing 1821 g. The bacteria (PMO and MMO) were incubated at 37ºC, 4ºC, -5ºC, and -16ºC for 72 h and then at 20-22ºC for 30 min and intraperitoneally injected to animals in doses of 1000×106, 500×106, and 250×106 m.b. in 0.5 ml physiological saline. Control animals received physiological saline (0.5 ml) preincubated at the same temperatures. PMO groups consisted of 12 animals, MMO groups consisted of 8 animals and control groups consisted of 6 specimens. The animals were monitored for 10 days, autopsy of 1-2 animals from each group was conducted on days 1, 3, 5, 7, and 10 for visual examination of internal organs. The effects of PMO on functional activity of the immune system were studied on F1 CBA/Black-6 mice (n=88) weighting 18-21 g and randomized into 11 equal groups. Before the study, the bacteria were incubated at -5ºC for 72 h followed by thawing at 2022ºC for 30 min. They were intraperitoneally injected to mice in doses of 50×106, 5×106, 0.5×106, 0.05×106, and 0.005×106 m.b. per mouse in 0.5 ml of 0.9% NaCl. Control animals received 0.5 ml lavage of 0.9% NaCl from non-inoculated nutrition medium. On day 14, the indexes of internal organs (organ weight to body weight ratio, %), engulfing (EA, %) and metabolic (NBT-test, %) activities of spleen macrophages, activity of cellular immunity in the delayed-type hypersensitivity test (DTH test) by Crowle method, and activity of humoral immunity estimated by the number of antibody-forming cell (AFC) in the spleen by Cunningham method were measured. All studies were carried out in accordance with Regulations for Studies on Experimental Animals (Order No. 755 of the Ministry of Health of the USSR of August 12, 1977) and European Convention of Vertebrate Protection (ETSN No. 123, Strasbourg, 1986). Significance of between-group differences was estimated using Student’s t test (SPSS 11.5 software).
RESULTS
Fig. 1. Concentration of microorganisms in vitro.
Preincubation of bacteria for 72 h at positive (37ºC, 4ºC) and negative temperatures (-5 ºC, -16ºC) did not change significantly the number and viability of PMO in vitro (Fig. 1). Preincubation temperature affected biological activity of bacteria in vivo (Table 1). Minimum viability
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 IMMUNOLOGY AND MICROBIOLOGY
774
TABLE 1. Viability (%) of Animals over 1 Day Group Physiological saline PMO
6
1000×10 m.b. 6
MMO
37оС
4 оС
-5оС
-16оС
100
100
100
100
0
0
0
0
500×10 m.b.
62.5
50
37.5
25
250×106 m.b.
100
100
100
100
1000×106 m.b.
8.4
16.7
41.7
33.4
6
58.4
66.7
91.7
83.4
6
100
100
100
100
500×10 m.b. 250×10 m.b.
of animals was found 24 h after administration of PMO incubated at 37ºC and injected in a dose of 1000×106 m.b. (8.4%). Maximum viability of animals was observed after injection of PMO incubated at -5ºC: 41.7, 91.7, and 100% after administration of bacteria in the doses of 1000×106, 500×106, 250×106 m.b., respectively. No animal deaths were recorded. Minimum level of animal viability was found after administration of MMO incubated at -5ºC and maximum – after injection of MMO incubated at 37ºC (Table 1). Insignificant hair loss, changes in the consistence and color of stool, decrease in motor activity and appetite, and apathetic behavior were found in survivors during the first three days after administration of PMO in a dose of 1000×106 m.b. and MMO in a dose of 500×106 m.b. irrespective of preincubation temperature. Autopsy 1 day later showed intermittent congestion of the liver, spleen, and changes in the lymphoid system of the intestine. Minor changes in behavioral reactions were observed on day 1 in animals receiving PMO and MMO in a dose of 250×106 m.b. Visual
examination revealed no changes in the internal organs of these animals. Taking these results into account, bacteria incubated at -5ºC for 72 h (protocol with minimum animal lethality) were injected to animals for investigation of PMO effects on structural and functional parameters of immune system. MMO was also preincubated at -5ºC for 72 h (Table 2). After bacteria administration, the animal weight did not significantly differ from the control level (Table 2). Negative feedback was not found between indexes of the thymus and the adrenal glands, which can reflect the absence of stress-inducing effect in both strains in study doses. Administration of the strains in a dose of 0.005×106 m.b. had different effects. Under these conditions PMO promoted simultaneous increase in structural parameters of the immune system (thymus and spleen indexes, p
772
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 IMMUNOLOGY AND MICROBIOLOGY
Effects of Temperature on Biological Activity of Permafrost Microorganisms L. F. Kalyonova, M. A. Novikova, A. M. Subbotin, and A. S. Bazhin Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 737-741, December, 2014 Original article submitted April 25, 2013 The number and viability of microorganism specimens Bacillus spp. isolated from permafrost soil remained unchanged after incubation at temperatures of -16-37ºC. Experiments on F1 CBA/Black-6 mice showed that incubation of bacteria at -5ºC for 72 h promotes a decrease in their toxicity and an increase in their immunostimulating effect. Key Words: permafrost bacteria; incubation temperature; immune system
Temperature is one of the most important environmental factors that strongly affects biological properties of microorganisms. Freeze–thaw cycles can be stressful for several types of bacteria and can modulate growth intensity, metabolism, and pathogenicity [2,4-6]. However, negative temperatures are a stressful factor not for all bacteria. For example, psychrophilic bacteria can growth at temperatures from 5 to -10ºC. These bacteria growth faster at higher temperatures, but do not growth at temperatures above 27ºC [4,6]. Permafrost soil is the unique ecosystem characterized by constant physical, chemical, and temperature parameters that did not change for a long time (from several thousands to millions years). Viable bacteria were found in samples of permafrost formations from Central Yakutia aging 3.5 million years [1]. Probably, bacteria are “conserved” in the formation, are the same age as the formation, and are evolutionary adapted to constant negative temperatures. Degradation of the permafrost zone due to natural (cyclic changes of climate on the Earth, water and wind erosion) and artificial (increased anthropogenic stress) reasons can be followed by the release of microorganisms from relict permafrost to the environment and enter biological circles. New life conditions (e.g. temperature) can be a stressful factor for released ancient microorganisms and can result in the changes of their functional activTyumen Research Center, Siberian Division of the Russian Academy of Sciences, Russia. Address for correspondence: lkalenova@mail. ru. L. F. Kalyonova
ity. It is practically impossible to estimate the potential role of relict microorganisms in human life, as little is known about their biological potential. Here we studied the effects of temperature on biological activity of Bacillus spp. from permafrost soil under in vitro and in vivo conditions.
MATERIALS AND METHODS Experiments were performed on MG8 strain of Bacillus spp. isolated from the samples of permafrost soil from Central Yakutia. Mean temperature of the soil is -5ºC. Bacillus spp. (PMO) was stored in polystyrene tubes in liquid nitrogen at -180ºC in doses of 106 microbial cells (m.b.) in 1 ml distilled water. PMO were defrosted for 30 min at 20-22ºC before culturing. Preliminary studies have demonstrated that optimal temperature for the growth of MG8 bacteria after inoculation to nutrient mediums is 20-26ºC; bacterial growth was decelerated with decreasing of culturing temperature. At -5ºC, signs of colony growth were found in 2-3 months and at -16ºC they were not observed for 6 months. In our experiment, we estimated the effects of preincubation temperature on biological activity of bacteria in vitro (number and viability) and in vivo (viability and reactivity of the immune system of warmblooded organism). Modern JP 5832 strain (MMO) of Bacillus spp. from medicinal substance Baktisubtil was used as the control. MMO in aliquots of 106 cells in 1 ml distilled water were kept in liquid nitrogen for
0007-4888/15/15860772 © 2015 Springer Science+Business Media New York
L. F. Kalyonova, M. A. Novikova, et al.
1 week before analysis. Further manipulations were simultaneously performed on both MMO and PMO. At the first stage, PMO were divided into aliquots of 2000×106, 1000×106, 500×106, and 250×106 m.b. in 2 ml of physiological saline (0.9% NaCl) and put into sterile polystyrene tubes. Bacterial aliquots were incubated at positive temperature (37ºC), temperatures
773 similar to permafrost temperature and freeze–thaw cycle (4ºC and -5ºC), and negative temperature (-16ºC) for 72 h. Then they were incubated at room temperature (20-22ºC) for 30 min, inoculated to solid mediums (5 Petri dishes per dose), and cultured at 26ºC (optimal temperature for PMO growth). The number of growth colonies was calculated after 18 h. The effects of PMO on animal viability was studied on F1 CBA/Black-6 mice (n=264) weighing 1821 g. The bacteria (PMO and MMO) were incubated at 37ºC, 4ºC, -5ºC, and -16ºC for 72 h and then at 20-22ºC for 30 min and intraperitoneally injected to animals in doses of 1000×106, 500×106, and 250×106 m.b. in 0.5 ml physiological saline. Control animals received physiological saline (0.5 ml) preincubated at the same temperatures. PMO groups consisted of 12 animals, MMO groups consisted of 8 animals and control groups consisted of 6 specimens. The animals were monitored for 10 days, autopsy of 1-2 animals from each group was conducted on days 1, 3, 5, 7, and 10 for visual examination of internal organs. The effects of PMO on functional activity of the immune system were studied on F1 CBA/Black-6 mice (n=88) weighting 18-21 g and randomized into 11 equal groups. Before the study, the bacteria were incubated at -5ºC for 72 h followed by thawing at 2022ºC for 30 min. They were intraperitoneally injected to mice in doses of 50×106, 5×106, 0.5×106, 0.05×106, and 0.005×106 m.b. per mouse in 0.5 ml of 0.9% NaCl. Control animals received 0.5 ml lavage of 0.9% NaCl from non-inoculated nutrition medium. On day 14, the indexes of internal organs (organ weight to body weight ratio, %), engulfing (EA, %) and metabolic (NBT-test, %) activities of spleen macrophages, activity of cellular immunity in the delayed-type hypersensitivity test (DTH test) by Crowle method, and activity of humoral immunity estimated by the number of antibody-forming cell (AFC) in the spleen by Cunningham method were measured. All studies were carried out in accordance with Regulations for Studies on Experimental Animals (Order No. 755 of the Ministry of Health of the USSR of August 12, 1977) and European Convention of Vertebrate Protection (ETSN No. 123, Strasbourg, 1986). Significance of between-group differences was estimated using Student’s t test (SPSS 11.5 software).
RESULTS
Fig. 1. Concentration of microorganisms in vitro.
Preincubation of bacteria for 72 h at positive (37ºC, 4ºC) and negative temperatures (-5 ºC, -16ºC) did not change significantly the number and viability of PMO in vitro (Fig. 1). Preincubation temperature affected biological activity of bacteria in vivo (Table 1). Minimum viability
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 IMMUNOLOGY AND MICROBIOLOGY
774
TABLE 1. Viability (%) of Animals over 1 Day Group Physiological saline PMO
6
1000×10 m.b. 6
MMO
37оС
4 оС
-5оС
-16оС
100
100
100
100
0
0
0
0
500×10 m.b.
62.5
50
37.5
25
250×106 m.b.
100
100
100
100
1000×106 m.b.
8.4
16.7
41.7
33.4
6
58.4
66.7
91.7
83.4
6
100
100
100
100
500×10 m.b. 250×10 m.b.
of animals was found 24 h after administration of PMO incubated at 37ºC and injected in a dose of 1000×106 m.b. (8.4%). Maximum viability of animals was observed after injection of PMO incubated at -5ºC: 41.7, 91.7, and 100% after administration of bacteria in the doses of 1000×106, 500×106, 250×106 m.b., respectively. No animal deaths were recorded. Minimum level of animal viability was found after administration of MMO incubated at -5ºC and maximum – after injection of MMO incubated at 37ºC (Table 1). Insignificant hair loss, changes in the consistence and color of stool, decrease in motor activity and appetite, and apathetic behavior were found in survivors during the first three days after administration of PMO in a dose of 1000×106 m.b. and MMO in a dose of 500×106 m.b. irrespective of preincubation temperature. Autopsy 1 day later showed intermittent congestion of the liver, spleen, and changes in the lymphoid system of the intestine. Minor changes in behavioral reactions were observed on day 1 in animals receiving PMO and MMO in a dose of 250×106 m.b. Visual
examination revealed no changes in the internal organs of these animals. Taking these results into account, bacteria incubated at -5ºC for 72 h (protocol with minimum animal lethality) were injected to animals for investigation of PMO effects on structural and functional parameters of immune system. MMO was also preincubated at -5ºC for 72 h (Table 2). After bacteria administration, the animal weight did not significantly differ from the control level (Table 2). Negative feedback was not found between indexes of the thymus and the adrenal glands, which can reflect the absence of stress-inducing effect in both strains in study doses. Administration of the strains in a dose of 0.005×106 m.b. had different effects. Under these conditions PMO promoted simultaneous increase in structural parameters of the immune system (thymus and spleen indexes, p
