WO2008103790A2

WO2008103790A2 - Bacillus anthracis selective medium

Info

Publication number: WO2008103790A2
Application number: PCT/US2008/054520
Authority: WO
Inventors: Cindy Allan; Michael Cassler; Jeanette Coffin; John David; Meredith Kroehl; Robert O'shea
Original assignee: Midwest Research Institute
Current assignee: Midwest Research Institute
Priority date: 2007-02-21
Filing date: 2008-02-21
Publication date: 2008-08-28
Anticipated expiration: 2009-08-21
Also published as: WO2008103790A3

Abstract

An improved medium for selectively culturing and detecting B. anthracis is provided. The medium comprises at least one polymyxin antibiotic, typically colistin, in combination with at least one dihydrofolate reductase inhibitor, typically trimethoprim, to provide the selectivity necessary to differentially culture B. anthracis from complex environmental samples. Methods for making the improved medium, as well as methods for utilizing the improved medium to selectively culture B. anthracis are also provided.

Description

BACILLUS ANTHRACIS SELECTIVE MEDIUM

TECHNICAL FIELD An improved medium for selectively culturing and detecting B. anthracis is provided.

The medium comprises at least one polymyxin antibiotic, typically colistin, in combination with at least one dihydrofolate reductase inhibitor, typically trimethoprim, to provide the selectivity necessary to differentially culture B. anthracis from complex environmental samples. Methods for making the improved medium, as well as methods for utilizing the improved medium to selectively culture B. anthracis are also provided.

BACKGROUND ART

Bioterrorism, especially the use of Bacillus anthracis (hereinafter B. anthracis) as a biological weapon, has become a major concern. B. anthracis is a spore-forming gram positive bacterium. While cultured B. anthracis quickly multiplies to form visually detectable colonies, it is difficult to differentially culture B. anthracis except under highly controlled laboratory conditions.

Bioterrorist attacks are, by their nature, difficult to prevent and identify. The longer the period of time between discovery and identification of the exact pathogen, the greater the potential for severe casualties. Government agencies, including the DOD, FBI and DHS, as well as private industry are expending considerable resources toward researching and developing means of preventing, identifying, and ameliorating bioterrorist attacks. These government agencies and private contractors are also involved in the detection and bioremidiation of B. anthracis in environmental samples. Unfortunately, identifying B. anthracis has been proven difficult thus far. The organism's similarity to its near neighbors, including Bacillus cereus and Bacillus thuringiensis, results in false positives by the most commonly-used identification tests. The conventional B. anthracis cultivation methods, including the use of PLET, SBA and RF Chromogenic culture media, were developed for use in clinical sample analysis and are less useful for complex environmental samples. In addition to being labor intensive and complicated to make (such as with PLET), it is difficult to sufficiently reduce non-target background when utilizing these methods. Further, one of these methods relies on time-developed color change, which can be ambiguous, especially with complex samples. Therefore, these conventional methods do not provide the best combination of selectivity and differentiation. New methods that would increase the ability to culture material from complex environmental as well as clinical samples are highly desirable.

Tests that provide a high degree of selectivity and can often effectively differentiate B. anthracis from its near neighbors do exist, for example, PCR-based assays. However, these methods require highly trained technicians, expensive equipment, and do not assess viability, which is often a critical factor. Immunological methods such as ELISA often have poor selectivity and also do not assess viability.

Therefore, a simple method that can be utilized by minimally-skilled personnel, in a laboratory and/or in the field, and provides suitable viable isolates is needed. A simple selective culturing medium that allows B. anthracis to form colonies while restricting the growth of other organisms, and that can be used effectively with complex environmental samples would be greatly beneficial. DISCLOSURE OF INVENTION

In one of many illustrative, non-limiting aspects of the present invention, there is provided a medium, and a method of making a medium, selective for culturing and detecting B. anthracis. The medium includes at least one polymyxin antibiotic in combination with at least one dihydrofolate reductase inhibitor.

In another of many illustrative, non-limiting aspects of the present invention, there is provided a method for culturing and detecting B. anthracis bacteria. The method includes forming a medium comprising at least one polymyxin antibiotic in combination with at least one dihydrofolate reductase inhibitor; inoculating the medium with B. anthracis bacteria; incubating the inoculated medium; and visually analyzing the selective incubated medium.

BEST MODE FOR CARRYING OUT THE INVENTION

There is provided herein an improved selective medium for the culture and detection of B. anthracis bacteria and spores from complex environmental samples. The improved selective medium is easy to prepare and use and is highly selective and exhibits good differential capability, even from r B. anthracis near neighbors. The efficient isolation of B. anthracis from complex samples by the improved selective medium can be used to reduce downstream analysis and decrease sample indentification times.

The improved selective medium has been successfully shown to aid in the isolation of B. anthracis from complex environmental samples. The new medium reduces the time spent having to sort through more B. anthracis-likβ colonies because B. anthracis is often visually similar to other microorganisms on culturing plates. The increase in selectivity coupled with reduction in time of sample analysis addresses market requirements that have not been adequately met by conventional methods.

Selective culturing media typically utilize an antibiotic or antibiotics that allow the target organism to multiply, while preventing or at least reducing the growth of non-target organisms. The novel combination of antibiotic additives utilized in the medium of the present invention has resulted in a medium with improved selectivity for B. anthracis as compared to prior art media. Additional differential additives, including culturing nutrient sources, pH indicators and blood, either alone or in combination, make distinguishing B. anthracis from near neighbors faster and more reliable. While the commercially-available conventional selective media perform well on less complex samples, the improved selective medium of the present invention shows a marked improvement over conventional media when used to identify complex environmental samples. The improved selective medium disclosed and claimed herein utilizes a combination of at least one polymyxin antibiotic and at least one dihydrofolate reductase inhibitor that is selective for bacteria enzymes. Polymyxins are a group of antibiotics active against Gram negative organisms. Polymyxins are polypeptides with a long hydrophobic tail which act as cation detergents binding the bacterial cell membrane and resulting in leakage of the cytoplasmic contents. Illustrative polymyxins include, but are not limited to, colistin, polymyxin B, surfactin, and mixtures thereof. Colistin is presently preferred for use in the present improved selective medium. In an illustrative example, the improved selective medium preferably includes from about 12.5 μg/mL to 50.0 μg/mL, more preferably from about 18-40 μg/mL and, most preferably from about 20-30 μg/mL, of at least one polymyxin antibiotic. Dihydrofolate reductase inhibitors interfere with cellular folate uptake thereby interfering with RNA and DNA replication. Trimethoprim [2,4-diamino-5-(3,4,5-trimethoxybenzyl) pyrimidine] is an illustrative dihydrofolate reductase inhibitor that exhibits a selective affinity for bacterial enzymes. Other dihydrofolate reductase inhibitors suitable for use in the present invention include, but are not limited to, epiroprim [2,4-diamino-5[3,5-diethoxy-4-(l-pyrrolyl)- benzylj-pyrimidine], brodimoprim [2,4-diamino-5-(4 bromo-3,5-dimethoxybenzyl) pyrimidine], K-130 [2,4-diamino-diphenyl sulphone-substituted2,4-diamino-4-benzyl-pyrimidine], and dapsone [4,4'-diaminodiphenyl sulphone]. In an illustrative example, the improved selective medium preferably includes from about 50.0 μg/mL to 150.0 μg/mL, more preferably from about 75-125 μg/mL and, most preferably from about 95-105 μg/mL of at least one dihydrofolate reductase inhibitor.

The improved selective medium typically includes a gelling material, thereby providing a solid medium to be plated, as is well known in the art. Suitable gelling materials include agar, agaroses, gelatin and mixtures thereof. A suitable commercially available agar is Lab-Lemco Agar. In an illustrative example, the improved medium preferably includes from about 7.5 g to 30 g, more preferably from about 10-25 g and, most preferably from about 12-20 g, of a gelling material.

The improved selective medium may further include one or more culture nutrients to aid B. anthracis growth. These types of nutrients are well known for their use in culturing media. Illustrative nutrients include protein sources such as peptones and tryptones, yeast extracts, sugar alcohols such as glycerol, and combinations thereof. The improved selective medium preferably includes from about 6 g to 55 g, more preferably from about 15-45 g and, most preferably from about 30-40 g, of a culture nutrient. In an illustrative example, the improved selective medium includes from about 6.2 g to 25 g peptone, and 7.5 g to 30 g glycerol, although any culture nutrient or combination thereof may be utilized so long as it does not interfere with the selectivity of the improved selective medium.

A salt or salts may be added to the improved selective medium, as is also well known in the art. Suitable salts are well known in the art and are preferably present in an amount of from about 2.5 g to 1O g, more preferably from about 3-8 g and, most preferably, from about 4-6 g. In an illustrative example, sodium chloride is added to the improved selective medium, but suitable salts include, but are not limited to, potassium chloride, magnesium chloride, calcium chloride, and combinations thereof.

An illustrative example of a solid formulation of the improved selective medium of the present invention is shown in Table 1 below: Table 1. Improved Selective Medium

The growth of B. anthracis colonies on the improved selective medium can be visually detected with an unaided eye. However, in an alternate illustrative embodiment, the visual identification of B. anthracis colonies is enhanced by the inclusion of a pH indicator in an amount of from about 30 mg to 120 mg, more preferably from about 35-90 mg and, most preferably, from about 45-65 mg. Suitable pH indicators are capable of indicating a pH 5.0 to pH 9.0, and include, but are not limited to, phenol red, methyl red, litmus, bromcresol purple, chrophenol red, bromothymol blue, neutral red, tumaric curcumin and mixtures thereof. An illustrative embodiment of this alternative embodiment of the improved selective medium, including a pH indicator, is illustrated in Table 2 below. Table 2. Improved Selective Medium Including pH Indicator

m another illustrative embodiment, the visual identification of B. anthracis colonies is enhanced by the inclusion of from about 25 mL to 100 mL, more preferably from about 35-75 mL, and most preferably from about 45-55 mL, of defibrinated blood in the improved medium. Sheep blood is the current standard; however, horse, rabbit and human blood, as well as mixtures thereof, can also be utilized effectively. An illustrative embodiment of this alternative embodiment of the improved selective medium, including a blood indicator, is illustrated in Table 3 below. Table 3. Improved Selective Medium Including Sheep Blood

The various embodiments of the improved selective medium of the present invention are prepared by methods typical in the art. The agar is suspended in distilled/deionized water which is then heated until the agar is completely dissolved. The agar solution and any heat-resistant components are then sterilized in the autoclave. Any heat-sensitive components are then added. The improved selective medium, including antibiotic components, can be plated, allowed to solidify and used immediately, or stored in a sealed container under refrigeration for several weeks. In the alternative, the improved selective medium can be stored for longer periods of time as a two-part system, a gelled aqueous growth medium and a dry antibiotic blend. The gelled medium is melted, cooled, and mixed with the powdered antibiotic blend. The resulting improved selective medium can then be plated and allowed to solidify.

The improved selective medium is especially useful for the following applications: 1. The direct plating of liquid environmental extracts in order to isolate and identify B. anthracis bacteria or spores; 2. The production of sub-cultures or replica plates from general media to reduce non-target background and isolate and/or identify B. anthracis bacteria or spores; and

3. The characterization of suspected B. anthracis colonies to determine if suspected isolates are/are not B. anthracis. EXAMPLES

In the following examples, the following commercially-available components were used:

Example 1 : General support of target organism on prior art media

Five plates of each TSA, RF Chromogenic and MRI (improved selective medium of the present invention) media were prepared according to manufacturer's instructions.

Approximately 200 cfu of target B. anthracis was inoculated onto each plate. The MRI inventive medium was comprised of Lab-Lemco Agar (15 mg/mL), NaCl (5 mg/niL), peptone (12.5 mg/mL), glycerol (15 mg/mL), colistin (25 μg/mL), trimethoprim (100 μg/mL) and phenol red (60 μg/mL.) All plates were incubated at 3O⁰C overnight and were counted the following morning. Table 4. Comparison of Three Media for General Support of Target

The recovery on RF was the highest but MRI recovery was not significantly (p=0.31 , T-test) lower that that noted for RF. Colonies were visible in 18-24 hours on MRI and TSA compared to 48 for RF. Colonies were easier to identify on the improved selective MRI media as compared to both other media.

Example 2: Support of multiple strains of target organism

The MRI improved selective medium of the present invention was prepared as described in Example 1. Fifteen plates of the improved selective medium were plated and inoculated with 15 unique strains of B. anthracis; 14 strains were ATCC strains including: Bacillus anthracis

14578, Bacillus anthracis 9660, Bacillus anthracis 240, Bacillus anthracis 10, Bacillus anthracis 937, Bacillus anthracis 8705, Bacillus anthracis 4728, Bacillus anthracis 6602, Bacillus anthracis 11949, Bacillus anthracis 6603, Bacillus anthracis 938, Bacillus anthracis 4229, Bacillus anthracis 11966, Bacillus anthracis Vollum, and Bacillus anthracis Sterne; one was a lab strain. All 15 strains exhibited significant observable colony growth on the improved selective media.

Example 3: Selective nature of media

A cocktail of organisms containing B. anthracis, B. thuringiensis, and Ochrobactrum anthropii were grown up in brain heart infusion broth until stationary growth phase was achieved. Next, each culture was enumerated and known concentrations of each organism were combined in a microcentrifuge tube and vortexed prior to being inoculated on tile. Concentrations of each target on the tile were B. anthracis (40 cfu), B. thuringiensis (50 cfu), and O. anthropii (50 cfu). The MRI inventive medium for this example was composed of Lab- Lemco Agar (15 mg/mL), NaCl (5 mg/mL), peptone (12.5 mg/mL), glycerol (15 mg/mL), colistin (25 μg/mL), trimethoprim (100 μg/mL), and defibrinated sheep's blood (5%). Results after plating of the MRI improved selective medium and incubation showed B. anthracis (8 cfu), B. thuringiensis (4 era), and O. anthropii (0 cfu). All cm values in the results were averages of 3 plates. Colony types between B. anthracis and B. thuringiensis were easily discriminated.

Example 4: Reduction of background in field samples

A soil sample known to contain a low amount of target organism was extracted with 30 mL of PBS/ 0.5% Tween 20/ 0.1 %BSA buffer. Following extraction, the sample was then inoculated on three different media, RF, TSA w/SB, and MRI, prepared as described in Example 3. Total colony counts were noted.

Table 5. Background Organism Reduction in Field Sample

The medium formulations were shown to selectively grow B. anthracis and B. cereus/B. thuringiensis near neighbors; however the phenol red/glycerol or blood is used to differentiate the B. anthracis from the near neighbors. Each formulation has been shown to support growth of a variety of virulent B. anthracis strains and used to successfully isolate B. anthracis from complex environmental samples.

Having described the invention in detail, those skilled in the art will appreciate that modifications may be made of the invention without departing from the spirit and scope thereof. Therefore, it is not intended that the scope of the invention be limited to the specific embodiments described. Rather, it is intended that the appended claims and their equivalents determine the scope of the invention.

Claims

CLAIMS We claim:

1. A medium selective for culturing and detecting B. anthracis, the medium comprising: at least one polymyxin antibiotic; and at least one dihydrofolate reductase inhibitor.

2. The medium of Claim 1 wherein said polymyxin antibiotic is selected from the group consisting of colistin, polymyxin B, surfactin, and mixtures thereof.

3. The medium of Claim 1 wherein said dihydrofolate reductase inhibitor is selected from the group consisting of trimethoprim, epiroprim, brodimoprim, K-130, dapsone and mixtures thereof.

4. The medium of Claim 1 wherein said polymyxin antibiotic comprises from about 12.5 μg/mL to 50.0 μg/mL of said medium.

5. The medium of Claim 1 wherein said dihydrofolate reductase inhibitor comprises from about 50 μg/mL to 150 μg/mL of said medium.

6. The medium of Claim 1 further comprising from about 7.5-30 g of at least one gelling material, thereby providing a solid medium.

7. The medium of Claim 6 wherein said gelling material is selected from the group consisting of agar, agaroses, gelatins and mixtures thereof.

8. The medium of Claim 1 further comprising from about 6-55 g of at least one culturing nutrient.

9. The medium of Claim 8 wherein said culturing nutrient is selected from the group consisting of peptone, glycerol, tryptone, yeast extract, and mixtures thereof.

10. The medium of Claim 1 further comprising from about 30-120 mg of a pH indicator capable of indicating a pH of 5.0 to 9.0.

11. The medium of Claim 10 wherein said pH indicator is selected from the group consisting of phenol red, methyl red, litmus, bromcresol purple, chrophenol red, bromothymol blue, neutral red, tumaric curcumin, and mixtures thereof.

12. The medium of Claim 1 further including from about 25-100 g of blood.

13. The medium of Claim 12 wherein said blood is selected from the group consisting of sheep blood, horse blood, rabbit blood, human blood and mixtures thereof.

14. A nutrient medium selective for culturing and detecting B. anthracis, the nutrient media comprising: at least one polymyxin antibiotic; at least one dihydrofolate reductase inhibitor; at least one nutrient; and at least one gelling material.

15. The nutrient medium of Claim 14 wherein said polymyxin antibiotic is selected from the group consisting of colistin, polymyxin B, surfactin and mixtures thereof.

16. The nutrient medium of Claim 14 wherein said dihydrofolate reductase inhibitor is selected from the group consisting of trimethoprim, epiroprim, brodimoprim, K-130, dapsone and mixtures thereof.

17. The nutrient medium of Claim 14 wherein said nutrient is selected from the group consisting of is selected from the group consisting of peptone, glycerol, tryptone, yeast extract, and mixtures thereof.

18. The nutrient medium of Claim 14 wherein said gelling material is selected from the group consisting of agar, agaroses, gelatins and mixtures thereof.

19. The nutrient medium of Claim 14 further comprising at least one salt.

20. The nutrient medium of Claim 19 wherein said salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, and mixtures thereof.

21. The nutrient medium of Claim 14 wherein the medium comprises: from about 12.5 μg/mL to 50.0 μg/mL of said polymyxin antibiotic; from about 50.0 μg/mL to 150 μg/mL of said dihydrofolate reductase inhibitor; from about 6 g to 55 g of said nutrient; from about 7.5 g to 30 g of said gelling material; and from about 2.5 g to 10 g of a salt.

22. The nutrient medium of Claim 14 further comprising from about 30 mg to 120 mg of at least one pH indicator capable of indicating a pH of 5.0 to 9.0.

23. The nutrient medium of Claim 22 wherein said pH indicator is selected from the group consisting of phenol red, methyl red, litmus, bromcresol purple, chrophenol red, bromothymol blue, neutral red, tumaric curcumin, and mixtures thereof.

24. The nutrient medium of Claim 14 further from about 25 g to 100 g of a blood.

25. The nutrient medium of Claim 24 wherein said blood is selected from the group consisting of sheep blood, horse blood, rabbit blood, human blood, and mixtures thereof.

26. A nutrient medium selective for culturing and detecting B. anthracis, the nutrient media comprising:

12.5 μg/mL to 50.0 μg/mL colistin; 50.0 μg/mL to 150 μg/mL trimethoprim; 6.2 g to 25 g peptone;

7.5 g to 30 g glycerol; 7.5 g to 30 g agar; 2.5 g to 10 g of a salt; and 30 mg to 120 mg phenol red.

27. A nutrient medium selective for culturing and detecting B. anthracis the nutrient media comprising:

12.5 μg/mL to 50.0 μg/mL colistin;

50.0 μg/mL to 150 μg/mL trimethoprim; 6.2 g to 25 g peptone;

7.5 g to 30 g glycerol;

7.5 g to 30 g agar;

2.5 g to 10 g of a salt; and

25 mL to 10O mL blood.

28. A method of making a medium selective for culturing and detecting B. anthracis bacteria and spores, the method comprising the steps of: combining at least one polymyxin antibiotic with at least one dihydrofolate reductase inhibitor.

29. The method of Claim 28 wherein said polymyxin antibiotic is selected from the group consisting of colistin, polymyxin B, surfactin and mixtures thereof.

30. The method of Claim 28 wherein said dihydrofolate reductase inhibitor is selected from the group consisting of trimethoprim, epiroprim, brodimoprim, K-130, dapsone and mixtures thereof.

31. The method of Claim 28 wherein said polymyxin antibiotic comprises from about 12.5 μg/mL to 50.0 μg/mL of the medium, and said dihydrofolate reductase inhibitor comprises from about 50 μg/mL to 150 μg/mL of the medium.

32. The method of Claim 28 further comprising the step of adding from about

7.5 g to 30 g of at least one gelling material, thereby providing a substantially solid medium.

33. The method of Claim 32 wherein said gelling material is selected from the group consisting of agar, agaroses, gelatins and mixtures thereof.

34. The method of Claim 28 further comprising the step of adding from about 6 g to 55 g of at least one nutrient.

35. The method of Claim 34 wherein said nutrient is selected from the group consisting of peptone, glycerol, tryptone, yeast extract and mixtures thereof.

36. The method of Claim 28 further comprising the step of adding from about 30 mg to 120 mg of at least one pH indicator.

37. The method of Claim 36 wherein said pH indicator is selected from the group consisting of phenol red, methyl red, litmus, bromcresol purple, chrophenol red, bromothymol blue, neutral red, tumaric curcumin, and mixtures thereof.

38. The method of Claim 28 further comprising the step of adding from about 25 g to 100 g of at least one defibrinated blood.

39. The method of claim 38 wherein said blood is selected from the group consisting of sheep blood, horse blood, rabbit blood, human blood and mixtures thereof.

40. A method comprising combining: at least one polymyxin antibiotic; at least one dihydrofolate reductase inhibitor; at least one nutrient; and at least one gelling material to form a solid nutrient medium selective for culturing and detecting B. anthracis bacteria and spores.

41. The method of Claim 34, wherein the polymyxin antibiotic includes colistin; the dihydrofolate reductase inhibitor includes trimethoprim; the nutrient includes peptone and glycerol; and the gelling material includes agar; wherein the medium further includes at least one salt.

42. The method of Claim 35 wherein the medium includes: 12.5 μg/mL to 50.0 μg/mL colistin;

50.0 μg/mL to 150 μg/mL trimethoprim; 6.2 g to 25 g peptone; 7.5 g to 30 g glycerol;

7.5 g to 30 g agar; and 2.5 g to 10 g of a salt.

43. A method for culturing and detecting B. anthracis bacteria, the method comprising: forming a medium comprising at least one polymyxin antibiotic in combination with at least one dihydrofolate reductase inhibitor; inoculating the medium with B. anthracis bacteria; incubating the inoculated medium; and visually analyzing the selective incubated medium.

44. A method of detecting B. anthracis bacteria and spores present in an environmental sample, the method comprising: forming a medium comprising at least one polymyxin antibiotic in combination with at least one dihydrofolate reductase inhibitor; inoculating the medium with the environmental sample; incubating the medium; and visually analyzing the medium to determine if B. anthracis bacteria or spores are present in the environmental sample.