US20160053221A1 - Methods and compositions for increasing toxin production - Google Patents

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.

Description

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 (Tones 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; Torres 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 (Tones 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); #6 α-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: #l-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); #6 α-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
  Ingredient	per liter	Acceptable range 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)
  NaHCO 3	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
  Ingredient	per liter	Acceptable range 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)
  NaHCO 3	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 arc 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 (C_{12 hrs 22}FeO₁₄)
• 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 (MnCl₂.4H₂O), 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		Compound solution
  2 g/L	Natural pH	1 g/L	Natural pH

  Ammonium ferric	5.0	Calcium chloride	4.7
  citrate
  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 #CO210091
  • 6. Mouse anti Toxin B Lot #030904
Results
• 1. The following table shows the amount of seed growth (OD_{600 nm}) as measured by DU700.

• 	Seed-1	Seed-2
  	1.73	2.31

• 2. The following table shows the amount of cell growth (OD_{600 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 3x to 5x 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

• 	Antibiotics		100x
  Antibiotics	powder	Sterile di water	Concentration
  (mg/L)	(mg)	(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	10x 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.

• 	10x
  	Solution	Sterile di water	Total
  	(mL)	(mL)	(mL)

  Antibiotics (mg/L)
  Control (without antibiotics)	0	5	5
  Ciprofloxacin 2 mg/L (100 μg/50 mL)	1	4	5
  Ciprofloxacin 10 mg/L (500 μg/50 mL)	5	0	5
  Clindamycin 0.5 mg/L (25 μg/50 mL)	1	4	5
  Clindamycin 2.5 mg/L (125 μg/50 mL)	5	0	5
  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 #CO210091
  • 6. Mouse anti Toxin B Lot #030904
Results
• 1. The following table shows the amount of seed growth (OD_{600 nm}) as measured by DU700.

• 	Seed-1	Seed-2
  	2.32	2.82

• 2. The following table shows the amount of cell growth (OD_{600 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 x longer than normal cells.
  		Some cells were curved.
  Clindamycin	0.5	Normal
  	2.5	Most sizes of the cells were 2x smaller than
  		normal. Cells grow very slow
  Vancomycin	0.1	Some cells were 2x longer than normal cells
  	0.5	Some cells were 2x 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 pM Biotin (12.2 ng/L)
• 5 μM Azaserine (43.25 μg/50 mL)+1 mM Adenosine (13.35 mg/50 mL)+50 pM 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.

• 	Chemical	Sterile di water	50x 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 water	500 μM d-Biotin
  Test component	(mg)	(mL)	(mg/L)
  d-Biotin	2.44	20	122
  (50 nM = 12.2 μg/L)

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

• 	Vitamin	Sterile	50x
  	B12	di water	Concentration
  Test component	(mg)	(mL)	(mg/L)
  Vitamin B12 (50 nM = 67.8 mg/L)	33.9	10	3390 (2.5 μM)

• 	50x		10x
  	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 to	Aza	Ade	Bio	water	Total
  50 mL SYS medium	(mL)	(mL)	(mL)	(mL)	(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	Total
  	(mL)	(mL)	(mL)	(mL)	(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	Total
  	(mL)	(mL)	(mL)	(mL)	(mL)
  #5-5 μM Azaserine +	1	1	0.5	2.5	5
  1 mM Adenosine +
  50 pM Biotin

  	0.5 nM
  	Biotin	5 nM	50 nM Biotin	Total
  	(mL)	Biotin (mL)	(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 (67.77 μg/L)	5	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 #CO210091
  • 6. Mouse anti Toxin B Lot #030904
Results
• 1. The following table shows the amount of seed growth (OD_{600 nm}) as measured by DU700.

• 	Seed-1	Seed-2
  	2.53	2.49

• 2. The following table shows the amount of cell growth (OD_{600 nm}) in cultures with the indicated compound.

• Test	12 hours	24 hours
  Control	2.71	2.39
  #1-50 μM Azaserine + 1 mM Adenosine + 50 nM	1.19	0.42
  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 + 50 nM	10391	19000
  Biotin
  #2-15 μM Azaserine + 1 mM Adenosine + 50 nM	8525	10423
  Biotin
  #3-15 μM Azaserine	9333	10838
  #4-5 μM Azaserine + 1 mM Adenosine + 50 nM	7471	7816
  Biotin
  #5-5 μM Azaserine + 1 mM Adenosine + 50 pM	9933	10811
  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 + 50 nM	6198	11940
  Biotin
  #2-15 μM Azaserine + 1 mM Adenosine + 50 nM	3430	3880
  Biotin
  #3-15 μM Azaserine	3589	4112
  #4-5 μM Azaserine + 1 mM Adenosine + 50 nM	2665	2694
  Biotin
  #5-5 μM Azaserine + 1 mM Adenosine + 50 pM	3345	3813
  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 (C₆H₄O₂.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.

• 			10x
  	Arginine	Sterile 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
  		di water	10x Concentration
  Test component		(mL)	(g/L)
  	Cysteine (g)
  Cysteine (33 mM = 4 g/L)	1.6	40	40
  	10x 33 mM
  	Cys. (mL)
  Cysteine (3.3 mM = 400	4	36	4
  mg/L)
  Cysteine (0.33 mM = 40	4	36	0.4
  mg/L)

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

• 	Tyrosine	Sterile di water	10x 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.

• 	Ascorbic	Sterile di	10x Concentration
  Test component	acid (g)	water (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	10x Concentration
  Test component	(mL)	water (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.

• 			Sterile
  		10x	di
  		Solution	water	Total
  Medium #	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 #CO210091
  • 6. Mouse anti Toxin B Lot #030904
Results
• 1. The following table shows the amount of seed growth (OD_{600 nm}) as measured by DU700.

• 	Seed-1	Seed-2
  	2.65	2.53

• 2. The following table shows the amount of cell growth (OD_{600 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 to 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), C_{12 hrs. 22}O₁₁.H₂O, FW 360.3, Sigma M9171 Lot #80K10101 soluble in water
• Sucrose: C_{12 hrs. 22}O₁₁, FW 342.3, Sigma, S3929, Lot #127K0093 soluble in water
• α-Lactose: C_{12hrs. 22}O₁₁.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:

• 		10x
  		Solution	Sterile di	Total
  Medium #	Test compound	(mL)	water (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	5	0	5
  	Monohydrate (6 g/L)
  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 (I072506A)
  • 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 #CO210091
  • 6. Mouse anti Toxin B Lot #030904
Results
• 1. The following table shows the amount of seed growth (OD_{600 nm}) as measured by DU700.

• 	Seed-1	Seed-2
  	2.57	2.561

• 2. The following table shows the amount of cell growth (OD_{600 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/	30
  		19685
  	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/	30
  		19685
  	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:

• 	Manufacturer/Lot #	Part #	Qty. Added

  Working Cell Bank	In-house	n/a	0.5 (2 ml)
  vial, WCB-A, 4.5 ml
  Anaerobic Gas		RM-0024	20
  mix (80% N2/10%
  CO2/10% H2)
  5N Sodium Hydroxide	J T Baker/E17507	5671-06	1 L
  1N Hydrochloric Acid	J T Baker/B08510	5618-02	200 ml

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 2L 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 IN 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. 3^rd 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 B
  Sample	OD@600 nm	Toxin A (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 (3^rd 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 spargcd with an appropriate gas for 30 minutes at 300 mL/min (see Table 6).
  • 3. 3^rd 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 B
  Sample (Vessel No.)	OD@600 nm	Toxin A (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 7.0 21 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 NaHCO₃ 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 m1/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/	30
  		19685
  	Yeast Extract	BD Bacto Part	20
  		#21270/Lot
  		#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 NaHCO ₃ 2 g/L and one with 5 g/L NaHCO₃).
Methods
• III. Production Bioreactor
  • 1. 5×1 L vessels (3^rd 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. 3^rd 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 CO 2 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 (3^rd 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. 3^rd 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	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	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 (3^rd 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. 3^rd stage cultures were incubated at the applicable temperature with no gassing for 21 hours.

• TABLE 9
  Vessel	Volume	Inoc.	Temp	pH	Sodium		Sparge	Culture
  No.	(ml)	(ml)	(° C.)	Control	Bicarb (g/L)	Degas Gas	Gas	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	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).

• Sample	OD@600 nm	Toxin A (ng/ml)	Toxin B (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-	30
  	00/18
  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 (3^rd 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. 3^rd stage cultures were incubated at the applicable temperature for 24 hours.

• TABLE 10
  					Sodium		Sparge
  Vessel	Volume	Inoc	Temp	pH	Bicarb	Degas	Gas	Culture
  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 stage
  				6.5
  QA2	900	50	37	Low end	2	Nitrogen	(Nitrogen)	3rd stage
  				6.5
  QA3	900	100	37	Low end	2	Nitrogen	(Nitrogen)	3rd stage
  				6.5
  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 13 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	Toxin B Concentration by
  	Measurement	By 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 (600nm).
• 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 (10)

1-85. (canceled)

86. A medium for culturing a Clostridium difficile bacterium comprising soy peptone, yeast extract, a buffering agent, a phosphate buffer, and at least one additive 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, wherein the medium is at a pH of between 6.35 and 7.45.

87. The medium of claim 86, wherein said the phosphate buffer comprises sodium phosphate, dibasic and potassium phosphate, monobasic.

88. The medium of claim 86, wherein the at least one additive comprises:

(a) 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;

(b) adenosine at a concentration of 1 mM, biotin at a concentration of 50 nM, and azaserine at a concentration of 50 μM;

(c) D-sorbitol;

(d) D-sorbitol at a concentration between 6 g/L and 20 g/L;

(e) D-sorbitol at a concentration of 12 g/L;

(f) chromium trioxide at a concentration of between 40 and 60 mg/L;

(g) chromium trioxide at a concentration of 50 mg/L;

(h) clindamycin at a concentration between 0.4 and 0.6 mg/L;

(i) clindamycin at a concentration of 0.5 mg/L;

(j) ascorbic acid at a concentration between 2.5 g/L and 10 g/L;

(k) ascorbic acid at a concentration selected from 2.5 g/L and 10 g/L;

(l) butyric acid at a concentration between 30 mM and 60 mM;

(m) butyric acid at a concentration selected from 30 mM and 60 mM;

(n) D(+)xylose at a concentration between 6 g/L and 10 g/L; or

(o) D(+)xylose at a concentration of 6 g/L.

89. A bacterial culture comprising Clostridium difficile and culture medium, wherein said culture medium comprises at least one additive 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.

90. The bacterial culture of claim 89, wherein said culture medium comprises:

(a) D-sorbitol;

(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;

(l) D(+)xylose at a concentration between 6 and 10 g/L.

(m) D(+)xylose at a concentration of 6 g/L; or

(n) soy peptone, yeast extract, KH₂PO₄, Na₂HPO₄, and NaHCO₃, and wherein the culture is at a pH of between 6.35 and 7.45.

91. The bacterial culture of claim 89, wherein said culture medium comprises at least two of said additives, optionally wherein:

(a) said medium comprises 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 (b) said medium comprises adenosine at a concentration of 1 mM, biotin at a concentration of 50 nM, and azaserine at a concentration of 50 μM.

92. A method of culturing Clostridium difficile comprising inoculating culture medium with Clostridium difficile, said medium comprising at least one additive 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.

93. The method of claim 92, wherein said culture medium comprises:

(a) at least two of said additives;

(b) D-sorbitol;

(c) D-sorbitol at a concentration between 6 g/L and 20 g/L;

(d) D-sorbitol at a concentration of 12 g/L;

(e) chromium trioxide at a concentration between 40 and 60 mg/L;

(f) chromium trioxide at a concentration of 50 mg/L;

(g) clindamycin at a concentration between 0.4 and 0.6 mg/L;

(h) clindamycin at a concentration of 0.5 mg/L;

(i) ascorbic acid at a concentration between 2.5 g/L and 10 g/L;

(j) ascorbic acid at a concentration selected from 2.5 g/L and 10 g/L;

(k) butyric acid at a concentration between 30 mM and 60 mM;

(l) butyric acid at a concentration selected from 30 mM and 60 mM;

(m) D(+)xylose at a concentration between 6 and 10 g/L;

(n) D(+)xylose at a concentration of 6 g/L;

(o) 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

(p) adenosine at a concentration of 1 mM, biotin at a concentration of 50 nM, and azaserine at a concentration of 50 μM.

94. The method of claim 93, wherein said medium further comprises soy peptone, yeast extract, KH₂PO₄, Na₂PO₄, and NaHCO₃, and wherein the culture is at a pH of between 6.35 and 7.45.

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