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Growth parameters of Borrelia burgdorferi sensu stricto at various temperatures
Zent.bl. Bakteriol. 288, 451-455 (1998) © Gustav Fischer Verlag
Zentralblatt fUr
Growth Parameters of Borrelia burgdorferi sensu stricto at Various Temperatures M. Heroldova}, M. Nemec}, and Z. Hubalek 2 1 2
Department of Microbiology, Faculty of Science, Masaryk University, Brno Laboratory of Medical Zoology, Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Brno
Received June 15, 1998 . Revision received August 10, 1998 . Accepted September 10, 1998
Summary Growth of Borrelia burgdorferi sensu stricto (prototype strain B-31) was studied in Barbour-Stoenner-Kelly BSK-H liquid medium, supplemented with 4.5 % rabbit serum and antibiotics (phosphomycin, rifampicin), at various temperatures to early stationary growth phase. The number of cells was determined by darkfield microscopy. In the range of cultivation temperatures of 25°C to 37 °C, generation time was between 8.26 and 12.36 h (the shortest one at 33 0q, and the specific growth rate between 0.056 and 0.083 h- 1 (the highest one at 33 0q. The optimum growth temperature for B. burgdorferi was 33°C, although good growth was also observed at 28 °C, 30°C, 35 °C and 37°C. The strain grew well but slowly at the temperature of 25°C, whereas no growth was observed at 20 0c.
Introduction The causative agent of Lyme disease, Borrelia burgdorferi sensu lato (s.1.), is a spirochete "shuttling" between two organisms: arthropod vector (ixodid tick) and homeotherm vertebrate host. This life cycle requires the ability of the microorganism to multiply at different temperatures and environments: a) at ambient temperature (in the vector tick); b) at 32-3rc (temperature of body surface of the vertebrate host); c) at 37-41 °c (temperature of internal organs of the vertebrate host - mammal or bird). The knowledge about growth abilities of B. burgdorferi and its growth parameters in vitro are limited because of its generally slow growth and high cul-
452
M. Heroldova, M. Nemec, and Z. Hubalek
tivation demands. The spirochete is usually cultivated in vitro in BarbourStoenner-Kelly (BSK) liquid medium in the 30-3re temperature range (1). Nevertheless, detailed data about the growth curve of B. burgdorferi have not yet been published. A better knowledge about growth of this microorganism is important for ecology, epidemiology as well as for clinical practice.
Materials and Methods The prototype strain B-31 (ATCC 35210) of Borrelia burgdorferi sensu stricto (s. s.) was tested. This strain was isolated from Ixodes scapularis collected on Shelter Island, New York, and passaged> 20 times in vitro, including 10 passages done at the laboratory of the Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, in BSK liquid medium at 33 ec. The growth of spirochetes was studied in BSK-H liquid medium (Sigma), supplemented with 4.5 % rabbit serum (Sigma) and the antibiotics, phosphomycin (50 Ilg/mi) and rifampicin (50 Ilg/mi), at temperatures of 20 ec, 25 ec, 28 ec, 30 ec, 33 ec, 35 ec and 37 ec. At each temperature, a set of plastic tubes containing 5 ml of culture medium was used. About 5 X 104 cells from the logarithmic phase of growth (5-6 day-old culture, with a cell density of 6-8 X 10 7 per ml and motility > 95 %) were inoculated into each tube. The tubes were tightly stoppered and incubated at various temperatures; the temperature fluctuation did not exceed ± 0.2 ec in any of the incubators. The growth was observed daily, for 9 days at least, to the early stationary growth phase. Cells were counted by darkfield microscopy (x 400) in 10 III volumes after appropriate dilution. A growth curve was constructed and the following growth parameters were determined: growth rate (R(h- 1) = 1I10g2 [(logNz10gNJl/t], where Nl is the number of bacteria present at an early stage in exponential growth phase, N2 is the number of bacteria present at some time of the exponential growth phase and t is the time interval between Nl and N 2; generation time G(h) = lIR; specific growth rate ll(h- 1 ) = 0,693/G.
Results and Discussion The course of the growth curve of B. burgdorferi s. s. is shown in Figures 1-3. Fig. 1 shows that at 20 °e, the number of spirochetes did not change during the period of observation (17 days). At 25 °e, the growth was slow, while cells of this strain grew well at 28°C. The growth was faster at 33 °e than at 30 °e (Fig. 2). The lag period was approximately one day longer at 30 °e (2.5 day) than at 33 °e (1.5 day). Fig. 3 shows that growth at 35 °e was faster than at 37°C. The shortest generation time (8.26 h) and the highest growth rate (0.12 h- 1) were observed at 33°C. The longest generation time (12.36 h) and the lowest growth rate (0.081 h- 1) were at 25°C. The growth was also slow at 37°e: the generation time was 12.01 h and the growth rate 0.083 h- 1• All growth parameters are given in Table 1. The lag period ranged between 36 h (at 33°C) and 225 h (at 25°C). At all tested temperatures except 20 °e, the cell density reached about 107/ml in the early stationary phase.
Borrelia burgdorferi, growth parameters
7 ••' . 6
453
......•.. .... 25°C
.... . ................. '
z .s"" 4·
•
•
•
•
•
2
o ~---+----~------~~------~--------~--~ 14 18 10 12 16 4 6 o days
Fig. 1. Growth of B. burgdorferi at 20°C, 25 °C and 28°C (N- number of cells per ml).
.
33°C
,.'
6
• ~4
......... ..
oS
2
o L------+------~----~r_-----r----~ 4 o \0 days
Fig. 2. Growth of B. burgdorferi at 30°C and 33°C (N- number of cells per ml).
454
M. Heroldova, M. Nemec, and Z. Hubalek 8
r--------------------------------------,
7 6
z
OIl
.Si
..
""
4
2
O~------r_------r_----~------~------~
o
2
6
4
8
10
days
Fig. 3. Growth of B. burgdorferi at 35°C and 37 °C (N- number of cells per ml).
Table 1. Growth parameters of B. burgdorferi at various temperatures Temperatures
Parameters
G(h) R (h- 1) f! (h-1)
25°C
28°C
30°C
33°C
35°C
3rC
12.360 0.081 0.056
11.290 0.089 0.061
11.120 0.090 0.062
8.260 0.121 0.083
9.640 0.104 0.072
12.050 0.083 0.058
The optimum temperature for growth of the strain of B. burgdorferi studied was 33 °e, although a very good growth was also observed at 28,30,35 and 37°C. At 25 °e, growth was well but slow, which might correspond to the ability of replication of B. burgdorferi in ixodid ticks during the summer season in the temperature climate zone. Spirochetes did not survive at 41 °e (i. e., at the body temperature of birds), as found in another study (3). The optimum temperature for in vitro growth of B. burgdorferi s.l. has been given as 30-35 °e and the spirochete is usually cultivated at 33 °e, but often also at 34°e (4, 5, 6, 8). Depending on culture conditions including temper-
Borrelia burgdorferi, growth parameters
455
ature (33-37 0c), the generation time of B. burgdorferi ranges between 10 and 12 h (6), or 7 and 20 h (8). The lag phase varies at different temperatures from about 2 weeks at 23°C to 4-6 days at 35 °C (9). The maximum cell density of B. burgdorferi is reported by various authors as 10 6 to 108/ml (2, 7, 8). Our results have supplied new data on comparision of the growth rates of B. burgdorferi at different temperatures in a wide range of 20°C to 41 0c. In contrast to other authors, we estimated the growth parameters quantitatively, in terms of generation time (G), specific growth rate (r-t) and growth rate (R). Acknowledgements. We are very grateful to Dr. ]. F. Anderson (Connecticut Agriculture Experiment Station) who provided the type strain B-31 of Borrelia burgdorferi and Dr. J. Halouzka for his help and advice. This study was founded by the Grant Agency of the Academy of Sciences of the Czech Republic (A6087601).
References 1. Barbour, A. G.: Isolation and cultivation of Lyme disease spirochetes. Yale
J.
BioI.
~ed.57(1984)4535-4539
2. Cluss, R. G., A. S. Goel, H. L. Rehm, J. G. Schoenecker, and]. T. Boothby: Coordinate synthesis and turnover of heat shock proteins in Borrelia burgdorferi: degradation ofDnaK during recovery from heat shock. Infect. Immun. 64 (1996) 1736-1743 3. Hubalek, Z.,]. Halouzka, and M. Heroldova: Growth temperature ranges of Borrelia burgdorferi sensu lato strains. J. ~ed. ~icrobiol. 47 (1998) in press 4. Johnson, S. E., G. C. Klein, G. P. Schmid, G. S. Bowen, ]. c. Feeley, and T. Schulze: Lyme disease: a selective medium for isolation of the suspected etiological agent, a spirochete. J. Clin. ~icrobiol. 19 (1994) 81-82 5. Morshed, M.]., H. Konishi, T. Nishimura, and T. Nakazana: Evaluation of agents for use in medium for selective isolation of Lyme disease and relapsing fever Borrelia species. Eur. J. Clin. ~icrobiol. Infect. Dis. 12 (1993) 512-518 6. Padgett, P.J., M.J. Golden, and D.]. Pekala: Improving the growth response Borrelia species. Can. J. ~icrobiol. 41 (1996) 1031-1034 7. Pollack, R.]., S. K. Telford, and A. Spielman: Standardization of medium for culturing Lyme disease spirochetes. J. Clin. ~icrobiol. 31 (1993) 1251-1255 8. Preac-Mursic, V. and B. Wilske: Biology of Borrelia burgdorferi. In: Aspects of Lyme borreliosis, (K. Weber and W. Burgdorfer, eds.), pp.44-58. Springer Verlag, Berlin (1993) 9. Stevenson, B., T. G. Schwan, and P. A. Rosa: Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi. Infect. Immun. 63 (1995)4535-4539
Corresponding author: ~gr. Monika Heroldova, Department of ~icrobiology, ~asa ryk University, Tvrdeho 14, Brno CZ-60200, Czech Republic
Zentralblatt fUr
Growth Parameters of Borrelia burgdorferi sensu stricto at Various Temperatures M. Heroldova}, M. Nemec}, and Z. Hubalek 2 1 2
Department of Microbiology, Faculty of Science, Masaryk University, Brno Laboratory of Medical Zoology, Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Brno
Received June 15, 1998 . Revision received August 10, 1998 . Accepted September 10, 1998
Summary Growth of Borrelia burgdorferi sensu stricto (prototype strain B-31) was studied in Barbour-Stoenner-Kelly BSK-H liquid medium, supplemented with 4.5 % rabbit serum and antibiotics (phosphomycin, rifampicin), at various temperatures to early stationary growth phase. The number of cells was determined by darkfield microscopy. In the range of cultivation temperatures of 25°C to 37 °C, generation time was between 8.26 and 12.36 h (the shortest one at 33 0q, and the specific growth rate between 0.056 and 0.083 h- 1 (the highest one at 33 0q. The optimum growth temperature for B. burgdorferi was 33°C, although good growth was also observed at 28 °C, 30°C, 35 °C and 37°C. The strain grew well but slowly at the temperature of 25°C, whereas no growth was observed at 20 0c.
Introduction The causative agent of Lyme disease, Borrelia burgdorferi sensu lato (s.1.), is a spirochete "shuttling" between two organisms: arthropod vector (ixodid tick) and homeotherm vertebrate host. This life cycle requires the ability of the microorganism to multiply at different temperatures and environments: a) at ambient temperature (in the vector tick); b) at 32-3rc (temperature of body surface of the vertebrate host); c) at 37-41 °c (temperature of internal organs of the vertebrate host - mammal or bird). The knowledge about growth abilities of B. burgdorferi and its growth parameters in vitro are limited because of its generally slow growth and high cul-
452
M. Heroldova, M. Nemec, and Z. Hubalek
tivation demands. The spirochete is usually cultivated in vitro in BarbourStoenner-Kelly (BSK) liquid medium in the 30-3re temperature range (1). Nevertheless, detailed data about the growth curve of B. burgdorferi have not yet been published. A better knowledge about growth of this microorganism is important for ecology, epidemiology as well as for clinical practice.
Materials and Methods The prototype strain B-31 (ATCC 35210) of Borrelia burgdorferi sensu stricto (s. s.) was tested. This strain was isolated from Ixodes scapularis collected on Shelter Island, New York, and passaged> 20 times in vitro, including 10 passages done at the laboratory of the Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, in BSK liquid medium at 33 ec. The growth of spirochetes was studied in BSK-H liquid medium (Sigma), supplemented with 4.5 % rabbit serum (Sigma) and the antibiotics, phosphomycin (50 Ilg/mi) and rifampicin (50 Ilg/mi), at temperatures of 20 ec, 25 ec, 28 ec, 30 ec, 33 ec, 35 ec and 37 ec. At each temperature, a set of plastic tubes containing 5 ml of culture medium was used. About 5 X 104 cells from the logarithmic phase of growth (5-6 day-old culture, with a cell density of 6-8 X 10 7 per ml and motility > 95 %) were inoculated into each tube. The tubes were tightly stoppered and incubated at various temperatures; the temperature fluctuation did not exceed ± 0.2 ec in any of the incubators. The growth was observed daily, for 9 days at least, to the early stationary growth phase. Cells were counted by darkfield microscopy (x 400) in 10 III volumes after appropriate dilution. A growth curve was constructed and the following growth parameters were determined: growth rate (R(h- 1) = 1I10g2 [(logNz10gNJl/t], where Nl is the number of bacteria present at an early stage in exponential growth phase, N2 is the number of bacteria present at some time of the exponential growth phase and t is the time interval between Nl and N 2; generation time G(h) = lIR; specific growth rate ll(h- 1 ) = 0,693/G.
Results and Discussion The course of the growth curve of B. burgdorferi s. s. is shown in Figures 1-3. Fig. 1 shows that at 20 °e, the number of spirochetes did not change during the period of observation (17 days). At 25 °e, the growth was slow, while cells of this strain grew well at 28°C. The growth was faster at 33 °e than at 30 °e (Fig. 2). The lag period was approximately one day longer at 30 °e (2.5 day) than at 33 °e (1.5 day). Fig. 3 shows that growth at 35 °e was faster than at 37°C. The shortest generation time (8.26 h) and the highest growth rate (0.12 h- 1) were observed at 33°C. The longest generation time (12.36 h) and the lowest growth rate (0.081 h- 1) were at 25°C. The growth was also slow at 37°e: the generation time was 12.01 h and the growth rate 0.083 h- 1• All growth parameters are given in Table 1. The lag period ranged between 36 h (at 33°C) and 225 h (at 25°C). At all tested temperatures except 20 °e, the cell density reached about 107/ml in the early stationary phase.
Borrelia burgdorferi, growth parameters
7 ••' . 6
453
......•.. .... 25°C
.... . ................. '
z .s"" 4·
•
•
•
•
•
2
o ~---+----~------~~------~--------~--~ 14 18 10 12 16 4 6 o days
Fig. 1. Growth of B. burgdorferi at 20°C, 25 °C and 28°C (N- number of cells per ml).
.
33°C
,.'
6
• ~4
......... ..
oS
2
o L------+------~----~r_-----r----~ 4 o \0 days
Fig. 2. Growth of B. burgdorferi at 30°C and 33°C (N- number of cells per ml).
454
M. Heroldova, M. Nemec, and Z. Hubalek 8
r--------------------------------------,
7 6
z
OIl
.Si
..
""
4
2
O~------r_------r_----~------~------~
o
2
6
4
8
10
days
Fig. 3. Growth of B. burgdorferi at 35°C and 37 °C (N- number of cells per ml).
Table 1. Growth parameters of B. burgdorferi at various temperatures Temperatures
Parameters
G(h) R (h- 1) f! (h-1)
25°C
28°C
30°C
33°C
35°C
3rC
12.360 0.081 0.056
11.290 0.089 0.061
11.120 0.090 0.062
8.260 0.121 0.083
9.640 0.104 0.072
12.050 0.083 0.058
The optimum temperature for growth of the strain of B. burgdorferi studied was 33 °e, although a very good growth was also observed at 28,30,35 and 37°C. At 25 °e, growth was well but slow, which might correspond to the ability of replication of B. burgdorferi in ixodid ticks during the summer season in the temperature climate zone. Spirochetes did not survive at 41 °e (i. e., at the body temperature of birds), as found in another study (3). The optimum temperature for in vitro growth of B. burgdorferi s.l. has been given as 30-35 °e and the spirochete is usually cultivated at 33 °e, but often also at 34°e (4, 5, 6, 8). Depending on culture conditions including temper-
Borrelia burgdorferi, growth parameters
455
ature (33-37 0c), the generation time of B. burgdorferi ranges between 10 and 12 h (6), or 7 and 20 h (8). The lag phase varies at different temperatures from about 2 weeks at 23°C to 4-6 days at 35 °C (9). The maximum cell density of B. burgdorferi is reported by various authors as 10 6 to 108/ml (2, 7, 8). Our results have supplied new data on comparision of the growth rates of B. burgdorferi at different temperatures in a wide range of 20°C to 41 0c. In contrast to other authors, we estimated the growth parameters quantitatively, in terms of generation time (G), specific growth rate (r-t) and growth rate (R). Acknowledgements. We are very grateful to Dr. ]. F. Anderson (Connecticut Agriculture Experiment Station) who provided the type strain B-31 of Borrelia burgdorferi and Dr. J. Halouzka for his help and advice. This study was founded by the Grant Agency of the Academy of Sciences of the Czech Republic (A6087601).
References 1. Barbour, A. G.: Isolation and cultivation of Lyme disease spirochetes. Yale
J.
BioI.
~ed.57(1984)4535-4539
2. Cluss, R. G., A. S. Goel, H. L. Rehm, J. G. Schoenecker, and]. T. Boothby: Coordinate synthesis and turnover of heat shock proteins in Borrelia burgdorferi: degradation ofDnaK during recovery from heat shock. Infect. Immun. 64 (1996) 1736-1743 3. Hubalek, Z.,]. Halouzka, and M. Heroldova: Growth temperature ranges of Borrelia burgdorferi sensu lato strains. J. ~ed. ~icrobiol. 47 (1998) in press 4. Johnson, S. E., G. C. Klein, G. P. Schmid, G. S. Bowen, ]. c. Feeley, and T. Schulze: Lyme disease: a selective medium for isolation of the suspected etiological agent, a spirochete. J. Clin. ~icrobiol. 19 (1994) 81-82 5. Morshed, M.]., H. Konishi, T. Nishimura, and T. Nakazana: Evaluation of agents for use in medium for selective isolation of Lyme disease and relapsing fever Borrelia species. Eur. J. Clin. ~icrobiol. Infect. Dis. 12 (1993) 512-518 6. Padgett, P.J., M.J. Golden, and D.]. Pekala: Improving the growth response Borrelia species. Can. J. ~icrobiol. 41 (1996) 1031-1034 7. Pollack, R.]., S. K. Telford, and A. Spielman: Standardization of medium for culturing Lyme disease spirochetes. J. Clin. ~icrobiol. 31 (1993) 1251-1255 8. Preac-Mursic, V. and B. Wilske: Biology of Borrelia burgdorferi. In: Aspects of Lyme borreliosis, (K. Weber and W. Burgdorfer, eds.), pp.44-58. Springer Verlag, Berlin (1993) 9. Stevenson, B., T. G. Schwan, and P. A. Rosa: Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi. Infect. Immun. 63 (1995)4535-4539
Corresponding author: ~gr. Monika Heroldova, Department of ~icrobiology, ~asa ryk University, Tvrdeho 14, Brno CZ-60200, Czech Republic