Preparation of small colony variants of therapeutic bacteria
The invention relates to methods of differentiation, isolation, propagation, and storage of small colony variants (SCVs) of E. coli , preferably E. coli 83972 or E. coli HU2117, or modified or variant forms thereof, and methods for using the prepared SCV bacteria to establish probiotic biofilms in treated subjects and/or on treated medical devices.
1. A method of culturing probiotic bacteria, comprising:
a) isolating an E. coli small colony variant (SCV) bacterium to form a preparation of SCV E. coli bacterial cells that is free of large colony variant (LCV) E. coli bacterial cells, wherein said E. coli SCV bacterium is selected from E. coli 83972 and E. coli HU2117, or a variant or derivative thereof;
b) inoculating a liquid growth medium with said preparation of SCV E. coli bacterial cells, wherein said liquid growth medium is a supplemented minimal medium comprising:
i) a buffer solution;
ii) a sugar or sugar alcohol; and
iii) cysteine, methionine, serine, and lysine,
wherein said liquid growth medium does not comprise added adenine, cytosine, guanine, uracil, yeast extract, or an enzymatic digest of complex protein;
c) incubating said liquid growth medium inoculated with said preparation of SCV E. coli bacterial cells to produce a liquid culture of SCV E. coli bacterial cells, wherein fewer than 50% of E. coli bacterial cells in the liquid culture are LCV E. coli bacterial cells.
2. The method of claim 1 , wherein said sugar or sugar alcohol comprises glycerol.
3. The method of claim 2 , wherein said sugar or sugar alcohol consists of glycerol as the sole added carbon source in said liquid growth medium.
4. The method of claim 1 , wherein in said liquid culture of SCV E. coli bacterial cells, fewer than 30% of E. coli bacterial cells in the liquid culture are LCV E. coli bacterial cells.
5. The method of claim 1 , wherein said liquid culture of SCV E. coli bacterial cells is free of LCV E. coli bacterial cells.
6. The method of claim 1 wherein said isolating comprises isolating an SCV bacterium from urine.
7. The method of claim 1 , wherein said isolating comprises growing an SCV bacterium in urine, wherein said urine comprises one or more of natural urine and synthetic urine.
8. The method of claim 1 , wherein said isolating comprises growing said SCV bacterium on a solid culture medium.
9. The method of claim 8 , wherein said solid culture medium is MacConkey's agar.
10. The method of claim 1 , wherein said buffer solution is a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer solution.
11. The method of claim 10 , wherein said MOPS buffer is MOPS/tricine.
12. The method of claim 1 , wherein said liquid growth medium further comprises one or more amino acids selected from asparagine, aspartic acid, glycine, phenylalanine, and tryptophan.
13. The method of claim 1 , wherein said liquid growth medium comprises one or more of ferrous sulfate, ammonium chloride, potassium sulfate, calcium chloride, magnesium chloride, and sodium chloride.
14. The method of claim 1 , wherein said liquid growth medium further comprises one or more of ammonium molybdate, boric acid, cobalt chloride, cupric sulfate, manganese chloride and zinc sulfate.
15. The method of claim 1 , wherein said liquid growth medium consists essentially of:
MOPS
40
mM
(3-(N-morpholino)-
4
mM
propanesulfonic acid)
Tricine
Iron Sulfate
10
μM
Ammonium Chloride
9.5
mM
Potassium Sulfate
276
μM
Calcium Chloride Monohydrate
0.5
μM
Magnesium Chloride
525
μM
Sodium Chloride
50
mM
Ammonium Molybdate
2.92 × 10 −9
M
Boric Acid
4 × 10 −7
M
Cobalt Chloride
3.02 × 10 −8
M
Cupric Sulfate
9.62 × 10 −9
M
Manganese Chloride
8.08 × 10 −8
M
Zinc Sulfate
9.74 × 10 −9
M
Potassium Phosphate, Dibasic
1.32
mM
Alanine
0.798
mM
Arginine HCl
5.2
mM
Asparagine
0.4
mM
Aspartic Acid, Potassium Salt
0.4
mM
Cysteine Monohydrate HCl
0.1
mM
Glutamic Acid, Potassium Salt
0.7
mM
Glutamine
0.6
mM
Glycine
0.8
mM
Histidine monohydrate HCl
0.2
mM
Isoleucine
0.4
mM
Leucine
0.8
mM
Lysine DiHydrochloride
0.4
mM
Methionine
0.2
mM
Phenylalanine
0.4
mM
Proline
0.4
mM
Serine
10.0
mM
Threonine
0.4
mM
Tryptophane
0.1
mM
Tyrosine
0.2
mM
Valine
0.6
mM
Thiamine HCl
0.01
mM
Calcium Pantothenate
0.01
mM
ρ-aminobenzoic acid
0.01
mM
ρ-hydroxybenzoic acid
0.01
mM
2,3-dihydroxybenzoic acid
0.01
mM
Glycerol
0.4%
(w/v)
Water