U.S. patent application number 10/868592 was filed with the patent office on 2004-11-18 for microbial exopolysaccharide.
Invention is credited to Bulla, Lee A. JR., Candas, Mehmet.
Application Number | 20040229207 10/868592 |
Document ID | / |
Family ID | 32599565 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229207 |
Kind Code |
A1 |
Bulla, Lee A. JR. ; et
al. |
November 18, 2004 |
Microbial exopolysaccharide
Abstract
A novel microorganism producing a nontoxic, non-antigenic
exopolysaccharide is taught. The exopolysaccharide has neutral
sugars migrating at the same rate as mannose, fucose, fructose and
galactose, acidic sugars migrating at the same rate as fucose and
amine sugars migrating at the same rate as glucose and fucose, and
wherein the ratio of galactose:fucose:glucose:mannose is about
1:2:3:6. The microbe and the exopolysaccharide have uses as a
biofilm in geologic applications and have several consumer uses as
food and drug polymers and use as a plasma extender.
Inventors: |
Bulla, Lee A. JR.; (Tioga,
TX) ; Candas, Mehmet; (Dallas, TX) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
32599565 |
Appl. No.: |
10/868592 |
Filed: |
June 14, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10868592 |
Jun 14, 2004 |
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09696929 |
Oct 25, 2000 |
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6759230 |
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60161588 |
Oct 26, 1999 |
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60161391 |
Oct 26, 1999 |
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Current U.S.
Class: |
435/2 ; 514/54;
536/123 |
Current CPC
Class: |
C08B 37/006 20130101;
C12N 1/36 20130101; A01N 63/20 20200101; C12P 19/04 20130101; C08J
2305/00 20130101; C08J 5/18 20130101; B09C 1/10 20130101; C12R
2001/01 20210501; C12N 1/205 20210501; B09C 1/00 20130101 |
Class at
Publication: |
435/002 ;
514/054; 536/123 |
International
Class: |
A61K 031/715; A01N
001/02 |
Claims
What is claimed is:
1. An isolated exopolysaccharide that consists of (a) neutral
sugars migrating at the same rate as mannose, fucose, fructose and
galactose, (b) acidic sugars migrating at the same rate as fucose,
and (c) amine sugars migrating at the same rate as glucose and
fucose, wherein the sugar ratio of sugars migrating as
galactose:fucose:glucose:mannose is about 1:2:3:6.
2. The exopolysaccharide of claim 1 which is that produced by ATCC
No. PTA-2500.
3. The exopolysaccharide of claim 1 which is in purified form.
4. A nutrient for plant or animal growth, which comprises the
exopolysaccharide of claim 3.
5. A plasma expander comprising the exopolysaccharide of claim
1.
6. The plasma expander of claim 5, wherein the exopolysaccharide is
produced by ATCC No. PTA-2500.
7. The plasma expander of claim 5, which further comprises sodium
ion at 100 to 120 mEq/1, calcium ion at about 5 mEq/1, potassium
ion at 0 to 3 mEq/1, and magnesium ion at 0 to 0.9 mEq/1.
8. The plasma expander of claim 5, which further comprises at least
one buffer and a nutrient, and a nutrient.
9. The plasma expander of claim 8 which further comprises vitamin
K.
10. The plasma expander of claim 8 which further comprises human
serum albumin.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 09/696,929, filed 25 Oct. 2000 which claims the benefit of
priority of U.S. Provisional Application for Patent Ser. No.
60/161,588 filed 26 Oct. 1999, and to Provisional Application for
Patent Ser. No. 60/161,391 filed. 26 Oct. 1999. The contents of
these applications are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates in general to a novel
non-pathogenic microbe that produces a nontoxic, non-antigenic
exopolysaccharide. The use of the microbe and exopolysaccharide in
environmental engineering, agricultural, geologic, consumer and
medical applications is described.
[0003] Inhibition and control of mucoidy exopolysaccharide is also
described.
BACKGROUND OF THE INVENTION
[0004] The invention pertains to a novel non-pathogenic microbe
that produces a non-toxic, non-antigenic exopolysaccharide. The
exopolysaccharide can be used as a biofilm in environmental
engineering and agricultural applications and as a filler or
polymer in consumer and medical applications. Biofilm applications
are described first, then particular medical applications are
described.
[0005] The term "biofilm" is used to describe an organic material
that includes microorganisms embedded in a polymer matrix of their
own making. The matrix consists largely of exopolysaccharides and
is a tough, elastic, mucoidal material that adheres strongly to
soil particles. Growth of a biofilm in a sandy soil is achieved by
injecting a bacterial and nutrient solution into soil specimens.
The resulting biofilm treatment is used to clog soil pores, thereby
reducing the ability of the soil to transmit fluids.
[0006] Examples of biofilms are produced by certain strains of
Klebsiella pneumoniae and Pseudomonas species. A problem with the
use of K. pneumoniae is that Klebsiella is a genus that includes a
number of human pathogens. Furthermore, the pathogenicity of K.
pneumoniae itself is associated with its ability to create a
mucoidal exopolysaccharide used in attachment and colonization that
helps the pathogen evade both the non-specific and specific immune
clearing defensive mechanisms.
[0007] Another example of a biofilm is described in U.S. Pat. No.
4,800,959, by Costerton, which discloses the use of a microbial
process for selectively plugging a subterranean formation. In the
process taught, a highly permeable stratum or zone in a
subterranean reservoir is plugged using Klebsiella or Pseudomonas
bacteria that were starved to reduce their size prior to being
injected into the target zone. The bacteria regain full cell size,
proliferate and commence production of biofilm-forming
exopolysaccharides upon exposure to minimal nutrient containing
media. The biofilm produced by these bacteria selectively seal off
the high permeability zones of a formation and reduce aqueous flow
through the zone.
[0008] In addition to the above described biofilm uses, there has
been a need for perfusion solutions and blood substitutes.
Currently available and approved compounds, however, have so far
failed to meet the increasing demands on our blood provider system.
A number of blood substitutes have been developed over the last few
years to attempt to meet the increasing demand for blood, blood
substitutes and plasma expanders. Unfortunately, many of the plasma
expanders that are currently in use fail as the small molecules on
which they depend to provide osmotic pressure readily traverse
capillary beds as a consequence of the negative osmotic pressure
found in post-arterial capillary beds. The loss of osmotic
potential, makes the long-term use of current plasma expanders for
maintaining proper ionic or fluid balance or plasma volume in a
mammalian subject unsatisfactory.
[0009] Those blood substitutes that have an impermeable substance
to maintain volume use human serum albumin or a mixture of plasma
proteins as the oncotic agent. These substitute plasma proteins
depend on the same blood and plasma supply as our current blood
provider system, therefore failing to meet the increased demand for
these products.
[0010] A number of patents have issued to Segall that are directed
to blood and plasma substitutes. U.S. Pat. No. 4,923,442, and the
reissue thereof, discloses a number of solutions used in blood
substitution of living subjects all of which include at least some
concentration of a cardioplegia agent, usually potassium ion. U.S.
Pat. No. 4,923,442 discloses surgical methods, particularly in
respect to instrument placement and the control of pulmonary wedge
pressure generally applicable to perfusion of subjects. U.S. Pat.
No. 5,130,230 discloses a blood substitute that may be used as a
system of solutions in which a number of solutions, are used
sequentially to completely replace the blood of living subjects.
U.S. Pat. No. 5,130,230 discloses that the blood substitute
comprises "an aqueous solution of electrolytes at physiological
concentration, a macromolecular oncotic agent, a biological buffer
having a buffering capacity in the range of physiological pH,
simple nutritive sugar or sugars, and magnesium ion in a
concentration sufficient to substitute for the flux of calcium
across cell membranes."
[0011] In addition to the patented inventions described above, a
number of commercially available products have been used for the
treatment of hypovolemic patients. These include: HESPAN.TM. (6%
hetastarch in 0.9% sodium chloride injection, PENTASPAN.TM. (10%
pentastarch in 0.9% sodium chloride injection [both by DUPONT
PHARMACEUTICALS.TM., Wilmington Dela.]), MACRODEX.TM. (6% dextran
70 in 5% dextrose injection or 6% dextran 70 in 0.9% sodium
chloride injection [PHARMACIA, INC..TM., Piscataway, N.J.]) and
RHEOMACRODEX.TM. (10% dextran 40 in 5% dextrose injection or 10t
dextran 40 in 0.9% sodium chloride injection [PHARMACIA, INC..TM.,
Piscataway, N.J.]). All of these products, however, depend on
compounds that are polymeric and that often dissociate or are
broken down by natural physiologic enzymes with time.
Alternatively, bacteria may take advantage of these newly supplied
nutrient sources, causing severe septicemia in patients that are
infected by pathogens at the time of injury. Thus, a need remains
for a better oncotic agent.
[0012] The ability to produce mucoidal exopolysaccharides in
medically important bacteria is critical to attachment to surfaces,
resulting in increased resistance to drug treatments. Both chemical
and physical treatments have been developed to control biofilm
formation. Methods in Enzymology, Vol. 310, Biofilms, Ed. Ron J.
Doyle, Academic Press, 1999. However, because biofilms are
associated with pathogenicity, persistent and resistant bacterial
infections and bio-corrosion of industrial structures, there is
need for additional simple and efficient methods to control
biofilms.
SUMMARY OF THE INVENTION
[0013] The newly discovered bacterium LAB-1, deposited at ATCC No.
PTA-2500, possesses a number of potential commercial biofilm
applications. These include, but are not limited to: (1) subsurface
biofilm cutoff wall formation; (2) subsurface liners that include
compacted, biofilm treated soil; (3) in-situ biofilm liners; (4)
barriers made by treating geotextiles with biofilm materials; (5)
improved ability of sand to retain moisture; (6) reclamation of
poor soils and conversion into agriculture land; (7) significantly
increased soil biomass in the form of polymers that function as a
nutrient supply for plant growth and/or help retain nutrients and
water; and (8) providing cohesion to otherwise cohesionless soils
(such as sand dunes), thus making the soil more resistant to
erosion by wind and/or water.
[0014] It has been found that the prior art methods and biofilms
fail to provide biologically and environmentally safe and
efficacious water, soil and waste retention characteristics. A
significant problem with existing technology is the pathogenicity
of the bacteria used to produce the biofilms. The present
invention, therefore, is directed to a non-pathogenic bacterium
that produces a biofilm made of exopolysaccharide that is
essentially made of neutral sugars that migrate at the same rate
as: mannose, fucose, fructose and galactose, acidic sugars that
migrate at the same rate as fucose and amine sugars that migrate at
the same rate as glucose and fucose. More particularly, the
bacterium is a LAB-1 strain. The biofilm producing bacterium may be
further defined as being capable of growth between about pH 4 and
11 and between about 150.degree. and 450.degree. C. The LAB-1
strain is capable of growth in minimal growth media, or may be
grown in an aqueous nutrient medium that includes yeast, peptone
and mineral salt ingredients. LAB-1 is a gram-negative, rod-shaped
bacterium of about 0.2.times.0.8 .mu.m that secretes the
exopolysaccharide described herein.
[0015] In one embodiment of the present invention, the LAB-1 strain
is used in plugging a permeable subterranean stratum by providing
LAB-1 bacteria in a nutrient-containing solution into the target
stratum. The nutrient-containing solution is generally adapted to
provide substantial and uniform growth conditions for the LAB-1.
Sufficient biofilm is produced under these conditions to
effectively plug the stratum. For example, the bacterium in situ
can yield a saturated hydraulic conductivity equal to or less than
1.5.times.10.sup.-5 cm/sec, equal to or less than
1.0.times.10.sup.-7 cm/sec or even equal to or less than
1.5.times.10.sup.-8 cm/sec.
[0016] Alternatively, the bacteria may be preincubated in culture
in an aqueous suspension medium with agitation for an incubation
period sufficient to initiate bacterial exopolysaccharide
production before injection into the stratum. The method of
plugging the subterranean stratum may also include draining
nutrient deficient suspension medium from the reservoir, and
recharging the reservoir with aqueous nutrient medium to maintain
bacterial growth for an elapsed time period sufficient to establish
a biofilm of prescribed saturated hydraulic conductivity. The
draining and recharging steps with aqueous nutrient medium may be
conducted at least once every 48 hours of elapsed time period. The
step of pre-incubating the bacteria may be, e.g., for at least
about 72 hours. These growth conditions permit for the
establishment of a biofilm having a population between about
10.sup.5-10.sup.15 bacterial Colony Forming Units per square
centimeter on a slide surface.
[0017] The biofilm may be used to plug open conduits, deposited in
a subsurface biofilm cutoff wall, used to enhance the water
retaining ability of subsurface liners or even for improving the
water retention capabilities of compacted, semi-compacted or
loosened biofilm treated soil. When used in a liner, the biofilm
may be deposited in-situ. The biofilm may also be used along with
and/or to enhance environmental barriers by treating geotextiles
with the biofilm.
[0018] Another important aspect of this polymer is its lack of
antigenicity and toxicity in an animal system. This suggests
several consumer/medical applications, including: (1) use a food
additive or food thickening or filler agent; (2) use as plasma
expander; (3) use in polymer industry; (4) use as chromatography
matrix support for purification of chemicals; (5) use in scientific
research as suspension solution instead of ficol and the like; (6)
use in determining the gene content of the organism, especially
those coding for the biosynthesis of the exopolysaccharide polymer;
(7) use of the polymer materials in the cosmetic field; (8) use to
augment insect or animal diets; (9) use as an additive in tissue
culture media; (10) for use as a semi-solid to solid matrix, e.g,
gel electrophoresis; (11) for use as an additive in toothpaste,
ointments, creams and lotions; (12) for mixing with dyes, stains,
paints and varnishes; (13) for inclusion in dialysis; (14) for use
in composite materials, e.g., bricks, tile, mortars; (15) for use
as part of a sealant; (16) viscosity modifier for oils, waxes &
greases; (17) use as a filler, thickener or extender in
pharmaceutical preparations; (18) use of the polymer in
bioscaffolding applications, including wound-healing applications;
and (19) use as a bacteriostatic (biostat) agent to inhibit or at
least fail to support bacterial growth, and even possibly as a
biocide.
[0019] In particular, a compound is needed for use as a plasma
extender that serves to increase blood volume and that is
impermeable at blood capillaries. The compound must not readily
dissociate or be rapidly broken down by natural physiologic enzymes
with time. Furthermore, the compound and its use as a plasma
expander must not provide bacteria with an exogenous nutrient
source, which may lead to accentuating already severe septicemia in
patients that are infected by pathogens at the time of the injury
that is causing hypovolemia.
[0020] More particularly, the present invention is an
exopolysaccharide produced by the LAB-1 bacterial stain. The
exopolysaccharaide does not appear to easily support bacterial
growth. This was determined by testing the ability of E. coli or B.
indica to grow on the exopolysaccharide and no growth was observed.
Further, the exopolysaccharide is not antigenic as tested by
injection into mice. Thus, the product appears to satisfy some of
the basic parameters required for a plasma expander.
[0021] The exopolysaccharide is secreted into the cell culture
medium and collected for use in, e.g., a plasma expander. When used
as a plasma expander alone, or in combination with other elements,
the exopolysaccharide will be provided in an isotonic solution. In
one embodiment, a blood-free plasma expander and blood substitute
for use in a subject in need thereof includes a single solution
with at least two water soluble oncotic agents, one of which is a
water soluble polysaccharide oncotic agent and one of which is
serum albumin, wherein the exopolysaccharide consisting essentially
of mannose, fucose, fructose and galactose, acidic fucose and amine
containing glucose and fucose.
[0022] The plasma expander and blood substitute may have a ratio of
water soluble exopolysaccharide oncotic agent to serum albumin
between 1:1 and 1:2, weight to weight. The combined percentage of
water-soluble exopolysaccharide oncotic agent and serum albumin in
a solution of the plasma expander and blood substitute may be in
the range of between about 4%-6% weight to volume.
[0023] The plasma expander and blood substitute may also include a
number of cations, alone or in combination. For example, the
cations may be provided in the following concentrations: Na.sup.+
at 110 to 120 mEq/1, Ca.sup.++ at about 5 mEq/1, K.sup.+ at 0 to 3
mEq/1, and Mg.sup.++ at 0 to 0.9 mEq/1. These cations may be
supplied as dissolved chloride salts. The plasma expander and blood
substitute may also include at least one buffer, for example, a
lactate and/or bicarbonate buffer. When buffered, the plasma
expander will generally be a biological buffer having a buffering
capacity in the pH range of about 6.8 to 7.8.
[0024] When used in hypovolemic patients, e.g., those that have
lost a large volume of blood due to trauma, additional agents may
be included in the plasma expander to aid in recovery. Such agents
may include, Vitamin K in a concentration of about 1-4 mg/l,
amylase, clotting factors, t-PA or even erythropoietin.
[0025] In non-medical uses, the exopolysaccharide of the present
invention may be used as a chromatography matrix support for
purification of chemicals. One such use will be as a suspension
solution for use in centrifugation. The exopolysaccharide may even
be used in solution as a suspension solution for use in size
separation.
[0026] The present invention may also be used as a biologically
stable, non-toxic material for use in coated plates for a number of
biological and analytical uses. Examples of such uses include the
coating of tissue culture plates for maintaining the growth, in
vitro, of cells. Cells that may be grown on the surface of the
exopolysaccharide include prokaryotic and eukaryotic cells. In an
analytical setting, the exopolysaccharide disclosed herein may be
used as a coating for instrumentation, such as biosensors, that
require the maintenance of a biologically compatible
environment.
[0027] Compositions containing propionic acid and ibuprofen when
incorporated into liquid or solid growth media of the LAB-1 strain
at a concentration range of 0.1-1.0% (w/v) differentially inhibits
its growth, development, cell attachment and biofilm production.
Growth of the newly discovered LAB-1 strain as well as its
production of mucoidal exopolysaccharide and biofilm may be
inhibited or controlled by propionic acid, derivatives of propionic
acid, compounds with related chemical structures or backbones such
as 2-(4-isobutylphenyl)-propionic acid, otherwise known as
ibuprofen, and solutions, mixtures, suspensions and other kinds of
preparations comprising such compounds singly or in combination
with other materials and compounds. It would be apparent to one of
ordinary skill in the art to apply the above methods to inhibit the
production of mucoid compounds and biofilm in any mucoid
organism.
[0028] A more complete appreciation of the present invention and
the scope thereof can be obtained from the accompanying drawings
which are briefly summarized below, the following detailed
description of the presently-preferred embodiments of the
invention, and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] A more complete understanding of the method and apparatus of
the present invention may be obtained by reference to the following
Detailed Description when taken in conjunction with the
accompanying Drawings wherein:
[0030] FIG. 1 is a photograph of Gram stained LAB-1 at 100 .times.
magnification.
[0031] FIG. 2 is a Coomassie stained SDS-PAGE gel of the total
protein content of LAB-1 and B. indicia grown on solid culture.
[0032] FIG. 3 is a Coomassie stained SDS-PAGE gel of the total
protein content of LAB-1 and B. indicia grown in liquid
culture.
[0033] FIG. 4 is the data from gas chromatography of fatty acids in
LAB-1.
[0034] FIG. 5 is a FACE gel showing the sugars identified in the
exopolysaccharide produced by LAB-1. Lanes: 1--MONO Ladder Standard
2 (100 pmol ea. monosaccharide); 2--Amine hydrolysis reaction
products; 3 [S]--MONO Ladder Standard 2 (100 pmol ea.
monosaccharide; scanned for trace shown in [S] Scan); 4--Neutral
hydrolysis reaction products; 5--Sialic acid hydrolysis reaction
products; 6--NANA labeling control 1 (100 pmol); 7--MONO
composition control; 8--MONO Ladder Standard 2 (100 pmol ea.
monosaccharide).
[0035] FIG. 6 is a MALDI trace of the exopolysaccharide produced by
LAB-1.
[0036] FIG. 7 is a drawing of a cross sectional view of a barrier
created with the biofilm of the present invention.
[0037] FIG. 8 is a graph of the hydraulic conductivity versus time
of a LAB-1 containing biofilm.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EXEMPLARY
EMBODIMENTS
[0038] The present invention will now be described more fully
hereinafter with reference to the accompanying drawings, in which
preferred embodiments of the invention are shown. This invention
may, however, be embodied in many different forms and should not be
construed as limited to the embodiments set forth herein; rather,
these embodiments are provided so that this disclosure will be
thorough and complete, and will fully convey the scope of the
invention to those skilled in the art.
EXAMPLE 1
Characterization of Lab-1 Organism and Exopolysaccharide
[0039] Samples of LAB-1 have been deposited under ATCC No.
PTA-2500. LAB-1 is a Gram-negative, rod-shaped bacterium isolated
by the present inventor from constructed soil samples in the state
of Wyoming. The soil sample was constructed by J. Turner at the
University Wyoming and contained a contaminant in a background of a
Beijerinckia indica. The contaminant, LAB-1, was isolated and
studied because of its excessive slime production.
[0040] A variety of biochemical tests were performed in order to
identify the species and the genus of organism. The tests included:
assessment of growth conditions, culture appearance, cell
appearance and staining characteristics, optimal temperature and pH
growth range, oxygen requirements, antibiotic sensitivity testing,
tests for catalase and oxidase (using TAXOS N.TM. disks), amino
acid utilization as carbon source, nitrogen fixation, motility
test, and a variety of commercially available tests from
MICRO-ID.TM., OXYFERM.TM., ENTEROTUBE.TM. and BIOLOG GN
MICROPLATE.TM..
[0041] Because the organism was a contaminant in soil amended with
a B. indica culture, B. indica and the LAB-1 organism were grown on
plates and total protein profiles were compared by SDS-PAGE. Fatty
acid analysis was performed by Microbial ID (MIDI) of Newark, Dela.
who used the high resolution MIDI SHERLOCK SYSTEM.TM. to identify
fatty acids with high resolution gas chromatography. The same
laboratory also sequenced the 16S gene and compared it against the
proprietary MICROSEQ.TM. database (PE APPLIED BIOSYSTEM.TM.).
[0042] The exopolysaccharide was further analyzed by GLYKO.TM.
(NOVATO.TM., CA) fluorophore-assisted carbohydrate electrophoresis
(FACE), and matrix-assisted laser desorption/ionization mass
spectrometry (MALDI). Immunogenicity of the exopolysaccharide was
also tested in mice.
[0043] Colonies of LAB-1 on solid medium were irregular with an
undulate edge. They exhibited convex elevation, a smooth glistening
surface, were white in color and translucent to opaque. The
consistency of the colonies was that of a very tenacious and
elastic slime. The LAB-1 exopolysaccharide exhibits tremendous
tenacity, extending without breakage when pulled with a glass rod
over a foot. Due to the production of this exopolysaccharide, it
was found difficult to lift the colonies from agar plates.
[0044] AB13 culture medium may be used to grow the LAB-1 strain and
is made as follows: Per liter of water add 20 g glucose, 1 g NaCl,
1 g yeast extract or 2 g NaNO.sub.3, 8 g K.sub.2HPO.sub.4, 0.2 g
KH.sub.2PO.sub.4, 0.5 g MgSO.sub.4, and 150 .mu.l 5% FeCl.sub.2. To
make solid medium, 15 g of agar are added to the medium. This
simplified end medium makes large-scale production of the
polysaccharide polymer affordable.
[0045] Growth in liquid AB13 medium was perfuse and the turbidity
was dense and uniform. When the LAB-1 bacterium is grown in liquid
medium, the entire solution becomes very viscous with a consistency
ranging from that of heavy corn syrup to that of egg whites,
depending on the stage of growth. Cultures grown without shaking
showed a flocculent deposit along with smooth surface growth. The
cultures had a very distinct odor, somewhat sweet smelling, but not
pleasant.
[0046] The bacteria were bacilliary with rounded ends and parallel
sides and were determined to be Gram negative. There were some
irregularities observed in the cell population in cell size, due
mainly to length differences, but the average size was about
0.2.times.0.8 .mu.m. The arrangement of cell packets seemed to be
irregular, although a large percentage of cells were aligned
side-to-side. The clumping of cells was believed to be due to the
tenacious slime layer. The exopolysaccharide could be seen in Gram
staining as a light cloud surrounding the cells. FIG. 1 shows the
Gram stained cells at 100.times.. The spore stain showed only red
vegetative cells, no spores were observed. The capsule stain showed
no capsules, but rather an indefinite exopolysaccharide surrounding
the cells. Observation of the stab culture showed that the organism
was motile.
[0047] Colony morphology on plates grown at 26.degree., 30.degree.,
37.degree. C. and 45.degree. C. was the same, although growth was
optimal at 37.degree. C. Elasticity of the slime layer was also
unchanged. Growth was unchanged in liquid AB13 cultures ranging
from pH 4, 9 and 11. Growth was not observed at pH 2. Colony
morphology on plates was also unchanged over this pH range. The
LAB-1 organism was determined to be a facultative anaerobe.
[0048] Various biochemical test results are summarized in the
following tables:
1TABLE 1 Morphology of LAB-1 Morphological Characteristics Results
Gram reaction negative Cell Shape bacilli Spores none Growth temp.
26-37.degree. C. pH range 4-11 Growth in peptone + Motility +
[0049]
2TABLE 2 Antibiotic Profile of LAB-1 Antibiotic Susceptibility
Results Amikacin sensitive Ampicillin resistant Erythromycin
resistant Neomycin sensitive Novobiocin sensitive Penicillin
resistant Polymyxin B sensitive Streptomycin resistant Taxos A
resistant Tetracycline sensitive
[0050]
3TABLE 3 Biochemistry of LAB-1 Biochemical Properties Results
Adonitol fermentation + Ammonification + Arabinose fermentation +
Arginine dihydrolase + Beta-galactosidase - Catalase + Citrate
utilization + Cytochrome oxidase - Esculin hydrolysis - Glucose
fermentation + Aerobic glucose + Anaerobic glucose + Hydrogen
sulfide - Indole (tryptophanase) - Inositol fermentation + Lactose
fermentation + Lysine decarboxylase - Malonate utilization -
Maltose oxidation + Mannitol oxidation + N2 gas production -
Nitrate reductase - Nitrogen fixation - Ornithine decarboxylase -
Phenylalanine deaminase - Sorbitol fermentation + Sucrose oxidation
- Urease + VP + Xylose oxidation +
[0051]
4TABLE 4 Carbon Utilization of LAB-1 Carbon Utilization Result
Carbon Utilization Result A-cyclodextrin + Itaconic acid - Dextrin
+ A-keto-butyric acid - Glycogen + A-keto-glutaric acid - Tween 40
+ A-keto-valeric acid - Tween 80 + D, L-lactic acid + N-acetyl
galactosamine + Malonic acid + N-acetyl-D-glucosamine + Propionic
acid - Adonitol + Quinic acid + L-arabinose + D-saccharic acid +
D-arabitol + Sebacic acid - D-cellobiose + Succinic acid +
I-erythritol - Bromo Succinic acid + D-fructose + Succinamic acid
+/- L-fucose + Glucuronamide + D-galactose + Alaninamide +/-
Gentiobiose +/- D-alanine + A-D-glucose +/- L-alanine + M-inositol
+ L-alanyl-glycine + A-D-lactose + L-asparagine + Lactulose +
L-aspartic acid + Maltose + L-glutamic acid + D-mannitol +/-
Glycyl-L-aspartic acid + D-mannose + Glycyl-L-glutamic acid +
D-melibiose + L-histidine + B-methyl-d-glucoside +
Hydroxy-L-proline + D-psicose + L-leucine - D-raffinose +
L-ornithine - L-rhamnose + L-phenylalanine - D-sorbitol + L-proline
+ Sucrose + L-pyroglutamic acid - D-trehalose + D-serine + Turanose
+ L-serine + Xylitol + L-threonine - Methyl pyruvate + Carnitine -
Methyl succinate + G-amino Butyric acid + Acetic acid + Urocanic
acid - Cis-aconitic acid + Inosine + Citric acid + Uridine - Formic
acid + Thymidine + D-galactonic acid + Phenylethylamine - lactone
D-galacturonic acid + Putrescine - D-gluconic acid + 2-amino
ethanol - D-glucosaminic acid + 2,3-butanediol - D-glucuronic acid
+ Glycerol + A-hydroxybutyric acid - D, L-A- glycerol + phosphate
B-hydroxybutyric acid + Glucose-1-phosphate + G-hydroxybutyric acid
- Glucose-6-phosphate + P-hydroxphenyl acetic + acid
[0052] Bacteriological information was compiled into the chart
shown in Tables 1 through 4. This information was used to search
the 9.sup.th edition (1994) of Bergey's Manual of Determinative
Bacteriology. The Bergey's profiles of all Gram negative
facultative anaerobic bacilli were researched in an attempt to find
potential matches. Special attention was paid to members of the
genus Klebsiella because this was the closest match provided by
BIOLOG.TM., and, to the genus Beijerinckia because LAB-1 was
isolated as a contaminant amongst a lawn of Beijerinckia
colonies.
[0053] Although the information contained in Tables 1-4 was
considered valuable, this particular method of exploration was
quickly proven to be inconclusive. No exact matches were
identified, and, the partial matches found were too numerous to
consider them all. Therefore, other more concrete methods of
identification were utilized.
[0054] Because it was entirely possible that LAB-1 could have been
a Beijerinckia species, protein profiles of each organism were
compared. For the gel in FIG. 2, the colonies of each organism were
harvested from AB13 plates and sheared by vortexing with glass
beads for five minutes. One volume of running buffer and one volume
of bromophenol blue tracking dye was added to the lysate, and the
result boiled for five minutes. After a brief spin, the samples
were overlaid twice with powdered sucrose to remove cell debris and
100 .mu.l were loaded onto a 4.5 stacking, 10% resolving
polyacrylamide gel and run at 30-35 mAmp.
[0055] Samples used for FIG. 3 were grown in liquid AB13 culture.
One ml was harvested by pelleting for five minutes, washed with 1%
NaCl and again with dH.sub.2O. Pellets were resuspended in 180
.mu.l of 50 mM Tris, and 10 mM EDTA, pH 8.0. Sixty .mu.l of PSS
(4.times.PSS is 700 .mu.l of 1 M Tris-HCl, pH 6.8, 4.3 g sucrose, 4
mg bromophenol blue, 1.45 .mu.l of 20% SDS, 44 .mu.l of 0.5 M EDTA,
10 mg DTT and dH.sub.2O to 10 ml) were added to the cells and the
mixture was boiled for five minutes. Samples were overlaid with
powdered sucrose to remove cell debris and run as above.
[0056] The Coomassie blue stained SDS-PAGE gels shown in FIGS. 2
and 3 clearly demonstrate that there are dissimilarities between
the two organisms. It was concluded that the unknown organism LAB-1
was not a Beijerinckia species.
[0057] Fatty acid analysis by gas chromatography by MIDI LABS.TM.,
Newark, Dela., was employed to identify the organism. The growth
conditions were standardized, the MIDI SHERLOCK SYSTEM.TM. was
fully automated, and the data compared against proprietary
databases containing the fatty acid profiles of more than 1,900
bacteria. The results are shown in FIG. 4. The best match, Kluyvera
cryocrescens, had a similarity of 0.835. This match would have been
considered very good if there had been a separation of at least
0.100 between this first choice and the second choice, Enterobacter
taylorae, with a similarity of 0.753.
[0058] Using these two species, reference was made to the 9.sup.th
edition (1994) of Bergey's Manual of Determinative Bacteriology to
compare their characteristics with those listed in Tables 1-4. Also
compared was the third choice, Kluyvera ascorbata. There were many
key differences in the biochemical profiles of LAB-1 and each of
the three matches.
[0059] The properties found to be different amongst these four
organisms are shown in Table 5. This information, along with the
fatty acid analysis results, led to the conclusion that the
organism was not a member of the genus Kiuyvera.
5TABLE 5 Differences between LAB-1 and K. cryocrescens, K.
ascorbata, E. Taylorae LAB-1 K. cryo. K. ascorb. E. Taylorae
Adonitol + - - - fermentation Arginine + - - + dihydrolase Esculin
hydrolysis - + + + Indole - + + - (tryptophanase) Inositol + - - -
fermentation Lactose + + + - fermentation Lysine - - + -
decarboxylase Melibiose + + + - fermentation MR - + + - VP + - - +
Nitrate reductase - + + + Ornithine - + + + decarboxylase Raffinose
+ + + - fermentation Sorbitol + + + - fermentation Urease + - -
-
[0060] The next logical step was to determine the 16S rRNA gene
sequence. This technique currently is the method of choice for
identification purposes. The gene sequence is shown in SEQ ID NO:
1. The identification based on the 16S rRNA gene sequence was
determined to be Leclercia adecarboxylata. The difference in
sequence homology between LAB-1 and L. adecarboxylata was only
0.59%. Stackebrandt & Goebel, INT'L J. SYSTEM. BACTERIOL. 44:
846 (1994) would consider this a species level match, however the
confidence limits of the data obtained by MIDI LABS.TM. allowed
identification only at the genus level. When the biochemical
characteristics of LAB-1 and of L. adecarboxylata (9.sup.th edition
Bergey's Manual of Determinative Bacteriology 1994) are compared,
there are yet again, numerous differences. The differences led to
the questions regarding this method.
6TABLE 6 Differences between LAB-1 and L. adecarboxylata LAB-1 L.
adecarb. Arginine dihydrolase + - Citrate utilization + - Esculin
hydrolysis - + Glycerol utilization + - Indole (tryptophanase) - +
Inositol fermentation + - Malonate utilization - + MR - + VP + -
Nitrate reductase - + Sorbitol fermentation + - Urease + -
[0061] Because the same laboratory (MIDI LABS.TM.) produced two
different identifications, the validity of both identities was
suspect. However, 16S rRNA gene sequencing is considered to be the
most reliable method currently available, and, therefore, the
identification of Leclercia adecarboxylata must be further
investigated. With all of the discrepancies that have been
encountered, it is reasonable to conclude that this unknown
organism (LAB-1) has not been previously identified.
[0062] Analysis of the exopolysaccharide produced by LAB-1 was by
FACE (sugar content) and MALDI (size). Judging from the intensity
of the bands shown on the FACE gel in FIG. 5, the amount of fucose
present is approximately twice that of galactose; the amount of
glucose is approximately 2.5 times that of galactose; and, the
amount of mannose is approximately 3 times that of fucose.
Therefore, the ratio of galactose:fucose:glucose:mannose is
approximately 1:2:3:6. Further investigations regarding the nature
and type of linkages and molecular weight determination of the
polymer may be undertaken, as will be known to those of skill in
the art.
[0063] Results of the MALDI study of the exopolysaccharide are
shown in FIG. 6. The data indicates that polymerization and
depolymerization of the polysaccharide occurred readily as
evidenced by the large range of molecular weights found. All
monosaccharides identified were neutral sugars that migrate at the
same rate as: mannose, fucose, fructose and galactose, acidic
sugars that migrate at the same rate as fucose and amine sugars
that migrate at the same rate as glucose and fucose. All are
six-carbon sugars. This fact makes it impossible to determine the
composition of the polymer when only the molecular weight is known.
Those substances with molecular weights below 180 are likely
breakdown products of the polymer. The largest polymer, molecular
weight of 1066.38, was comprised of approximately six 6C
sugars.
[0064] It was later discovered that the initial exopolysaccharide
sample analyzed for immunogenicity in mice contained residual amino
acids. The small amount of protein present was from the yeast
extract in the media and from dead cells. Even with these amino
acids present, it was determined that the exopolysaccharide was
non-immunogenic, although a very small immunogenic reaction was
observed. This reaction made it necessary to further purify the
exopolysaccharide so that it was protein-free.
[0065] It was determined that the best way to free the
exopolysaccharide of amino acids was to (i) modify the medium to
contain no amino acids and (ii) limit growth of the organism to
prevent cell death and breakdown. After implementing these two
modifications, the exopolysaccharide was found to be free of
protein (not shown). The protein-free exopolysaccharide was
provided to WASHINGTON BIOTECHNOLOGY.TM. [St Louis, Mo.] to perform
more in-depth studies to determine immunogenic properties. The
results were gratifying and the exopolysaccharide immunized rabbits
maintained antibody titers of less that 1:100, or non-detectable,
for the entire 12 week experiment. In contrast, the inactivated
cell-immunized rabbits reached antibody titers as high as
1:1,638,400. These results are very encouraging because a
non-immunogenic biopolymer with tremendous elasticity such as the
one characterized in this study probably has numerous industrial,
agricultural and biomedical applications.
[0066] Much information has been gathered about LAB-1.
Unfortunately, its complete identity remains indeterminate.
Reliable identification methods have been employed but the results
do not agree with each other. It is highly likely that this
organism has not been previously identified.
[0067] More research is warranted before a definitive
identification can be made. Further studies should include direct
comparison of LAB-1 with Klebsiella, Kluyvera cryocrescens and
Leclercia adecarboxylata. Methods important to compare these
organisms include protein profile determinations and DNA analysis.
Results obtained from these approaches will provide good evidence
of any phylogenetic relationships.
[0068] To further characterize the exopolysaccharide, the
monosaccharide linkages and branching of the polysaccharides should
be determined. Also, it would be very useful to determine the
nature of its overall polymerization. The localization of the
gene(s) coding for the polysaccharides may be determined, as will
be known to those of skill in the art of molecular biology. Even
further studies may be conducted to identify LAB-1, and may
include: chromosomal DNA fingerprinting, random primer PCR
profiling, rRNA or other gene sequencing, determination of the G+C
% content, lipid analysis and BIOLOG.TM. analysis (a more
comprehensive biochemical analysis). Also, detailed studies
regarding the chemistry of the polysaccharide will be
completed.
EXAMPLE 2
Applications to Engineered Waste Containment and Treatment
[0069] The LAB-1 strain may be used to construct environmental
biofilm barriers for containment and treatment of contaminated soil
and groundwater. The purpose of containment barriers is to control
the transport of chemical contaminants from waste disposal
facilities or from areas which have become contaminated by spills,
industrial processes, illegal dumping or other sources. Several
different types of barriers are possible, including the following:
(1) subsurface biofilm cutoff wall; (2) subsurface liners
consisting of compacted, biofilm treated soil; (3) in-situ biofilm
liners; and (4) barriers made by treating geotextiles with
biofilm.
[0070] The results disclosed herein demonstrate that soil hydraulic
conductivity (k) may be reduced by several orders of magnitude by
the addition of the biofilm-producing bacterium disclosed herein.
The reductions of k obtained using the LAB-1 strain are sufficient
to meet Environmental Protection Agency (EPA) criteria for barrier
materials, defined as a k value of 10.sup.-7 cm/sec or less. The
low hydraulic conductivity persists when the soil is permeated with
a variety of chemical solutions, suggesting that a biofilm barrier
may be compatible with a wide range of contaminants. The biofilm
disclosed herein may also be useful for controlling contaminant
transport mechanisms, such as diffusion, adsorption and
biodegradation.
[0071] Solutions of biofilm and nutrient are pumped into the
subsurface through a series of closely-spaced vertical wells.
Formation of biofilm in the soil around the wells causes a decrease
in soil permeability and decrease in contaminant transport
sufficient to form an engineered barrier to contaminant migration.
Specific design parameters such as well depth and spacing, pumping
pressures, composition of bacterial and nutrient solutions, and
time of pumping, are site-specific and depend upon site geology,
type and extent of subsurface contamination, ground water
conditions, and other variables which must be considered on a
case-by-case basis.
[0072] FIG. 7 shows one use of the present invention for the
formation of subsurface liners for the containment of wastes in
engineered disposal facilities, such as landfills. A landfill 10 is
depicted in cross-sectional view. Waste 12 is disposed within a
subsurface liner 14. If the liner 14 is being placed during the
creation of the landfill 10, a biofilm liner may be used prior to
deposition of the liner 14. In addition, a containment wall may be
erected that surrounds the waste site, and additional layers of
decontaminating biofilm barriers may be included.
[0073] In preexisting landfills, such as the one depicted in FIG.
7, waste may leach in the form of a leachate 16 into subsurface
strata 18 and 20. A biofilm barrier wall 28 is created that
surrounds the waste 12 and captures the leachate 16. The biofilm
barrier wall 28 is constructed so as to reach into strata 22, 24
into which the waste 12 does not leach. One advantage of the
biofilm of the present invention is that it permits such remedial
application to existing landfills that may be leaking and even
prevents leachate 16 from reaching a subterranean water layer
26.
[0074] Current technology for a biofilm barrier wall 28, for
example, may employ fine-grained soils that are field compacted to
achieve a hydraulic conductivity of less than 10.sup.-7 cm/sec
(commonly referred to as "clay liners"). At many sites, such soil
is not readily available and must be transported from off-site,
increasing substantially the cost of compacted soil liners. Using
the LAB-1 strain of the present invention soil, containment
conditions may be met by treating readily available soils with the
biofilm in order to achieve the low hydraulic conductivity required
for compacted soil liners.
[0075] One specific field of use for the LAB-1 biofilms is creating
subsurface biofilm liners that include spreading untreated soil in
loose (uncompacted) lifts using conventional soil spreading
equipment (e.g., bulldozers). Loose lifts will generally be 150 to
225 cm thick. A solution that includes water, LAB-1, and nutrients
are applied to the soil, using, e.g., conventional equipment used
to apply water to soil (e.g., a truck-mounted water tank with
sprinkler hoses). The soil is then compacted using conventional
equipment (e.g., sheeps foot rollers) to achieve the specified
density, typically resulting in a compacted lift thickness of 100
to 150 cm. The required number of lifts and liner total thickness
are site-specific design parameters which are determined by
analysis of contaminant transport and regulatory requirements for
containment.
[0076] Compacted clay liners typically range from 0.6 to 1.3 meters
thick. The proposed procedure is similar to field construction of
clay liners, except that the soil is treated with a
biofilm-producing solution. Alternatively, solutions of strain
LAB-1 and nutrients are injected into the ground at a specified
depth to create in situ biofilm liners. This type of liner is
particularly useful at sites contaminated by accidental spills.
Alternatively, previously grown biofilm may be mixed directly into
or onto the soil.
[0077] Subsurface liners may also be constructed by treating
geotextiles with biofilm. Geotextiles are generally made of
synthetic fibers that are either woven or matted together, yielding
a porous fabric that is used for soil separation, reinforcement,
filtration or drainage. Containment barriers can be created by
spraying bitumen, rubber-bitumen or other polymeric mixtures into a
properly deployed geotextile that contains the LAB-1 produced
biofilm disclosed herein. One particular example for use of the
LAB-1 biofilm is in the application of a liquid solution containing
strain LAB-1 and nutrients to geotextiles to clog the pore spaces
and reduce permeability, creating a barrier to flow.
EXAMPLE 3
Materials: Soil and Bacteria
[0078] Soil used by the present inventors to analyze waste
containment capability is a naturally occurring, easily attainable
sand. Based on its grain size distribution and Atterberg limits,
this soil is classified as SM, or silty sand of low plasticity, in
the Unified Soil Classification System. Permeability tests yield a
saturated hydraulic conductivity (k) of approximately
1.5.times.10.sup.-5 cm/sec when compacted to maximum dry density.
This value of k would make the soil unsuitable for use as a waste
containment barrier. Initial studies indicated that k could be
reduced to values on the order of 10.sup.-8 to
10.sup.-.differential.cm/s- ec, which is in the range required for
waste containment, by treating this soil with the biofilm-producing
bacterial strain LAB-1.
[0079] The operational procedure for use of the LAB-1 bacterium to
form a biofilm that may be used to test water permeability may
include the following steps: (1) compacting soil into a cylindrical
specimen which is placed in a flexible wall permeameter, (2)
permeating the specimen with a solution containing LAB-1, and (3)
measuring the soil hydraulic conductivity while the specimen is
permeated first with nutrient solution, then by water, as is taught
by Dennis M. L. and Turner, J. P. J. GEOTECHNICAL &
GEOENVIRONMENTAL ENG. 124: 120-127 (1988) (in which a similar
procedure was used with the bacterium B. indica).
[0080] Using the biofilm produced by the LAB-1 strain disclosed
herein, hydraulic conductivity was reduced from
k=1.5.times.10.sup.-5 to approximately k=5.times.10.sup.-8 cm/sec
upon establishment of a plugging biofilm, which required permeation
with nutrient solution for approximately one week. Most of this
decrease occurred within 1 to 2 days, during which the k was
reduced to less than 10.sup.-7 cm/sec.
[0081] FIG. 8 is a graph that shows hydraulic conductivity versus
time for a specimen treated with LAB-1. The low hydraulic
conductivity persisted for over 160 days, even though the nutrient
solution was discontinued after 6 days.
EXAMPLE 4
Applications to Agriculture
[0082] Large areas of the earth include desert lands that are not
arable without large-scale reclamation. Reclamation in the context
of desert lands requires not only irrigation, but extensive soil
modification. The economical and social impact of successfully
converting non-productive desert land into productive agricultural
land is enormous and provides significant benefits to mankind.
[0083] The use of LAB-1 as a biologically and environmentally sound
source of support and nutrients for soil treatment improves the
agricultural properties of sandy soils as described herein. Many
naturally existing desert soils are aeolian (wind-deposited) and
consist of sand and silt sized particles with little or no organic
content. Such soils are considered poor for agricultural
development because they are highly porous, which promotes rapid
infiltration and seepage of irrigation water away from the surface
where it is most needed for crops. Lack of organic material
generally corresponds to low nutrient content. Many desert areas
are active aeolian environments in which wind is the dominant agent
of sediment transport.
[0084] Agricultural development is severely impacted when topsoil
is eroded and transported by wind. Considering the characteristics
of sandy desert soils versus the requirements of soils for
agriculture, the present invention includes the use of the LAB-1
derived biofilm for the treatment of agricultural soils to improve
the following soil agricultural properties: (1) improved water
retention characteristics; (2) enhanced ability to establish and
support plant growth; and (3) improved erosion resistance. These
improvements may be obtained by adding complete or dried and
pulverized biofilm, or by the application of LAB-1 strain in
bacterial/nutrient solutions using conventional soil watering
equipment (e.g., a truck-mounted tank with sprinkler hoses or
conventional irrigation systems).
[0085] The biofilm of the present invention has been used for the
treatment of soil. The biofilm altered the soil's properties in
many ways that enhanced the soil's ability to support agriculture.
These include the following: (1) an improved ability of sand to
retain moisture; (2) an increased biomass in the form of
polysaccharides that function as a nutrient supply for plant
growth; (3) improved soil cohesion; and (4) increased resistance of
soil to erosion.
EXAMPLE 5
Medical Applications
[0086] The biofilm of bacterium LAB-1 possesses a number of
characteristics that are of potential commercial application in
medical devices and treatment, including its low antigenicity,
non-toxicity, and its biodegradable nature. For these consumer
applications, the exopolysaccharide may be purified, e.g., by the
addition of concentrated NaOH to the cell culture at a final
concentration of 0.2 M, followed by the addition of 3 volumes of
ethanol to precipitate the polymer and other materials. The
precipitate is collected and redissolved in half the original
volume of water. Protein is removed by either extracting twice with
phenol or by ultra-filtration. The aqueous phase is dialyzed,
lyophilized and ground to yield a fine white powder as will be
known to those skilled in the art.
[0087] As the exopolysaccharide is not cell-bound, the
exopolysaccharide produced by LAB-1 may, alternatively, be purified
without using the alkali treatment or the phenol extraction. Not
only is the purification process thereby simplified, it may prevent
the removal of alkali-labile acetyl moieties from the purified
LAB-1 exopolysaccharide.
[0088] Total carbohydrate concentration in culture broths and
polymer solutions may be determined by the phenol reaction,
described by Gerhardt in Manual of Methods for General Bacteriol.
(Amer. Soc. Microbiol., Washington, D.C., 1991). Glucose, galactose
and xanthan gum (SIGMA CHEMICAL CO..TM., St. Louis, Mo.) may be
used as standards. Total protein concentration in culture broths
and polymer solutions may be determined using the BIO-RAD.TM.
protein assay (BIO-RAD LABORATORIES.TM., Richmond, Calif.).
Lysozyme may be used as the standard. Cellular protein may be
released by boiling the cells in 0.2 M NaOH.
[0089] Purified polysaccharide may be further hydrolyzed in 1 M
trifluoroacetic acid at 120.degree. C. for times varying between 30
minutes and 2 hours. Monosaccharides in the polysaccharide
hydrolysate may be separated using, e.g., a WATERS.TM. HPLC
equipped with a BROWNLEE.TM. polypore PB, lead loaded cation
exchange column, operated at 85.degree. C., with water as the
eluent. Detection may be performed by refractive index using, e.g.,
a WATERS.TM. Model 401 Differential Refractometer.
[0090] The polysaccharide may be further characterized by proton
NMR spectroscopy and infrared spectroscopy. Infrared analysis,
along with the monosaccharide composition data, may be compared to
the composition and IR scans of polysaccharides from mutant or
genetically manipulated strains to detect changes in structure.
[0091] Being either non- or weakly-immunogenic, the biofilm of the
present invention may be used as a vaccine adjuvant or carrier that
provides a reservoir for antigens. An adjuvant may be prepared
using the LAB-1 derived biofilm of the present invention. Adjuvants
may be synthesized by any one of a number of established methods,
as has been described by M. Bodansky, et al., "Peptide Synthesis,"
second edition, Wiley, New York 1976 and R. W. Roeske, Peptides
(N.Y.) 3, 102 (1981).
[0092] A particularly useful method is the methanesulfonic acid
catalyzed esterification procedure described by C. Penney, et al.,
J. Organic Chemistry 50, 1457-1459 (1985). During the preparation
of an adjuvant, it may be desirable to temporarily protect reactive
functional groups. For example, amines may be protected by
urethane-type groups, alcohols by t-butyl or benzyl groups, and
acids by ester groups.
[0093] The adjuvant may be purified by any of the techniques
described previously. One such purification technique is silica gel
chromatography, in particular the "flash" (rapid) chromatographic
technique, as described by W. Clark Still, et al., J. Organic
Chemistry, 43, 2923-2925 (1978). Other chromatographic methods,
however, including HPLC, may be used for purification of the
adjuvant. Crystallization may also be used to purify the adjuvant.
In some cases, no purification is required as a product of
analytical purity is obtained directly from the synthesis. Methods
of formulating the adjuvant into vaccine preparations are well
known in the art, and not detailed herein.
[0094] As the base bacterium of the present invention does not
appear to be a human pathogen, and its exopolysaccharide is not
toxic or antigenic and does not readily support bacterial growth,
the exopolysaccharide provides an ideal source for oncotic plasma
expanders. Furthermore, the exopolysaccharaide's lack of intrinsic
antigenicity makes it an ideal candidate for use in, e.g., all
mammals. Thus, the present invention includes a mixture of
components that, when placed in aqueous solution, may be used to
expand the plasma volume of a subject in need thereof.
[0095] For purposes of the further description of the invention,
the mixture of plasma expanding components according to the
invention will be discussed as an aqueous solution. From the
following description of the invention it is expected that one
ordinarily skilled in the art would be enabled to provide the
mixture as a dry mixture and make the adjustments to amounts of
sodium chloride, fluid and/or dextrose, etc. as necessary.
[0096] The exopolysaccharide oncotic agents of the foregoing
mixture of components are ones that are generally water
semi-soluble. By water semi-soluble oncotic agent is meant
partially-water soluble molecules that when dissolved in the fluid
phase of circulating plasma in a living subject are of a size
sufficient to prevent their immediate loss from the circulation by
traversing the fenestration of the capillary bed into the
interstitial spaces of the tissues of the body. The term
polysaccharide oncotic agent thus does not include such
polysaccharides as chitin, because chitin is not soluble in
water.
[0097] The plasma expander is made by solubilizing the purified
exopolysaccharide in a biocompatible fluid. Plasma expander
solutions according to the invention may also contain additional
ingredients, including but not limited to, sodium ions (110-120
mEq/1), calcium ions (5 mEq/1), potassium ions (0-3 mEq/1),
magnesium ions (0-0.9 mEq/1), and vitamin K (0-10 mg per subject or
0-3 mg/l). Further, the expander may be buffered to about pH 7.4
and may provide assimilable sugar (e.g.,, dextrose or glucose at
5-10 mM). Preferably, the expander is provided as a sterile
solution, but it may also be provided as a powder that can be
reconstituted in a sterile manner, or sterilized after
reconstitution.
[0098] While this invention has been described in reference to
illustrative embodiments, this description is not intended to be
construed in a limiting sense. Various modifications and
combinations of the illustrative embodiments, as well as other
embodiments of the invention, will be apparent to persons skilled
in the art upon reference to the description. It is therefore
intended that the appended claims encompass any such modifications
or embodiments. All references cited herein are hereby expressly
incorporated by reference.
* * * * *