Comparison of Growth of Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto at Five Different Temperatures

Lyme borreliosis is caused by the spirochete Borrelia burgdorferi sensu lato, a fastidious bacterium that replicates slowly and requires special conditions to grow in the laboratory. Borrelia isolation from clinical material is a golden standard for microbiological diagnosis of borrelial infection. Important factors that affect in vitro borrelia growth are temperature of incubation and number of borrelia cells in the sample. The aim of the study was to assess the influence of temperature on borrelia growth and survival by evaluation and comparison of growth of 31 different borrelia strains at five different temperatures and to determine the influence of different inoculums on borrelia growth at different temperatures. Borreliae were cultured in the MKP medium; the initial and final number of spirochetes was determined by dark field microscopy using Neubauer counting chamber. The growth of borrelia was defined as final number of cells/mL after three days of incubation. For all three Borrelia species, the best growth was found at 33°C, followed by 37, 28, and 23°C, while no growth was detected at 4°C (P<0.05). The growth of B. afzelii species was weaker in comparison to the other two species at 23, 28, 33 and 37°C (P<0.05), respectively. There was no statistically significant difference between the growth of B. garinii and B. burgdorferi sensu stricto at 28, 33, and 37°C (P>0.05), respectively. Inoculum had statistically significant influence on growth of all three Borrelia species at all tested temperatures except at 4°C.


Introduction
Lyme borreliosis is a multisystem disease caused by the spirochetes of the Borrelia burgdorferi sensu lato complex that are transmitted by the hard ticks of the Ixodes species complex [1,2].
In Europe, at least four Borrelia species (B. afzelii, B. garinii, B. burgdorferi sensu stricto, B. spielmanii) can cause disease in humans, but some other species have also been reported to be a rare (B. bissettii and B. lusitaniae) or potential (B. valaisiana) causes of human disease. In contrast, B. burgdorferi sensu stricto is the only known species that cause human disease in North America [1,3,4].
Spirochetes can be isolated from skin, blood, cerebrospinal fluid (CSF), and other clinical materials during early as well as late Lyme borreliosis [1,5,6]. The clinical material for isolation should be transported to the laboratory as soon as possible; if feasible, specimens such as skin and CSF, must be inoculated immediately into the culture medium. Isolation, as well as cultivation are demanding procedures that are performed in a limited number of laboratories [6,7].
The in vitro generation time of borrelia ranges from 7 to 20 hours; it is influenced by available nutrients, conditions of cultivation and adaptation of borrelia to the artificial medium [9] Cultivation from clinical material may last up to 12 weeks, which is much longer than for the majority of other human bacterial pathogens [5,7,14]. Borrelia requires complex liquid media for in vitro cultivation, due to inability to synthesize any amino acids, nucleosides, nucleotides, fatty acids, and severalother cellular building blocks [15]. For a routine laboratory work, modified Kelly-Pettenkofer (MKP), Barbour-Stoenner-Kelly II (BSK-II) and commercially available BSK-H (Sigma, USA) are the most commonly used media [10,16,17].
In addition, temperature during clinical material transportation from patient to the laboratory is important for borrelia survival. Room temperature was reported as suitable for transport of samples infected with borrelia during the period from one to 11 days, while refrigerator temperature (5°C) was described as inadequate [18][19][20].
The aim of the study was to assess and compare the growth of B. afzelii, B. garinii, and B. burgdorferi sensu stricto strains at five different temperatures (4, 23, 28, 33, and 37°C) and to examine the influence of different inoculum on the growth at different temperatures.

Borrelia strains
Thirty-one strains, 10 B. afzelii, 10 B. garinii, and 11 B. burgdorferi sensu stricto were randomly selected from the collection of strains of the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Slovenia. Isolates were obtained from 31 patients diagnosed with different clinical manifestations of Lyme borreliosis at the Department of Infection Diseases of the University Medical Center Ljubljana, Slovenia. Borrelia species of the isolated strains was determined by MluI-restriction fragment length polymorphism (Mlu-RFLP) as described previously [5,21,22]. Data regarding the origin of strains are listed in Table 1. Stock cultures of these low passage isolates had been stored at -80°C; for the study, we inoculated and cultured them in the MKP medium [5,23].

Cultivation and numbering of spirochetes
The study was carried out in aseptic conditions provided by laminar flow box in order to reduce the risk of contamination. An aliquot of frozen spirochetes was thawed at room temperature, transferred into starting tubes with 7 mL of MKP medium and incubated at 33°C. Borrelia strains were allowed to multiply to cell density of about 10 6 /mL (4-day-old cultures, with a cell motility >95%) that was determined by dark filed microscopy using Neubauer counting chamber.
The study was conducted using starting tubes with borrelia cells/mL in the range: 1x10 5 -2.5x10 7 for B. afzelii, 3x10 5 -5x10 7 for B.garinii, and 8.5x10 5 -4.8x10 7 for B. burgdorferi sensu stricto strains. Within the range of individual species different concentrations of borrelia cells/mL were distinguished and further processed; generally, these concentrations were reported as low and high. Experiment with individual borrelia strain at particular temperature was repeated at least 10 times with low and high concentration, respectively.
After establishing the exact number of cells/mL in starting tubes, we inoculated aliquots of 50 μL into five new tubes with 7 mL of fresh MKP medium and determined the initial number of cells/mL in inoculated tubes. The tubes were incubated at five different temperatures: 4, 23, 28, 33 and 37°C. After 3 days of incubation we checked the number of spirochetes (final number). The initial and the final number of spirochetes were determined by dark filed microscopy using Neubauer counting chamber. Growth was defined as an increase in the number of cells/ mL comparing the initial number and the number of cells/mL after three days of incubation (final number).

Statistical analysis
All statistical tests were performed using SigmaPlot 11.0 (Systat. Software Inc., Richmond, CA, USA). All graphics were created using MS Excel 2007.
Correlation between the initial and final number of borrelia cells/mL at particular temperature was assessed using Spearman's rho correlation coefficient. A linear regression analysis was applied to evaluate the influence of initial number of cells/mL on the growth of borrelia.
Kruskal-Wallis and Mann-Whitney tests were used to compare growth of Borrelia species at five and two different temperatures, respectively. Values were expressed as median, 25th percentile (P25) and 75th percentile (P75). P values less than 0.05 were considered statistically significant to all statistical analyses.

Ethics Statement
The study was approved by the National Medical Ethics Committee of the Republic of Slovenia (No: 35p/10/12).

Results
Results of the present study are based on the growth analysis of 31 different borrelia strains at five different temperatures.
Moreover, positive β coefficient indicated a statistically positive relationship between the initial and final number of cells/mL for all three Borrelia species growing at 23, 28, 33 and 37°C, but not at 4°C. Results are presented in Table 3.
The Mann Whitney test that compares growth at two particular temperatures showed statistically significant differences in B. afzelii growth between particular and all other tested temperatures (P <0.05).
Comparison of B. garinii growth at two particular temperatures with Mann Whitney test showed statistically significant differences in growth between particular and all other tested temperatures (P <0.05).
Comparison of growth at particular temperatures according to Borrelia species (B. afzelii, B. garinii, and B. burgdorferi sensu stricto) Comparison of growth of B. afzelii, B. garinii, and B. burgdorferi sensu stricto strains at different temperatures is shown on Fig 4A-4E.
The Mann-Whitney test demonstrated statistically significant differences between the growth of B. afzelii and B. garinii strains (P = 0.023), as well as between B. afzelii and B. burgdorferi sensu stricto strains (P = 0.001), while no statistically significant difference was found comparing the growth of B. garinii and B. burgdorferi sensu stricto strains (P = 0.807).
The Kruskal-Wallis test showed statistically significant differences in the growth at 28°C between three Borrelia species (B. afzelii, B. garinii, and B. burgdorferi sensu stricto) after three days of incubation (P<0.001).
The Mann-Whitney test demonstrated statistically significant difference between the growth of B. afzelii and B. garinii (P<0.001) as well as B. burgdorferi sensu stricto (P<0.001), while no significant difference was found between B. garinii and B. burgdorferi sensu stricto (P = 0.868).
The Mann-Whitney test demonstrated statistically significant difference comparing the growth of B. afzelii and B. garinii (P<0.001) as well as B. afzelii and B. burgdorferi sensu stricto strains (P<0.001), while no significant difference was found comparing the growth of B. garinii and B. burgdorferi sensu stricto strains (P = 0.490).

Discussion
Several factors influence in vitro borrelia growth such as medium ingredients and its pH, temperature of incubation, the presence of contaminants, sample's cell density, number of borrelia strains in the sample, antecedent antibiotic therapy, capacity of particular Borrelia species to grow, etc. [10,12,14,22,24,25]. Since there are many differences between bacterial growth in vivo and in vitro, the in vitro conditions and consequently the findings of the in vivo studies are only a rough approximation of the in vivo situation [26]. Therefore, the results of the in vitro studies can be used as a basis for understanding of the in vivo events but not as a proof that in vitro and in vivo findings are equivalent.
In the present study, we analyzed in vitro growth of 10 B. afzelii, 10 B. garinii, and 11 B. burgdorferi sensu stricto strains at different temperatures within the temperature range from 4 to 37°C. Even though similar in vitro studies have been reported previously [12,13] their findings were limited due to a very small number of examined strains.
Temperature is one of the most important factors influencing bacterial growth. The optimal temperature for borrelia in vitro growth was reported to be in a rather wide range from 30 to 37°C [10]; borreliae are normally cultivated at temperatures 30 to 34°C, most often at 32 to 33°C [9,13,14,16,18,22,27].
Hubálek et al. [12] reported on differences in optimal growth temperatures for distinct Borrelia species. The authors tested only three borrelia strains (one B. afzelii, B. garinii and B. burgdorferi sensu stricto, respectively) and found that the optimal temperature for strain B31 (B. burgdorferi sensu stricto) was 33°C, for strain BR75 (B. afzelii) 35°C, and for strain BR14 (B. garinii) 37°C. Heroldová et al. [13] tested the same B31 strain and confirmed that 33°C is the optimal growth temperature for the strain, but the strain showed good growth also at 28, 30, 35 and 37°C. Since results based on one strain may not represent a typical finding for individual Borrelia species we analyzed larger number of strains that included 10 B. afzelii, 10 B. garinii, and 11 B. burgdorferi sensu stricto strains ( Table 1). The best growth for all three Borrelia species was ascertained at 33°C; a good growth was observed at 37°C, while growth was weak at 28 and 23°C, and not detectedat 4°C (Figs 1-3). The finding that all three Borrelia species survived at 23, 28, 33, and 37°C during three days' incubation suggests that temperature range 23 to 37°C can be suitable for transport of clinical material to the laboratory and that these temperatures should not have a substantial negative impact on the survival of borreliae in the sample as well as on their further isolation and cultivation.
Among three Borrelia species, the growth of B. afzelii was weaker than the growth of B. garinii or B. burgdorferi sensu stricto, while the growth of B. garinii and B. burgdorferi sensu stricto was comparable. These findings were valid for temperatures 23, 28, 33 and 37°C. As expected, no growth was observed at 4°C (Fig 4A-4E).
It is well known that B. afzelii and B. garinii are mostly associated with skin manifestations (erythema migrans and acrodermatitis chronica atrophicans) and nervous system involvement (Lyme neuroborreliosis), respectively, while B. burgdorferi sensu stricto is associated with joint involvement (Lyme arthritis), which is more often observed in North America than in Europe [1,2]. Some authors suggested that differences in Borrelia species organotropism may be due to distinct tissue temperature [11,12]. Hubalek et al. [12] tested in vitro growth of three different pathogenic Borrelia species at different temperatures and showed that B. garinii was the most thermotolerant species, followed by B. afzelii, while B. burgdorferi sensu stricto was the least theromtolerant. Thus, "heat-stable" Borrelia species affect warmer regions of the body (e.g. B. garinii is mostly associated with Lyme neuroborreliosis), while "heat-sensitive" borrelia strains involve body regions with lower temperature (e.g. B. afzelii is mostly associated with skin manifestations) [11]. However, the results of the present study do not support previous hypothesis that different Borrelia species organotropism could be a consequence of distinct tissue temperature.
Generally, difference in growth between borrelia strains at different temperatures may be interpreted as natural borrelia characteristic.
We demonstrated that the initial number of borrelia cells/mL had statistically significant positive influence on the growth of all three Borrelia species (B. afzelii, B. garinii, and B. burgdorferi sensu stricto) at all tested temperatures, except at 4°C (Table 3). This findings suggest that a large number of borrelia cells/mL present in a clinical sample has good chances to survive, adapt and grow during in vitro incubation while specimen with small number of borrelia/mL may adversely influence borrelial isolation rate and the outcome of cultivation [14,22].

Conclusions
Our study revealed that 23, 28, 33 and 37°C were suitable for borrelia growth and survival. For all three Borrelia species (B. afzelii, B. garinii, and B. burgdorferi sensu stricto), the optimal temperature was 33°C, followed by 37, 28, and 23°C. There was no statistical significant difference between the growth of B. garinii and B. burgdorferi sensu stricto at 23, 28, 33 and 37°C, respectively, while the growth of B. afzelii species was weaker in comparison to the other two species. The initial number of borrelia cells/mL influenced on growth of all three Borrelia species at all tested temperatures, except at 4°C, suggesting that the large number of borrelia is important for successful cultivation. Reported difference in growth of borrelia strains at different temperature may be interpreted as natural borrelia characteristic.