ABSTRACT:
The purpose of this research project was to find an economical means for producing amphotericin B in the laboratory at Stephen F. Austin State University Science Research Center. Following the protocol in patent 6,312,993, and characterization methods from patent 2,908,611, Streptomyces nodosus was successfully cultured and amphotericin B was produced with distinct peaks in the 362-405nm region in an ultraviolet-spectrum analysis.

INTRODUCTION
Background of Streptomyces nodosus:
Amphotericin B is produced by Streptomyces nodosus (ATCC 14899), which was found by Gold, et al. in 1955 along the Orinoco River in Tembladora, Venezuela (3). In 1959, Dutcher, et al produced the first patent of amphotericin B production and in 1962, Trejo and Bennett officially characterized S. nodosus according to the International Code of Nomenclature of Bacteria and Viruses, thus providing the full name Streptomyces nodosus Trejo. (1,2).

Characterization of Streptomyces nodosus
Streptomyces nodosus is a gram-positive bacteria, which can be identified by several methods. Along with a positive gram stain, the mycelium are very distinct when viewed under 640X magnification showing open and closed spirals (2). The spores can also be viewed with an electron microscope to identify a semi-spiral shaped structure as seen in Fig. 1 (2).

Another characteristic is the color of the mycelium and growth characteristics when cultured on different media (1,2). For example, when S. nodosus is grown on a Glucose-asparagine agar slant, a deep olive-gray colony results with a water-insoluble liquid deposited at the base of the slant (1,2). However, when this liquid is mixed with methanol, it is soluble. In this study, such tests were performed on a potato slant, skim milk, a Saubaraud agar slant, and the Glucose-Asparagine slant and compared with the results of both Trejo (2) and patent 2,908,611.

Amphotericin B
Amphotericin B is one of two polyketides synthesized by S. nodosus through the polyketide synthesis pathway (6). As shown in Fig. 1, the compound is classified as a heptaene-M polyketide with the "M" referring to the mycosamine sugar attached glycosidically to the end of the heptaene chain (4,5). Amphotericin A is a highly toxic homolog of amphotericin B and must be removed from the crude fermentation product to less than 5% to be achieve an acceptable therapeutic index (6). While it is difficult to find the exact structure of amphotericin A, it is suggested that the structure is exactly the same except a reduced double bond at carbons 28 and 29 along the heptaene chain classifying it as a tetraene and diene(6). This reduction may come from tailoring enzymes since some intermediates in the formation of amphotericin B serve soley as precursors to the formation of amphotericin A, however the pathway for this is still unknown (6). For production purposes, the similarity in amphotericin structures can make chemical separation of the two difficult and costly (6).

The antifungal property of amphotericin B comes partly from the amphoteric structure of the polyketide. It attaches preferentially to the ergosterol of fungi over mammalian sterol and creates a hole in the cell membrane allowing contents to leak out (3). This change in osmolarity ultimately kills the cell (3).

While this mode of attack makes amphotericin extrememly effective, binding to human sterols, such as cholesterol, does occur (3). Binding to cholesterol in renal epithelial cells accounts for the high nephrotoxicity known for Amphotericin B Deoxycholate, the standard derivative administerd today (3,7). Many of the current amphotericin B phamaceutical products, such as Ambisome, are designed to lower this toxicity through lipid derivatives, however it has also been found that with different derivatives of the molecule, different areas of the body, such as the brain or kidney, are affected (3).

Production: Original and New Method
When Dutcher, et al. published the first patent for amphotericin B, amphotericin A had to be chemically separated after both were produced in whole broth culture (1). These methods both proved costly, time consuming, and the final yield of amphotericin B was low. Schaffner, et al., from Rutgers University, discovered that the post-modification of amphotericin B into amphoterin A could be stopped through additions of protein syntesis inhibitor to the whole broth culture during the production phase. This eliminates the need for any chemical separation reducing cost and greatly increases purity of amphotericin B (6).

MATERIALS AND METHODS Materials and Equipment: 500ml baffled shake flask, incubator, 6" test tubes, petri dishes, autoclave, desktop centrifuge (Biorad; model: 14-K), 2ml microfuge tubes Chemicals and Reagents: Dextrose (Becton Dickson; 211864), BactoPeptone (Difco; 21167), CaCO3 (Sigma), MnCl2 (Sigma), FeSO4 (Sigma; 80K3150), Streptomycin salt (Sigma), Protease Peptone (Difco; D122-17-4), Methanol, Na2HPO4 (Amresco; 2716A66), Dimethyl Sulfoxide (Sigma), Agar (Difco; 214530), Saubaraud Agar (Difco; 0109-17-1), K2HPO4 (Amresco; 2156A33), Asparagine (Sigma; 85H5703), Streptomyces nodosus Trejo culture (ATCC; 14899)

PROCEDURE:
Cultivation:
GYM agar media was obtained and enough YM agar media was prepared for at least two agar plates as described on the container instructions. The media was autoclaved at 125°C for 15 minutes and poured into two petri dishes. The plates were at 37°C overnight to assure no contamination was present. Streptomyces nodosus Trejo, obtained from a glycerol stock culture in -70°, was thawed and, using a sterile loop, streaked onto the GYM plates. These plates were then stored in an incubator at 25°C for 3 days or until sufficient isolated colonies could be seen. The YM Broth media was obtained and 150ml was prepared as described on the container in a 500ml baffled shake flask. The broth was autoclaved at 125°C for 15 minutes and capped with a cotton plug. After cooling to room temperature, the broth media was inoculated with a colony from the YM agar plates and set into a rotary shaker at 250rpm at 30°C for three days. The YM broth culture media was then used to inoculate characterization test media, the production broth media, and glycerol stock solutions as described below.

Production:
A YED production medium (5% dextrose, 1%BactoPeptone, 1% Calcium Carbonate, 0.001% MnCl2. 4H2O, 0.001%FeSO4. 7H2O, in distilled water and adjusted to pH 7.2) was made to a total volume of 150ml and autoclaved at 125°C for 15 minutes in a 500ml baffled shake flask. This was then capped with a cotton plug and allowed to cool to room temperature. The YED production medium was then inoculated using a sterile loop full of YM broth culture medium and set into a rotary shaker at 250rpm and 30°C. Streptomycin sulfate was then obtained and diluted to a final concentration of 0.4 .mu.g/ml. in distilled water.

Characterization Tests:
A white, spherical colony was isolated from the YM broth culture medium and heat dried onto a slide. A gram stain was performed and it was viewed under oil immersion at 1000X magnification.

A Glucose-asparagine agar slant was prepared (1% dextrose, 0.05% K2HPO4, 0.05% asparagine, 1.5% agar: w/v in distilled water) along with a Saubaraud agar slant (prepared as described on the container), a potato plug slant, and a hydrogen sulfide test medium (2% peptone, 0.1% dextrose, 0.2% Na2HPO4, 0.2% agar: w/v di water). All test media were prepared in 6" test tubes, autoclaved at 125°C for 15 minutes, and capped with cotton plugs. They were then stored in an incubator at 25°C and checked periodically for observations.

Antibiotic Additions: Sterile additions of 20 .mu.g of the prepared streptomycin sulfate solution per 50ml of production medium were added at hours 24 and 96 after the time of inoculation.

Analysis:
On the eigth day after inoculation of the production medium, an analysis was performed to determine the presence of amphotericin A or B. Two 2ml. microfuge tubes were obtained and 1ml of 20% production broth in dimethyl sulfoxide was pippetted into each one. They were then shaken vigorously for 60 minutes and placed in a desktop centrifuge set to high speed for 10 minutes. One ml of 20% supernatant in methanol was pipetted into two new 2ml microfuge tubes and again centrifuged as before. This solution was analyzed in a ultra-violet spectrometer between 250nm and 450nm.

RESULTS:
Because this experiment was run three times before successfully producing amphotericins, valuable observations were made in each step of the process of what helped as well as what mistakes should be avoided when reproducing this experiment.

Cultivation:
After 2 to 3 days light brown to tan, circularly shaped colonies formed on the second and third attempts. In the first attempt, it was observed that incubating the petri dishes with the cap on top allows the internal condensation to contaminate the agar media.

The bacterial colonies formed in the YM broth media are very characteristic. In the first experiment, regular 300ml Erlenmeyer flasks were used with solid caps instead of cotton plugs. Large white spheres about 1mm in size formed as seen in Fig. 4. In the third attempt, the baffled shake flask with cotton plugs produced much more agitation formed a thick foamy layer. Although the colonies were still white and spherical, the size was reduced drastically.

Production:
Colony growth was similar in the production media to that of the YM broth media due to the different flask and caps used as described above. However, in the first two attempts, only 0.1% CaCO2 was used resulting in a translucent yellow-brown broth. In the third attempt, adding the full 1% CaCO2 produced a much cloudier solution with some white powder settled on the bottom. Also, the pH was not adjusted from 6.8 to 7.2 as was done in the third attempt. The production medium in the third attempt also produced a thick foamy layer.

Characterization Tests:
A gram stain of the mycelium taken from a white, spherical colony in the YM broth culture media showed deeply twisted branching mycelium, which folded back on themselves in spirals. The gram stain was positive as seen in Figure 6.

   
The Glucose-asparagine agar slant showed heavy growth after 4 to 5 days. The growth was very distinctive with a deep olive-gray color coupled with production of an amber colored liquid at the bottom of the slant (Fig. 7, 8). The liquid was found to be soluble in methanol.

Colonies grew on the Saubaraud agar slant after 3 to 4 days in small lightly clear-yellow in color. After 15 to 20 days, a thick wrinkled layer formed very closely resembling the gyri and sulcus of a brain (Fig. 9).

The hydrogen sulfide test media showed dense colony growth by day 4. After 8 days, the colonies took the shape of light yellow flowers with white spores in the middle as shown in Figure 10.

Analysis:

Peaks in the ultraviolet spectrum appeared at 273nm, 294nm, 304nm, 318nm, 345nm, 362nm, 382nm, and 406.5nm..

DISCUSSION
Several factors help to conclude the presence of amphotericin B in the production medium for the third attempt. First, the gram stain and characterization tests were positive according to Dutcher, et al. (1) and Trejo (2). Second the original YM agar plates showed only one colony type without any contaminants. Third, every peak from the ultraviolet spectrum matched the expected values. In an ultraviolet spectrum, amphotericin A produces two significant peaks at 304nm and 318nm, while amphotericin B produces two significant peaks at 382nm and 405nm. The smaller peaks also matched those listed.

While the production of amphotericin B was successful, amphotericin A was also produced. It is possible that the streptomycin sulfate was incorrect in either the dilution or simply needed to be added earlier than 24 hours after production medium inoculation. In order to produce amphotericin B in a pure enough form for therapuetical use, this area should be tested more to inhibit the amphotericin A.

REFERENCES
1. Dutcher JD et al. Amphotericin B, its production and its salts. U.S. Pat. 2,908,611 dated Oct. 13, 1959
2. Trejo, W.; Bennett, R.. Streptomyces nodosus sp. n., the amphotericin-producing organism. J. Bacteriol. 85: 436-439
3. http://www.doctorfungus.org/thedrugs/Ampho_Deoxycholate.htm
4. www.ucd.ie/~ofrss/science/industr/1158.html
5. Creuger, W. ; Crueger, A. Biotechnology: A Textbook of Industrial Microbiology. 2nd Edition. Sinaur Assoc., Inc., Sutherland, MA 01375. pg. 260
6. Schaffner, C., Kientzler, D. "Process for the production of amphotericin B inhibiting production of amphotericin A." U.S. Pat. 6,132,993 dated Oct. 17, 2000
7. http://www.thedrugmonitor.com/DCAB.html
8. http://www.ambisome.com/introfr.htm

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