# Patent

## Title
Methods and compositions for increasing toxin production

## Abstract

The invention provides methods and compositions (such as for example, culture media) for culturing Clostridium difficile and producing the C. difficile Toxins A and B.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national stage filing under 35 U.S.C. §371 of international application PCT/US2009/058268, filed Sep. 24, 2009, which claims benefit of Provisional Patent Application. 61/099,759, filed Sep. 24, 2008.
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. Provisional Patent Application No. 61/099,759, filed on Sep. 24, 2008, which is incorporated herein by reference in its entirety.


BACKGROUND OF THE INVENTION
Clostridium difficile (C. difficile) Toxins A and B are responsible for C. difficile-associated disease (CDAD), which manifests itself as nosocomial diarrhea and pseudomembranous colitis (Kuijper et al., Clinical Microbiology and Infection 12(Suppl. 6):2-18, 2006; Drudy et al., International Journal of Infectious Diseases 11(1):5-10, 2007; Warny et al., Lancet 366(9491):1079-1084, 2005; Dove et al., Infection and Immunity 58(2):480-488, 1990; Barroso et al., Nucleic Acids Research 18(13):4004, 1990).
Toxins A and B are encoded by two separate but closely linked (and highly homologous) genes. Toxins A and B are produced simultaneously in C. difficile strain VPI 10463 (ATCC 43255), and the ratio of the produced toxins is usually 3:1, respectively (Karlsson et al., Microbiology 145:1683-1693, 1999). The toxins begin to be formed during the exponential growth phase, and are usually released from the bacteria between 36 and 72 hours of culture. Toxins present within the bacteria can be released earlier by sonication or by use of a French pressure cell.
Treatment of the toxins with formaldehyde results in the corresponding Toxoids A and B, which are completely inactivated and retain at least partial immunogenicity (Torres et al., Infection and Immunity 63(12):4619-4627, 1995). It has been shown that vaccination employing both toxoids is effective in hamsters, healthy adults, and patients with recurrent CDAD (Torres et al., Infection and Immunity 63(12):4619-4627, 1995; Kotloff et al., Infection and Immunity 69(2):988-995, 2001; Sougioultzis et al., Gastroenterology 128(3):764-770, 2005; Tones et al., Vaccine Research 5(3):149-162, 1996). Additionally, the administration of both free and aluminum salt (adjuvant) bound toxoids leads to appropriate immune responses (Torres et al., Vaccine Research 5(3):149-162, 1996; Giannasca et al., Infection and Immunity 67(2):527-538, 1999).
The administration of both toxoids simultaneously is more effective than administration of the individual proteins alone (Kim et al., Infection and Immunity 55(12):2984-2992, 1987). A toxoid composition found effective in inducing protective immune responses against toxin A and toxin B in patients with recurrent CDAD included both toxoids, at a ratio of 1.5:1, A:B (Sougioultzis et al., Gastroenterology 128(3):764-770, 2005).
Both the A and B toxoids are thus candidates for vaccine development. Greater production efficiency of Toxins A and B is desired to facilitate vaccine production.
SUMMARY OF THE INVENTION
In one aspect, the invention features a culture medium (e.g., for culturing a Clostridium difficile bacterium) at a pH of between 6.35 and 7.45 (e.g., 6.5, 7.28, or between 6.35 and 6.65) including peptone (e.g., soy peptone), a yeast extract (e.g., Difco Bacto Yeast extract), a buffering agent (e.g., NaHCO3), and a phosphate buffer (e.g., sodium phosphate, dibasic and potassium phosphate, monobasic). This culture medium can also include at least one additive (e.g., 2, 3, or more additives) selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, D-sorbitol, sucrose, and a combination of azaserine, adenosine, and biotin.
In another aspect, the invention features a bacterial culture including Clostridium difficile and culture medium including at least one additive (e.g., 2, 3, or more additives) selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, D-sorbitol, sucrose, and a combination of azaserine, adenosine, and biotin. This medium can also include peptone (e.g., soy peptone), yeast extract (e.g., Difco Bacto Yeast extract), sodium phosphate, dibasic, potassium phosphate, monobasic, and NaHCO3, and the culture medium can be at a pH of between 6.35 and 7.45 (e.g., 6.5, 7.28, or between 6.35 and 6.65).
In another aspect, the invention features a method of culturing Clostridium difficile including inoculating culture medium with Clostridium difficile, with the medium including at least one additive (e.g., 2, 3, or more additives) selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, D-sorbitol, sucrose, and a combination of azaserine, adenosine, and biotin. This medium can also include peptone (e.g., soy peptone), yeast extract (e.g., Difco Bacto Yeast extract), sodium phosphate, dibasic, potassium phosphate, monobasic, and NaHCO3, and the culture medium can be at a pH of between 6.35 and 7.45 (7.28 or between 6.35 and 6.65). Preferably the culture medium is at a pH of 6.5.
In another aspect, the invention features a method for obtaining or preparing one or more C. difficile toxins including by preparing an aqueous growth medium including soy peptone, inoculating the medium with a C. difficile bacterium (e.g., using an aqueous C. difficile culture), culturing the inoculated medium (e.g., at a pH of 6.5, 7.28, between 6.35 and 7.45, or between 6.35 and 6.65)) under conditions which facilitate growth of bacterium and toxin production (e.g., at a temperature between 37° C. to 41° C.), and isolating the one or more C. difficile toxins from growth medium (e.g., by removing from the growth medium viable C. difficile organisms and spores, separating the one or more toxins from the growth media, and purifying the one or more toxins). This culture medium can also include yeast extract, NaHCO3, sodium phosphate, dibasic, potassium phosphate, monobasic, and D-sorbitol. This culturing can be carried out, e.g., under anaerobic conditions. The steps of inoculating the medium with a C. difficile bacterium (e.g., using an aqueous C. difficile culture) and culturing the inoculated medium can be repeated more than once, with inoculation into fresh growth medium with each repeat. This method can also include detoxifying the isolated one or more C. difficile toxins to prepare one or more toxoids (e.g., by reacting the one or more toxins by the addition of formaldehyde).
In another aspect, the invention features a method of enhancing the production of Toxin B from a C. difficile culture by preparing an aqueous growth medium including soy peptone, inoculating the medium with a C. difficile bacterium, culturing the inoculated medium at 37° C. to 41° C. and at a pH between pH 6.35 and pH 6.65 (e.g., at 37° C. and at a pH of 6.5). The pH and/or temperature can be held constant or vary during the culturing. The growth media can further include yeast extract, NaHCO3; sodium phosphate, dibasic, potassium phosphate, monobasic, and D-sorbitol. Toxin B production can be enhanced relative to Toxin A production, producing, e.g., ratios of Toxin A relative to Toxin B of less than 3:1, 2:1, 1.5:1, or less.
In any of the foregoing aspects, yeast extract can be between 10 and 30 g/L, the NaHCO3 can be between 2 and 5 g/L; the sodium phosphate, dibasic can be between 1 and 10 g/L, and the potassium phosphate, monobasic can be between 1 and 10 g/L. The adenosine can be present at a concentration of between 0.8 and 1.2 mM (e.g., 1 mM), the biotin at a concentration of between 40 and 60 nM (e.g., 50 nM), and the azaserine at a concentration between 15 and 50 μM (e.g., 50 μM). The concentration of D-sorbitol can be between 6 g/L and 20 g/L or between 8 g/L and 18 g/L (e.g., 12 g/L). The chromium trioxide can be present at a concentration of between 40 and 60 mg/L (e.g., 50 mg/L). The clindamycin can be present at a concentration between 0.4 and 0.6 mg/L (e.g., 0.5 mg/L). The ascorbic acid can be present at a concentration between 2.5 g/L and 10 g/L (e.g., 2.5 g/L and 10 g/L). The butyric acid can be present at a concentration between 30 mM and 60 mM (e.g., 30 mM and 60 mM). The D(+)xylose can be at a concentration between 6 g/L and 10 g/L (e.g., 6 g/L).
The invention provides several advantages. For example, the media and the methods of the invention allow increased production of Clostridium difficile toxins, which leads to increased efficiency and decreased costs in the production of toxin-based products such as vaccines. Other features and advantages of the invention will be apparent from the following Detailed Description, the Drawings, and the Claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-G are graphs showing the amount of production of the indicated toxin in cultures containing the indicated additive at the indicated concentration.
FIG. 2A is an SDS PAGE gel showing the amount of Toxin A produced in cells cultured with the indicated compounds at 12 hours.
FIG. 2B is an SDS PAGE gel showing the amount of Toxin A produced in cells cultured with the indicated compounds at 24 hours.
FIG. 3A is an SDS PAGE gel showing the amount of Toxin A produced in cells cultured with the indicated compounds at 12 hours.
FIG. 3B is an SDS PAGE gel showing the amount of Toxin A produced in cells cultured with the indicated compounds at 24 hours.
FIG. 4A is an SDS PAGE gel showing the amount of Toxin A produced in cells cultured with the indicated compounds at 12 hours. The lanes were loaded with samples from cultures including the following compounds: Control; #1 Arginine (50 mM); #2 Cysteine (0.33 mM); #3 Cysteine (3.3 mM); #4 Cysteine (33 mM); #5 Tyrosine (50 mg/L); #6 Ascorbic acid (2.5 g/L); #7 Ascorbic acid (10 g/L); #8 Butyric acid (30 mM); #9 Butyric acid (60 mM).
FIG. 4B is an SDS PAGE gel showing the amount of Toxin A produced in cells cultured with the indicated compounds at 24 hours. The lanes were loaded with samples from cultures including the following compounds: #1 Arginine (50 mM); #2 Cysteine (0.33 mM); #3 Cysteine (3.3 mM); #4 Cysteine (33 mM); #5 Tyrosine (50 mg/L); #6 Ascorbic acid (2.5 g/L); #7 Ascorbic acid (10 g/L); #8 Butyric acid (30 mM); #9 Butyric acid (60 mM).
FIG. 5A is an SDS PAGE gel showing the amount of Toxin A produced in cells cultured with the indicated compounds at 12 hours. The lanes were loaded with samples from cultures including the following compounds: #1 D(−)Fructose (6 g/L); #2-D(+)Galactose (6 g/L); #3 Mannose (6 g/L); #4 Maltose Monohydrate (6 g/L); #5 Sucrose (6 g/L); #6α-Lactose (6 g/L); #7 D(+)Xylose (6 g/L); #8 D-Sorbitol (6 g/L); #9 myo-Inositol (6 g/L).
FIG. 5B is an SDS PAGE gel showing the amount of Toxin A produced in cells cultured with the indicated compounds at 24 hours. The lanes were loaded with samples from cultures including the following compounds: #1-D(−)Fructose (6 g/L); #2 D(+)Galactose (6 g/L); #3 Mannose (6 g/L); #4 Maltose Monohydrate (6 g/L); #5 Sucrose (6 g/L); #6α-Lactose (6 g/L); #7 D(+)Xylose (6 g/L); #8 D-Sorbitol (6 g/L); #9 myo-Inositol (6 g/L).
FIG. 6A is a graph showing the amount of Toxin A produced (ng/ml) in cultures subject to the indicated pH control.
FIG. 6B is a graph showing the amount of Toxin B produced (ng/ml) in cultures subject to the indicated pH control.
FIG. 7A is a graph showing the amount of Toxin A produced (ng/ml) in cultures subject to the indicated pH control.
FIG. 7B is a graph showing the amount of Toxin B produced (ng/ml) in cultures subject to the indicated pH control.
FIG. 7C is a graph showing specific Toxin A productivity produced (ng/ml per OD unit) in the cultures subject to the indicated pH control.
FIG. 7D is a graph showing specific Toxin B productivity produced (ng/ml per OD unit) in the cultures subject to the indicated pH control.
FIG. 7E is a graph comparing the specific Toxin A and Toxin B produced (ng/ml) in cultures subject to the indicated pH control in Examples 6 and 7.
FIG. 8A is a graph showing the amount of Toxin A produced (ng/ml) in cultures subject to the indicated pH control and the indicated amount of sodium bicarbonate.
FIG. 8B is a graph showing the amount of Toxin B produced (ng/ml) in cultures subject to the indicated pH control and the indicated amount of sodium bicarbonate.
FIG. 9A is a graph showing the amounts of Toxin A and B produced (ng/ml) in 18 hour cultures subject to the indicated pH control in comparison to cell growth (OD600).
FIG. 9B is a graph showing the amounts of Toxin A and B produced (ng/ml) in 22 hour cultures subject to the indicated pH control in comparison to cell growth (OD600).
FIG. 10A is a graph showing the amount of Toxin A produced (ng/ml) in cultures subject to the indicated temperature.
FIG. 10B is a graph showing the amount of Toxin B produced (ng/ml) in cultures subject to the indicated temperature.
FIG. 10C is a graph showing specific Toxin A productivity produced (ng/ml per OD unit) in the cultures subject to the indicated temperature.
FIG. 10D is a graph showing specific Toxin B productivity produced (ng/ml per OD unit) in the cultures subject to the indicated temperature.
FIG. 11 is a graph showing the amount of Toxin A and Toxin B produced (ng/ml) in comparison to cell growth (OD600) in cultures subject to the indicated pH condition and inoculated with the indicated % of inoculum.



DETAILED DESCRIPTION
In general, the invention features methods and compositions (such as for example, culture media) for culturing C. difficile and producing the C. difficile Toxins A and B. These two toxins can be used individually or in combination, in the preparation of toxoids.
As discussed further below, the culturing of C. difficile in the media of the invention leads to enhanced Toxin A and Toxin B production. Similarly, as discussed further below, enhanced toxin production is seen by culturing C. difficile in accordance to the methods of the invention.
Basal Media
The compositions and methods of the invention feature the use of a basal medium in conjunction with certain medium additives. In one example, the basal medium is comprised of peptone (e.g., 20-40 g/L), yeast extract (e.g., 10-30 g/L), a phosphate buffer (such as for example, potassium phosphate monobasic (e.g., 0.5-1.5 g/L) and sodium phosphate dibasic (e.g., 1-10 g/L)) and a buffering agent (such as for example, sodium bicarbonate (e.g., 1-10 g/L)). The peptone used may be soy-based or animal-derived (such as for example, tryptone).
In one example, the basal medium is SYS media. SYS medium contains the ingredients listed in Table 1A at the indicated concentrations. The basal medium may be titrated to a pH of between 6.35 and 7.45 (for example, 6.5, 7.28, or between 6.35 and 6.65). Exemplary ranges of concentrations for each of the indicated ingredients are also indicated.









TABLE 1A






Grams
Acceptable range


Ingredient
per liter
of grams per liter


















Soy peptone A3
30
20-40
(e.g., 25-35 and 29-31)


Difco Bacto Yeast extracts
20
10-30
(e.g., 15-25 and 19-21)


KH2PO4
0.9
0.5-1.5
(e.g., 0.8-1.0)


Na2HPO4
5
1-10
(e.g., 2-8 and 4-5)


NaHCO3
5
1-10
(e.g., 2-8 and 4-5)









Table 1B sets forth an alternative basal media useful in the compositions and methods of the invention, named TYS.









TABLE 1B






Grams
Acceptable range


Ingredient
per liter
of grams per liter


















Difco Bacto Tryptone
30
20-40
(e.g., 25-35 and 29-31)


Difco Bacto Yeast extracts
20
10-30
(e.g., 15-25 and 19-21)


KH2PO4
0.9
0.5-1.5
(e.g., 0.8-1.0)


Na2HPO4
5
1-10
(e.g., 2-8 and 4-5)


NaHCO3
5
1-10
(e.g., 2-8 and 4-5)









In substitution of the Soy peptone A3 and the Difco Bacto Tryptone any peptone (e.g., any soy peptone) can be utilized. Examples of soy peptones that can be used in the basal media (and their sources) include the following:
Kerry Biosciences: HyPer 1510,
IPS: Hy-Soy Kosher, and
Becton Dickinson: BD Select Phytone UF
In substitution of the Difco Bacto Yeast Extract, any yeast extract can also be used in the basal media. Examples of suitable yeast extracts (and their sources) are readily known to those skilled the art.
The suitability of a particular peptone or yeast extract for use in the invention can be determined using the experimental methods described herein. The invention also includes use of other bacterial growth media, in combination with the additives described below.
Additives
The invention also features the use of certain additives with a basal media (e.g., SYS media). Exemplary additives of the invention are set forth in Table 2, which also includes the exemplary concentration ranges for the indicated additives, as well as a single exemplary concentration. Additives include:
Chromium trioxide (Chromium(VI) oxide CrO3). Chromium trioxide is the acid anhydride of chromic acid. Chromium trioxide is a strong oxidant, highly toxic, corrosive, and carcinogenic compound.
Clindamycin (C18H33ClN2O5S). Clindamycin is a lincosamide antibiotic and is indicated for Clostridium difficile-associated diarrhea (the most frequent cause of pseudomembranous colitis). Clindamycin has a bacteriostatic effect. It interferes with bacterial protein synthesis by binding preferentially to the 50S subunit of the bacterial ribosome.
Azaserine (C5H7N3O4). Azaserine is a naturally occurring serine derivative diazo compound and is a known carcinogen. Azaserine is a glutamine analogue that irreversibly inhibits glutamine phosphoribosyl amidotransferase, which is involved in the biosynthesis of inosine monophosphate (IMP). IMP is an important precursor to the purine nucleotides which include adenosine monophosphate (AMP) and guanosine monophosphate (GMP).
Ascorbic acid (C6H8O6). Ascorbic acid is a sugar acid with antioxidant properties. L-Ascorbic acid is also known as vitamin C.
Butyric acid (C4H8O2). Butyric acid is a carboxylic acid and a short chain fatty acid. Butyric acid has been associated with the ability to inhibit the function of histone deacetylase enzymes, thereby favoring an acetylated state of histones in the cell.
Xylose (C5H10O5). Xylose (wood sugar) is a five-carbon monosaccharide. Xylose can be metabolized into useful products by a variety of organisms, e.g., Clostridium difficile. 
Sorbitol (C6H14O6). Sorbitol, also known as glucitol, is a sugar alcohol. Sorbitol also is an osmotic stress agent (osmotic shock is induced by 0.5 M sorbitol).







TABLE 2







Additives









Compounds




(Concentration)
Concentration Range
Single Concentration













Chromium trioxide
40-60 mg/L
50
mg/L


Clindamycin
0.1-10 mg/L (e.g., 0.4-0.6 mg/L)
0.5
mg/L













Azaserine,
15-50 μM
0.5-1. mM
40-60 nM
50 μM
1 mM
50 nM


Adenosine, and

(e.g., 0.8-1.2 mM)






Biotin
















Ascorbic acid
2.5-10 g/L
2.5
g/L


Butyric acid
30-60 nM
60
mM


D(+)Xylose
1-15 g/L (e.g., 6-10 g/L)
6
g/L


D-Sorbitol
6-20 g/L (e.g., 8-18 g/L)
12
g/L









Methods

Growth of C. difficile according to the methods of the invention proceeds in at least two phases: seed growth and fermentation. The seed growth phase, as described further below, may proceed in one or more seed culture stages (e.g, two stages or three stages).
A seed culture is first grown by inoculating seed medium with a sample from a stock culture (e.g., a working cell bank (WCB)). A sample of this seed culture is used either to inoculate a second seed culture or to inoculate a relatively large fermentation culture. Such seed cultures are typically carried out to allow the quantity of the microorganism from a stored culture (e.g., WCB) to be exponentially increased (scaled-up). Seed cultures can also be used to rejuvenate relatively dormant microbes in stored cultures. As is well understood in the art, more than one seed culture (e.g., two or three cultures or stages) can be used to scale-up the quantity of C. difficile for inoculation into the fermentation medium.
The number of seed cultures (or stages) used depends on, for example, the size and volume of the fermentation step. For example, the culture process may involve two seed cultures: a first seed culture is grown from an inoculation of a WCB (stage one seed culture), a sample of this seed culture is used to inoculate a second seed culture (stage two seed culture), and a sample from this second culture is used to inoculate a fermentation culture (fermentation stage). In a preferred embodiment of the present invention, the first and second seed cultures are grown in SYS media.
In stage one, a culture of C. difficile is suspended in seed medium and is incubated at a temperature between 30-40° C., preferably at 37±1° C., for 18 hours in an anaerobic environment. In stage two, a sample of the stage one seed medium is used to inoculate a stage two seed medium for further growth. After inoculation, the stage two medium is incubated at a temperature between 30-40° C., preferably at 37±1° C., for approximately 10 hours, also in an anaerobic environment. Preferably, growth in seed media at any stage does not result in cell lysis before inoculation of fermentation media. Additional growth in a third (fourth, etc.) stage seed culture can also be carried out.
In the fermentation stage, an appropriate concentration of seed culture, which can range from, e.g., 0.1-10%, is used to inoculate the fermentation media. Preferably, concentrations of 1.0% or 5.0% can be used. Most preferably, concentrations of 10% are used.
Fermentation is preferably carried out in an anaerobic chamber at approximately 35° C. to 45° C. and preferably at a temperature between 37° C. to 41° C. (e.g., 37° C.). The pH of the fermentation may be controlled at a pH between pH 6.35 to 7.45 (e.g., between 6.35 to 6.65, and preferably, at pH 6.5). Alternatively, the pH of the culture media is uncontrolled and is allowed to decrease naturally during the fermentation process.
C. difficile can be cultivated by fermentation with continuous exposure to a suitable gas or gas mixture (such as, for example, 80% nitrogen/10% CO2/10% hydrogen, 100% CO2, or 100% nitrogen). Such gases or gas mixtures may also be sparged (i.e., bubbled) through the medium during fermentation. As an alternative to sparging (or in addition to it), a gas mixture (e.g., 80% nitrogen/10% CO2/10% hydrogen) or a gas (e.g., CO2 or nitrogen) may be applied to the culture media as an overlay to degas the media throughout the fermentation process. The fermentation culture is preferably sparged prior to inoculation with either a mixture of 80% nitrogen/10% CO2/10% hydrogen, 100% CO2 or 100% nitrogen to remove any residual oxygen in the medium. During the fermentation process the culture may be sparged periodically. Alternatively, an overlay of a gas mixture or a gas (e.g., 100% nitrogen) may be applied to the culture.
Fermentation proceeds for approximately 16 to 24 hours (e.g., 18 to 21 hours). Preferably, agitation (e.g., 100 rpm) is applied to the culture medium during the fermentation process (and/or during stages one and two of seed cultures). Growth can be monitored by measuring the optical density (O.D.) of the medium.
C. difficile toxins can be isolated and purified from fermentation cultures using purification methods well known in the art such as for example, Kotloff et al., Infect. Immun 2001; 69:988-995, Coligan et al., “Current Protocols in Protein Science,” Wiley & Sons; Ozutsumi et al., Appl. Environ. Microbiol. 49:939-943, 1985; and Kim et al., Infection and Immunity 55:2984-2992, 1987; which are incorporated herein by reference. The purified toxins can then, for example, be inactivated by chemical treatments known in the art (e.g., formaldehyde treatment).
All references cited within this disclosure are hereby incorporated by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way.
EXAMPLES
The basal media and additives of the invention were used to culture Clostridium difficile and produce Clostridium difficile Toxins A and B. Tables 3 and 4 (and FIGS. 1A-1G) summarize the amount of toxin produced by Clostridium difficile cells cultured in SYS media with the indicated additive at the indicated concentration after the indicated amount of time. Throughout the examples, Clostridium difficile, ATCC No. 43255, ATCC Lot# 2888434, was cultured.







TABLE 3







Percent increase in Toxins A and B at time points 12 and 24 hours


following growth in the presence of the listed compounds









Compounds
Toxin A
Toxin B











(Concentration)
12 hrs.
24 hrs.
12 hrs.
24 hrs.














Chromium trioxide (50 mg/L)

18.67

80.45


Clindamycin (0.5 mg/L)

27.52

94.21


50 μM Azaserine + 1 mM
7.69
80.03
96.2
238.34


Adenosine + 50 nM Biotin






Ascorbic acid (2.5 g/L)

4.65
5.26
24.42


Ascorbic acid (10 g/L)

1.68
18.54
49.17


Butyric acid (30 mM)
13.8
10.07
33.6
60.95


Butyric acid (60 mM)



88.6


D(+)Xylose (6 g/L)

10.06
8.49
45.83


D-Sorbitol (6 g/L)
49.19
86.29
68.03
153.03
















TABLE 4A







Total measured production of Toxins A and B at time points 12 and 24


hours following growth in the presence of the listed compounds










Toxin A
Toxin B


Compounds
(ng/mL)
(ng/mL)











(Concentration)
12 hrs.
24 hrs.
12 hrs.
24 hrs.














Control (CD-2284)
5974
6809
2436
2645


Chromium trioxide
3880
8080
2188
4773


(50 mg/L)






Increase (%)

18.67

80.45


Control (CD-2304)
10083
10475
3392
3469


Clindamycin (0.5 mg/L)
611
13358
189
6737


Increase (%)

27.52

94.21


Control (CD-2353)
9649
10554
3159
3529


50 μM Azaserine,
10391
19000
6198
11940


1 mM Adenosine,






50 nM Biotin






Increase (%)
7.69
80.03
96.20
238.34


Control (CD-2380)
10822
11515
3825
3992


Ascorbic acid (2.5 g/L)
10789
12050
4026
4967


Increase (%)

4.65
5.26
24.42


Ascorbic acid (10 g/L)
9957
11708
4534
5955


Increase (%)

1.68
18.54
49.17


Butyric acid
12315
12674
5110
6425


(30 mM = 2.75 mL/l)






Increase (%)
13.80
10.07
33.60
60.95


Butyric acid
5335
10681
3063
7529


(60 mM = 5.5 mL/l)






Increase (%)



88.60


Control (CD-2401)
10108
10508
3756
3832


D(+)Xylose (6 g/L)
10135
11565
4075
5588


Increase (%)

10.06
8.49
45.83


D-Sorbitol (6 g/L)
15080
19575
6311
9696


Increase (%)
49.19
86.29
68.03
153.03
















TABLE 4B







Total measured production of Toxins A and B at time points 12 and 24


hours following growth in the presence of the listed compounds










Toxin A
Toxin B


Compounds
(ng/mL)
(ng/mL)











(Concentration)
12 hrs.
24 hrs.
12 hrs.
24 hrs.














50 μM Azaserine +
10391
19000
6198
11940


1 mM Adenosine + 50 nM Biotin






15 μM Azaserine +
8525
10423
3430
3880


1 mM Adenosine + 50 nM Biotin






 5 μM Azaserine +
7471
7816
2665
2694


1 mM Adenosine + 50 nM Biotin









Example 1
This Example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various metallic ions.
Materials
The following are the Example 1 test compounds, along with the compound formula and source.

    AFC-Ammonium ferric citrate (C6H8O7.nFe.nH3N), USB Cat. 15751 Lot. 121753
    FC-Ferric citrate (C6H5FeO7), FW 244.95, MB BIomedicals LLC, Cat. 195181, Lot R23927
    FG-Ferrous gluconate Hydrade (C12 hrs.22FeO14)
    FS-Ferric sulfate (FeSO4.7H2O), FW 278.02 CA-Calcium chloride Anhydrous (CaCl2), FW 110.98, J. T. Baker Cat. 1311-01, Lot. A13602
    CC-Cobalt chloride 6 Hydrate Crystal (CoCl2.6H2O), FW 237.93, Mallinckrodt Chemicals Cat. 4535-02
    CT-Chromium trioxide Crystal (CrO3), FW 99.99, J. T. Baker Cat. 1638-04, Lot.
    MS-Magnesium sulfate (MgSO4.7H2O), FW 246.50
    MC-Manganese chloride (MnCl24H2O), FW 197.90, J. T. Baker Cat. 2540-04, Lot E37335


The following table indicates the natural pH of the indicated compound in solution at the indicated concentration.













Compound solution
Natural
Compound solution



2 g/L
pH
1 g/L
Natural pH


















Ammonium ferric citrate
5.0
Calcium chloride
4.7


Ferric citrate
3.0
Cobalt chloride
4.8


Ferrous gluconate
4.5
Chromium trioxide
<2.5


Ferric sulfate
4.7
Magnesium sulfate
5.0




Manganese chloride
5.0









Methods

The following methods were used to test the production of Toxin A and B by Clostridium difficile when cultured in the presence of the above-listed additives.
I. Medium Preparations:


    
    
        1. Prepare 1000 mL SYS medium in 2 L beaker.
        2. Transfer SYS to media bottles and degas for over 30 minutes with 10% H2+10% CO2+80% N2.
        3. Before transferring the medium, fill gas (10% H2+10% CO2+80% N2) from the fill port of the Flexboy bag into the bag to remove oxygen, then empty the gas from the bag. Connect the filling system manifold with the bags.
        4. For seed medium in 50 mL Flexboy bags, pump 30 mL medium into the bag from the fill port with a flow speed at 100 mL/minute.
        5. For fermentation medium in 250 mL Flexboy bags:
        
            i) Put the bag on a balance before filling with the medium and adjust to “0.”
            ii) Pump the medium into the bag from the fill port with a flow speed at 100 mL/min until the balance show 50 g, stop the pumping.
        
        
        6. Move the bag for seed-1 to 37° C. CO2 incubator to warm overnight. Keep bag for seed-2 and fermentation at 4° C. until use.
        7. Move the bags to 37° C. CO2 incubator to warm up overnight before use.
        8. For different compounds:
        
            i) Prepare 40 mL solutions of different compounds with the concentrations at 2.0 g/L (80 mg compound+40 mL di water (pH)).
            ii) Prepare 40 mL solutions of different compounds with the concentrations at 1.0 g/L (40 mg compound+40 mL di water (pH)).
            iii) For all compounds but ferric citrate, filter the solution using Millipore 50 mL disposable vacuum filtration system with 0.22 μm Millipore Express Plus membrane. The ferric citrate was autoclaved.
            iv) Before transfer of seed-2 to fermentation bags add the compound solutions as the following concentrations listed in the following table:
        
        
    
    















Compounds

Sterile di water
Total


(mg/L)

(mL)
(mL)



















2 g/L solution





(mL)




Control (without any compound)
0
2.5
2.5


100 mg/L (5 mg/50 mL)
2.5
0
2.5



1 g/L solution





(mL)




 50 mg/L (2.5 mg/50 mL)
2.5
0
2.5









II. Fermentation Process:

    
    
        1. First stage seed culture: 1 mL WCB, containing 50% glycerol, was transferred into a 50 mL Flexboy bag containing 30 mL SYS medium and incubated at 37±1° C. for 24 hours.
        2. Second stage seed culture: 1.5 mL of first stage seed culture at inoculums of 5% were transferred into the 50 mL Flexboy bag containing 30 mL SYS medium and incubated at 37±1° C. for 22 hours.
        3. Fermentation: 2.5 mL of second stage seed culture was inoculated at 5% for each 250 mL Flexboy bag containing 50 mL of SYS medium and incubated at 37° C.±1° C. for 24 hours.
        4. Take samples at 12 hours and 24 hours. Cell growth was measure at 600 nm. The blank of fermentation media was used as zero for the spectrophotometer. The cell concentration was diluted 10×.
        5. The toxin production is measured by capture ELISA.

III. Capture ELISAs:

        1. Toxin A standard Lot# CD-2062 (1072506A)
        2. Goat anti-Toxin A, Lot# CD-2017
        3. Mouse MAb to C. difficile Toxin A (PCG4)
        4. Toxin B standard Lot#QC06329
        5. Goat anti-Toxin B, Lot# C0210091
        6. Mouse anti Toxin B Lot#030904

Results

    
    
    1. The following table shows the amount of seed growth (OD600 nm) as measured by DU700.















Seed-1
Seed-2








1.73
2.31









    2. The following table shows the amount of cell growth (OD600 nm) in cultures with the indicated compound.
















Test
12 hours
24 hours



















Control
2.56
2.25



Ammonium ferric citrate
2.58
2.22*



Ferric citrate
2.38
2.48*



Ferrous gluconate
2.73
2.51*



Ferric sulfate
2.55
2.73*



Calcium chloride
2.39
2.20



Cobalt chloride
2.34
1.89*



Chromium trioxide
1.48
1.02**



Magnesium sulfate
2.37
2.03



Manganese chloride
2.52
1.97





*The broth became dark green because iron reacted with other compounds


**A lot of cells showed 3× to 5× longer than the normal cells in 24 h broth






    3. The following table shows the amount of Toxin A produced (ng/mL) in cultures with the indicated compound (FIGS. 2A and 2B).
















Test
12 hours
24 hours








Control
5974
6809



Ammonium ferric citrate
5752
6634



Ferric citrate
5580
6544



Ferrous gluconate
5453
6208



Ferric sulfate
5162
5706



Calcium chloride
6294
6563



Cobalt chloride
5252
6647



Chromium trioxide
3880
8080



Magnesium sulfate
5060
5527



Manganese chloride
4768
5449









    4. The following table shows the amount of Toxin B produced (ng/mL) in cultures with the indicated compound.
















Test
12 hours
24 hours








Control
2436
2645



Ammonium ferric citrate
2153
2503



Ferric citrate
2191
2550



Ferrous gluconate
2190
2082



Ferric sulfate
2068
2059



Calcium chloride
2494
2516



Cobalt chloride
2043
2721



Chromium trioxide
2188
4773



Magnesium sulfate
1873
1922



Manganese chloride
1784
1921









    5. The following table shows the amount of spore formation in 24 hour fermentation in cultures with the indicated compound. Broth was examined by microscope.















Test
24 hours








Control
No spore formation found



Ammonium ferric citrate
No spore formation found



Ferric citrate
No spore formation found



Ferrous gluconate
No spore formation found



Ferric sulfate
No spore formation found



Calcium chloride
No spore formation found



Cobalt chloride
No spore formation found



Chromium trioxide
No spore formation found



Magnesium sulfate
No spore formation found



Manganese chloride
No spore formation found









Conclusions

Chromium trioxide, when added to the SYS medium at 50 mg/L, caused increases in production of Toxin A (20%) and Toxin B (80%) after 24 hours in fermentation broth, but not after 12 hours in fermentation broth.
Example 2
This example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various antibiotics.
Materials
The following are antibiotics, along with the compound formula and source, which were tested in this example.

    Cip-Ciprofloxacin (C17H18FN3O3) FW 331.35, BioChemika, Lot#WA19781, soluble with 0.2 mL of 5 N HCl.
    Cli-Clindamycin hydrochloride (C18H33ClN3O5S.HCl) FW 461.44, Sigma C5269, Lot#37k1535, soluble in water.
    Van-Vancomycin hydrochloride (C66H75Cl2N9O24.HCl) FW 1485, Sigma V20029, Lot#037K0686 soluble in water.
    Pen G-Penicillin G Sodium salt (C16H17N2NaO4S) FW 356.4, Sigma P3032, Lot#057K04931, soluble in water.
    Fe-EDTA was also tested (Ethylenediaminetetraacetic acid, Ferric Sodium Salt, (C10H12FeN2NaO8) FW 421.10, Acros Organics 304680050, Lot#A0245953).


Antibiotics were tested at the following concentrations:
Ciprofloxacin (2 mg/L and 10 mg/L), Clindamycin (0.5 mg/L and 2.5 mg/L), Vancomycin (0.1 mg/L and 0.5 mg/L), and Penicillin G (0.1 mg/L and 0.5 mg/L).
Ethylenediaminetetraacetic acid Ferric Sodium Salt was tested at a concentration of 100 mg/L.
Materials
The following materials were used to test the production of Toxin A and B by Clostridium difficile when cultured in the presence of the above antibiotics and compounds.

    1. Make 100× concentration antibiotic solutions/10× Fe-EDTA solutions




















100×


Antibiotics
Antibiotics
Sterile di
Concentration


(mg/L)
powder (mg)
water (mL)
(mg/L)





Ciprofloxacin (10 mg/L)
40
40
1000


Clindamycin (2.5 mg/L)
5
20
250


Vancomycin (0.5 mg/L)
2
40
50


Penicillin G (0.5 mg/L)
2
40
50













Compounds
Compound
Sterile di water
10× Concentration


(mg/L)
(mg)
(mL)
(mg/L)





Fe-EDTA 100 mg/L
40
40
1000









    2. Make 10× concentration of antibiotic solution:


Take 4 mL of 100× concentration solution+36 mL di water.
Methods
I. Medium Preparations:


    
    
        1. Prepare 1000 mL SYS medium in 2 L beaker.
        2. Transfer SYS to media bottles and degas for over 30 minutes with 10% H2+10% CO2+80% N2.
        3. Before transferring the medium, fill gas (10% H2+10% CO2+80% N2) from the fill port of the Flexboy bag into the bag to remove oxygen, then empty the gas from the bag. Connect the filling system manifold with the bags.
        4. For seed medium in 50 mL Flexboy bags, pump 30 mL medium into the bag from the fill port with a flow speed at 100 mL/minute.
        5. For fermentation medium in 250 mL Flexboy bags:
        
            i) Put the bag on a balance before filling with the medium and adjust to “0”.
            ii) Pump the medium into the bag from the fill port with a flow speed at 100 mL/min until the balance show 50 g, stop the pumping.
        
        
        6. Move the bag for seed-1 to 37° C. CO2 incubator to warm overnight. Keep bag for seed-2 and fermentation at 4° C. until use.
        7. Move the bags to 37° C. CO2 incubator to warm up overnight before use.
        8. For different antibiotics:
        
            i) Prepare 40 mL solutions of different antibiotics with the concentrations at 10× (see above table).
            ii) Prepare 40 mL solutions of FE-EDTA with the concentrations at 1000 mg/L (40 mg compound+40 mL di water (pH)).
            iii) Filter the solution using the Millipore 50 mL Disposable Vacuum Filtration System with 0.22 μm Millipore Express Plus Membrane.
            iv) Before transfer of seed-2 to fermentation bags, add the compound solutions at the following concentrations listed in the following tables.
        
        
    
    
















10× Solution
Sterile di
Total



(mL)
water (mL)
(mL)







Antibiotics





(mg/L)





Control (without antibiotics)
0
5
5


Ciprofloxacin 2 mg/L
1
4
5


(100 μg/50 mL)





Ciprofloxacin 10 mg/L
5
0
5


(500 μg/50 mL)





Clindamycin 0.5 mg/L (25 μg/50 mL)
1
4
5


Clindamycin 2.5 mg/L
5
0
5


(125 μg/50 mL)





Vancomycin 0.1 mg/L (5 μg/50 mL)
1
4
5


Vancomycin 0.5 mg/L (25 μg/50 mL)
5
0
5


Penicillin G 0.1 mg/L (5 μg/50 mL)
1
4
5


Penicillin G 0.5 mg/L (25 μg/50 mL)
5
0
5


Compounds





(mg/L)





Fe-EDTA 100 mg/L (5 mg/50 mL)
5
0
5









II. Fermentation Process:

    
    
        1. First stage seed culture: 1 mL WCB, containing 50% glycerol, was transferred into a 50 mL Flexboy bag containing 30 mL SYS medium and incubated at 37±1° C. for 24 hours.
        2. Second stage seed culture: 1.5 mL of first stage seed culture at inoculums of 5% were transferred into the 50 mL Flexboy bag containing 30 mL SYS medium and incubated at 37±1° C. for 22 hours.
        3. Fermentation: 2.5 mL of second stage seed culture was inoculated at 5% for each 250 mL Flexboy bag containing 50 mL of SYS medium and incubated at 37° C.±1° C. for 24 hours.
        4. Take samples at 12 hours and 24 hours. Cell growth was measure at 600 nm. The blank of fermentation media was used as zero for the spectrophotometer. The cell concentration was diluted 10×.
        5. The toxin production is measured by capture ELISA.

III. Capture ELISAs:

        1. Toxin A standard Lot# CD-2062 (1072506A)
        2. Goat anti-Toxin A, Lot# CD-2017
        3. Mouse MAb to C. difficile Toxin A (PCG4)
        4. Toxin B standard Lot#QC06329
        5. Goat anti-Toxin B, Lot# C0210091
        6. Mouse anti Toxin B Lot#030904

Results

    
    
    1. The following table shows the amount of seed growth (OD600 nm) as measured by DU700.















Seed-1
Seed-2








2.32
2.82









    2. The following table shows the amount of cell growth (OD600 nm) in cultures with the indicated compound.

















Test
mg/L
12 hours
24 hours








Control
—
2.71
2.48



Ciprofloxacin
2
2.60
2.53




10
0.87
1.38



Clindamycin
0.5
1.24
2.53




2.5
0.11
0.75



Vancomycin
0.1
3.01
2.53




0.5
3.08
2.52



Penicillin G
0.1
2.69
2.49




0.5
2.64
2.79



Fe-EDTA
100
2.91
2.37









    3. The following table shows the amount of Toxin A produced (ng/mL) in cultures with the indicated compound (FIGS. 3A and 3B).

















Test
mg/L
12 hours
24 hours




















Control
—
10083
10475



Ciprofloxacin
2
6309
6953




10
1685
8614



Clindamycin
0.5
611
13358




2.5
496
445



Vancomycin
0.1
9142
9557




0.5
8328
9305



Penicillin G
0.1
8435
8871




0.5
8142
8746



Fe-EDTA
100
7256
8485









    4. The following table shows the amount of Toxin B produced (ng/mL) in cultures with the indicated compound.

















Test
mg/L
12 hours
24 hours




















Control
—
3392
3469



Ciprofloxacin
2
2135
2112




10
707
3448



Clindamycin
0.5
189
6737




2.5
146
131



Vancomycin
0.1
3131
3216




0.5
2770
3127



Penicillin G
0.1
2755
3133




0.5
2816
2664



Fe-EDTA
100
2427
2541









    5. The following table indicates cell morphological characteristics in cultures with the indicated compound.














Test
mg/L
12 hours







Control
—
Normal


Ciprofloxacin
2
Normal



10
Many cells were 2-4× longer than normal cells.




Some cells were curved.


Clindamycin
0.5
Normal



2.5
Most sizes of the cells were 2× smaller than




normal. Cells grow very slow


Vancomycin
0.1
Some cells were 2× longer than normal cells



0.5
Some cells were 2× longer than normal cells


Penicillin G
0.1
Normal



0.5
Normal


Fe-EDTA
100
Normal









    6. The following table shows the amount of spore formation in 24 h fermentation in cultures with the indicated compound. Broth was examined by microscope.
















Test
mg/L
24 hours








Control
—
No spore formation found



Ciprofloxacin
2
No spore formation found




10
No spore formation found



Clindamycin
0.5
No spore formation found




2.5
No spore formation found



Vancomycin
0.1
No spore formation found




0.5
No spore formation found



Penicillin G
0.1
No spore formation found




0.5
No spore formation found



Fe-EDTA
100
No spore formation found









Conclusions

Clindamycin, when added to SYS medium at 0.5 mg/L, caused increases in Toxin A (28%) and Toxin B (94%) after 24 hours in fermentation broth, but not after 12 hours in fermentation broth.
Example 3
This Example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various vitamins and antibiotics
The following are the Example 3 test compounds, along with the compound formula and source.

    Aza-Azaserine (C5H7N3O4) FW 173.10, (O-diaxoacetyl-L-serine) Fluka BioChemika, 11430 Lot#1301321, soluble in water.
    Ade-Adenosine (C10H13N5O4) FW 267.25, Sigma A4036, Lot#046K06612, soluble with 5N HCl.
    B12-Vitamin B12 (C63H88CoN14O14P) FW 1,355.37, Sigma, V6629Lot#124K17072, soluble in water.
    Bio-d-Biotin (C10H16N2O3S) FW 244, Supelco 4-7868, Lot#LB5668 soluble with 5N HCl.


The following combinations of compounds were also tested at the indicated concentrations.
#1-50 μM Azaserine (8650 μg/L)+1 mM Adenosine (267 mg/L)+50 nM Biotin (12.2 μg/L)
50 μM Azaserine (432.5 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 nM Biotin (610 ng/50 mL)
#2-15 μM Azaserine (2595 μg/L)+1 mM Adenosine (267 mg/L)+50 nM Biotin (12.2 μg/L)
15 μM Azaserine (129.75 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 nM Biotin (610 ng/50 mL)
#3-15 μM Azaserine (2595 μg/L), (129.75 μg/50 m/l)
#4-5 μM Azaserine (865 μg/L)+1 mM Adenosine (267 mg/L)+50 nM Biotin (12.2 μg/L)
5 μM Azaserine (43.25 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 nM Biotin (610 ng/50 mL)
#5-5 μM Azaserine (865 μg/L)+1 mM Adenosine (267 mg/L)+50 μM Biotin (12.2 ng/L)
5 μM Azaserine (43.25 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 μM Biotin (0.61 ng/50 mL)
#6-0.05 nM d-Biotin (12.2 ng/L), (0.61 ng/50 mL)
#7-0.5 nM d-Biotin (122 ng/L), (6.1 ng/50 mL)
#8-5 nM d-Biotin (1.22 μg/L), (61 ng/50 mL)
#9-50 nM Vitamin B12 (67.77 μg/L), (3.39 μg/50 mL)
Materials
1. Make 50× concentration solutions.


















50×



Chemical
Sterile di water
Concentration


Test component
(mg)
(mL)
(mg/L)





Azaserine
4.325
10
432.5 (2.5 mM)


(50 μM = 8.65 mg/L)





Adenosine
133.5
10
13350 (50 mM) 


(1 mM = 267 mg/L)














2.5 mM Aza
Sterile di water
250 μM Azaserine


Test component
(mL)
(mL)
(mL)





Azaserine
2
18
20


(5 μM = 865 μg/L)









2. Make d-Biotin (500 μM) solutions, then dilute to 2.5 μM, 50 nM, 5 nM, and 0.5 nM.














d-Biotin
Sterile di
500 μM d-Biotin


Test component
(mg)
water (mL)
(mg/L)







d-Biotin (50 nM = 12.2 μg/L)
2.44
20
122









3. Make 50× concentration solutions then dilute to 10×.

















Sterile di
50×



Vitamin B12
water
Concentration


Test component
(mg)
(mL)
(mg/L)





Vitamin B12
33.9
10
3390 (2.5 μM)


(50 nM = 67.8 mg/L)














50×

10×



concentration
Sterile di water
Concentration


Test component
(mL)
(mL)
(mg/L)





Vitamin B12
2
8
678 (500 nM)


(50 nM = 67.8 mg/L)









Methods

I. Medium Preparations:

    
    
        1. Prepare 1000 mL SYS medium in 2 L beaker.
        2. Transfer SYS to media bottles and degas for over 30 minutes with 10% H2+10% CO2+80% N2.
        3. Before transferring the medium, fill gas (10% H2+10% CO2+80% N2) from the fill port of the Flexboy bag into the bag to remove oxygen, then empty the gas from the bag. Connect the filling system manifold with the bags.
        4. For seed medium in 50 mL Flexboy bags, pump 30 mL medium into the bag from the fill port with a flow speed at 100 mL/minute.
        5. For fermentation medium in 250 mL Flexboy bags:
        
            i) Put the bag on a balance before filling with the medium and adjust to “0.”
            ii) Pump the medium into the bag from the fill port with a flow speed at 100 mL/minute until the balance show 50 g, stop the pumping.
        
        
        6. Move the bag for seed-1 to 37° C. CO2 incubator to warm overnight. Keep bag for seed-2 and fermentation at 4° C. until use.
        7. Move the bags to 37° C. CO2 incubator to warm up overnight before use.
        8. For different compounds:
        
            i) Prepare the solutions (see above table).
            ii) Filter the solution using the Millipore 50 mL Disposable Vacuum Filtration System with 0.22 μm Millipore Express Plus Membrane.
            iii) Before transfer of seed-2 to fermentation bags, add the compound solutions as the following concentrations listed in the following tables.
        
        
    
    




















2.5 mM
50 mM
2.5 μM
di



Chemical add
Aza
Ade
Bio
water



to 50 mL SYS medium
(mL)
(mL)
(mL)
(mL)
Total (mL)





Control
0
0
0
5
5


#1-50 μM Azaserine +
1
1
1
2
5


1 mM Adenosine +







50 nM Biotin






250 μM
50 mM
2.5 μM
di




Aza
Ade
Bio
water




(mL)
(mL)
(mL)
(mL)
Total (mL)





#2-15 μM Azaserine +
3
1
1
0
5


1 mM Adenosine +







50 nM Biotin







#3-15 μM Azaserine
3
0
0
2
5


#4-5 μM Azaserine +
1
1
1
2
5


1 mM Adenosine +







50 nM Biotin






50 μM
50 mM
5 nM
di




Aza
Ade
Bio
water




(mL)
(mL)
(mL)
(mL)
Total (mL)





#5-5 μM Azaserine +
1
1
0.5
2.5
5


1 mM Adenosine +







50 pM Biotin)















0.5 nM Biotin
5 nM Biotin
50 nM
Total



(mL)
(mL)
Biotin (mL)
(mL)





#6-0.05 nM Biotin
5
0
0
5


#7-0.5 nM Biotin
0
5
0
5


#8-5 nM Biotin
0
0
5
5















500 nM Vitamin B12
Total




(mL)
(mL)






#9-50 nM Vitamin B12
5
5



(67.77 μg/L)









II. Fermentation Process:

    
    
        1. First stage seed culture: 1 mL WCB, containing 50% glycerol, was transferred into a 50 mL Flexboy bag containing 30 mL SYS medium and incubated at 37±1° C. for 24 hours.
        2. Second stage seed culture: 1.5 mL of first stage seed culture at inoculums of 5% were transferred into the 50 mL Flexboy bag containing 30 mL SYS medium and incubated at 37±1° C. for 22 hours.
        3. Fermentation: 2.5 mL of second stage seed culture was inoculated at 5% for each 250 mL Flexboy bag containing 50 mL of SYS medium and incubated at 37° C.±1° C. for 24 hrs.
        4. Take samples at 12 hours and 24 hours. Cell growth was measure at 600 nm. The blank of fermentation media was used as zero for the spectrophotometer. The cell concentration was diluted 10×.
        5. The toxin production is measured by capture ELISA.

III. Capture ELISAs:

        1. Toxin A standard Lot# CD-2062 (1072506A)
        2. Goat anti-Toxin A, Lot# CD-2017
        3. Mouse MAb to C. difficile Toxin A (PCG4)
        4. Toxin B standard Lot#QC06329
        5. Goat anti-Toxin B, Lot# C0210091
        6. Mouse anti Toxin B Lot#030904

Results

    
    
    1. The following table shows the amount of seed growth (OD600 nm) as measured by DU700.















Seed-1
Seed-2








2.53
2.49









    2. The following table shows the amount of cell growth (OD600 nm) in cultures with the indicated compound.














Test
12 hours
24 hours







Control
2.71
2.39


#1-50 μM Azaserine + 1 mM Adenosine +
1.19
0.42


50 nM Biotin




#2-15 μM Azaserine + 1 mM Adenosine +
2.76
2.02


50 nM Biotin




#3-15 μM Azaserine
2.33
2.55


#4-5 μM Azaserine + 1 mM Adenosine +
2.84
2.48


50 nM Biotin




#5-5 μM Azaserine + 1 mM Adenosine +
2.49
2.47


50 pM Biotin




#6 0.05 nM Biotin
3.03
2.74


#7 0.5 nM Biotin
2.59
2.51


#8 5 nM Biotin
2.86
2.54


#9 50 nM Vitamin B12
2.82
2.88









    3. The following table shows the amount of Toxin A produced (ng/mL) in cultures with the indicated compound.














Test
12 hours
24 hours

















Control
9649
10554


#1-50 μM Azaserine + 1 mM Adenosine +
10391
19000


50 nM Biotin




#2-15 μM Azaserine + 1 mM Adenosine +
8525
10423


50 nM Biotin




#3-15 μM Azaserine
9333
10838


#4-5 μM Azaserine + 1 mM Adenosine +
7471
7816


50 nM Biotin




#5-5 μM Azaserine + 1 mM Adenosine +
9933
10811


50 pM Biotin




#6 0.05 nM Biotin
8708
9481


#7 0.5 nM Biotin
8601
9124


#8 5 nM Biotin
8573
8877


#9 50 nM Vitamin B12
5858
6286









    4. The following table shows the amount of Toxin B produced (ng/mL) in cultures with the indicated compound.














Test
12 hours
24 hours

















Control
3159
3529


#1-50 μM Azaserine + 1 mM Adenosine +
6198
11940


50 nM Biotin




#2-15 μM Azaserine + 1 mM Adenosine +
3430
3880


50 nM Biotin




#3-15 μM Azaserine
3589
4112


#4-5 μM Azaserine + 1 mM Adenosine +
2665
2694


50 nM Biotin




#5-5 μM Azaserine + 1 mM Adenosine +
3345
3813


50 pM Biotin




#6 0.05 nM Biotin
2616
3167


#7 0.5 nM Biotin
2717
3084


#8 5 nM Biotin
2756
2936


#9 50 nM Vitamin B12
1819
2030









Conclusions

50 μM Azaserine, 1 mM Adenosine, and 50 nM Biotin together, when added to the SYS medium, caused increases in production of Toxin A (80%) and Toxin B (238%) after 24 hours in fermentation broth.
Example 4
This Example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various amino acids and organic compounds.
Materials
The following are the Example 4 test compounds, along with the compound formula and source.

    
    
        Arg-L-Arginine Monohydrochloride (C6H4N4O2.HCl), FW. 210.67 Sigma A5131-500G, Lot# 016K0001, soluble in water.
        Cys-L-Cysteine (C3H7NO2S), FW 121.16, Sigma C-7352, Lot#082K0377, soluble in water.
        Tyr-L-Tyrosine (C9H11NO3), FW 181.19, Sigma T8566, Lot#107K0157, soluble in water with HCl.
        Asc-Ascorbic acid (C6H8O6) FW 176.12, Sigma A5960 Lot#043K0131, soluble in water.
        But-Butyric acid (C4H8O2) FW 88.11, Aldrich B103500 Lot#03511DA, soluble in water.
    
    


These compounds were tested using the following concentrations (10×):
L-Arginine Monohydrochloride (50 mM).
L-Tyrosine (50 mg/L).
L-Cysteine (0.33 mM, 10 mM, and 33 mM).
Ascorbic acid (2.5 g/L and 10 g/L).
Butyric acid (30 mM and 60 mM).

    1. Make 10× Arginine solutions.

















Sterile
10×



Arginine
di water
Concentration


Test component
(g)
(mL)
(g/L)







Arginine (50 mM = 10.5 g/L)
4.2
40
105









    2. Make 10× Cysteine solutions at 33 mM then dilute to 1 mM and 0.33 mM.



















Sterile
10×




di water
Concentration


Test component
Cysteine (g)
(mL)
(g/L)





Cysteine (33 mM = 4 g/L)
1.6
40
40






10×
Sterile
10×



33 mM Cys.
di water
Concentration


Test component
(mL)
(mL)
(g/L)





Cysteine (3.3 mM = 400 mg/L)
4
36
4






10×
Sterile
10×



3.3 mM Cys.
di water
Concentration


Test component
(mL)
(mL)
(g/L)





Cysteine (0.33 mM = 40 mg/L)
4
36
0.4









    3. Make 10× Tyrosine solutions at 50 mg/L.
















Tyrosine
Sterile di water
10× Concentration


Test component
(g)
(mL)
(g/L)







Tyrosine (50 mg/L)
0.02
40
1









    4. Make 10× Ascorbic acid solutions at 2.5 g/L and 10 g/L.

















Sterile 




Ascorbic acid
di water
10× Concentration


Test component
(g)
(mL)
(g/L)


















Ascorbic acid (2.5 g/L)
1
40
25


Ascorbic acid (10 g/L)
4
40
100









    5. Make 10× Butyric acid solutions at 30 mM.
















Butyric 





acid
Sterile di water
10× Concentration


Test component
(mL)
(mL)
(mL/l)


















Butyric acid
1.1
38.9
27.5


(30 mM = 2.75 mL/L)





Butyric acid
2.2
37.8
55


(60 mM = 5.5 mL/L)









Methods

I. Medium Preparations:

    
    
        1. Prepare 1000 mL SYS medium in 2 L beaker.
        2. Transfer SYS to media bottles and degas for over 30 minutes with 10% H2+10% CO2+80% N2.
        3. Before transferring the medium, fill gas (10% H2+10% CO2+80% N2) from the fill port of the Flexboy bag into the bag to remove oxygen, then empty the gas from the bag. Connect the filling system manifold with the bags.
        4. For seed medium in 50 mL Flexboy bags, pump 30 mL medium into the bag from the fill port with a flow speed at 100 mL/minute.
        5. For fermentation medium in 250 mL Flexboy bags:
        
            i) Put the bag on a balance before filling with the medium and adjust to “0”.
            ii) Pump the medium into the bag from the fill port with a flow speed at 100 mL/min until the balance show 50 g, stop the pumping.
        
        
        6. Move the bag for seed-1 to 37° C. CO2 incubator to warm overnight. Keep bag for seed-2 and fermentation at 4° C. until use.
        7. Move the bags to 37° C. CO2 incubator to warm up overnight before use.
        8. For different antibiotics:
        
            i) Prepare 40 mL solutions of different antibiotics with the concentrations at 10× (see above table).
            ii) Prepare 40 mL solutions of FE-EDTA with the concentrations at 1000 mg/L (40 mg compound+40 mL di water (pH)).
            iii) Filter the solution using the Millipore 50 mL Disposable Vacuum Filtration System with 0.22 μm Millipore Express Plus Membrane.
            iv) Before transfer of seed-2 to fermentation bags add the compound solutions at the concentrations listed in the following tables.
        
        
    
    


















10×
Sterile di



Medium

Solution
water
Total


#
Test compound
(mL)
(mL)
(mL)







0
Control (without antibiotics)
0
5
5


1
Arginine (50 mM = 10.5 g/L)
5
0
5


2
Cysteine (0.33 mM = 40 mg/L)
5
0
5


3
Cysteine (3.3 mM = 400 mg/L)
5
0
5


4
Cysteine (33 mM = 4 g/L)
5
0
5


5
Tyrosine (50 mg/L)
5
0
5


6
Ascorbic acid (2.5 g/L)
5
0
5


7
Ascorbic acid (10 g/L)
5
0
5


8
Butyric acid (30 mM = 2.75 mL/l)
5
0
5


9
Butyric acid (60 mM = 5.5 mL/l)
5
0
5









II. Fermentation Process:

    
    
        1. First stage seed culture: 1 mL WCB, containing 50% glycerol, was transferred into a 50 mL Flexboy bag containing 30 mL SYS medium and incubated at 37±1° C. for 24 hours.
        2. Second stage seed culture: 1.5 mL of first stage seed culture at inoculums of 5% were transferred into the 50 mL Flexboy bag containing 30 mL SYS medium and incubated at 37±1° C. for 22 hours.
        3. Fermentation: 2.5 mL of second stage seed culture was inoculated at 5% for each 250 mL Flexboy bag containing 50 mL of SYS medium and incubated at 37° C.±1° C. for 24 hrs.
        4. Take samples at 12 hours and 24 hours. Cell growth was measure at 600 nm. The blank of fermentation media was used as zero for the spectrophotometer. The cell concentration was diluted 10×.
        5. The toxin production is measured by capture ELISA.

III. Capture ELISAs:

        1. Toxin A standard Lot# CD-2062 (1072506A)
        2. Goat anti-Toxin A, Lot# CD-2017
        3. Mouse MAb to C. difficile Toxin A (PCG4)
        4. Toxin B standard Lot#QC06329
        5. Goat anti-Toxin B, Lot# C0210091
        6. Mouse anti Toxin B Lot#030904

Results

    
    
    1. The following table shows the amount of seed growth (OD600 nm) as measured by DU700.















Seed-1
Seed-2








2.65
2.53









    2. The following table shows the amount of cell growth (OD600 nm) in cultures with the indicated compound.
















Test
12 hours
24 hours








Control
2.61
2.61



#1 Arginine (50 mM = 10.5 g/L)
2.89
2.55



#2 Cysteine (0.33 mM = 40 mg/L)
2.43
2.36



#3 Cysteine (3.3 mM = 400 mg/L)
2.38
2.59



#4 Cysteine (33 mM = 4 g/L)
1.67
1.74



#5 Tyrosine (50 mg/L)
2.41
2.34



#6 Ascorbic acid (2.5 g/L)
2.49
2.19



#7 Ascorbic acid (10 g/L)
2.16
1.98



#8 Butyric acid (30 mM = 2.75 mL/l)
2.37
2.00



#9 Butyric acid (60 mM = 5.5 mL/l)
1.38
1.98









    3. The following table shows the amount of Toxin A produced (ng/mL) in cultures with the indicated compound (FIGS. 4A and 4B).
















Test
12 hours
24 hours



















Control
10822
11515



#1 Arginine (50 mM = 10.5 g/L)
6007
6616



#2 Cysteine (0.33 mM = 40 mg/L)
9691
10365



#3 Cysteine (3.3 mM = 400 mg/L)
9828
10741



#4 Cysteine (33 mM = 4 g/L)
897
853



#5 Tyrosine (50 mg/L)
11394
11624



#6 Ascorbic acid (2.5 g/L)
10789
12050



#7 Ascorbic acid (10 g/L)
9957
11708



#8 Butyric acid (30 mM = 2.75 mL/l)
12315
12674



#9 Butyric acid (60 mM = 5.5 mL/l)
5335
10681









    4. The following table shows the amount of Toxin B produced (ng/mL) in cultures with the indicated compound.
















Test
12 hours
24 hours



















Control
3825
3992



#1 Arginine (50 mM = 10.5 g/L)
2300
2626



#2 Cysteine (0.33 mM = 40 mg/L)
3446
3581



#3 Cysteine (3.3 mM = 400 mg/L)
3017
3185



#4 Cysteine (33 mM = 4 g/L)
339
322



#5 Tyrosine (50 mg/L)
3752
4462



#6 Ascorbic acid (2.5 g/L)
4026
4967



#7 Ascorbic acid (10 g/L)
4534
5955



#8 Butyric acid (30 mM = 2.75 mL/l)
5110
6425



#9 Butyric acid (60 mM = 5.5 mL/l)
3063
7529









    5. The following table indicates cell morphological characteristics in cultures with the indicated compound.















Test
12 hours/24 hours








Control
Normal



#1 Arginine (50 mM = 10.5 g/L)
Normal



#2 Cysteine (0.33 mM = 40 mg/L)
Normal



#3 Cysteine (3.3 mM = 400 mg/L)
Normal



#4 Cysteine (33 mM = 4 g/L)
Cells show gray color



#5 Tyrosine (50 mg/L)
Normal



#6 Ascorbic acid (2.5 g/L)
Normal



#7 Ascorbic acid (10 g/L)
Normal



#8 Butyric acid (30 mM = 2.75 mL/l)
Normal



#9 Butyric acid (60 mM = 5.5 mL/l)
Normal









    6. The following table shows the amount of spore formation in 24 hour fermentation in cultures with the indicated compound. Broth was examined by microscope.















Test
Spore formation








Control
No spore found



#1 Arginine (50 mM = 10.5 g/L)
Very a few spores found



#2 Cysteine (0.33 mM = 40 mg/L)
Very a few spores found



#3 Cysteine (3.3 mM = 400 mg/L)
No spore found



#4 Cysteine (33 mM = 4 g/L)
No spore found



#5 Tyrosine (50 mg/L)
No spore found



#6 Ascorbic acid (2.5 g/L)
No spore found



#7 Ascorbic acid (10 g/L)
No spore found



#8 Butyric acid (30 mM = 2.75 mL/l)
No spore found



#9 Butyric acid (60 mM = 5.5 mL/l)
No spore found









Conclusions

Ascorbic acid, when added to SYS medium at 10 g/L, caused increases in Toxin B of 19% after 12 hours and 49% after 24 hours of incubation in fermentation broth.
Butyric Acid, when added to SYS medium at 30 mM, caused increases in Toxin A of 14% and Toxin B of 34% after 12 hours of incubation in fermentation broth. It caused increases in Toxin A of 16% and Toxin B of 61% after 24 hours of incubation in fermentation broth. Butyric Acid, when added to SYS medium at 60 mM, caused increases in Toxin B of 89% after 24 hours of incubation in fermentation broth.
Example 5
This Example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various concentrations of carbohydrates.
The following table summarizes the data regarding toxin increases (%) following the addition of increasing concentrations of D-sorbitol.












D-Sorbitol
Toxin A
Toxin B











Concentration
12 hours
24 hours
12 hours
24 hours














 6 g/L
25.55
53.98
45.99
112.30


 8 g/L
25.09
74.99
43.39
148.98


10 g/L
45.47
140.89
51.76
216.31


12 g/L
46.80
150.04
59.21
295.51


14 g/L
23.50
127.69
27.77
254.23


16 g/L
14.91
117.43
21.49
244.44


18 g/L
34.90
136.02
30.00
236.74


20 g/L
8.57
118.43
9.90
212.09









Materials

The following are the Example 5 test compounds, along with the compound formula and source.
D(−)Fructose: (contained <0.05% glucose) C6H12O6, FW 180.2, Sigma F0127 Lot#60K0013
D(+)Galactose: C6H12O6, FW 180.2, Sigma G0625, Lot#102K0169 soluble in water (1 g/1.7 mL)
D(+)Mannose: C6H12O6, FW 180.16, Sigma M6020 Lot# soluble in water (50 mg/mL)
D(+)Maltose Monohydrate: (contained <0.3% glucose), C12 hrs.22O11.H2O, FW 360.3, Sigma M9171 Lot#80K10101 soluble in water
Sucrose: C12 hrs.22O11, FW 342.3, Sigma, 53929, Lot#127K0093 soluble in water

    
    
        α-Lactose: C12 hrs.22O11.H2O, FW 360.3, Sigma L2643, Lot# soluble in water (0.2 g/mL)
    
    


D(+)Xylose: C5H10O5, FW 150.132, Sigma X3877 Lot# soluble in water (1 g/0.8 mL)
D-Sorbitol: C6H14O6, FW 182.2, Sigma 53889, Lot#042K01355 soluble in water.
myo-Inositol: C6H12O6, FW 180.16, Sigma 17508, Lot# soluble in water (50 mg/mL)
10× solutions of the above carbohydrates were produced.
Methods
I. Medium Preparations:


    
    
        1. Prepare 1000 mL SYS medium in 2 L beaker.
        2. Transfer SYS to media bottles and degas for over 30 minutes with 10% H2+10% CO2+80% N2.
        3. Before transferring the medium, fill gas (10% H2+10% CO2+80% N2) from the fill port of the Flexboy bag into the bag to remove oxygen, then empty the gas from the bag. Connect the filling system manifold with the bags.
        4. For seed medium in 50 mL Flexboy bags, pump 30 mL medium into the bag from the fill port with a flow speed at 100 mL/minute.
        5. For fermentation medium in 250 mL Flexboy bags:
        
            i) Put the bag on a balance before filling with the medium and adjust to “0.”
            ii) Pump the medium into the bag from the fill port with a flow speed at 100 mL/min until the balance show 50 g, stop the pumping.
        
        
        6. Move the bag for seed-1 to 37° C. CO2 incubator to warm overnight. Keep bag for seed-2 and fermentation at 4° C. until use.
        7. Move the bags to 37° C. CO2 incubator to warm up overnight before use.
        8. For different compounds test:
        
            i) Prepare the solutions with different chemicals (see above table)
            ii) Filter the solution using Millipore 50 mL Disposable Vacuum Filtration System with 0.22 μm Millipore Express Plus Membrane.
            iii) Before transfer of seed-2 to fermentation bags, add the compound solutions as follows:
        
        
    
    


















10×
Sterile





Solution
di water
Total


Medium #
Test compound
(mL)
(mL)
(mL)







0
Control (without carbohydrate
0
5
5



additive)





1
D(−)Fructose (6 g/L)
5
0
5


2
D(+)Galactose (6 g/L)
5
0
5


3
D(+)Mannose (6 g/L)
5
0
5


4
D(+)Maltose Monohydrate (6 g/L)
5
0
5


5
Sucrose (6 g/L)
5
0
5


6
α-Lactose (6 g/L)
5
0
5


7
D(+)Xylose (6 g/L)
5
0
5


8
D-Sorbitol (6 g/L)
5
0
5


9
myo-Inositol (6 g/L)
5
0
5









II. Fermentation Process:

    
    
        1. First stage seed culture: 1 mL WCB, containing 50% glycerol, was transferred into a 50 mL Flexboy bag containing 30 mL SYS medium and incubated at 37±1° C. for 24 hours.
        2. Second stage seed culture: 1.5 mL of first stage seed culture at inoculums of 5% were transferred into the 50 mL Flexboy bag containing 30 mL SYS medium and incubated at 37±1° C. for 22 hours.
        3. Fermentation: 2.5 mL of second stage seed culture was inoculated at 5% for each 250 mL Flexboy bag containing 50 mL of SYS medium and incubated at 37° C.±1° C. for 24 hours
        4. Take samples at 12 hours and 24 hours. Cell growth was measure at 600 nm. The blank of fermentation media was used as zero for the spectrophotometer. The cell concentration was diluted 10×.
        5. The toxin production is measured by capture ELISA.

III. Capture ELISAs:

        1. Toxin A standard Lot# CD-2062 (1072506A)
        2. Goat anti-Toxin A, Lot# CD-2017
        3. Mouse MAb to C. difficile Toxin A (PCG4)
        4. Toxin B standard Lot#QC06329
        5. Goat anti-Toxin B, Lot# C0210091
        6. Mouse anti Toxin B Lot#030904

Results

    
    
    1. The following table shows the amount of seed growth (OD600 nm) as measured by DU700.















Seed-1
Seed-2








2.57
2.561









    2. The following table shows the amount of cell growth (OD600 nm) in cultures with the indicated compound.














Test
12 hours
24 hours







Control (without carbohydrate additive)
2.78
2.57


#1 D(−)Fructose (6 g/L)
3.42
2.21


#2 D(+)Galactose (6 g/L)
2.92
2.51


#3 D(+)Mannose (6 g/L)
5.14
2.84


#4 D(+)Maltose Monohydrate (6 g/L)
2.91
2.80


#5 Sucrose (6 g/L)
2.74
2.62


#6 α-Lactose (6 g/L)
2.57
2.36


#7 D(+)Xylose (6 g/L)
3.34
3.53


#8 D-Sorbitol (6 g/L)
4.00
3.35


#9 myo-Inositol (6 g/L)
2.61
2.53









    3. The following table shows the amount of Toxin A produced (ng/mL) in cultures with the indicated compound (FIGS. 5A and 5B).
















Test
12 hours
24 hours



















Control
10108
10508



#1 D(−)Fructose (6 g/L)
1031
2146



#2 D(+)Galactose (6 g/L)
8596
9154



#3 D(+)Mannose (6 g/L)
3568
5288



#4 D(+)Maltose Monohydrate (6 g/L)
8741
8764



#5 Sucrose (6 g/L)
11042
10881



#6 α-Lactose (6 g/L)
9258
10167



#7 D(+)Xylose (6 g/L)
10135
11565



#8 D-Sorbitol (6 g/L)
15080
19575



#9 myo-Inositol (6 g/L)
8189
8743









    4. The following table shows the amount of Toxin B produced (ng/mL) in cultures with the indicated compound.
















Test
12 hours
24 hours



















Control
3756
3832



#1 D(−)Fructose (6 g/L)
319
755



#2 D(+)Galactose (6 g/L)
3071
3379



#3 D(+)Mannose (6 g/L)
1518
2123



#4 D(+)Maltose Monohydrate (6 g/L)
3079
3022



#5 Sucrose (6 g/L)
3902
4235



#6 α-Lactose (6 g/L)
3126
3655



#7 D(+)Xylose (6 g/L)
4075
5588



#8 D-Sorbitol (6 g/L)
6311
9696



#9 myo-Inositol (6 g/L)
2743
3196









    5. The following table indicates cell morphological characteristics in cultures with the indicated compound.
















Test
12 hours/24 hours









Control
Normal



#1 D(−)Fructose (6 g/L)
Cell lyse at 24 hours



#2 D(+)Galactose (6 g/L)
Normal



#3 D(+)Mannose (6 g/L)
Cell lyse at 24 hours



#4 D(+)Maltose Monohydrate (6 g/L)
Normal



#5 Sucrose (6 g/L)
Normal



#6 α -Lactose (6 g/L)
Normal



#7 D(+)Xylose (6 g/L)
Normal



#8 D-Sorbitol (6 g/L)
Cell lyse at 24 hours



#9 myo-Inositol (6 g/L)
Normal










    6. The following table shows the amount of spore formation in 24 h fermentation in cultures with the indicated compound. Broth was examined by microscope.
















Test
Spore formation









Control
No spore found



#1 D(−)Fructose (6 g/L)
No spore found



#2 D(+)Galactose (6 g/L)
No spore found



#3 D(+)Mannose (6 g/L)
No spore found



#4 D(+)Maltose Monohydrate (6 g/L)
No spore found



#5 Sucrose (6 g/L)
No spore found



#6 α -Lactose (6 g/L)
No spore found



#7 D(+)Xylose (6 g/L)
No spore found



#8 D-Sorbitol (6 g/L)
No spore found



#9 myo-Inositol (6 g/L)
No spore found










Conclusions

D(+)Xylose, when added to SYS medium at 6 g/L, slightly increased cell growth and Toxin A production. D(+)Xylose increased Toxin B production 9% after 12 hours of incubation in fermentation broth and 46% after 24 hours of incubation in fermentation broth.
D-Sorbitol, when added to SYS medium at 6 g/L, markedly increased cell growth and toxins production. Cell growth was increased 44% after 12 hours of incubation in fermentation broth. Toxin A production was increased 49% after 12 hours of incubation in fermentation broth and 86% after 24 hours of incubation in fermentation broth. Toxin B production was increased 68% after 12 hours of incubation in fermentation broth and 153% after 24 hours of incubation in fermentation broth.
Example 6
This example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media supplemented with D-Sorbitol (12 g/L) under pH controlled conditions.
Materials
The following materials and equipment were used in this example.
A Biostat B Plus Fermentation System (Sartorius) was used for the first stage seed culture and second stage seed culture, using 2×2 L fermenters (Sartorius), one for each seed culture seed. A Biostat Q Plus Fermentation System (Sartorius) was used for the third stage seed culture using 4×1 L fermenters (Sartorius). A peristalitic pump (Masterflex) was used to transfer media for both systems. Media composition for the seed stages were as follows:










SYS media













Formulation



Component
Manufacturer/Lot #
(g/L)















KH2PO4
J T Baker/E29H22
0.9



Na2PO4
J T Baker/A12145
5



NaHCO3
J T Baker/A13668
5



Soy Peptone A3 SC
Organotechnie/19685
30



Yeast Extract
BD Bacto/7109497
20










Media composition for the production stage was as follows:











SYS media + Sorbitol













Formulation



Component
Manufacturer/Lot #
(g/L)















KH2PO4
J T Baker/E29H22
0.9



Na2PO4
J T Baker/A12145
5



NaHCO3
J T Baker/A13668
5



Soy Peptone A3 SC
Organotechnie/19685
30



Yeast Extract
BD Bacto/7109497
20



D-Sorbitol(70%)*
Spectrum/WJ1030
17.1 ml







*17.1 ml of D-Sorbitol (70%) represents 12 g/L






The SYS media and SYS media+Sorbitol were each prepared using reverse osmosis deionized water (RODI-water).

Additional materials included:


















Qty.



Manufacturer/Lot #
Part #
Added




















Working Cell
In-house
n/a
0.5
(2 ml)


Bank vial,


WCB-A, 4.5 ml


Anaerobic Gas

RM-0024
20


mix(80% N2/10%


CO2/10% H2)


5N Sodium
J T Baker/E17507
5671-06
1
L


Hydroxide


1N Hydrochloric
J T Baker/B08510
5618-02
200
ml


Acid









Methods

The following methods were used to test the production of Toxin A and B when cultured under pH controlled conditions.

I. Seed Bioreactor 1

    1. A 2 L vessel was prepared with a ring sparger and a pitched blade impeller on the bottom of the shaft set at a 45° angle.
    2. A pH probe was calibrated according to Sartorius procedures and installed in the bioreactor.
    3. The bioreactor was then autoclaved on a dry cycle for 30 min with 10 min pre and post-vacuum cycles.
    4. After sterilization, the sterile vessel was connected to the Biostat B Plus System.
    5. SYS medium was prepared as described above
    6. 1600 mL of medium were aseptically transferred to the sterile bioreactor.
    7. Vessel temperature and agitation were set to 37° C. and 100 rpm, respectively.
    8. Prior to inoculation, the bioreactor was de-gassed by sparging with anaerobic gas mix at 300 mL/min for 15 minutes.
    9. 4 mL of WCB-A was aseptically transferred to the bioreactor to initiate the culture.
    10. During the culture, the bioreactor was sparged with anaerobic gas mix at 100 mL/min and incubated for 18 h.
    11. At end of 18 hr, a 5 ml sample was taken for OD measurement.

II. Seed Bioreactor 2

    1. A 2 L vessel was prepared with a ring sparger and a pitched blade impeller on the bottom of the shaft set at a 45° angle.
    2. A pH probe was calibrated according to Sartorius procedures and installed in the bioreactor.
    3. The bioreactor was then autoclaved on a dry cycle for 30 min with 10 min pre and post-vacuum cycles.
    4. After sterilization, the sterile vessel was connected to the Biostat B Plus System.
    5. SYS medium was prepared as described above
    6. 1800 mL of medium were aseptically transferred to the sterile bioreactor.
    7. Vessel temperature and agitation were set to 37° C. and 100 rpm, respectively.
    8. Prior to inoculation, medium in vessel was sparged with anaerobic gas mix at 300 mL/min for 15 minutes.
    9. 100 mL of the 1st stage culture was aseptically transferred the 2nd stage.
    10. During the culture, the bioreactor was sparged with anaerobic gas mix at 100 mL/min and incubated for 10 h.
    11. At end of 10 hr, a 5 ml sample was taken for OD measurement.

III. Production Bioreactor

    1. 4×1 L vessels were prepared each with a ring sparger and a pitched blade impeller on the bottom of the shaft set at  ̃45° angle.
    2. A pH probe for each bioreactor was calibrated according to Sartorius procedures and installed in each bioreactor.
    3. All bioreactors were then autoclaved on a dry cycle for 30 min with 10 min pre and post-vacuum cycles.
    4. After sterilization, the bioreactors were connected to the Biostat Q Plus System.
    5. SYS medium+Sorbitol was prepared as described above
    6. 900 mL of medium was aseptically transferred to each bioreactor.
    7. Acid and base bottles were autoclaved, aseptically filled with sterile filtered 1N HCl and 5N NaOH, respectively, and attached to the bioreactors.
    8. Agitiaton was set to 100 rpm for all bioreactors.
    9. The desired temperature and pH control set points were implemented (see Table 5).
    10. Prior to inoculation, bioreactors were de-gassed by sparging with appropriate gas (see Table 5) for 30 minutes at 300 mL/min.
    11. Each vessel was inoculated with  ̃100 mL of culture from Seed Bioreactor 2.
    12. 3rd stage cultures were incubated at 37° C. with no additional sparging for 18 hours.
    13. Samples ( ̃5 mL) were taken at appropriate times for OD and toxin measurements, typically between 14.5 to 18 hrs post-inoculation.
    14. For ELISA, 2×1 mL of sample were spun in 1.8 mL microcentrifuge tubes at 10,000 g for 1 min, then decanted and 0.2 μm filtered. The samples were stored at 2-8° C. until tested.


















TABLE 5





Vessel
Volume
Inoc
Temp
Stirrer
pH
NaHCO3
Degas
Sparge
Culture


No.
(ml)
(ml)
° C.
(rpm)
Control*
(g/L)
Gas
Gas
Stage







B1
800
 2
37
100
None
5
Gas mix
Gas mix
1st stage


B2
900
100
37
100
None
5
Gas mix
Gas mix
2nd stage


QA1
900
100
37
100
6.5
5
Gas mix
None
3rd stage


QA2
900
100
37
100
7.2
5
Gas mix
None
3rd stage


QA3
900
100
37
100
8.0
5
Gas mix
None
3rd stage


QB3
900
100
37
100
None
5
Gas mix
None
3rd stage





*pH control using 1N HCl, 5N NaOH






Results

The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated pH control (FIGS. 6A, 6B).


















Toxin A
Toxin B



Sample
OD@600 nm
(ng/ml)
(ng/ml)









1 stage @18 hrs
1.96
N/A
N/A



2nd stage @10 hrs
2.71
N/A
N/A



Control @14.5 hrs
4.02
27006
11296



Control @16.25 hrs
4.37
29141
11916



Control @18 hrs
4.32
32247
14522



pH 6.5 @14.5 hrs
3.08
32144
14400



pH 6.5 @16.25 hrs
3.32
34301
13731



pH 6.5 @18 hrs
3.56
36511
15578



pH 7.2 @14.5 hrs
5.17
16447
 6258



pH 7.2 @16.25 hrs
5.12
17739
 6609



pH 7.2 @18 hrs
5.12
21214
 7368



pH 8.0 @14.5 hrs
4.56
1095
  191*



pH 8.0 @16.25 hrs
4.38
1451
  318*



pH 8.0 @18 hrs
3.61
1500
  281*







*Below LOQ at dilution tested






Conclusions

The highest yields of both Toxin A and Toxin B were produced by maintaining the pH of the culture at a low pH (i.e., 6.5). The control culture, which was subject to no pH control also showed significantly more toxin production than those cultures subjected to a controlled pH 7.2 or pH 8.0. Lacking pH control, the pH of the control culture declined naturally (typically, declining from a starting pH of approximately pH 7.3 to a final pH of approximately 6.3).
SDS-Page gels showed similar bands and intensities for the control and pH 6.5, with the only differences being in the intensity of a band in the 100 kDa range.
Example 7
This example includes data on the amount of toxin produced when Clostridium difficile is cultured under pH controlled conditions in SYS basal media having a reduced sodium bicarbonate concentration of 2 g/L and supplemented with D-Sorbitol (12 g/L).
The materials and methods utilized in this experiment were as set out in Example 6, except as noted below.
Materials
SYS media+Sorbitol included 2 g/L NaHCO3 (reduced from 5 g/L used in Example 6)
Methods
III. Production Bioreactor


    1. 5×1 L vessels (3rd stage vessels: QB2, QB3, QA1, QA2, QA3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH control setpoints were implemented (see Table 6).
    2. Prior to inoculation, bioreactors were sparged with an appropriate gas for 30 minutes at 300 mL/min (see Table 6).
    3. 3rd stage cultures were incubated at 37° C. with no gassing for 21 hours.


















TABLE 6





Vessel
Volume
Inoc
Temp
Stirrer
pH
NaHCO3
Degas
Sparge
Culture


No.
(ml)
(ml)
° C.
(rpm)
Control*
(g/L)
Gas
Gas
Stage







B1
1600
 4
37
100
None
5
Gas mix
Gas mix
1st stage


B2
1800
200
37
100
None
5
Gas mix
Gas mix
2nd stage


QA1
 900
100
37
100
7.0
2
Gas mix
None
3rd stage


QA2
 900
100
37
100
7.5
2
Gas mix
None
3rd stage


QA3
 900
100
37
100
None
2
Gas mix
None
3rd stage


QB2
 900
100
37
100
6.0
2
100%
None
3rd stage









CO2




QB3
 900
100
37
100
6.5
2
100%
None
3rd stage









CO2





*pH control using 1N HCl, 5N NaOH






Results

    1. The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated pH control (FIGS. 7A, 7B).




















Toxin A
Toxin B



Sample (Vessel No.)
OD@600 nm
(ng/ml)
(ng/ml)









1st stage 18 h
1.59
N/A
N/A



2nd stage 10 h
2.64
N/A
N/A



pH 6.0 15 h (QB2)
1.55
10364
6670



pH 6.5 15 h (QB3)
2.82
26012
16119



pH 7.0 15 h (QA1)
4.73
24928
12383



pH 7.5 15 h (QA2)
4.93
7688
2292



Control 15 h (QA3)
3.25
30259
20561



pH 6.0 18 h (QB2)
1.49
9047
5785



pH 6.5 18 h (QB3)
3.21
33477
20031



pH 7.0 18 h (QA1)
5.39
24702
10588



pH 7.5 18 h (QA2)
4.55
7694
1882



Control 18 h (QA3)
3.72
46454
22015



pH 6.0 21 h (QB2)
1.75
13473
5254



pH 6.5 21 h (QB3)
3.53
38631
17972



pH 7.02 1 h (QA1)
4.83
29123
10538



pH 7.5 21 h (QA2)
4.96
7484
1816



Control 21 h (QA3)
4.01
41521
20046










    2. Specific Toxin A productivity produced (ng/ml per OD unit) in the cultures subject to the indicated pH control is set out in FIG. 7C. Specific Toxin B productivity produced (ng/ml per OD unit) in the cultures subject to the indicated pH control is set out in FIG. 7D.

Conclusions



Lowering the sodium bicarbonate to 2 g/L in the SYS+Sorbitol medium allowed for a lower starting pH with less acid and/or CO2 sparged. It is possible to achieve a pH of 6.5 without the addition of acid, by sparging with CO2.
The uncontrolled pH condition in this experiment had at least equivalent total Toxin B production and slightly higher total Toxin A production than the pH 6.5 condition. Specific toxin production was similar for the uncontrolled and pH 6.5 conditions.
FIG. 7E depicts a comparison of the results from this experiment and that set out in Example 6. FIG. 7E shows total toxin concentration at 18 h for various conditions over the 2 experiments. A clear drop-off in toxin production is seen in cultures at pH 6.5 to pH 6.0 and a more gradual decline in toxin production in the higher pH conditions. The optimal pH is slightly higher than 6.5.
Example 8
This example includes data on the effect of different concentrations of sodium bicarbonate (i.e., 0 g/L, 2 g/L, and 5 g/L) and carbon dioxide sparing on the pH of SYS basal media supplemented with D-Sorbitol (12 g/L).
Materials
The following materials and equipment were used in this example.
The Biostat Q Plus Fermentation System (Sartorius) was used for the first, second, and third stage cultures, using three 1 L fermenters (Sartorius). The composition of the SYS media supplemented with D-Sorbitol (SYS media+Sorbitol) was as described in Example 6, except that no NaHC03 was added to the initial 4 L batch prepared.
Methods
The following methods were used to test changes in pH.

    1. 3 pH probes were calibrated on the Biostat Q Plus system
    2. 4 L of SYS media with sorbitol was made without sodium bicarbonate and 1 L was added to a 1 L fermenter.
    3. 6 g of sodium bicarb was added to the remaining 3 L of media for a bicarb concentration of 2 g/L. 1 L of the media was added to a 1 L fermenter.
    4. 6 g of sodium bicarb was added to the remaining 2 L of media for a bicarb concentration of 5 g/L. 1 L of the media was added to a 1 L fermenter.
    5. All fermenters were mixed at 100 rpm and the pH probes were installed
    6. 100% CO2 was sparged at 500 ml/min and the data acquisition software was started to generate pH curves
    7. After  ̃3.5 hours, 5 ml of 5N HCl was added to each fermenter.

Results

    1. The following table shows the pH changes noted using different concentrations of sodium bicarbonate.

















Lowest pH with
pH with 5 ml 5N


Condition
Starting pH
CO2 sparging
HCl added


















0 g/L Bicarb
7.15
6.18
5.72


2 g/L Bicarb
7.15
6.28
5.94


5 g/L Bicarb
7.12
6.4
6.1









Conclusions

A lower final pH can be achieved in SYS medium with a lower sodium bicarbonate concentration when gassing with CO2. Using 2 g/L bicarb can lower the pH by 0.12 units with CO2 sparge alone compared to 5 g/L.
Example 9
This example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media supplemented with D-Sorbitol (12 g/L) with different concentrations of sodium bicarbonate (i.e., 0 g/L, 2 g/L, and 5 g/L) and spared with carbon dioxide or an anaerobic gas mix (80% N2/10% CO2/10% H2).
The materials and methods utilized in this experiment were as set out in Example 6, except as noted below.
Materials
The composition of the SYS media for the first stage seed cultures was as described in Example 6. For the third stage cultures, an SYS media supplemented with Sorbitol was prepared having the following composition and using RODI-water:










SYS media + Sorbitol











Formulation


Component
Manufacturer/Lot #
(g/L)












KH2PO4
J T Baker/E29H22
0.9


Na2PO4
J T Baker/A12145
5


NaHCO3
J T Baker/A13668
0


Soy Peptone A3 SC
Organotechnie/19685
30


Yeast Extract
BD Bacto Part#21270/Lot
20



#8352570


D-Sorbitol (70%)
Spectrum/WJ1030
17.1 ml









Two separate batches of SYS media+Sorbitol culture media were also prepared having the same composition but with a different concentration of NaHCO3 (i.e., one with NaHCO3 2 g/L and one with 5 g/L NaHCO3).

Methods

III. Production Bioreactor

    1. 5×1 L vessels (3rd stage vessels: QB2, QB3, QA1, QA2, QA3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH control setpoints were implemented (see Table 7).
    2. Prior to inoculation, bioreactors were sparged with an appropriate gas for 30 minutes at 300 mL/min (see Table 7).
    3. 3rd stage cultures were incubated at 37° C. with no gassing for 21 hours.


















TABLE 7





Vessel
Volume
Inoc
Temp
Stirrer
pH
NaHCO3
Degas
Sparge
Culture


No.
(ml)
(ml)
° C.
(rpm)
Control*
(g/L)
Gas
Gas
Stage







B1
1600
 4
37
100
None
5
Gas mix
Gas mix
1st stage


B2
1800
200
37
100
None
5
Gas mix
Gas mix
2nd stage


QA1
 900
100
37
100
None
0
Gas mix
None
3rd stage


QA2
 900
100
37
100
None
2
Gas mix
None
3rd stage


QA3
 900
100
37
100
None
5
Gas mix
None
3rd stage


QB2
 900
100
37
100
6.5
2
100%
None
3rd stage









CO2




QB3
 900
100
37
100
6.5
2
Gas mix
None
3rd stage





*pH control using 1N HCl, 5N NaOH






Results

    1. The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated pH control (FIGS. 8A, 8B).

















Toxin A
Toxin B


Sample
OD@600 nm
(ng/ml)
(ng/ml)


















1st stage 18 h
0.76
N/A
N/A


2nd stage 10 h
2.27
N/A
N/A


QB2- pH 6.5 CO2 16 h
2.82
18519
10519


QB3- pH 6.5 gas mix 16 h
2.71
22109
11051


QA1- 0 g/L bicarb 16 h
2.73
22898
10217


QA2- 2 g/L bicarb 16 h
3.22
29048
15099


QA3- 5 g/L bicarb 16 h
4.02
25579
12087


QB2- pH 6.5 CO2 18 h
2.98
22820
13695


QB3- pH 6.5 gas mix 18 h
2.97
25185
14463


QA1- 0 g/L bicarb 18 h
3.15
25688
11576


QA2- 2 g/L bicarb 18 h
3.5
34927
18500


QA3- 5 g/L bicarb 18 h
3.86
28656
15256









    2. The 1st stage cell growth in this experiment was lower than typically seen in this experiment. There was not a significant difference in the growth or toxin production of the fermentations controlled at pH 6.5 with either CO2 or anaerobic gas mix sparging. A sodium bicarbonate concentration of 2 g/L sodium bicarbonate provided a higher specific and total toxin A and B productivity compared to concentrations of 0 g/L and 5 g/L.

Conclusions



The use of CO2 for degassing the media is an option when controlling pH at 6.5 because of the comparable toxin yields to the anaerobic gas mix degassed fermentation.
Example 10
This example includes data on the amount of toxin produced when Clostridium difficile is cultured under a range of temperatures (37-41° C. with a midpoint of 39° C.) and a range of pH (6.35 to 6.65 with a midpoint of 6.5) in SYS basal media supplemented with D-Sorbitol (12 g/L).
The materials and methods utilized in this experiment were as set out in Example 6, except as noted below.
Materials
The composition of the SYS media for the first stage seed cultures was as described in Example 6. For the third stage cultures, an SYS media supplemented with Sorbitol was prepared having the following composition and using RODI-water:










SYS media + Sorbitol











Formulation


Component
Manufacturer/Part #/Lot #
(g/L)












KH2PO4
J T Baker/3248-07/Y48478
0.9


Na2PO4
J T Baker/3827-01/B08143
5


NaHCO3
J T Baker/3509-05/E05589
2


Soy Peptone A3 SC
Organotechnie/130-127-
30



00/19685


Yeast Extract
BD Bacto/212720/8352570
20


D-Sorbitol (70%)
Spectrum/S0220/WJ1030
17.1 ml









Methods

III. Production Bioreactor

    1. 6×1 L vessels (3rd stage vessels: QB1, QB2, QB3, QA1, QA2, QA3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH control setpoints were implemented (see Table 8).
    2. Prior to inoculation, bioreactors were sparged with an appropriate gas for 30 minutes at 300 mL/min (see Table 8).
    3. 3rd stage cultures were incubated at the applicable temperature with no gassing for 21 hours.


















TABLE 8





Vessel
Volume
Inoc
Temp
Stirrer
pH
NaHCO3
Degas
Sparge
Culture


No.
(ml)
(ml)
° C.
(rpm)
Control
(g/L)
Gas
Gas
Gas







B1
1600
 4
37
100
None
5
Gas mix
Gas mix
1st stage


B2
1800
200
37
100
None
5
Gas mix
Gas mix
2nd stage


QA1
 900
100
39
100
None
2
Gas mix
none
3rd stage


QA2
 900
100
39
100
6.5 
2
Gas mix
none
3rd stage


QA3
 900
100
37
100
6.35
2
Gas mix
none
3rd stage


QB1
 900
100
37
100
6.65
2
Gas mix
none
3rd stage


QB2
 900
100
41
100
6.35
2
Gas mix
none
3rd stage


QB3
 900
100
41
100
6.65
2
Gas mix
none
3rd stage





Gas mix utilized was 80% N2/10% CO2/10% H2






Results

    1. The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated temperature and the indicated pH control (FIGS. 9A, 9B).

















Toxin A
Toxin B


Sample
OD@600 nm
(ng/ml)
(ng/ml)







1st stage 18 h
0.84
N/A
N/A


2nd stage 10 h
1.92
N/A
N/A


QA1 39° C. uncontrolled 18 h
3.23
29892
18321


QA2 39° C. pH 6.5 18 h
3.24
32565
18445


QA3 37° C. pH 6.35 18 h
2.83
21827
9173


QB1 37° C. pH 6.65 18 h
3.32
33149
18508


QB2 41° C. pH 6.35 18 h
2.43
21537
13522


QB3 41° C. pH 6.65 18 h
3.14
25924
16784


QA1 39° C. uncontrolled 21 h
3.26
27314
13886


QA2 39° C. pH 6.5 21 h
3.20
30509
13658


QA3 37° C. pH 6.35 21 h
3.04
34317
16935


QB1 37° C. pH 6.65 21 h
3.41
24851
14450


QB2 41° C. pH 6.35 21 h
2.90
21176
16561


QB3 41° C. pH 6.65 21 h
2.95
28002
20790









    2. Cell growth was higher in lower temperature and higher pH conditions. Toxin A production was higher in the low temperature (37° C.) and low pH (6.35) conditions. Toxin B production was higher in the high temperature (41° C.) and high pH (6.65) conditions. Lower toxin A yields were seen in high temperature and low pH conditions and lower toxin B yields were seen in low temperature and high pH conditions.

Conclusions



Optimal conditions for production of Toxin A and B are different. Since Toxin B availability is a limiting factor for the production of a vaccine product comprising Toxoids A and B (e.g., in a ratio of 3:2), conditions which favor Toxin B production may be preferred. Higher temperature (41° C.) and higher controlled pH (6.65) are the best conditions for Toxin B production. While these conditions are not the most optimal for Toxin A yields, Toxin A is produced and at a level above other conditions.
Example 11
This example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media supplemented with D-Sorbitol (12 g/L) under a controlled a pH 6.5, and at various temperatures (33, 35, 37, 39, 41, 43° C.) and 2 g/L sodium bicarbonate.
The materials and methods utilized in this experiment were as set out in Example 6, except as noted below.
Materials
The composition of the SYS media for the first stage seed cultures was as described in Example 6. For the third stage cultures, an SYS media supplemented with Sorbitol was prepared having the following composition and using RODI-water:










SYS media + Sorbitol











Formulation


Component
Manufacturer/Part #/Lot #
(g/L)












KH2PO4
J T Baker/3248-07/E29H22
0.9


Na2PO4
J T Baker/3827-01/B08143
5


NaHCO3
J T Baker/3509-05/E05589
2


Soy Peptone A3 SC
Organotechnie/130-127-
30



00/102630


Yeast Extract
BD Bacto/212720/8352570
20


D-Sorbitol (70%)
Spectrum/S0220/WJ1030
17.1 ml









Methods

III. Production Bioreactor

    1. 6×1 L vessels (3rd stage vessels: QA1 to QA3, QB1 to QB3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH control setpoints were implemented (see Table 9).
    2. Prior to inoculation, bioreactors were sparged with an appropriate gas for 30 minutes at 300 mL/min (see Table 9).
    3. 3rd stage cultures were incubated at the applicable temperature with no gassing for 21 hours.

















TABLE 9










Sod-










ium





Vess-
Vol-



Bi-


Cul-


el
ume
Ionc.
Temp
pH
carb

Sparge
ture


No.
(ml)
(ml)
(° C.)
Control
(g/L)
Degas Gas
Gas
Gas







B1
1600
 4
37
None
5
Gas mix
Gas mix
1st 










stage


B2
1800
200
37
None
5
Gas mix
Gas mix
2nd 










stage


QA1
 900
100
33
6.5 
2
100% CO2
None
3rd 










stage


QA2
 900
100
35
6.5*
2
100% CO2
None
3rd 










stage


QA3
 900
100
37
6.5 
2
100% CO2
None
3rd 










stage


QB1
 900
100
39
6.5 
2
100% CO2
None
3rd 










stage


QB2
 900
100
41
6.5 
2
100% CO2
None
3rd 










stage


QB3
 900
100
43
6.5 
2
100% CO2
None
3rd 










stage





*pH control for QA2 was not activated due to a problem with the base tubing. Gas mix utilized was 80% N2/10% CO2/10% H2






Results

    1. The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated temperature and the indicated pH control (FIGS. 10A, 10B, 10C, 10D).




















Toxin A
Toxin B



Sample
OD@600 nm
(ng/ml)
(ng/ml)









1st stage 18 h
1.31
N/A
N/A



2nd stage 10 h
1.90
N/A
N/A



QA1 19 h
2.40
10956
5853



QA2 19 h
2.42
15409
9487



QA3 19 h
2.95
19723
13736



QB1 19 h
2.12
18425
16929



QB2 19 h
2.53
18465
18184



QB3 19 h
1.71
9981
9110



QA1 22 h
2.57
12200
8218



QA2 22 h
2.73
16895
10681



QA3 22 h
2.74
29124
22679



QB1 22 h
2.20
17686
16658



QB2 22 h
2.46
18730
22104



QB3 22 h
1.76
10160
9091










    2. Cell growth decreases at temperatures higher than 37° C. Toxin A production is highest and similar within the range of 37-41° C. Toxin B yield increases almost linearly with increasing temperature from 37-41° C.

Conclusions



Culturing C. difficile at 37-41° C. is optimal for both Toxin A and B production. Culturing C. difficile at temperatures at the higher end of the 37-41° C. range favors increased Toxin B production.
Example 12
This example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media supplemented with D-Sorbitol (12 g/L) using different inoculum concentrations (1%, 5%, and 10% of initial bioreactor volume) and under different pH conditions (controlled pH 6.5 and controlled at pH 6.5 with base-only).
The materials and methods utilized in this experiment were as set out in Example 6, except as noted below.
Materials
The composition of the SYS media for the first stage seed cultures was as described in Example 6. For the third stage cultures, an SYS media supplemented with Sorbitol was prepared having the following composition and using RODI-water:










SYS media + Sorbitol











Formulation


Component
Manufacturer/Part #/Lot #
(g/L)












KH2PO4
J T Baker/3248-07/Y48478
0.9


Na2PO4
J T Baker/3827-01/B08143
5


NaHCO3
J T Baker/3509-05/E05589
2


Soy Peptone A3 SC
Organotechnie/130-127-00/18
30


Yeast Extract
BD Bacto/212720/8352570
20


D-Sorbitol (70%)
Spectrum/S0220/WJ1030
17.1 ml (12 g/L)









Methods

III. Production Bioreactor

    1. 6×1 L vessels (3rd stage vessels: QA1 to QA3, QB1 to QB3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH setpoints were implemented (see Table 11). For vessels QA1, QA2, and QA3, pH was set at 6.5 for control with base-only (5N NaOH). Base-only control involves the addition of base to the culture to adjust the culture pH to pH 6.5 in the event the culture pH becomes lower than 6.5. Under such control, the pH of the culture naturally decreases from the initial media pH (approximately pH 7.4) to pH 6.5.
    2. Prior to inoculation, bioreactors were sparged with the applicable gas for 30 minutes at 300 mL/min (see Table 10) and then an overlay of nitrogen gas was added to the applicable vessels.
    3. 3rd stage cultures were incubated at the applicable temperature for 24 hours.

















TABLE 10










Sod-










ium





Vess-
Vol-



Bi-

Sparge
Cul-


el
ume
Inoc
Temp
pH
carb
Degas
Gas
ture


No.
(ml)
(ml)
(° C.)
Control
(g/L)
Gas
(overlay)
Stage







B1
1600
 4
37
none
5
Gas mix
Gas mix
1st 










stage


B2
1800
200
37
none
5
Gas mix
Gas mix
2nd 










stage


QA1
 900
 10
37
Low end
2
Nitrogen
(Nitrogen)
3rd 






6.5



stage


QA2
 900
 50
37
Low end
2
Nitrogen
(Nitrogen)
3rd 






6.5



stage


QA3
 900
100
37
Low end
2
Nitrogen
(Nitrogen)
3rd 






6.5



stage


QB1
 900
 10
37
6.5
2
CO2
(Nitrogen)
3rd 










stage


QB2
 900
 50
37
6.5
2
CO2
(Nitrogen)
3rd 










stage


QB3
 900
100
37
6.5
2
CO2
(Nitrogen)
3rd 










stage









Results

The following table shows total cell growth (OD 600 nm) and the amount of Toxin A produced (ng/ml) and Toxin B produced (ng/ml) in the cultures subject to the indicated temperature and the indicated pH control (FIG. 11).














Cell Growth by OD600
Toxin A Concentration By
Toxin B Concentration by



Measurement
ELISA
ELISA

















Sample
0 h
16 h
20 h
24 h
16 h
20 h
24 h
16 h
20 h
24 h





QA1 low end
 0.013
4.24
4.75
4.46
12840
23135
24056
7006.1
15679.21
16010.07


pH 6.5, 1%












inoc












QA2 low end
 0.089
4.71
4.64
4.62
17831
24387
26873
 9599.37
13386.5 
17642.25


pH 6.5, 5%












inoc












QA3 low end
 0.182
4.49
4.76
4.39
19978
34845
29240
12300.11
18254.64
16550.53


pH 6.5, 10%












inoc












QB1 pH 6.5,
−0.016
3.11
4.08
4.25
13757
28501
34720
 9002.91
16828.93
25691.37


1% inoc












QB2 pH 6.5,
 0.062
3.22
4.29
4.59
19815
33186
34216
11500.96
21603.89
21967.57


5% inoc












QB3 pH 6.5,
 0.153
3.56
4.5 
4.46
26469
35068
44541
16546.46
21293.34
34246.13


10% inoc









In this experiment, a 10 L vessel (Sartorius) was also utilized. The vessel was autoclaved and connected to the Biostat system and the following conditions were set: 37° C. and agitation (stirring) at 100 rpm. Culture pH was not controlled. The vessel was filled with 9 L of the SYS media also utilized in filling the 1 L fermenters (i.e., SYS media with 12 g/L sorbitol and 2 g/L Na2HCO3). The vessel was then de-gassed using Nitrogen gas and inoculated with 1 L of the Seed Bioreactor 2 culture. Toxin production and cell growth (OD) was measured following an 18 hour incubation: Toxin A (24533 ng/ml); Toxin B (14837 ng/ml); 2.94 OD(600 nm).
A 10 L vessel was also included in two of the experiments set out above (i.e., Examples 10 and 11) and was prepared, inoculated, and cultured similarly (except de-gassing was done with gas mix 80% N2/10% CO2/10% H2 and agitation was set at 75 rpm). The measured toxin production and cell growth following an 18 hour incubation was as follows: in Example 10, Toxin A (29605 ng/ml); Toxin B (10732 ng/ml); 2.95 OD(600 nm); in Example 11, Toxin A (25681 ng/ml); Toxin B (24898 ng/ml); 3.17 OD (600 nm). In a separate experiment, toxin production and cell growth in a 10 L culture with SYS media (with 12 g/L sorbitol and 2 g/L Na2HCO3) under similar conditions (i.e., a 10% inoculum concentration, culture temperature of 37° C. and 50 rpm agitation) was similar: Toxin A (21090 ng/ml); Toxin B (12228 ng/ml) and 3.02 OD (600 nm).
Conclusions
Similar toxin yields may be achieved by using inoculum rates lower than 10% although the culture duration may need to be increased. Inoculations of 1% and 5% achieved toxin yields >30 μg/ml for toxin A and >15 μg/ml of toxin B after 20 hours.
Other Embodiments
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference in their entirety.

## Claims

1. A method for obtaining one or more Clostridium difficile (C. difficilel) toxins comprising the steps of:
(a) preparing an aqueous growth medium comprising soy peptone and D-sorbitol;
(b) inoculating the medium with a C. difficile bacterium;
(c) culturing the inoculated medium under conditions which facilitate growth of bacterium and toxin production; and
(d) isolating the one or more C. difficile toxins from the growth medium.

2. The method of claim 1, wherein:
(a) in step (a) the growth medium comprises: (i) between 10 and 20 g/L soy peptone; (ii) between 10 and 30 g/I yeast extract; between 2 and 5 g/L NaHCO3; between 1 and 10 g/L sodium phosphate, dibasic; between 1 and 10 g/L potassium phosphate, monobasic; and between 6 and 20 g/L D-sorbitol;
(b) in step (c) the inoculated medium is at pH of between 6.35 and 6.65 during culturing;
(c) in step (c) the culturing of the inoculated medium takes place at 37° C. to 41° C.;
(d) the culturing of the inoculated media is carried out under anaerobic conditions; or
(e) in step (b) the medium is inoculated with an aqueous C. difficile culture.

3. The method of claim 1, further comprising the step of detoxifying the isolated one or more C. difficile toxins to prepare one or more toxoids.

4. The method of claim 3, wherein:
(a) steps (b) and (c) are repeated more than once, with inoculation into fresh growth medium in each repeat;
(b) wherein step (c) takes place at 37° C. to 41° C.;
(c) step (d) comprises: removing from the growth medium viable C. difficile organisms and spores, separating the one or more toxins from the growth media, and purifying the one or more toxins; or
(d) step (e) comprises reacting the one or more toxins with the addition of formaldehyde.

5. The method of claim 1, wherein:
(a) the growth medium further comprises:
at least one additive selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, sucrose, and a combination of azaserine, adenosine, and biotin.

6. The method of claim 5, wherein the growth medium comprises:
(a) at least two of said additives;
(b) D-sorbitol at a concentration between 6 g/L and 20 g/L;
(c) D-sorbitol at a concentration of 12 g/L;
(d) chromium trioxide at a concentration between 40 and 60 mg/L;
(e) chromium trioxide at a concentration of 50 mg/L;
(f) clindamycin at a concentration between 0.4 and 0.6 mg/L;
(g) clindamycin at a concentration of 0.5 mg/L;
(h) ascorbic acid at a concentration between 2.5 g/L and 10 g/L;
(i) ascorbic acid at a concentration selected from 2.5 g/L and 10 g/L;
(j) butyric acid at a concentration between 30 mM and 60 mM;
(k) butyric acid at a concentration selected from 30 mM and 60 mM;
(I) D(+)xylose at a concentration between 6 and 10 g/L;
(m) D(+)xylose at a concentration of 6 g/L;
(n) adenosine at a concentration of between 0.8 and 1.2 mM, biotin at a concentration of between 40 and 60 nM, and azaserine at a concentration between 15 and 50 μM; or
(o) adenosine at a concentration of 1 mM, biotin at a concentration of 50 nM, and
azaserine at a concentration of 50 μM.

7. The method of claim 6, wherein the growth medium comprises soy peptone, yeast extract, KH2PO4, Na2HPO4, and NaHCO3, and wherein the culture is at a pH of between 6.35 and 7.45.

8. A method of enhancing the production from a C. difficile culture of Toxin B relative to the production of Toxin A comprising the steps of:
(a) preparing an aqueous growth medium comprising soy peptone and D-sorbitol;
(b) inoculating the medium with a C. difficile bacterium; and
(c) culturing the inoculated medium at 37° C. to 41° C.

9. The method of claim 8, wherein:
(a) step (c) takes place at 37° C. and at a pH of pH 6.5;
(b) the pH of step (c) is controlled at a pH between pH 6.35 to pH 6.65; or
(c) the aqueous growth medium of step (a) further comprises between 10 and 20 g/L soy peptone; and between 6 and 20 g/L D-sorbitol.

10. The method of claim 8, wherein:
(a) the production of Toxin A relative to Toxin B is less than 3:1;
(b) the production of Toxin A relative to Toxin B is less than 2:1; or
(c) the production of Toxin A relative to Toxin B is equal to or less than 1.5:1.