Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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85. WO03047353 - 05.06.2003
PROBIOTIC FORMULATION AND METHOD FOR REDUCTION OF PATHOGENIC BACTERIA

URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=WO03047353


Inventor(s): KEETON JIMMIE A (US); WILLIAMS DIANE P (US)
IP Class 4 Digits: A61K; C12N; A01N
IP Class: C12N1/20; A61K35/74; A01N63/02
E Class: A61K35/74+M; A61K45/06; A01N63/00; A01N63/02; A61K33/00+M
Application Number: US20010006102 (20011203)
Priority Number: US20010006102 (20011203)
Family: WO03047353
Equivalent: AU2002346642
Abstract:

A COMPOSITION AND METHOD FOR REDUCING THE LEVELS OF PATHOGENIC BACTERIA IN AN AQUATIC ENVIRONMENT. THE COMPOSITION INCLUDES ISOLATED BACILLUS CEREUS WHICH ARE ABLE TO REDUCE A NUMBER OF WELL KNOWN PATHOGENIC BACTERIUM FROM AQUATIC ENVIRONMENTS.Description:

FIELD OF THE INVENTION

[0001] The invention relates generally to a novel formulation for the control of pathogenic bacteria and more particularly to a probiotic formulation for the control of Aeromonas, Pseudomonas, Vibrio, Streptococcus and other pathogenic bacteria to fish, shellfish and other aquatic life. Furthermore, the invention relates to methods for controlling the levels of pathogenic bacteria, for promoting a healthy aquatic environment, and for promoting the health of aquatic organisms including particularly the reduction of fish morbidity and mortality. The compositions and methods are applicable in both freshwater and saltwater aquatic environments. The invention also has application for reducing pathogenic microbes in sewage or wastewater treatment facilities, specifically Escherichia coli or other pathogens not yet identified.


BACKGROUND OF THE INVENTION

[0002] Bacterial pathogens represent a substantial threat to aquatic environments, especially where the environment is heavily populated by fish, shellfish and other aquatic life. Aquatic environments having populations of pathogens are suboptimal for the health and development of various forms of aquatic life. Examples of such aquatic environments include a number of aquaculture type industries including, fish production facilities, where over 2.5 million pounds of fish are produced each year; freshwater and saltwater tropical fish aquariums, especially at the wholesale level where large populations of fish are combined for delivery and sale to potential customers; and koi and other outdoor ponds. Also included are intensive recirculating aquaculture systems, closed system aquaculture or other related intensive aquatic production systems containing aquatic life forms.

[0003] Prominent bacterial pathogens within the aquaculture industry include bacteria from the genus Aeromonas, Vibrio, Pseudomonas, Streptococcus, and Columnaris. These pathogens can lead to illness and death of the fish, shellfish or other aquatic life in the affected environment. The cost associated with these losses, and the potential health risk to those who consume the infected aquatic life is significant, and represents a major concern within these industries. Severe infections with pathogenic microbial species can result in mortalities approaching 80-90 percent.

[0004] Presently, bacterial pathogens are controlled by widespread use of substances such as chemicals and antimicrobial agents including antibiotics. However, widespread resistance to such treatments represents a major threat to the fish and shellfish populations as well as to those who consume or are associated with the resistant bacteria. Pathogenic bacterial resistance to chemicals and antimicrobials, as well as the lack of other effective treatments is of grave concern to the aquaculture industry. Against this backdrop the present invention has been developed.


SUMMARY OF THE INVENTION

[0005] In one aspect, compositions of the present invention include the isolation of an organism or organisms useful in the reduction of other bacteria, and preferably pathogenic bacteria, in an aquatic environment. One preferred isolate, strain EHC 100 is identified as belonging to the Bacillus cereus species of bacteria.

[0006] In another aspect, a method for reducing the levels of a bacteria, and preferably a pathogenic bacteria, in an aquatic environment includes providing the compositions of the present invention to the aquatic environment and monitoring the levels of pathogenic bacteria in the aquatic environment.

[0007] In another aspect, compositions of the present invention are used in the prevention of fish and shellfish mortality and in the treatment of fish and shell fish having a bacterially related disease.

[0008] These and various other features as well as advantages which characterize the present invention will be apparent from a reading of the following detailed description and a review of the associated figures.

BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1 illustrates a reduction in pathogenic bacteria levels in an aquatic environment treated with a composition in accordance with the present invention.

[0010] FIG. 2 illustrates a reduction in Tilapia fingerling mortality in an aquatic environment treated with a composition in accordance with the present invention.

[0011] FIG. 3 illustrates a reduction in pathogenic bacteria levels in a koi pond treated with a composition in accordance with the present invention.

[0012] FIG. 4 illustrates a reduction in pathogenic bacteria levels in a koi pond treated with a composition in accordance with the present invention.

[0013] FIG. 5 illustrates a reduction in pathogenic bacteria levels in a koi pond treated with a composition in accordance with the present invention.

[0014] FIG. 6 illustrates a reduction in pathogenic bacteria levels in an aquaculture with a Bacillus EHC 100 strain of the present invention.

[0015] FIG. 7 illustrates a reduction in vibrio pathogens in 0.8 ha shrimp ponds using the Bacillus EHC 100 strain of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Probiotic Organism

[0017] As described more fully in the Examples below, a probiotic organism that reduces the levels of pathogenic bacteria in aquatic environments has been identified and characterized. The identified organism belongs to the Bacillus genus of bacteria, and in particular, belongs to the Bacillus cereus species of bacteria. According to the Biolog system of classification, the probiotic organism falls into Group 3 Gram-positive spore-forming rods. In precisely classifying the organism taxonomically, there may be instances of comparison where the identified organism appears similar to other organisms. For example, Bacillus thuringiensis (producer of insecticidal toxins) and Bacillus anthracis (etiological agent of anthrax) may be closely related to the identified organism. There is significant similarity among these organisms according to several aspects such as fatty acid analysis, and DNA sequence analysis including data regarding the intergenic space for ribosomal genes. The identified organism, however, is distinct in having the properties of significantly affecting the level of pathogenic bacteria in an aquatic environment and affecting fish health in aquaculture systems.

[0018] The probiotic organism of the present invention was identified using an activity assay, where a previously described waste and sludge reducer (K.I. Waste & Sludge Reducer, Keeton Industries, Fort Collins, Colo.) (hereinafter K.I. Reducer) was analyzed for its effects on Aeromonas and Pseudomonas levels in an aquatic environment. K.I. Reducer itself is used for control of accumulated organic waste and sludge in ponds, lakes, ornamental ponds, aquaria, and aquaculture facilities; also, it reduces nutrient levels through accelerated microbial decomposition of organic wastes on the pond bottom. In addition, K.I. Reducer also decreases the negative effects of eutrophication, the bio-oxygen-demand status, organic waste digestion and the rapid overgrowth of some bacteria and algae in aquatic environments.

[0019] From selected batches of the K. I. Reducer, smaller aliquots were prepared and tested for the ability to cause a decrease in pathogenic bacteria levels. Standard microbial isolation techniques and identifications using fatty acid analysis were performed on samples having the greatest effects on pathogenic bacteria reduction. Samples from a batch having the greatest activity were selected for further testing. Further testing showed that an isolated organism from K.I. Reducer showed significant activity in reducing pathogenic bacteria levels in aquatic environments. This isolate, designatedstrain EHC 100, was identified as a strain of the species Bacillus cereus by standard microbiological techniques and fatty acid analysis. This isolate has been deposited with the Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria Ill., having accession number ______. Note that as used herein, "isolated" refers to an organism of the present invention that has been separated from at least one contaminant found in the organisms natural environment or found in K.I. Reducer.

[0020] Formulations Of Bacillus EHC 100 strain were fermented and blended in solutions of 2 to 5% sodium. These blends were tested for the ability to reduce a level of pathogenic bacteria. Note that other like salts may be substituted for sodium.

[0021] The Bacillus EHC 100 strain compositions can be blended into other compositions that contain other species of beneficial microorgnisms. A blend can retain one or more than one property such as having the ability to reduce pathogenic bacteria in an aquatic environment and the ability to improve fish health.

[0022] Here, the terms "reduce" and "control" are used interchangeably and refer to a statistically significant difference in the level of a pathogenic bacteria in a target environment, or in the overall level of bacteria other than any composition used for treatment, when the difference relates to a level prior to treatment compared to a level following treatment.

[0023] Some conditions may affect the effectiveness of the compositions in an aquatic environment to reduce pathogenic bacteria or to improve fish health. In some cases, the effectiveness of any of the compositions can be dependent on the total alkalinity of the aquatic environment, or the temperature of the aquatic environment. For example, an aquatic environment having a temperature of about 50[deg.] F. to 62[deg.] F. may require an enhanced dose of the composition to produce the same effects as an aquatic environment at about 63[deg.] F. to 68[deg.] F. Therefore, there is likely a sliding scale of how much of the composition is required to produce the same effects on the aquatic environment dependent on the temperature of the aquatic environment. Other conditions that may affect effectiveness of compositions include whether a pond is heavily stocked or has poor filtering. Regardless, the dose of the composition may be varied to suit the conditions of the aquatic environment and produce the expected results.

[0024] Mechanisms of Action

[0025] The mechanisms of action are currently under investigation with regard to the ability of the compositions of the present invention to affect levels of pathogenic bacteria or to affect the health of aquatic organisms. All possible mechanisms of action for the present invention are not intended to limit the scope of the invention in any way and are all believed to be within the scope of the present invention.

[0026] One potential mechanism of action is for a composition of the present invention to directly compete with other bacteria, such as pathogenic species of bacteria for resources, such as nutrients in the aquatic environment. The compositions of the present invention may prevent other bacteria from maintaining or increasing their population levels. Such direct competition is effective in cases where the organisms, for example EHC100, in the composition are present initially with greater numbers, or in a case where an organism in 115 the composition grows at a greater rate than one or more other species of bacteria in the target aquatic environment.

[0027] A second potential mechanism involves indirect competition, where an organism in the composition fosters an environment that affects a third party organism in the aquatic environment. For example, the third party organism may then be in direct competition with the pathogenic species of bacteria in the aquatic environment.

[0028] A third potential mechanism involves the production of a molecule by an organism in the composition, for example EHC100, where the molecule acts either directly or indirectly upon a species of pathogenic bacteria in the aquatic environment. Such molecules could be peptides that function as toxins, or enzymes.

[0029] A fourth potential mechanism involves the production of a molecule by an organism in the composition, for example ECH100, that induces pathogen resistance in the aquatic organism population (similar to Systemic Acquired Resistance (SAR) in plants).

[0030] The possibility exists that some level of each mechanism is involved, independently or in combination, to give rise to the activities of the compositions described herein. It is also anticipated that an as of yet undiscovered mechanism of action may also be involved with the mechanisms of action of the present invention.

[0031] Culture Conditions

[0032] Initial compositions were isolated as pure cultures in standard media as known in the art such as nutrient agar or trypticase soy agar. Compositions that are embodiments of the present invention are grown in liquid broth cultures. The media can be a conventional media known in the art such as trypticase soy broth (TSB). Other enrichment media may also be used to grow compositions.

[0033] At the end of culturing, the density of the target organism is preferably between 4*10and 6*10cfu/mL, although greater or smaller concentrations may be attained and used according to the invention. In the cultures, about at least 80% of the colony forming units can be spores. In growing the cultures, a range of about 10[deg.] C. to 37[deg.] C. is used, preferably a range of 20[deg.] C. to 34[deg.] C. is used, and most preferably, a range of 28[deg.] C. to 30[deg.] C. is used. The cultures are incubated for about 12 to about 72 hours, and preferably for an amount of time required to reach the preferred cell density in a given culture medium.

[0034] A completed liquid culture can be stored at refrigeration temperatures or at room temperatures. Preferably, the cultures are stored at from about 3[deg.] C. to about 25[deg.] C. In another embodiment, the liquid culture may be dehydrated for storage and re-hydrated upon use. In yet another embodiment, the liquid culture may be lyophilized and stored. A culture may retain effectiveness for applications when stored for several months at room temperature and for over a year at refrigeration temperature.

[0035] Note that compositions may be used in aquatic applications in the presence of other products, for example, antibiotics which are well known in the art.

[0036] Applications

[0037] The organisms in compositions of embodiments of the invention are effective at reducing pathogenic bacteria levels in aquatic environments. In the methods of the invention, the pathogen-reducing effects of the present invention are achieved by treating an aquatic environment with one or more doses of a composition.

[0038] Dose sizes can range from about 20 mL to 60 mL of approximately 4*10to approximately 6*10organisms per 1,000 gallons of aquatic medium. Doses can be adjusted depending on a variety of factors including level of stocking, level of filtering, temperature, and alkalinity. Note that doses can be repeated as needed to achieve the desired result.

[0039] Other methods are envisioned to be within the scope of the present invention, including methods for treating a population of aquatic organisms such as fish or shellfish, methods for preventing bacterially transmitted disease in aquatic organisms, methods for improving an aquatic environment; methods for isolating an organism used to reduce pathogenic bacteria levels in an aquatic environment, where the desirable properties of the target organism are used to screen for the targeted organism; and the like. With respect to the present invention, "treatment" of fish or shellfish in an aquatic environment is meant to refer to any reduction in the percentage of fish or shellfish infected with a sufficient level of pathogenic bacterium that the fish or shellfish exhibit symptoms of the infection, for example, exhibit sores or blisters. A treatment, as such, is accomplished if a detectable number of fish or shellfish in the aquatic environment exhibit either fewer symptoms then before or have been alleviated of symptoms, by the compositions of the present invention being added to the environment.

[0040] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
EXAMPLE 1
Probiotic Solution Decreases Pathogenic Bacteria

[0041] As shown in FIG. 1, addition of a volume of about 10-20 mL of product per 1,000 gallons system volume with a concentration of about 4*10cfu/mL Of Bacillus EHC 100 strain to an aquaculture system having up to approximately 105 colonies per mL of Streptococcus, Pseudomonas and Aeromonas resulted in a dramatic decrease in the pathogens concentrations. Pathogens were substantially eliminated over the course of one month of treatment.

[0042] Similar results are shown in Table 1, where treatment of multiple strains of hemolytic streptococci with the probiotic composition reduced the level of hemolytic streptococci to undetectable levels as measured over a two-week period. Consistent results were observed a second experiment, also shown in Table 1. These experiments involved a commercial closed system Aquaculture Facility. A volume of about 10-20 mL of product per 1000 gallons system volume with a concentration of about 4*10cfu/mL of the probiotic composition was used for an aquaculture environment having a volume capacity of about two million gallons. All analytical microbiological tests were performed by a certified laboratory using conventional methods as known in the art.

[0043] The data illustrates that the probiotic solutions of the invention are associated with a reduction in target pathogenic bacteria in an aquaculture environment.

[0044] Table 1 illustrates treatment with Bacillus EHC 100 strain in intensive aquaculture system. Beta hemolytic streptococcus was isolated in two production sytems at 2000 cfu/ml in system I and 1000 cfu/ml in system II, respectively. The systems were treated with three applications of strain EHC100 at the recommended dosage of 10-20 ml, Bacillus EHC 100 strain per 1000 gallons of system volume. Two weeks after the third application, no pathogenic beta hemolytic Streptococcus bacteria were found in the fish tank growout system.

[0045] The aquaculture tanks were screened for Steptococcus organisms only, as prior testing had confirmed that fish were infected with Streptococcus.

[0046] Further evidence that the probiotic strain EHC 100 causes a reduction in pathogenic organisms is provided in FIGS. 2 and 3. FIG. 2 illustrates a reduction of pathogenic organisms in outdoor shrimp ponds following treatments with Bacillus EHC 100 strain. Pathogenic organisms where isolated and identified as Vibrio, Pseudomonas aeruginosa, Enterobacter sp. And Proteus sp at the start of testing. Applications of Bacillus EHC 100 strain were added at a rate of 1 liter per 100,000 gallons of pond water, 1 treatment each 3 days for nine days followed by one weekly treatment for a 28 day period. Pathogenic organisms were decreased from 15,000 CFU/ml to 13 CFU/ml at the end of 28 day period. All tests were performed by qualified state pathologist at the Government Laboratory Of Diagnostic Veterinarial Science.

[0047] FIG. 3 provides a graph that depicts reduction of Vibrio bacterial pathogens in a 0.8 hectare intensive culture outdoor shrimp pond in the Republic of Ecuador. Vibrio was completely controlled by normal applications of Bacillus cereus, EHC 100 strain, during the entire 120 day growout cycle for shrimp Paneaus vannemei (White Shrimp)

TABLE 1

Effect of B. cereus, EHC 100 strain on control of



pathogenic bacteria

Results


SiteControl (cfu/mL)Treated (cfu/mL)

Experiment 12 * 102000 hemolyticdetected two weeks after

streptococci/mltreatment. Detection limit of

before treatment1 CFU per mL of hemolytic

streptococcus

Experiment 111 * 101000 hemolyticdetected after two weeks after

streptococci/mltreatment detection limit of 1

before treatmentCFU/ml streptococcus


EXAMPLE 2
Probiotic Solution Decreases Tilapia Mortality

[0048] As shown in FIG. 4, probiotic solution treatment of Tilapia fingerlings resulted in a significant decrease in mortality. Approximately one liter of probiotic solution having a concentration of 4*10bacillus of the invention was added per 100,000 gallons of system volume three times in 9 days followed by weekly treatments over a four week period. Fingerling mortality was monitored over a four week period.

[0049] The data illustrates that the probiotic solutions of the invention have a positive effect in reducing the mortality of treated aquatic populations.
EXAMPLE 3
Probiotic Solution Decreases Pathogenic Bacteria

[0050] As shown in FIGS. 5, 6, and 7, and Table 2, addition of a probiotic solution of the present invention to various aquatic systems including ponds resulted in a significant decrease in the pond water levels of certain target pathogenic bacteria.

[0051] In these examples, the concentration of bacteria of the composition in the initial 20 ml dose was about 4*10CFU/mL to 6*10CFU/mL.

[0052] FIG. 5 shows the results of treating Pond A with a composition at a dose rate of 20 mL per 1,000 gallons of aquatic medium. Pond A water initially having approximately 25,000 to 30,000 Aeromonas and Pseudomonas cfu/mL was treated with a probiotic solution of the invention. A period of treatment lasting about 19 days resulted in a significant decrease in the pathogens levels: below 3,000 cfu/mL for Aeromonas and undetectable levels for Pseudomonas.

[0053] FIG. 6 shows the results of treating Pond B water with a composition at a dose rate of 20 mL per 1,000 gallons of aquatic medium. Pond B water initially had levels of bacteria as follows: Aeromonas sobria at 10,000 cfu/mL, Aeromonas hydrophilia at 20,000 cfu/mL, and Pseudomonas at 160,000 cfu/mL. A period of treatment lasting about 32 days resulted in a significant decrease in the pathogens levels. At the end of the treatment period, the following levels were observed: Aeromonas sobria was undetectable, Aeromonas hydrophilia at 3,000 cfu/mL, and Pseudomonas was undetectable.

[0054] FIG. 7 shows the results of treating a koi system with a composition at a dose rate of 20 mL per 1,000 gallons of aquatic medium and at a dose rate of 60 mL per 1,000 gallons. The koi system water initially had levels of bacteria as follows: Aeromonas sobria/hydrophilia at 500,000 cfu/mL, and Burkholderia cepacia (Pseudo) at an undetectable level. Several treatment periods were examined, including periods of 18, 48, and 97 days. After the initial treatment at a dose rate of 20 mL per 1,000 gallons, an assessment was made at 18 days. At 18 days, the following levels were observed: Aeromonas sobria/hydrophilia at 500,000 cfu/mL, and Burkholderia cepacia (Pseudo) at an undetectable level.

[0055] After the 18 day period, the dose rate was increased to 60 ml per 1,000 gallons. After 48 days, a significant change was noted in at least one population of bacteria. At 48 days, the following levels were observed:Aeromonas sobria/hydrophilia at 35,000 cfu/mL, and Burkholderia cepacia (Pseudo) at level of 60,000 cfu/mL. After 97 days, the following levels were observed: Aeromonas sobria/hydrophilia at 16,000 cfu/mL, and Burkholderia cepacia (Pseudo) at an undetectable level. Thus the treatment ultimately resulted in a reduction of more than 10-fold for Aeromonas species after 48 days, with a reduction of more than 25-fold after 97 days.

[0056] Treatments during the first 18 days were standard dosing of 20 ml/1000 gallon system water, one treatment every 3 days for 9 days, followed by once per week.

[0057] Some of these experiments involved an outdoor pond. All analytical microbiological tests were performed by a certified laboratory using conventional methods as known in the art.

TABLE 2


Data supporting FIGS. 5, 6, and 7.

BeforeAfter

treatment,treatment,

OrganismDay 0Day 19

.Aeromonas30,0003,000

.Pseudomonas25,000none

.detected

BeforeAfter

treatment,treatment,

OrganismDay 0Day 32

.Aeromonas10,000none

sobriadetected

.Aeromonas20,0003,000

hydrophilia

.Pseudomonas160,000none

detected


BeforeAfterAfterAfter

treatment,treatment,treatment,treatment,

OrganismDay 0Day 18Day 48Day 97

.Aeromonas500,000500,00035,00018,000

sobria +

Aeromonas

hydrophilia

Burkholderianonenone60,000none

cepaciadetecteddetecteddetected

(Pseudo)
EXAMPLE 4


EHC100 has a Fatty Acid Composition Consistent with that of Bacillus cereus

[0058] The fatty acid composition of an individual species of bacteria can serve as a reliable identifying characteristic. In general, a sample of unknown bacteria is cultured and its fatty acids extracted for separation by gas chromatography. A computer generated profile of the separated fatty acids (having from 9 to 20 carbons in length) is compared to profiles for over 2600 species in a Microbial Identification System (Sherlock Libraries). Matches are determined through statistical probability software.

[0059] Fatty acid analysis on a target sample can be performed by a commercial laboratory, for example, Acculab, 223 Lake Drive, Pancader Corporate Center, Newark, Del. Briefly, Acculab receives a target culture, grows and harvests the bacteria, and places the bacteria in an extraction tube. One ml of a saponification solution (90 g sodium hydroxide mixed with 300 ml methanol and 300 ml dH2O) is added to the sample and the sample is heated in a boiling water bath for 5 minutes. Samples are removed from the water bath and vortexed for about 5 seconds. The samples are returned to the water bath for an additional 25 minutes at which time it is removed and cooled in tap water. Two mls of methylation solution (405 mls of 6 N hydrochloric acid added to 345 ml of methyl alcohol) is added to the sample and the sample is heated for about 9 to 11 minutes at 79 to 81[deg.] C. The sample is removed and cooled in tap water. Approximately 1.5 mls of extraction solution (400 ml of hexane mixed with 400 ml of methyl tert-butyl ether) is then added to the sample and the sample is gently tumbled for about 10 minutes. The aqueous lower phase is discarded and 3 mls of base wash (9 g sodium hydroxide dissolved in 750 ml dH2O) is added for an additional 5 minute incubation. Approximately [2/3] of the organic phase is removed and analyzed by gas chromatography (MIDI BIO-GC, Hewlett-Packard) using a fused silica capillary column (coated with 5% phenyl methyl silicone). GC generated data is analyzed using MIDI BIO-GC software.

[0060] Results are computed to provide a numeric value which expresses how closely the fatty acid composition of the unknown sample compares with the mean fatty acid compositions of the strains used to create the Sherlock Library. A value with a similarity above 0.5 or higher and more than 0.1 separation between the first and second entry is considered a good library comparison.

[0061] As shown in Table 3, EHC100 has a fatty acid composition that likely corresponds to the fatty acid composition of Bacillus cereus. A sample of EHC100 was isolated using methods in accordance with the present invention and supplied to Acculab. The EHC100 sample showed a 0.752 value, which was over 0.1 greater than the next entry -0.619 (Bacillus canadensis), indicating that there is a good likelihood that EKC100 is a Bacillus cereus.

TABLE 3


Acculab Generated Profile and Similarity Index for EHC100

RTAreaAr/HtResponECLName%Comment 1Comment 2

1.5974470114370.0287.014SOLVENT PEAK1.698173500.0167.2221.78177840.1337.3944.2474930.0281.06011.60812:0 ISO0.36ECL deviates -0.001Reference 0.006

5.279105410.0321.01412.61413:0 ISO7.39ECL deviates -0.000Reference 0.006

5.37814910.0341.01112.70313:0 ANTEISO1.04ECL deviates -0.001Reference 0.008

6.53566170.0350.98113.61914:0 ISO4.49ECL deviates -0.000Reference 0.006

7.04430130.0380.97214.00014:02.03ECL deviates -0.000Reference 0.006

7.983466310.0390.96014.62315:0 ISO30.96ECL deviates -0.000Reference 0.005

8.11983020.0390.95814.71315:0 ANTEISO5.50ECL devialea -0.000Reference 0.005

8.5533870.0350.95415.00015:00.26ECL deviates -0.000Reference 0.005

9.18819510.0400.94915.38916:1 w7c alcohol1.28ECL deviates -0.002

9.34449710.0410.94815.484Sum In Feature 23.26ECL deviates -0.00416:1 IS0 I/14:0 3OH

9.577112220.0460.94615.62616:0 ISO7.35ECL deviates -0.001Reference 0.003

9.7936850.0420.94515.75816:1 w11c0.45ECL deviates -0.001

9.956158000.0440.94415.858Sum In Feature 310.32ECL deviates -0.00615:0 ISO 2OH/16:1w7c

10.19060790.0440.94316.00016:03.97ECL deviates -0.000Reference 0.004

10.56216060.0450.94216.21815:0 20H1.05ECL deviates -0.001

10.85566130.0450.94016.390ISO 17:1 w10c4.30ECL deviates -0.002

10.97978240.0470.94016.462ISO 17:1 w5c5.09ECL deviates -0.001

11.11820780.0440.94016.54317:1 ANTEISO A1.35ECL deviates -0.003

11.267125730.0430.93916.63117:0 SIO8.17ECL deviates -0.001Reference 0.005

11.42421410.0440.93916.72317:0 ANTEISO1.39ECL deviates -0.000Reference 0.004

4971SUMMED FEATURE 23.2612:0 ALDE?unknown 10.928

16:1 ISO I/14:0 3OH14:0 3OH/16:1 ISO I

15800SUMMED FEATURE 310.3216:1 w7c/15 iso 20H15:0 ISO 2OH/16:w7c

Solvent ARTotal AreaNamed Area% NamedTotal AmntNbr RefECL DeviationRef ECL Shift

447011437151017151017100.00144550120.0020.005

Similarity Index

TSBA40 (Rev 4.10) Bacillus0.752

B. cereus0.752

B.c.GC subgroup A*0.752

B. thuringiensis canadensis sv.**0.619

B. thuringiensis kurstakii**0.523

[0062] It is understood for purposes of this disclosure, that various changes and modifications may be made to the invention that are well within the scope of the invention. Numerous other changes may be made which will readily suggest themselves to those skilled in the art and which are encompassed in the spirit of the invention disclosed herein.

[0063] The specification contains citations to references such as patents, patent applications, and publications. Each is hereby incorporated by reference for all purposes.Claims:

What is claimed is:


1. A probiotic composition for the reduction of bacterium in an aquatic environment comprising:

an isolated bacteria of the genus Bacillus.


2. The composition of claim 1 wherein the bacteria is further defined as Bacillus cereus.
3. The composition of claim 1 wherein the isolated Bacillus is strain EHC 100 having ATCC deposit accession number ______.
4. The composition of claim 1 wherein the bacterium being reduced is a pathogenic bacterium selected from the group consisting essentially of Streptococcus, Psuedomonas and Aeromonas.
5. The composition of claim 3 wherein the bacterium being reduced is a pathogenic bacterium selected from the group consisting essentially of Streptococcus, Psuedomonas and Aeromonas.
6. The composition of claim 1 further comprising from 2 to 5% sodium.
7. The composition of claim 2 wherein the isolated Bacillus cereus has a density of from 4*10to 6*10colony forming units per milliliter.
8. The composition of claim 7 wherein the colony forming units of the Bacillus cereus are less than 99% spores.
9. The composition of claim 7 wherein the colony forming units of the Bacillus cereus are less than 80% spores.
10. The composition of claim 1 wherein the aquatic environment is a koi pond.
11. The composition of claim 1 wherein the aquatic environment is saltwater.
12. The composition of claim 1 wherein the aquatic environment is a shrimp pond.
13. The composition of claim 1 wherein the aquatic environment is freshwater.
14. A method for reducing levels of pathogenic bacteria in an aquatic environment comprising:

adding a sufficient amount of the composition of claim 2 to the aquatic environment to cause a reduction in the pathogenic bacteria.


15. The method of claim 14 further comprising:

determining the levels of pathogenic bacteria in the aquatic environment before and after addition of the composition; and

adding a second dose of the composition to the aquatic environment to cause a further reduction in the pathogenic bacteria.
16. A method for reducing levels of pathogenic bacteria in an aquatic environment comprising:

adding a sufficient amount of the composition of claim 3 to the aquatic environment to cause a reduction in the pathogenic bacteria.


17. The method of claim 16 further comprising:

determining the levels of pathogenic bacteria in the aquatic environment before and after addition of the composition; and

adding a second dose of the composition to the aquatic environment to cause a further reduction in the pathogenic bacteria.
18. The method of claim 14 wherein 20 to 60 milliliters of the composition having from 4*10to 6*10of the Bacillus cereus is added per 1,000 gallons of aquatic environment.
19. The method of claim 16 wherein 20 to 60 milliliters of the composition having from 4*10to 6*10of the EHC 100 is added per 1,000 gallons of aquatic environment.
20. The method of claim 14 wherein the aquatic environment is a koi pond.
21. The method of claim 16 wherein the aquatic environment is a koi pond.
22. The method of claim 14 wherein the aquatic environment is saltwater.
23. The method of claim 16 wherein the aquatic environment is saltwater.
24. The method of claim 14 wherein the aquatic environment is freshwater.
25. The method of claim 16 wherein the aquatic environment is freshwater.
26. A method for reducing the levels of fish morbidity in an aquatic environment comprising:

adding a sufficient amount of the composition of claim 2 to the aquatic environment to cause a reduction in pathogenic bacterium in the aquatic environment.


27. A method for reducing the levels of fish morbidity in an aquatic environment comprising:

adding a sufficient amount of the composition of claim 3 to the aquatic environment to cause a reduction in pathogenic bacterium in the aquatic environment.


28. The method of claim 26 further comprising:

determining the levels of pathogenic bacteria in the aquatic environment before and after addition of the composition;

determining the approximate number of fish in the aquatic environment before and after the addition of the composition to the aquatic environment; and

adding a second dose of the composition to the aquatic environment to cause a further reduction in the pathogenic bacteria.


29. The method of claim 27 further comprising:

determining the levels of pathogenic bacteria in the aquatic environment before and after addition of the composition;

determining the approximate number of fish in the aquatic environment before and after the addition of the composition to the aquatic environment; and

adding a second dose of the composition to the aquatic environment to cause a further reduction in the pathogenic bacteria.


30. The method of claim 26 wherein the fish is Tilapia.
31. The method of claim 27 wherein the fish is Tilapia.
32. The method of claim 26 wherein 20 to 60 milliliters of the composition having from 4*10to 6*10of the Bacillus cereus is added per 1,000 gallons of aquatic environment.
33. The method of claim 27 wherein 20 to 60 milliliters of the composition having from 4*10to 6*10of the EHC 100 is added per 1,000 gallons of aquatic environment.
34. The method of claim 26 wherein the aquatic environment is a koi pond.
35. The method of claim 27 wherein the aquatic environment is a koi pond.
36. The method of claim 26 wherein the aquatic environment is a shrimp pond.
37. The method of claim 27 wherein the aquatic environment is a shrimp pond.
38. The method of claim 26 wherein the aquatic environment is saltwater.
39. The method of claim 27 wherein the aquatic environment is saltwater.
40. The method of claim 26 wherein the aquatic environment is freshwater.
41. The method of claim 27 wherein the aquatic environment is freshwater.
42. A method for treating fish infected with a pathogenic bacteria in an aquatic environment comprising:

adding a sufficient amount of the composition of claim 3 to the aquatic environment to cause a reduction in the percentage of fish infected with the pathogenic bacterium.


43. A method for treating shellfish infected with a pathogenic bacteria in an aquatic environment comprising:

adding a sufficient amount of the composition of claim 3 to the aquatic environment to cause a reduction in the percentage of shellfish infected with the pathogenic bacterium.


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