CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Patent Application No. 61/099,759, filed on September 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 l l(l):5-10, 2007; Warny et al., Lancet 366(9491): 1079-1084, 2005; Dove et al., Infection and Immunity 58(2):480-488, 1990; Barroso et al., Nucleic Acids Research 18(13):4004, 1990). Toxins A and B are encoded by two separate but closely linked (and highly homologous) genes. Toxins A and B are produced simultaneously in C. difficile strain VPI 10463 (ATCC 43255), and the ratio of the produced toxins is usually 3:1, respectively (Karlsson et al., Microbiology 145:1683-1693, 1999). The toxins begin to be formed during the exponential growth phase, and are usually released from the bacteria between 36 and 72 hours of culture. Toxins present within the bacteria can be released earlier by sonication or by use of a French pressure cell.

Treatment of the toxins with formaldehyde results in the corresponding Toxoids A and B, which are completely inactivated and retain at least partial immunogenicity (Torres et al., Infection and Immunity 63(12):4619-4627, 1995). It has been shown that vaccination employing both toxoids is effective in hamsters, healthy adults, and patients with recurrent CDAD (Torres et al., Infection and Immunity 63(12):4619-4627, 1995; Kotloff et al., Infection and Immunity 69(2):988-995, 2001 ; Sougioultzis et al, Gastroenterology 128(3):764-770, 2005; 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 (Torres et al., Vaccine Research 5(3):149-162, 1996; Giannasca et al., Infection and Immunity 67(2):527-538, 1999).

The administration of both toxoids simultaneously is more effective than administration of the individual proteins alone (Kim et al., Infection and Immunity 55(12):2984-2992, 1987). A toxoid composition found effective in inducing protective immune responses against toxin A and toxin B in patients with recurrent CDAD included both toxoids, at a ratio of 1.5: 1 , A:B (Sougioultzis et al., Gastroenterology 128(3):764-770, 2005).

Both the A and B toxoids are thus candidates for vaccine development. Greater production efficiency of Toxins A and B is desired to facilitate vaccine production.

SUMMARY OF THE INVENTION

In one aspect, the invention features a culture medium (e.g., for culturing a Clostridium difficile bacterium) at a pH of between 6.35 and 7.45 (e.g., 6.5, 7.28, or between 6.35 and 6.65) including peptone (e.g., soy peptone), a yeast extract (e.g., Difco Bacto Yeast extract), a buffering agent (e.g., NaHCO ₃ ), 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 NaHCO ₃ , 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 tri oxide, 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 NaHCO ₃ , 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 ⁰ C to 41 ⁰ 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, NaHCO ₃ , 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 ⁰ 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, NaHCO ₃ ; 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 NaHCO ₃ 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. IA-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. 3 A 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. 5 A 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 myoinositol (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 /nyo-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. 8 A 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. 1OA is a graph showing the amount of Toxin A produced (ng/ml) in cultures subject to the indicated temperature.

Fig. 1OB is a graph showing the amount of Toxin B produced (ng/ml) in cultures subject to the indicated temperature.

Fig. 1 OC is a graph showing specific Toxin A productivity produced (ng/ml per OD unit) in the cultures subject to the indicated temperature.

Fig. 1 OD 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 IA 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 IA

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

Table IB

hi 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 CrO ₃ ). Chromium trioxide is the acid anhydride of chromic acid. Chromium trioxide is a strong oxidant, highly toxic, corrosive, and carcinogenic compound.

Clindamycin (C _I8 H _S3 CIN ₂ O _S S). Clindamycin is a lincosamide antibiotic and is indicated for Clostridium <iz//?c?7e-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 5OS subunit of the bacterial ribosome.

Azaserine (C ₅ H- ₇ N ₃ O ₄ ). 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 (C ₆ HgO ₆ ). Ascorbic acid is a sugar acid with antioxidant properties. L-Ascorbic acid is also known as vitamin C.

Butyric acid (C ₄ HgO ₂ ). 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 (C ₅ H ] ₀ O ₅ ). 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 (CeHi ₄ Oe). 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

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 ⁰ 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 ⁰ 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 ⁰ C to 41 ⁰ C (e.g., 37 ⁰ 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% CO ₂ /10% hydrogen, 100% CO _2, 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% CO ₂ A 0% hydrogen) or a gas (e.g., CO ₂ 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% CO ₂ /10% hydrogen, 100% CO ₂ or 100% nitrogen to remove any residual oxygen in the medium. During the fermentation process the culture may be sparged periodically. Alternatively, an overlay of a gas mixture or a gas (e.g., 100% nitrogen) may be applied to the culture.

Fermentation proceeds for approximately 16 to 24 hours (e.g., 18 to 21 hours).

Preferably, agitation (e.g., 100 rpm) is applied to the culture medium during the fermentation process (and/or during stages one and two of seed cultures). Growth can be monitored by measuring the optical density (O.D.) of the medium.

C. difficile toxins can be isolated and purified from fermentation cultures using purification methods well known in the art such as for example, Kotloff et al., Infect. Immun 2001; 69:988-995, Coligan et al., "Current Protocols in Protein Science," Wiley & Sons; Ozutsumi et al., Appl. Environ. Microbiol. 49:939-943, 1985; and Kim et al., Infection and Immunity 55:2984-2992, 1987; which are incorporated herein by reference. The purified toxins can then, for example, be inactivated by chemical treatments known in the art (e.g., formaldehyde treatment).

All references cited within this disclosure are hereby incorporated by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way.

Examples

The basal media and additives of the invention were used to culture Clostridium difficile and produce Clostridium difficile Toxins A and B. Tables 3 and 4 (and Figs. 1 A-IG) 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 rowth in the resence of the listed com ounds

Table 4A

Total measured production of Toxins A and B at time points 12 and 24 hours followin rowth in the resence of the listed com ounds

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 (CeH ₈ O ₇ .nFe.nH ₃ N), USB Cat. 15751 Lot. 121753 FC-Ferric citrate (C ₆ H ₅ FeO ₇ ), FW 244.95, MB Biomedicals LLC, Cat. 195181, Lot R23927 FG-Ferrous gluconate Hydrade (C _{12 hrs.22} FeOi ₄ ) FS-Ferric sulfate (FeSO ₄ -7H ₂ O), FW 278.02 CA-Calcium chloride Anhydrous (CaCl ₂ ), FW 110.98, J.T. Baker Cat. 1311-01, Lot. A13602 CC-Cobalt chloride 6 Hydrate Crystal (CoCl ₂ -O H ₂ O), FW 237.93, Mallinckrodt

Chemicals Cat. 4535-02

CT-Chromium trioxide Crystal (CrO ₃ ), FW 99.99, J.T. Baker Cat. 1638-04, Lot.

MS-Magnesium sulfate (MgSO ₄ -7 H ₂ O), FW 246.50

MC-Manganese chloride (MnCl ₂ -4 H ₂ O), 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.

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% H ₂ + 10% CO ₂ + 80% N ₂ .

3. Before transferring the medium, fill gas (10% H ₂ + 10% CO ₂ + 80% N ₂ ) 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 5O g, stop the pumping. 6. Move the bag for seed-1 to 37 ⁰ C CO ₂ incubator to warm overnight. Keep bag for seed-2 and fermentation at 4 ⁰ C until use.

7. Move the bags to 37 ⁰ C CO ₂ 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:

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 ran. The blank of fermentation media was used as zero for the spectrophotometer. The cell concentration was diluted 10x.

5. The toxin production is measured by capture ELISA.

III. Capture ELlSAs:

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# C0210091

6. Mouse anti Toxin B Lot#030904

Results

1. The following table shows the amount of seed growth (OD ₆ oo mn) as measured by

DU700.

2. The following table shows the amount of cell growth (OD ₆ oonm) in cultures with the indicated compound.

*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).

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

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.

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 (Ci ₇ H ₁₈ FN ₃ O ₃ ) FW 331.35, BioChemika, Lot#WA19781, soluble with 0.2 mL of 5 N HCl. Cli-Clindamycin hydrochloride (Ci ₈ H ₃₃ ClN ₃ O ₅ S-HCl) FW 461.44, Sigma C5269, Lot#37kl535, soluble in water.

Van- Vancomycin hydrochloride (C ₆₆ H ₇₅ Cl ₂ N ₉ O ₂₄ -HCl) FW 1485, Sigma V20029, Lot#037K0686 soluble in water.

Pen G-Penicillin G Sodium salt (C ₁₆ H ₁₇ N ₂ NaO ₄ S) FW 356.4, Sigma P3032, Lot#057K04931 , soluble in water.

Fe-EDTA was also tested (Ethylenediaminetetraacetic acid, Ferric Sodium Salt, (Ci ₀ Hi ₂ FeN ₂ NaO ₈ ) 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 10Ox concentration antibiotic solutions/ 1Ox Fe-EDTA solutions

2. Make 10x concentration of antibiotic solution:

Take 4 mL of 10Ox concentration solution + 36 mL di water. Methods

1. 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% H ₂ + 10% CO ₂ + 80% N ₂ . 3. Before transferring the medium, fill gas (10% H ₂ + 10% CO ₂ + 80% N ₂ ) 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 ⁰ C CO ₂ incubator to warm overnight. Keep bag for seed-2 and fermentation at 4 ⁰ C until use. 7. Move the bags to 37 ⁰ C CO ₂ incubator to warm up overnight before use.

8. For different antibiotics: i) Prepare 40 mL solutions of different antibiotics with the concentrations at 10x

(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.

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 ran, The blank of fermentation media was used as zero for the spectrophotometer. The cell concentration was diluted 10x.

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# C0210091

6. Mouse anti Toxin B Lot#030904 Results

1. The following table shows the amount of seed growth (OD ₆ oo _mn ) as measured by DU700.

2. The following table shows the amount of cell growth (OD βoo _nm ) in cultures with the indicated compound.

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

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

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

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.

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 (C ₅ H ₇ N ₃ O ₄ ) FW 173.10, (O-diaxoacetyl-L-serine) Fluka BioChemika, 11430 Lot#1301321 , soluble in water.

Ade-Adenosine (Ci ₀ H ₁₃ N ₅ O ₄ ) FW 267.25, Sigma A4036, Lot#046K06612, soluble with 5N HCl.

Bi ₂ - Vitamin Bi ₂ (C ₆₃ H ₈₈ CoNi ₄ Oi ₄ P) FW 1 ,355.37, Sigma, V6629Lot#124K17072, soluble in water.

Bio-d-Biotin (Ci _O Hi ₆ N ₂ O ₃ S) 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/1) #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 niL) #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 50x concentration solutions.

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

3. Make 5Ox concentration solutions then dilute to 10x.

Methods

I. Medium preparations:

1. Prepare 1000 mL SYS medium in 2L beaker.

2. Transfer SYS to media bottles and degas for over 30 minutes with 10% H ₂ + 10% CO ₂ + 80% N ₂ .

3. Before transferring the medium, fill gas (10% H ₂ + 10% CO ₂ + 80% N ₂ ) 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 CO ₂ incubator to warm overnight. Keep bag for seed-2 and fermentation at 4°C until use.

7. Move the bags to 37°C CO ₂ 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.

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 ⁰ 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± l°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 1 Ox.

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# C0210091

6. Mouse anti Toxin B Lot#030904 Results

1. The following table shows the amount of seed growth (OD ₆ oo nm) as measured by

DU700.

2. The following table shows the amount of cell growth (OD ₆ oo _n m) in cultures with the indicated compound.

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

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

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 (C ₆ H ₄ N ₄ O ₂ -HCl), FW. 210.67 Sigma A5131-500G,

Lot# 016K0001 , soluble in water.

Cys-L-Cysteine (C ₃ H ₇ NO ₂ S), FW 121.16, Sigma C-7352, Lot#082K0377, soluble in water.

Tyr-L-Tyrosine (C ₉ H ₁₁ NO ₃ ), FW 181.19, Sigma T8566, Lot#107K0157, soluble in water with HCl. Asc-Ascorbic acid (C ₆ H ₈ O ₆ ) FW 176.12, Sigma A5960 Lot#043K0131, soluble in water.

But- Butyric acid (C ₄ H ₈ O ₂ ) FW 88.11, Aldrich B103500 Lot#0351 IDA, soluble in water.

These compounds were tested using the following concentrations (1Ox):

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 1Ox Arginine solutions.

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

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

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

5. Make 1Ox Butyric acid solutions at 30 mM.

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% H ₂ + 10% CO ₂ + 80% N ₂ .

3. Before transferring the medium, fill gas (10% H ₂ + 10% CO ₂ + 80% N ₂ ) 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 ⁰ C CO ₂ incubator to warm overnight. Keep bag for seed-2 and fermentation at 4 ⁰ C until use.

7. Move the bags to 37 ⁰ C CO ₂ incubator to warm up overnight before use. 8. For different antibiotics: i) Prepare 40 mL solutions of different antibiotics with the concentrations at 10x (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.

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± l°C for 24 hrs.

4. Take samples at 12 hours and 24 hours. Cell growth was measure at 600 ran. The blank of fermentation media was used as zero for the spectrophotometer. The cell concentration was diluted 10x.

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# C0210091

6. Mouse anti Toxin B Lot#030904

Results

1. The following table shows the amount of seed growth (OD ₆ oo _nm ) as measured by

DU700.

2. The following table shows the amount of cell growth (OD βoo _nm ) in cultures with the indicated compound.

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

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

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

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.

Conclusions Ascorbic acid, when added to SYS medium at 10 g/L, caused increases in Toxin B of 19% after 12 hours and 49% after 24 hours of incubation in fermentation broth.

Butyric Acid, when added to SYS medium at 30 mM, caused increases in Toxin A of 14% and Toxin B of 34% after 12 hours of incubation in fermentation broth. It caused increases in Toxin A of 16% and Toxin B of 61% after 24 hours of incubation in fermentation broth. Butyric Acid, when added to SYS medium at 60 mM, caused increases in Toxin B of 89% after 24 hours of incubation in fermentation broth.

Example 5

This Example includes data on the amount of toxin produced when Clostridium difficile is cultured in SYS basal media in the absence and presence of various concentrations of carbohydrates.

The following table summarizes the data regarding toxin increases (%) following the addition of increasing concentrations of D-sorbitol.

Materials

The following are the Example 5 test compounds, along with the compound formula and source. D(-)Fructose: (contained <0.05% glucose) C ₆ Hi ₂ O ₆ , FW 180.2, Sigma F0127

Lot#60K0013

D(+)Galactose: C ₆ H ₁₂ O ₆ , FW 180.2, Sigma G0625, Lot#102K0169 soluble in water (l g/1.7 mL)

D(+)Mannose: C ₆ H ₁₂ O ₆ , FW 180.16, Sigma M6020 Lot# soluble in water (50 mg/mL) D(+)Maltose Monohydrate: (contained <0.3% glucose), C _{12 hrs} . ₂₂ Oii- H ₂ O, FW 360.3,

Sigma M9171 Lot#80K10101 soluble in water

Sucrose: C ₁₂ ^ ₂₂ On, FW 342.3, Sigma, S3929, Lot#127K0093 soluble in water α-Lactose: Ci _{2 h} , _s . ₂₂ O _n - H ₂ O, FW 360.3, Sigma L2643, Lot# soluble in water (0.2 g/mL) D(+)Xylose: C ₅ Hi ₀ O ₅ , FW 150.132, Sigma X3877 Lot# soluble in water (1 g/0.8 mL)

D-Sorbitol: C ₆ H ₁₄ O ₆ , FW 182.2, Sigma S3889, Lot#042K01355 soluble in water. myo-Inositol: C ₆ Hi ₂ O ₆ , FW 180.16, Sigma 17508, Lot# soluble in water (50 mg/mL) 10x 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% H ₂ + 10% CO ₂ + 80% N ₂ .

3. Before transferring the medium, fill gas (10% H ₂ + 10% CO ₂ + 80% N ₂ ) 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 CO ₂ incubator to warm overnight. Keep bag for seed-2 and fermentation at 4°C until use.

7. Move the bags to 37°C CO ₂ 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:

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± I ⁰ 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 10x. 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# C0210091

6. Mouse anti Toxin B Lot#030904 Results

1. The following table shows the amount of seed growth (OD βoo _nm ) as measured by

DU700. 2. The following table shows the amount of cell growth (OD ₆ oo _nm ) ^m cultures with the indicated compound.

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

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

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

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.

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 x 2L 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 x IL 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

Media composition for the production stage was as follows:

SYS media + Sorbitol

* 17. ImI 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:

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 18h.

11. At end of 18 hr, a 5ml sample was taken for OD measurement.

II. Seed Bioreactor 2

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. 1800 niL 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 1Oh.

I I. At end of 10 hr, a 5ml sample was taken for OD measurement.

III. Production Bioreactor

1. 4 x IL 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 x 1 mL of sample were spun in 1.8 mL microcentrifuge tubes at 10,000g for lmin, then decanted and 0.2μm filtered. The samples were stored at 2- 8°C until tested.

Table 5

*pH control using IN 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).

* 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 10OkDa 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 NaHCO ₃ (reduced from 5 g/L used in Example 6) Methods

III. Production Bioreactor

1. 5x1 L vessels (3 ,r ^ra d stage vessels: QB2, QB3, QAl , QA2, QA3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH control setpoints were implemented (see Table 6).

2. Prior to inoculation, bioreactors were sparged with an appropriate gas for 30 minutes at 300 mL/min (see Table 6).

3. 3 ^rd stage cultures were incubated at 37°C with no gassing for 21 hours.

Table 6

*pH control using IN 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).

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

Conclusions

Lowering the sodium bicarbonate to 2g/L in the SYS + Sorbitol medium allowed for a lower starting pH with less acid and/or CO ₂ sparged. It is possible to achieve a pH of 6.5 without the addition of acid, by sparging with CO ₂ .

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. Figure 7E depicts a comparison of the results from this experiment and that set out in Example 6. Figure 7E shows total toxin concentration at 18h 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 IL fermenters (Sartorius). The composition of the SYS media supplemented with D-Sorbitol (SYS media + Sorbitol) was as described in Example 6, except that no NaHCO ₃ was added to the initial 4L 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. 4L of SYS media with sorbitol was made without sodium bicarbonate and 1 L was added to a IL fermenter.

3. 6g of sodium bicarb was added to the remaining 3L of media for a bicarb concentration of 2g/L. IL of the media was added to a IL fermenter.

4. 6g of sodium bicarb was added to the remaining 2L 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 lOOrpm and the pH probes were installed

6. 100% CO ₂ was sparged at 500ml/min and the data acquisition software was started to generate pH curves

7. After ~3.5 hours, 5ml of 5N HCl was added to each fermenter.

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

Conclusions

A lower final pH can be achieved in SYS medium with a lower sodium bicarbonate concentration when gassing with CO ₂ . Using 2g/L bicarb can lower the pH by 0.12 units with CO ₂ 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%N ₂ /10%CO ₂ /10%H ₂ ).

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 RODl-water:

SYS media + Sorbitol

Two separate batches of SYS media + Sorbitol culture media were also prepared having the same composition but with a different concentration OfNaHCO ₃ (i.e., one with NaHCO ₃ 2 g/L and one with 5 g/L NaHCO ₃ ).

Methods

III. Production Bioreactor

1. 5xlL vessels (3 ^rd stage vessels: QB2, QB3, QAl, 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 ⁰ C with no gassing for 21 hours.

Table 7

*pH control using IN 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).

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 CO ₂ or anaerobic gas mix sparging. A sodium bicarbonate concentration of 2g/L sodium bicarbonate provided a higher specific and total toxin A and B productivity compared to concentrations of Og/L and 5g/L.

Conclusions

The use of CO ₂ 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 ⁰ 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

Methods

III. Production Bioreactor

1. 6xlL vessels (3 ^rd stage vessels: QBl , QB2, QB3, QAl , 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 ^r stage cultures were incubated at the applicable temperature with no gassing for 21 hours.

Table 8

Gas mix utilized was 80%N ₂ /10%CO ₂ /10%H ₂ 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).

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 ⁰ 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 ⁰ C) and 2g/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

Methods

III. Production Bioreactor

1. 6xlL vessels (3 ^ld stage vessels: QAl to QA3, QBl 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 ^ld stage cultures were incubated at the applicable temperature with no gassing for 21 hours. Table 9

*- pH control for QA2 was not activated due to a problem with the base tubing. Gas mix utilized was 80%N ₂ /10%CO ₂ /10%H ₂

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. 1OA, 1OB, 1OC, 10D).

2. Cell growth decreases at temperatures higher than 37°C. Toxin A production is highest and similar within the range of 37-41 ⁰ C. Toxin B yield increases almost linearly with increasing temperature from 37-41 ⁰ 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 ⁰ 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

Methods

III. Production Bioreactor

1. 6xlL vessels (3 ^ld stage vessels: QAl to QA3, QBl to QB3) were prepared in 2 sets (of 2 and 3). The desired temperature and pH setpoints were implemented (see Table 11). For vessels QAl, 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. l id stage cultures were incubated at the applicable temperature for 24 hours.

Table 10

Results

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

In this experiment, a 1OL 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 9L of the SYS media also utilized in filling the IL fermenters (i.e., SYS media with 12 g/L sorbitol and 2 g/L Na ₂ HCOs). The vessel was then de-gassed using Nitrogen gas and inoculated with IL 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 1OL 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 degassing was done with gas mix 80%N ₂ /10%CO ₂ /10%H ₂ 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(600nm); in Example 11, Toxin A (25681 ng/ml); Toxin B (24898 ng/ml); 3.17 OD (600nm). In a separate experiment, toxin production and cell growth in a 1OL culture with SYS media (with 12g/L sorbitol and 2g/L Na ₂ HCO ₃ ) 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 (600nm). 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 >15μg/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.