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JoVE Journal Immunology and Infection

Immunology and Infection

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

Published: November 25, 2022 doi: 10.3791/64431
Automatic Translation
*1, *2, 2, 3, 1, 2, 3
1Department of Biology, Mt. Allison University, 2Department of Molecular Biology, Umeå University, 3Biology Centre Czech Academy of Sciences, Institute of Parasitology
* These authors contributed equally

Summary

In vitro culture is a direct detection method for the presence of living bacteria. This protocol describes methods for the culture of diverse Borrelia spirochetes, including those of the Borrelia burgdorferi sensu lato complex, relapsing fever Borrelia species, and Borrelia miyamotoi. These species are fastidious and slow growing but can be cultured.

Abstract

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently reclassified into Borreliella, the relapsing fever (RF) group Borrelia, and a third reptile-associated group of spirochetes. Culture-based methods remain the gold standard for the laboratory detection of bacterial infections for both research and clinical work, as the culture of pathogens from bodily fluids or tissues directly detects replicating pathogens and provides source material for research. Borrelia and Borreliella spirochetes are fastidious and slow growing, and thus are not commonly cultured for clinical purposes; however, culture is necessary for research. This protocol demonstrates the methodology and recipes required to successfully culture LB and RF spirochetes, including all recognized species from B. burgdorferi s.l. complex including B. afzelii, B. americana, B. andersonii, B. bavariensis, B. bissettii/bissettiae, B. burgdorferi sensu stricto (s.s.), B. californiensis, B. carolinensis, B. chilensis, B. finlandensis, B. garinii, B. japonica, B. kurtenbachii, B. lanei, B. lusitaniae, B. maritima, B. mayonii, B. spielmanii, B. tanukii, B. turdi, B. sinica, B. valaisiana, B. yangtzensis, and RFspirochetes, B. anserina, B. coriaceae, B. crocidurae, B. duttonii, B. hermsii, B. hispanica, B. persica, B. recurrentis, and B. miyamotoi. The basic medium for growing LB and RF spirochetes is the Barbour-Stoenner-Kelly (BSK-II or BSK-H) medium, which reliably supports the growth of spirochetes in established cultures. To be able to grow newly isolated Borrelia isolates from tick- or host-derived samples where the initial spirochete number is low in the inoculum, modified Kelly-Pettenkofer (MKP) medium is preferred. This medium also supports the growth of B. miyamotoi. The success of the cultivation of RF spirochetes also depends critically on the quality of ingredients.

Introduction

Borrelia is a genus of spirochete bacteria that encompasses three major clades: the Lyme borreliosis (LB) group, the relapsing fever (RF) group, and a less well-characterized group seemingly restricted to reptiles. Borrelia taxonomy is in flux with the advent of molecular methodologies that allow genome and proteome comparisons, as is true of most other taxonomic groups1,2,3,4,5,6,7. The LB group (also called the Lyme disease group) has traditionally been termed Borrelia burgdorferi sensu lato after its best-characterized member Borrelia burgdorferi sensu stricto. This paper uses the currently most widely used terminology: the LB, the RF, and the reptile-associated group, and describes the culture protocols for the LB and the RF groups.

As would be expected for a member of the family Spirochaetaceae, Borrelia can adopt the distinctively long and thin spiral shape, typically 20-30 µm long and 0.2-0.3 µm wide. However, Borrelia cells are highly pleomorphic and can adopt many other shapes in both culture and in vivo1,8 as a result of their complex cellular and genetic structure. In its spirochetal form, the planar sine wave morphology results from its axial endoflagella rotating in the periplasmic space between the inner and outer membranes. This structure enables the cells to be highly motile, with the outer membrane containing proteins that enable the cell to interact with host tissues9,10. The expression of the outer membrane proteins is tightly regulated and affect not only host tissue invasion but also interaction with the host immune system11. This complex gene expression allows the Borrelia cells to shuttle between the very different environments of vertebrate hosts and invertebrate vectors. The genome of Borrelia is unusual among prokaryotes, consisting of a linear rather than a circular chromosome. In addition to the linear chromosome, Borrelia species contain 7-21 plasmids, some linear and some circular. The plasmids harbor the majority of genes required for host adaptation and virulence, and the circular plasmids derived from prophages are thought to be responsible for the majority of horizontal gene flow between spirochetal cells12,13. Consistent with a role in host adaptation, some, possibly many or all, members of the Lyme borreliosis group lose plasmids in culture14. The best studied "lab adapted" strain of B. burgdorferi, B31, has only seven of the nine plasmids found in wild isolates of this species15. Similarly, B. garinii loses plasmids in culture16. Some studies have shown that RF species and B. miyamotoi retain plasmids when cultured14,17, but recent work demonstrates altered plasmids and infectivity with long-term in vitro cultivation18.

Culture-based methods remain the gold standard for the laboratory detection of bacterial infections, for both research and clinical work14,17. The culture of pathogens from bodily fluids or tissues directly detects replicating pathogens and provides source material for research14,17. This protocol demonstrates the methodology and recipes required to successfully culture the spirochetes of the LB group as well as RF Borrelia and B. miyamotoi. The basic medium for growing Borrelia spirochetes is the Barbour-Stoenner-Kelly medium (BSK-II or the commercially available BSK-H), with or without antibiotics to reduce the growth of contaminating prokaryotes. This medium was adapted from a medium originally used to support RF Borrelia19, further modified by Stoenner20 and then by Barbour21. Many modifications have since been developed, each having effects on bacterial physiology that can affect growth, infectivity, and pathogenicity22. This medium reliably supports the growth of spirochetes in established cultures and has been used to isolate spirochetes from tick, mammal, and clinical samples23. The more recently developed variation, modified Kelly-Pettenkofer (MKP) medium, can provide better isolation success, morphology, and motility when isolating new Borrelia isolates from environmental samples, when the number of spirochetes present in the sample available to seed the culture is low23,24. In all cases, the success of cultivation is critically dependent on the freshly prepared medium and the use of appropriate ingredients; not all commercial ingredients produce high-quality medium. Inoculated cultures can be conveniently incubated without shaking in a conventional 32-34 °C incubator in the presence of a small amount of residual ambient oxygen. Borrelia spirochetes are anaerobes but are exposed in nature to fluctuations in oxygen and carbon dioxide concentrations and respond with changes in gene expression26,27,28,29. Thus, gene expression, growth, and other metabolic studies should control for oxygen and carbon dioxide levels with the use of an oxygen-controlled incubator or anaerobic chamber. In culture, cultures are checked weekly, or more often, for the presence of spirochetes with either darkfield microscopy or phase contrast microscopy. Culture smears can be stained with either silver stains, immunohistochemistry, or through the use of fluorescently tagged strains29,30. PCR followed by DNA sequencing is a sensitive and specific method to detect and genetically identify or confirm the Borrelia species30,31,32,33.

Many minor variations on BSK-II exist, and some are commercially available. The protocol described here in section 1 has been adapted from Barbour (1984)21. Liquid MKP medium is a more recently developed medium and is described in section 2. It is prepared according to a previously reported protocol33,34, which, similarly to BSK medium, consists of two steps: preparation of basic medium and preparation of complete medium. Borrelia culture medium can be prepared with or without antibiotics, as described in section 3; the antibiotics act to reduce contaminating bacteria introduced when inoculating with clinical or environmental samples, as described in section 4; if inoculating with pure Borrelia sp. culture, antibiotics may not be needed. Making long-term Borrelia stocks is often important, and a protocol for doing so is described in section 5. Section 6 describes using these media to isolate pure Borrelia sensu lato clones from clinical or environmental samples. There are a number of approaches possible36; below is one found to be effective. The plating medium used in this protocol is a modification of BSK-II plating medium37 and MKP medium34 (with rabbit serum increased to 10%38).

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Protocol

All studies involving samples obtained from human subject were approved by the Institutional Review Board of the relevant University and/or medical facility, and written informed consent was obtained from participants before the samples were collected. All studies involving samples obtained from animals were approved and conducted under the guidelines of the Institutional Animal Ethics Committee. Where relevant, approvals have been obtained for environmental sampling.

NOTE: The success of culture is critically dependent on the quality of ingredients. Commercial BSK medium is available and robustly supports the growth of lab-adapted pure B. burgdorferi and related species where high dose inoculum can be used to seed the culture. For isolates of strains from primary biological material, freshly prepared medium is preferred and often necessary.

Borrelia sp. are known or suspected pathogens, and unscreened human or animal tissues can also be a biohazard. Handling of potentially infectious material requires personal protective equipment and sterile technique for investigator safety. Additionally, the medium is so nutrient-rich that the culture is easily contaminated. Biosafety level 2 containment is generally required for this work and all work with Borrelia sp. or samples should be conducted in a biological safety cabinet.

1. Instructions for making BSK-II medium

  1. Preparation of complete BSK-II medium
    1. Obtain reagents to prepare 1 L of the medium, as shown in Table 1.
      NOTE: BSA purity (or lack of purity) is critical for optimal growth. In particular, BSA purity differs between both manufacturer and lot. Please see Table 2 for a list of more versus less successful products. Obtaining several samples from different vendors, testing them for optimal growth, then buying a large quantity of the best lot is an effective strategy to mitigate this problem and produce a good medium.
    2. Start with dissolving the BSA in a beaker by stirring in the water, which will take at least half an hour.
    3. Add all the dry chemicals and let them dissolve. Then add the CMRL.
    4. Adjust the pH of the BSK-II medium to 7.6, if necessary, with 1 M NaOH.
    5. Add rabbit serum to the BSK-II medium to a concentration of 6%-10%. Filter sterilize (0.22 µm pore size) the BSK-II medium. The required amount of serum depends on the Borrelia species. Use 6%-7% for the RF species and strains, and for Borrelia burgdorferi sensu lato. If growth is not strong, add another 1%-2% sterile rabbit serum to the medium.
    6. Freeze the stock of BSK-II medium in 100 mL or 400 mL aliquots.
    7. For RF strains, gelatin is required. Add 100 mL of 7% sterile gelatin (slightly warmed to put it into a solution) to 400 mL BSK-II medium. Warm the BSK-II with gelatin at 37 °C to make the gelatin melt before inoculating the culture.
      NOTE: Medium can be frozen (-20 °C) prior to filter sterilization and the addition of rabbit serum and gelatin (for RF spirochetes) or after filter sterilization and serum (and gelatin) addition. The medium is stable when frozen for a long time. When stored in a fridge, use the medium within 1 month. RF species grow best with fresh medium, usually not more than 1 week old. If not frozen, visually inspect the quality of the medium for turbidity. Discard the medium that appears to be contaminated, precipitating ingredients, or otherwise dubious.
    8. If antibiotics are to be added to the medium, add them to freshly made medium or previously frozen aliquots; the latter is the more flexible approach. Use antibiotic concentrations as described by Oliver et al.39.
    9. While thawing a tube of the medium, weigh antibiotics into sterile (autoclaved) 1.7 mL tubes or sterile 15 mL conical tubes, using a precision balance. Prepare the stock solutions as follows: Dissolve 25 mg of amphotericin B in 10 mL of DMSO, 20 mg of phosphomycin in 1 mL of autoclaved distilled water, and 50 mg of rifampicin in 1 mL of DMSO.
    10. Filter sterilize (0.2 µm syringe filters) the antibiotics and transfer them into a new tube of appropriate size.
    11. Dilute the antibiotics in a ratio of 1:1000 of the medium to reach their final concentration. For 500 mL of BSK, add 0.5 mL of amphotericin B stock solution (2.5 mg/mL in DMSO), 0.5 mL of phosphomycin stock solution (20 mg/mL in sterile H2O), and 0.5 mL of rifampicin (US: rifampin) stock solution (50 mg/mL in DMSO).
    12. Use the antibiotic-supplemented medium or aliquot and freeze at -20 °C.
Constituents Amount (/L)
BSA fraction V 50 g
CMRL-1066 (10x) 100 mL
Neopeptone 5 g
Hepes 6 g
Citric acid trisodium salt dihydrate 0.7 g
Glucose 5 g
TC Yeastolate 2 g
Pyruvic acid (Na salt) 0.8 g
N-acetyl-D-glucoseamine 0.4 g
Sodium bicarbonate 2.2 g
Water (high purity) 900 mL

Table 1: Composition of 1 L of BSK-II medium.

Source Suitability
Serva GmbH, Heidelberg, Germany Yes
Proliant Biologicals, Boone IA, USA Yes
Sigma (Millipore-Sigma & Sigma- Aldrich), St. Louis, MO, United States No

Table 2: Sources of BSA and suitability for BSK-II medium.

2. Instructions for making MKP

  1. Preparation of basic medium
    1. Carefully weigh and dissolve all powdered components as listed below in Table 3 in 3/4 of the final volume of ddH2O (ca. 750 mL) by stirring until complete dissolving, which takes ~1 h.
    2. After complete dissolving, adjust the pH with NaOH to 7.6 and the volume to 1000 mL, and then sterilize by filtration (0.22 µm filter).
      NOTE: Basic medium can be stored at -20 °C for up to 3 months.
  2. Preparation of MKP complete medium
    1. Mix all components as listed in Table 4 in a sterile manner; sterilize the gelatin by autoclaving and handle in a sterile manner. Work in a sterile biological safety cabinet and use a sterile filtered serum.
    2. Aliquot MKP in 50 mL tubes. Put one small aliquot at 33 °C for several days, then check under a darkfield microscope for the absence of contamination.
      NOTE: Keep MKP complete medium at +4 °C for no more than 1 month, and do not freeze. The complete medium can be additionally sterilized by filtration using a 0.22 µm filter.
Constituents Amount (/L)
CMRL-1066 (10x without glutamine) 100 mL (if liquid)/9.7 g/L if powder
Neopeptone 3 g
Hepes 6 g
Citric acid 0.7 g
Glucose 3 g
Pyruvic acid 0.8 g
N-acetylglucoseamine 0.4 g
Sodium bicarbonate 2 g

Table 3: Composition of 1 L of basic MKP (Modified Kelly-Pettenkofer) medium.

Constituents Amount (mL)
Basic medium 500
7% gelatin (in H2O) (freshly autoclaved but not hot) 100
Rabbit serum 36
35% BSA 17.5

Table 4: Composition of MKP (Modified Kelly-Pettenkofer) complete medium.

3. Culture of Borrelia, with or without antibiotics

  1. Perform all experimental steps under sterile working conditions in a biological safety cabinet. Disinfect the surface and all materials with 70% ethanol, and transfer them immediately to the biological safety cabinet. UV sterilize all non-biological materials in the biological safety cabinet for 5 min before the start of the work.
  2. Use the freshest medium possible.
  3. To start a culture, pre-warm the medium (33 °C - 37 °C) in an incubator. Apply shaking if needed.
    NOTE: Depending on the volume of medium, this step may take several hours.
  4. Add ~1 mL of thawed Borrelia culture from a glycerol or similar stock, previously stored at -80 °C and thawed at room temperature (RT), as soon as it is liquid, to 5-15 mL of pre-warmed BSK medium. Inoculate the culture into a small volume (5-15 mL of BSK) using commercially available glass or plastic vials with screw caps. Fill the tubes almost full to make a micro- or anaerobic atmosphere. Close the tubes tightly; if cultured in this manner, CO2 is not needed.
  5. Grow RF species (and B. persica) at 37 °C in an incubator without stirring or agitation. Grow B. burgdorferi sensu lato species at 33-34 °C.
  6. Monitor the growth of the Borrelia culture using phase contrast or darkfield microscopy at 200x-400x magnification (Figure 3). To ensure even distribution of cells, mix the culture with a serological pipette or for more mixing with a 3 mL transfer pipette while pipetting up and down.
    NOTE: Borrelia are most visible at higher magnification (200X-400X), but are best countable at 200X magnification on a hemocytometer40. Borrelia movement and morphology are indicative of the presence of these bacteria.
  7. To quantify bacterial growth, count several (10) individual small squares in the sixteen-field grid pattern of counting area on both sides of the hemocytometer and average. Multiply the average cell number per small square with the conversion factor appropriate for the hemocytometer being used to determine the cell count per mL.
  8. Monitor the bacterial growth at 1-2 day intervals. The exponential growth of Borrelia is dependent on the viability and cell density and may take 1 week or longer at 34 °C. Dilute the exponential phase cultures at a ratio of 1:2 or 1:4 with the medium in a 1.7 mL tube before loading on the hemocytometer. Include appropriate consideration of the dilution factor when determining the cell count. After use, spray the surfaces and hemocytometer with diluted bleach (10%) and/or 70% ethanol.
    ​NOTE: Borrelia are in exponential growth when cell concentrations are 1-5 x 107 cells/mL. Within this range, the cells grow well.
  9. Extended culture will deplete nutrients and change medium pH so subculturing is required. Under sterile conditions in a biological safety cabinet, transfer 1/10 of the culture volume into a new tube and fill it with the new medium (a fresh aliquot of the same medium used to grow the previous passage). A 1:10 dilution is optimal. If culture volume is to be changed, adjust the inoculum amount accordingly, and continue incubation. The number of passages increases with each medium change.
  10. For extraction of DNA or isolation of Borrelia cells, centrifuge the exponential phase Borrelia for 10 min at 4000 x g to pellet the bacteria. Discard the supernatant in suitable biohazard waste. Process the pelleted bacteria with a commercial DNA extraction kit or resuspend the bacteria in an appropriate medium for the intended experiment.
  11. At the end of each manipulation, sterilize all equipment with ethanol and UV radiation, as appropriate for the equipment. Collect the liquid waste, including excess culture, in a waste bottle and add bleach to a final concentration of 10%. Place this bottle at -80 °C before discarding it. Alternatively, sterilize the liquid waste and culture without bleach or alcohol by autoclaving.

4. Long-term storage of Borrelia cultures

  1. Preparation of glycerol stock of Borrelia culture
    1. Take ~1.8 mL aliquots of exponential phase cultures, cell concentrations ~107-108 cells/mL, and add sterile glycerol to a final concentration of 10% (concentrations of 5%-20 % work).
    2. ​Place in 2 mL screw cap cryogenic vials. Store at -80 °C.
    3. Maintain a stock with at least 10 tubes of each strain in the freezer.
  2. An alternate storage recipe to produce a glycerol stock for permanent storage
    1. Mix 2/3 of Borrelia culture with 1/3 of 15% glycerol in Brucella broth. Use 2.8 g of Brucella broth dissolved in 100 mL of ddH2O, which was sterile filtered (0.22 µm).
    2. Keep the glycerol stock of samples at -70 °C to -80 °C.

5. Isolation of spirochetes from environmental or clinical sources

NOTE: If isolating material from human, animal, or environmental sources, research ethics, animal care, or ecological certification, as appropriate, must be obtained.

  1. Cultivation of Borrelia from hard ticks
    1. Collect adult females, males, and/or nymphal ticks.
    2. Surface sterilize all tick samples by dipping them into 70% ethanol for 2 min and air-dry in a biological safety cabinet. Dissect individually into several pieces with a sterile scalpel. Place the pieces into a 2 mL tube containing 1.5 mL of medium, supplemented with antibiotics.
    3. Incubate the cultures at 34 °C and check for spirochetes by darkfield or phase contrast microscopy twice weekly for the first 2 weeks and weekly thereafter for 6 weeks (more often for RF spirochetes). Subculture culture-positive samples in 5 mL of the same medium.
      NOTE: Contaminated cultures can be purified to some extent by filtration using a 0.2 µm filter; antibiotics may be required to fully resolve contamination.
  2. Cultivation of Borrelia from blood samples
    NOTE: The blood sample should be taken by a qualified medical, veterinary, or research professional, depending on the source of the material. Less than 24 h at RT should elapse between sample
    removal and arrival in the lab.
    1. Obtain 10 mL of blood, collected into glass blood collection tubes with acid citrate dextrose (ACD; "yellow top") as an anticoagulant. Leave at room temperature. It is recommended that less than 24 h pass between blood collection and inoculation.
    2. Centrifuge the blood with anticoagulant for 10 min at 200-400 x g to separate plasma from blood cells.
    3. Gently remove the plasma from the tube and inoculate 1 mL of plasma into 5 mL of pre-heated MKP complete medium. MKP can be supplemented with antibiotics. Incubate cultures at 33 °C with daily visual inspection and weekly darkfield or phase contrast microscopy until borrelial growth is noticed (more often for RF Borrelia), which can take up to 9 weeks.
      NOTE: If using larger or smaller inoculation volumes, maintain the 1:5 ratio of inoculant to medium. Use sera or plasma rather than whole blood.
  3. Cultivation of Borrelia from skin biopsy
    1. Surface sterilize the skin biopsy (ideally a 3 mm x 3 mm x 3 mm cube; i.e., with 70% ethanol and hydrogen peroxide). Place the biopsy sample in physiological saline.
      NOTE: A biopsy sample should be taken by a qualified medical, veterinary, or research professional, depending on the source of the material. Less than 24 h at RT should elapse between sample removal and arrival in the lab.
    2. Dice the sample into two to six smaller pieces using a sterile razor blade in a sterile glass Petri dish while immersed in the physiological saline. Transfer all of the samples and ~1 mL of the physiological saline in which the skin biopsy was stored into 5 mL of prewarmed medium supplemented with antibiotics and incubate at 33 °C.
    3. In case of overgrowth of contaminating bacteria, add antibiotics as in step 1.1.10 above, or by adding two tablets of sulfamethoxazole (23.75 mg; final concentration 5 mg/mL) + trimethoprim (1.25 mg; 0.25 mg/mL) or Bactrim (minimum inhibitory concentration >128 µg/mL), two tablets of Amikacin (2 x 30 µg; final concentration 12 µg/mL), and two tablets of Phosfomycine (2 x 200 µg; final concentration 80 µg/mL).
      NOTE: In all cases, be sure to remain below the minimum inhibitory concentration of that antibiotic for the Borrelia species for which culture is being attempted41,42,43.
    4. If the culture is positive for Borrelia, keep the culture for an additional 3-4 days or until the amount of Borrelia reaches 10+ spirochetes in a microscope field using a 40X objective. Create a glycerol stock for permanent storage.

6. Isolation of monoclonal populations of  B. burgdorferi  sensu lato spirochetes and  B. miyamotoi  from liquid cultures by cultivation on solid media

  1. For making plates, warm 300 mL of 1.5X MKP complete medium (without gelatin). Also, warm 200 mL of 1.7% agarose (autoclaved in deionized water, stored at RT, and microwaved before use) to 55 °C. Mix both liquids.
  2. Pipette 15 mL of this mixture into each of 16 deep Petri dishes to make the bottom agar oselayer.
  3. Retain the rest of the medium at 55 °C in a water bath.
    NOTE: The Borrelia are mixed into the top agarose layer.
  4. First, quantify Borrelia culture density using a hemocytometer and dilute to the desired density in a liquid medium.
    NOTE: 500, 200, 100, and 50 spirochetes per plate work well.
  5. After the bottom plates have solidified, add 10 mL of the remaining agarose mixture to a 15 mL tube containing the appropriate number of spirochetes.
  6. Pour the suspension onto the bottom agarose in the labeled Petri dish.
    NOTE: Since Borrelia spirochetes are sensitive to high temperature, care should be taken to ensure that the bacteria are not exposed to 55 °C for an extended period of time; pour the top agar immediately upon adding it to the bacterial cells in the medium.
  7. After the plates are completely solidified, close the Petri dishes and place them upside down at 34-35 °C in 2.5% CO2.
    NOTE: The colonies will appear 1 week after plating if the procedure is successful.
  8. To screen and expand the individual colonies, select single colonies from the plates and put them into 1.5 mL of modified liquid MKP, or other suitable media, in sterile 2 mL culture tubes.
  9. Incubate the cultures at 34 °C and examine for spirochetes by darkfield or phase microscopy. Use these cultures for analysis or stocks, made as described above.

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Representative Results

Borrelia culture media BSK and MKP, and variants, are rich culture media with ingredients that need to be prepared and sterilized sequentially. When correctly prepared, BSK medium should be red-orange and clear (Figure 1). Turbidity and precipitation that persist after warming indicate problematic ingredients, medium production, or contamination; such medium is best discarded. If gelatin is added to BSK and with MKP, the medium will be gelatinous when refrigerated; upon warming, it will be liquid, although slightly viscous.

The growth of pure Borrelia cultures will not change the turbidity of the medium. However, the growth of bacterial cultures, Borrelia, as well as contaminants, will change medium color as a result of the pH indicator. Overgrowth by other bacteria will produce turbidity and clumped materials (Figure 2).

Culture growth can be monitored by phase contrast or darkfield microscopy (Figure 3 and Figure 4). Borrelia cells in the exponential phase are typically slender and kinked, and motile to some extent. As the cultures go into the stationary phase, round bodies are increasingly evident (Figure 5). Clinical or environmental isolates frequently contain multiple Borrelia strains and/or species. To isolate pure clones, liquid cultures can be plated to isolate monoclonal colonies (Figure 6); sometimes, multiple rounds of colony isolation may be needed.

Figure 1
Figure 1: BSK and MKP medium. (A) BSK (with antibiotics) with a pure culture of Borrelia burgdorferi. With or without B. burgdorferi medium remains without visible turbidity and is orange-amber (color varies slightly with ingredients). (B) MKP medium, uninoculated, thawing. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Contaminated BSK and MKP medium with overgrowth of competing bacteria. Medium is discolored, turbid, and over time competing bacteria die and precipitate to the bottom of the tube. (A) Overgrown BSK tube. (B) Old BSK tube with precipitated contaminating bacteria. (C) MKP tube with (left) and without (middle) culture and contaminated culture (right). Please click here to view a larger version of this figure.

Figure 3
Figure 3: Use of a hemocytometer to monitor Borrelia growth. Borrelia burgdorferi strain B31 (a subset indicated by arrows) viewed on a hemocytometer (one line of the small grid is indicated by the arrowhead) under 200X phase contrast. This magnification and process allow monitoring of cultures and estimation of cell density. Scale bar is 10 µm. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Darkfield image of Borrelia burgdorferi. Darkfield image of Borrelia burgdorferi strain SCW-53, at high density in exponential phase after 5-7 days of cultivation in MKP medium (400X magnification). Scale bar is 20 µm. This strain was isolated from the hard tick Ixodes affinis, collected in South Carolina, USA. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Borrelia in old cultures. As Borrelia cells go into the stationary phase in old cultures, round body forms are increasingly seen to replace the spirochetal form. These may be dormant forms or dead cells. Left panel, Dieterle stain; middle panel, immunohistochemistry stain; and right panel, fluorescent in situ hybridization of a Borrelia culture (presumed to be B. burgdorferi based on origin but the species and strain was not molecularly identified) inoculated with canine urine. Collection of the data in Figure 5 was approved by the Mrt. Allison University Animal Care Committee, approval number 16-1. Please click here to view a larger version of this figure.

Figure 6
Figure 6: Borrelia burgdorferi sensu lato complex spirochetes. Colonies of Borrelia burgdorferi sensu lato complex spirochetes grown on solid medium in order to isolate pure clonal populations. Please click here to view a larger version of this figure.

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Discussion

The laboratory culture of bacteria is the springboard for research. The profound advantage conferred by the ability to culture is exemplified by the struggle, over more than a century culminating only recently in success, to culture Treponema pallidum, the spirochete that is the etiological agent of syphilis44. Borrelia spirochetes are also challenging to culture, but culture is possible23,24,34,44,45,46,47,48,49.

Borrelia cells are fastidious and each species and strain, and even isolate differs, both genetically and through adaptation to the host from which they have been isolated51,52,53,54,55. When making media, the use of appropriate ingredients make an enormous difference in the vigor of Borrelia culture or even the viability of the culture. The use of freshly prepared, highly enriched, and properly stored medium is essential to promote bacterial growth and is a well-established requirement for bacterial growth. For Borrelia, maintaining anaerobic or microaerobic conditions is required for good growth. This can be achieved most simply by filling tubes to the top, so there is little to no air remaining in the tube and growing the cultures without agitation; more stringent control of oxygen and carbon dioxide may be required for gene expression and related studies26,27,28,29,30. The addition of antibiotics to the bacterial culture medium may seem counter-intuitive; the use of low doses of antibiotics in the Borrelia medium is to discourage the growth of faster-growing bacteria. If the culture is inoculated with a pure Borrelia isolate, the addition of antibiotics is not needed; however the use of added antibiotics is essential to discourage overgrowth from competing bacteria when inoculating with clinical or environmental samples. BSK medium can also be supplemented with 3%-7% gelatin. This is needed for the growth of RF Borrelia species21. The use of BSK or MKP for the growth of Borrelia burgdorferi sensu lato species depends on the source of the isolate. Both media support the growth of established cultures, but MKP has been found to perform better when seeding new cultures with low numbers of Borrelia cells, such as occurs with clinical or environmental isolates23,24. This medium also allows the culture of B. miyamotoi56,57,58. Ultimately, there is an element of art to the science of growing fastidious microbes, but it is possible with care and persistence.

The culture of Borrelia underlies advances in biomedical research. The culture of Borrelia has allowed determination of the full genome and proteome of many Borrelia species, which has permitted researchers to start to unravel the evolution and biology of these spirochetes54,58,59. Similarly, access to pure cultures of Borrelia allows the understanding of the many complex interactions of Borrelia with the mammalian host and arthropod vector61,62,63,64,65, including interactions with the host immune system11,65, allows mechanisms of transmission to be inferred29,66, and provides the key investigative tool to distinguish culturable, hence necessarily living, cells from cellular debris, a distinction that is critical in unraveling the mechanisms of pathogenesis as well as effective treatment regimens8,67,68,69,70,71. While the in vitro Borrelia culture can take several weeks before spirochetes are sufficiently abundant for detection, limiting clinical diagnostic applications, fundamental understanding of Borrelia biology, genomics, host-pathogen interactions, and many more applications has relied on the ability to isolate pure Borrelia cultures from environmental isolates, and to amplify those isolates through culture to produce sufficient material for biochemical and genetic analysis. There is a societal push for rapid response to infections to support rapid diagnosis and treatment. The other component of this need, however, is the biomedical and biological research needed for a solid foundation upon which to appropriately and successfully treat and prevent disease. While challenging, in vitro cultivation of Borrelia spirochetes is possible and can support these needs.

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Disclosures

No conflicts of interest were declared.

Acknowledgments

This work was partially supported by the Natural Science and Engineering Research Council of Canada and the Canadian Lyme disease Foundation (AB and VL), the Swedish Research Council (SB, M-LF and IN), TAČR GAMA 2 project - "Support of application potential verification 2.0 at the Biology Centre CAS" (TP01010022) (MG and NR), and by a grant from Ministry of Health of the Czech Republic NV19-05-00191 (MG and NR). We thank S. Vranjes (2021, Rudenko lab) for the image in Figure 4 and J. Thomas-Ebbett (2021, Lloyd lab) for the images in Figure 5. We thank all researchers who have contributed to the field and apologize to those whose work we were not able to cite due to space limitations.

Materials

Name Company Catalog Number Comments
1.7 mL tubes VWR 87003-294 Standard item - any supplier will do
0.2 µm Sterile syringe filter  VWR 28145-501 Standard item - any supplier will do
10 µL barrier pipette tip Neptune BT10XLS3 Standard item - any supplier will do
10 mL Serological pipettes  Celltreat 229011B Standard item - any supplier will do
1000 µL barrier pipette tip Neptune BT1000.96 Standard item - any supplier will do
15 mL tube Celltreat 188261 Standard item - any supplier will do
20 µL barrier pipette tip Neptune BT20 Standard item - any supplier will do
20 mL Sterile syringe  BD 309661 Standard item - any supplier will do
200 µL barrier pipette tip Neptune BT200 Standard item - any supplier will do
25 mL Screw Cap Culture Tubes Fisher Scientific 14-933C Standard item - any supplier will do
25 mL Serological pipettes Celltreat 229025B Standard item - any supplier will do
3 mL Sterile syringe BD 309657 Standard item - any supplier will do
35% BSA  Sigma A-7409 Source is important - see note
5 mL Serological pipettes  Celltreat 229006B Standard item - any supplier will do
50 mL tube Celltreat 229421 Standard item - any supplier will do
6.5 ml MKP glass tubes  Schott Schott Nr. 26 135 115 Standard item - any supplier will do
Amikacine Sigma PHR1654 Standard item - any supplier will do
Amphotericin B Sigma A9528-100MG Standard item - any supplier will do
Bactrim/rimethoprim/sulfamethoxazole Sigma PHR1126-1G Standard item - any supplier will do
BBL Brucella broth  BD 211088 Standard item - any supplier will do
Biosafety Cabinet Labconco 302419100 Standard item - any supplier will do
Blood collection tubes (yellow top - ACD) Fisher Scientific BD Vacutainer Glass Blood Collection Tubes with Acid Citrate Dextrose (ACD) Standard item - any supplier will do
BSK-H Medium [w 6% Rabbit serum]  Darlynn biologicals BB83-500 Standard item - any supplier will do
centrifuge  Eppendorf model 5430 Standard item - any supplier will do
Citric acid TrisodiumSaltDihydrate Sigma C-8532 100 g Standard item - any supplier will do
CMRL Gibco BRL 21540 500 mL Standard item - any supplier will do
CMRL-1066 Gibco 21-510-018 Standard item - any supplier will do
Cryogenic Tubes (Nalgene) Fisher Scientific 5000-0020 Standard item - any supplier will do
Deep Petri with stacking ring 100 mm × 25 mm Sigma P7741 Standard item - any supplier will do
Digital Incubator VWR model 1545 Standard item - any supplier will do
DMSO ThermoFisher D12345 Standard item - any supplier will do
Filters for filter sterilization Millipore 0.22μm GPExpressPLUS Membrane SCGPU05RE Standard item - any supplier will do
Gelatin Difco BD 214340 500 g Standard item - any supplier will do
Glass Culture Tubes Fisher Scientific 99449-20 Standard item - any supplier will do
Glucose Sigma G-7021 1 kg Standard item - any supplier will do
Glycerol Sigma G5516 Standard item - any supplier will do
Hemafuge (Hematocrit & Immuno hematology centrifuge ) Labwissen Model 3220 Standard item - any supplier will do
HEPES Sigma  H-3784 100 g Standard item - any supplier will do
N-acetylglucoseamine Sigma  A-3286 25 g Standard item - any supplier will do
Neopeptone Difco  BD 211681 500 g Standard item - any supplier will do
Neubauer Hematocytometer Sigma  Z359629 Standard item - any supplier will do
Phase contrast microscope  Leitz Standard item - any supplier will do
Phosphomycin Sigma P5396-1G Standard item - any supplier will do
Phosphomycine Sigma P5396 Standard item - any supplier will do
Pipetboy Integra Standard item - any supplier will do
Precision Standard Balance OHAUS model TS200S Standard item - any supplier will do
Pyruvic acid (Na salt) Sigma P-8574 25 g Standard item - any supplier will do
Rabbit Serum  Gibco 16-120-032 Source is important 
Rabbit Serum  Sigma R-4505  100 mL Source is important 
Rifampicin Sigma R3501-1G Standard item - any supplier will do
Sodium bicarbonate Sigma S-5761     500 g Standard item - any supplier will do
Sufametaxazole  Sigma PHR1126 Standard item - any supplier will do
TC Yeastolate Difco  BD 255752 100 g Standard item - any supplier will do
Transfer Pipettes VWR 470225-044 Standard item - any supplier will do
Trimethoprim Sigma PHR1056 Standard item - any supplier will do

DOWNLOAD MATERIALS LIST

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Tags

Spirochetes Borrelia Burgdorferi Relapsing Fever Borrelia Culture Methods Bacteria Detection Lyme Borreliosis Group Relapsing Fever Group Reptile Borrelia Pathogenic Borrelia Species Bacterial Culture Body Fluid Culture Tissue Culture Spirochete Culture Borrelia Culture Protocol Culture And Preservation Methods Borrelia Miyamotoi Species Isolation And Culture Strain Isolation And Culture Environmental Isolates Mammalian Isolates BSK-II Medium Preparation Reagents For BSK-II Medium
Cultivation Methods of Spirochetes from <em>Borrelia burgdorferi</em> Sensu Lato Complex and Relapsing Fever <em>Borrelia</em>
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Berthold, A., Faucillion, M. L.,… More

Berthold, A., Faucillion, M. L., Nilsson, I., Golovchenko, M., Lloyd, V., Bergström, S., Rudenko, N. Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia. J. Vis. Exp. (189), e64431, doi:10.3791/64431 (2022).

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