DE102006045252B4 - Protein-free medium for the cultivation of Borrelia and its use - Google Patents

Abstract

Method for culturing bacteria of the Borrelia genus, characterized in that the bacteria are brought into contact with a protein-free culture medium which contains at least one chemically defined fatty acid or at least one chemically defined fatty acid ester and at least one chemically defined zinc (II) compound and supports the growth of bacteria.

Description

The present invention relates to protein-free culture media which are suitable for culturing, in particular propagation, of bacteria of the genus Borrelia, and their use for culturing, in particular propagation, of bacteria of the genus Borrelia. Furthermore, the present invention relates to culture media based on the described culture media, which can be obtained by supplementation with defined components, and their use for culturing, in particular propagation, of bacteria of the genus Borrelia.

State of the art:

Borrelia bacteria are described as causing several human diseases, in particular as Lyme disease and relapsing fever. For their proof as well as for the research z. As the infection mechanism and the development of vaccines, they are regularly propagated in vitro.

For the in vitro cultivation of Borrelia, essentially complex culture media are used which contain natural, chemically undefinable components such as proteinaceous mammalian serum fractions and complex protein mixtures, e.g. As proteolytically treated muscle extracts, yeast extracts or gelatin. As an embodiment, the commercially available variant with the name Complete BSK-H is mentioned (product information B8291, Sigma-Aldrich). Similar compositions are described in the literature as MKP and BSK II (Ruzic-Sabljic et al., 2004, Int J Med. Microbiol.).

Disadvantageously, the chemically undefinable components of these media are subject to large variations in their compositions from batch to batch, carry the risk of contamination with viruses or mycoplasma, are expensive to manufacture and complicate the study of secretion processes and effects of chemically defined components on the cultured Borrelia and the genetic modification of borrelia.

Accordingly, in vitro cultured Borrelia usually show, depending on the particular culture media used or depending on individual components of the culture media used in each case strong fluctuations in their growth rates and their gene expression. In particular, this affects genes which are expressed in vivo as a function of the respective host organism (Pollack et al., 1993, J. Clin. Microbiol, Yang et al., 2001, Infect. Immun.). This makes it difficult to compare the results of different laboratories cultivating Borrelia in particular.

The development of a reliable, chemically defined culture medium based on more standardized components is therefore necessary to facilitate the investigation of the influence of chemically defined components on Borrelia and their genetic modification and to standardize the results of different laboratories.

It has previously been attempted to culture Borrelia in a chemically defined, serum-free culture medium (Alban et al 2000, Microbiology). It was found that Borrelia with the described culture medium RPMI 1640 are not reproducible, but on the contrary, depending on the cultivation period, die off. Two weeks after inoculation of the medium with living Borrelia, no living Borrelia were detectable. Accordingly, this medium is not suitable for solving the problems described.

Description of the invention:

Surprisingly, it has now been found that the problems described can be solved by the subject matter of claims 1 to 15, in particular by culturing, in particular propagation, the Borrelia in serum-free, low-protein or protein-free media, so that they to a Any time after inoculation are mostly alive or able to live and reproduce. Prerequisite is the removal of spent and feeding fresh medium at appropriate intervals z. B. by centrifugation and resuspension. Those skilled in the art are aware of such needs in the cultivation of microorganisms.

The present invention makes it possible for the first time to cultivate, in particular multiply, borrelia in the absence of serum, in particular in the absence of proteins.

Borrelia were shown to be culturable in media corresponding to the compositions shown in Table 1 or Table 2 which were optionally supplemented with various chemically definable and chemically undefinable components such as phosphomycin, rifampicin, amphotericin B, bovine serum albumin (BSA) or rabbit serum , in particular reproducible, were. The growth rates were similar to those of Borrelia, which were cultured in Complete BSK-H.

In addition, it has been shown that the commonly used rabbit serum and the other chemically non-definable protein mixtures (muscle extract, yeast extract, gelatin) by a lipid mixture (L5146, Sigma-Aldrich), in addition to the cholesterol also defined in Complete BSK-H essentially fatty acids and fatty acid esters can be substituted.

Furthermore, it has been shown that the commonly used BSA can be substituted by zinc (II) compounds, in particular by zinc (II) chloride.

Evidence of the identity of the cultured organisms was obtained by Western blot analysis with rabbit rabbit antiserum specific for Outer Surface Protein C (OspC). It was found that the amount of OspC expressed was in each case approximately proportional to the amount of cultured Borrelia, which led to the conclusion that they were Borrelia.

Having described the exact composition of serum-free, low-protein or protein-free media suitable for cultivating, in particular propagating, Borrelia, it will be apparent to those of ordinary skill in the art that similar results may be obtained by modifying or altering the invention. in particular by modifying or altering the composition of the media used, without affecting the subject of the invention or any of its embodiments. Accordingly, such modifications or alterations are also subject matter of claims 1 to 15.

Examples

Example 1: Cultivation of Borrelia in serum-free medium

In each case 30 ml of a sterile-filtered medium of the composition according to Table 1 are inoculated with 2 × 10 ⁶ cells of Borrelia burgdorferi (strain B31), Borrelia afzelii (strain VS461) or Borrelia garinii (strain 20047) under the sterile safety cabinet and 37 ° C incubated in a closed vessel. During the incubation, the cell suspensions are vortexed at intervals of twelve hours each by gentle shaking. In parallel, the same number of respective organisms in each of 30 ml Complete BSK-H (B8291, Sigma-Aldrich) is inoculated and incubated identically. Samples are taken at different times and the cells counted using a suitable counting chamber in the dark field microscope.

In addition, extracted cells are resuspended in PBS (50 mM sodium hydrogen phosphate pH 7.4, 150 mM NaCl) and the amount of proteins contained analyzed by protein determination (DC assay, Biorad). Subsequently, proteins of the various borrelia samples are separated by SDS-PAGE and transfected onto a nitrocellulose membrane, stained first with Ponceau S and then incubated with an antiserum against rabbit OspC.

For specific immunological detection with antibodies, the membrane, if necessary cut into strips, is first blocked for 15 minutes with Universal buffer (EUROIMMUN) with 3% (w / v) milk powder. Subsequently, it is incubated for 3 hours with a human serum (1: 200 in universal buffer with 3% (w / v) milk powder). Then it is washed three times for 5 minutes each with universal buffer. In a second incubation step, the antibodies bound to the proteins in the positive case react with a conjugate solution (EUROIMMUN diluted 1:10 in universal buffer) containing alkaline phosphatase-labeled anti-rabbit IgG antibodies.

Subsequently, it is washed as after the serum incubation. In a third incubation step, the bound antibodies are then detected with an NBT / BCIP substrate solution (4-nitroblue tetrazolium chloride / chlorobromoindolyl phosphate, EUROIMMUN). Table 1 component Concentration (mg / L) glycine 50 in BSK-H contain Sigma-Aldrich B3528 Hydroxy L-proline 10 L-alanine 25 L-Argininehydrochlorid 58 L-aspartic acid 30 L-cysteine hydrochloride monohydrate 260 L-cystine 20 L-glutamic acid 75 L-histidine hydrochloride monohydrate 20 L-isoleucine 20 L-leucine 60 L-lysine hydrochloride 70 L-methionine 15 L-phenylalanine 25 L-proline 40 L-serine 25 L-threonine 30 L-tryptophan 10 L-tyrosine dihydrate disodium salt 40 L-valine 25 ascorbic acid 50 biotin 0.01 cholesterol 0.2 Cholinechlorid 0.5 D-calcium pantothenate 0.01 folic acid 0.01 i-Inositol 0.05 niacinamide 0,025 Nicotinic acid (niacin) 0,025 Para-Aminobenzoylsäure 0.05 pyridoxal hydrochloride 0,025 Continuation Table 1 component Concentration (mg / L) pyridoxine hydrochloride 0,025 in BSK-H contain Sigma-Aldrich B3528 riboflavin 0.01 thiamine hydrochloride 0.01 Calcium chlorides (CaCl ₂ ) (anhyd.) 200 Magnesium sulfate (MgSO ₄ ) (anhyd.) 97.7 Potassium chloride (KCl) 400 Sodium bicarbonate (NaHCO ₃ ) 2200 Sodium chloride (NaCl) 6800 Sodium dihydrogen phosphate monohydrate (NaH ₂ PO ₄ -H ₂ O) 122 2'-deoxyadenosine 10 2'-deoxycytidine 11.6 2'-deoxyguanosine 10 5-methyl-deoxycytidine 0.1 Co-carboxylase 1 Coenzyme A 2.5 D-glucose 6000 Diphosphopyridine nucleotide (NAD) 7 FAD (flavin adenine dinucleotide) 0.106 Glutathione (red.) 10 phenol 21.24 Sodium acetate trihydrate 50 Sodium glucuronate monohydrate 3.9 thymidine 10 Triphosphopyridine nucleotide (NADP) 1 Tween ^80® 5 Uridine 5'-triphosphate 1 N-acetyl-D-glucosamine 400 Citric acid dihydrate disodium salt 700 HEPES 6000 BSA 50000 Neopeptone 5000 yeast extract 2000 gelatin peptone 10000 Sigma-Aldrich 70951 Lipidmix (1000x) 2 [ml / L] Sigma-Aldrich L5146

Results:

In all cultures, both in the medium of the composition according to Table 1 and in Complete BSK-H, a discoloration of the culture medium turns from red to yellow after four to ten days. The color change indicates in each case an acidification of the medium by the metabolism of the contained organisms. At the same time, an increase in the turbidity of the suspension is observed by an increase in the number of organisms.

The number of organisms in all cultures each show a sigmoidal relationship to culture duration, typical of cell division-propagating organisms. The amount of organisms upon reaching the plateau phase amounts to 10 ⁸ to 10 ⁹ cells in a 30 ml culture and is approximately proportional to the total protein amount of the harvested cells (4-6 mg).

When normalized for protein analysis of the various samples in Western blot, combined with the incubation with an antiserum against OspC shows each a staining of a protein corresponding to 25 kDa, the calculated mass of OspC. The color, which is approximately the same in each case, indicates a ratio of the OspC which is approximately constant with respect to the protein amount.

Example 2: Cultivation of Borrelia in protein-free medium

The cultivation of the three Borrelia strains is carried out according to Example 1. Instead of the medium of the composition according to Table 1, however, a medium of the composition according to Table 2 is used.

The specific detection of Borrelia occurs by Western blot according to Example 1. Table 2 component Concentration (mg / L) glycine 50 in CMRL contain US Biologicals C5900 Hydroxy L-proline 10 L-alanine 25 L-Argininehydrochlorid 57.87 L-aspartic acid 30 L-cysteine hydrochloride monohydrate 260 L-cystine 20 L-glutamic acid 75 L-histidine hydrochloride monohydrate 20 L-isoleucine 20 L-leucine 60 L-lysine hydrochloride 70 L-methionine 15 L-phenylalanine 25 L-proline 40 L-serine 25 L-threonine 30 L-tryptophan 10 L-tyrosine dihydrate disodium salt 40 L-valine 25 ascorbic acid 50 biotin 0.01 cholesterol 0.2 Cholinechlorid 0.5 D-calcium pantothenate 0.01 folic acid 0.01 i-Inositol 0.05 niacinamide 0,025 Nicotinic acid (niacin) 0,025 Para-Aminobenzoylsäure 0.05 pyridoxal hydrochloride 0,025 Pyridoxinhydxochlorid 0,025 riboflavin 0.01 thiamine hydrochloride 0.01 Calcium chlorides (CaCl ₂ ) (anhyd.) 200 Magnesium sulfate (MgSO ₄ ) (anhyd.) 97.69 Potassium chloride (KCl) 400 Sodium chloride (NaCl) 6800 Sodium dihydrogen phosphate monohydrate (NaH ₂ PO ₄ -H ₂ O) 122 2'-deoxyadenosine 10 2'-deoxycytidine 11.6 2'-deoxyguanosine 10 5-methyl-deoxycytidine 0.1 Co-carboxylase 1 Coenzyme A 2.5 Diphosphopyridine nucleotide (NAD) 7 Continuation Table 2 Component Biologicals Concentration (mg / L) FAD (flavin adenine dinucleotide) 0.106 in CMRL contain US C5900 Glutathione (red.) 10 phenol 21.24 Sodium acetate trihydrate 50 Sodium glucuronate monohydrate 3.88 thymidine 10 Triphosphopyridine nucleotide (NADP) 1 Tween ^80® 5 Uridine 5'-triphosphate 1 L-cysteine 100 Sigma-Aldrich C7352 D-glucose 6000 Sigma-Aldrich G8769 N-acetyl glucosamine 400 Sigma-Aldrich A8625 HEPES 6000 Sigma-Aldrich H3537 Zinc (II) chloride 13.6 Sigma-Aldrich Z4875 Lipidmix (1000x) 2 [ml / L] Sigma-Aldrich L5146

Results:

The results essentially correspond to Example 1. In addition, observation of the bacteria in the dark field microscope shows that the borrelia cultured in the medium of the composition according to Table 2 are substantially smaller than when cultivated in Complete BSK-H or medium of the composition according to Table 1. In addition, the cell count per 30 ml of culture is increased by a factor of about five to ten, while the amount of protein per 30 ml of culture is approximately identical.

Example 3: Passage of borrelia in different media

The cultivation of the three Borrelia strains is carried out according to Example 1 or 2. Shortly before reaching the plateau phase, a 200 ul aliquot of each culture is removed under the safety cabinet and transferred to a new vessel with fresh medium. The process will continue over five passages (approximately four to seven weeks).

The specific detection of Borrelia is carried out by Western blotting according to Example 1.

Results:

The results essentially correspond to Example 1. The repeated transfer of Borrelia into fresh medium leads to renewed exponential growth. It does not matter from which spent medium (composition according to Table 1, composition according to Table 2, Complete BSK-H) in which fresh medium (composition according to Table 1, composition according to Table 2, Complete BSK-H) is inoculated. In addition to the increase in the number of organisms in the starting culture, this is further evidence of the viability of the majority of the bacteria contained.

Claims (12)

Process for the cultivation of bacteria of the genus Borrelia, characterized in that the bacteria are brought into contact with a protein-free culture medium containing at least one chemically defined fatty acid or at least one chemically defined fatty acid ester and at least one chemically defined zinc (II) compound and supports the growth of the bacteria. A method according to claim 1, characterized in that an originally protein-free culture medium according to claim 1 is supplemented with at least one chemically defined protein-containing component. A kit for the cultivation of bacteria of the genus Borrelia, characterized in that the kit contains at least one container with a protein-free culture medium containing at least one chemically defined fatty acid or at least one chemically defined fatty acid ester and at least one chemically defined zinc (II) Contains compound and supports the growth of bacteria. Protein-free culture media for the cultivation of bacteria of the genus Borrelia, characterized in that they contain at least one chemically defined fatty acid or at least one chemically defined fatty acid esters and at least one chemically defined zinc (II) compound and support the growth of the bacteria. Culture media according to claim 4, characterized in that they are present in a simple formulation. Culture media according to claim 4, characterized in that they are present in concentrated formulation. Culture media according to claims 4 and 6, characterized in that they are present in tenfold formulation. Culture media according to claims 4 to 7, characterized in that they contain at least one chemically defined fatty acid from the group of palmitic acid, stearic acid, arachidic acid, behenic acid, palmitoleic acid, oleic acid, icosenoic acid, linoleic acid, linolenic acid, arachidonic acid. Culture media according to Claims 4 to 8, characterized in that they contain at least one chemically defined ester which is obtained by esterification of one of the fatty acids from the group of palmitic acid, stearic acid, arachidic acid, behenic acid, palmitoleic acid, oleic acid, icosenoic acid, linoleic acid, linolenic acid, arachidonic acid, and in each case one of the alcohols of the group methanol, ethanol, propanol, butanol, pentanol, hexanol, glycerol and derivatives of these alcohols can be produced. Culture media according to claims 4 to 9, characterized in that the total amount of chemically defined fatty acids and fatty acid esters contained is between 1 mg / L and 200 mg / L. Culture media according to claims 4 to 10, characterized in that it comprises at least one chemically defined zinc (II) compound from the group zinc (II) chloride, zinc (II) sulfate, zinc (II) citrate, zinc (II) acetate, Zinc (II) bromide, zinc (II) nitrate, zinc (II) phosphate. Culture media according to claims 4 to 11, characterized in that they contain zinc (II) in a concentration of between 10 μM and 1000 μM.