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

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 groups^{1,2,3,4,5,6,7}. The LB group (also called the Lyme di....

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 cult.......

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 possible^{23,24,.......}

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