Enrichment medium for gram-negative bacteria

Abstract

Enrichment media useful for the repair and growth of gram-negative bacteria are described herein. The enrichment medium may include pyruvic acid (e.g., sodium pyruvate), magnesium (e.g., magnesium sulfate), yeast extract, and a selectivity agent that inhibits the growth of gram-positive bacteria.

Description

Attorney Docket No.: 234994-559211 Enrichment Medium for Gram-Negative Bacteria CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to and the benefits of U.S. Provisional Application No.63 / 626,116, filed January 29, 2024, the contents of which are herein incorporated by reference in their entirety. TECHNICAL FIELD

[0002] The present invention relates to an enrichment medium (e.g., enrichment broth),which is useful for growth of gram-negative bacteria, such as Salmonella and E. coli. BACKGROUND

[0003] Enrichment media are used to grow bacteria from a sample, including repairing andgrowing sublethally injured cells. Gram-negative bacteria, such as Salmonella and E. coli present safety concerns when present in food or environmental samples. To detect gram- negative bacteria in a sample, it is important to grow the bacteria to a level sufficient to detect the presence of the bacteria. Rapid growth is particularly desirable in order to rapidly detect the presence of the bacteria, e.g., by detection from the sample in the same day or same work shift that the sample was collected. SUMMARY OF THE INVENTION

[0004] The present invention provides an enrichment medium useful for repair and growthof gram-negative bacteria.

[0005] In one aspect, provided herein is an enrichment medium comprising pyruvic acid,magnesium, yeast extract, and water.

[0006] In another aspect, provided herein is an enrichment medium comprising sodiumpyruvate, magnesium sulfate, yeast extract, and water.

[0007] In another aspect, provided herein is an enrichment medium comprising pyruvicacid, magnesium, yeast extract, a selectivity agent that inhibits the growth of gram-positive bacteria, and water.

[0008] In another aspect, provided herein is an enrichment medium comprising sodiumpyruvate, magnesium sulfate, yeast extract, a selectivity agent that inhibits the growth of gram-positive bacteria, and water. 59099806.1Attorney Docket No.: 234994-559211

[0009] In another aspect, provided herein is an enrichment medium formulation (e.g., apowder) comprising a pyruvic acid salt, a magnesium salt, and yeast extract. The formulation can be mixed with water to prepare an enrichment medium.

[0010] In another aspect, provided herein is an enrichment medium formulation (e.g., apowder) comprising sodium pyruvate, magnesium sulfate, and yeast extract.

[0011] In another aspect, provided herein is an enrichment medium formulation (e.g., apowder) comprising a pyruvic acid salt, a magnesium salt, yeast extract, and a selectivity agent that inhibits the growth of gram-positive bacteria.

[0012] In another aspect, provided herein is an enrichment medium formulation (e.g., apowder) comprising sodium pyruvate, magnesium sulfate, yeast extract, and a selectivity agent that inhibits the growth of gram-positive bacteria.

[0013] In another aspect, provided herein is a method of growing gram-negative bacteria inan enrichment medium.

[0014] Various embodiments of these and other aspects may include one or more of thefeatures as described in the detailed description below. DETAILED DESCRIPTION

[0015] In the following description, numerous specific details are given to provide athorough understanding of the embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

[0016] Reference throughout this specification to "one embodiment," "an embodiment," or"embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0017] Unless indicated otherwise, when a range of any type is disclosed or claimed, it isintended to disclose or claim individually each possible number that such a range could reasonably encompass, including any sub-ranges encompassed therein. Moreover, when a range of values is disclosed or claimed, which Applicants intend to reflect individually each possible number that such a range could reasonably encompass, Applicants also intend for the 2 59099806.1Attorney Docket No.: 234994-559211 disclosure of a range to reflect, and be interchangeable with, disclosing any and all sub- ranges and combinations of sub-ranges encompassed therein.

[0018] The terms "comprises" and variations thereof do not have a limiting meaning wherethese terms appear in the description and claims.

[0019] As used herein, "a," "an," "the," "at least one," and "one or more" are usedinterchangeably. Thus, for example, a microorganism can be interpreted to mean "one or more" microorganisms.

[0020] The term "and / or" means one or all of the listed elements or a combination of anytwo or more of the listed elements.

[0021] The present invention provides an enrichment medium useful for repair and growthof gram-negative bacteria.

[0022] In one aspect, provided herein is an enrichment medium (e.g., enrichment broth)comprising pyruvic acid, magnesium, and yeast extract.

[0023] In another aspect, provided herein is an enrichment medium (e.g., enrichment broth)comprising sodium pyruvate, magnesium sulfate, and yeast extract.

[0024] In another aspect, provided herein is an enrichment medium (e.g., enrichment broth)comprising pyruvic acid, magnesium, yeast extract, a selectivity agent that inhibits the growth of gram-positive bacteria, and water.

[0025] In another aspect, provided herein is an enrichment medium (e.g., enrichment broth)comprising sodium pyruvate, magnesium sulfate, yeast extract, a selectivity agent that inhibits the growth of gram-positive bacteria, and water.

[0026] The combined use of pyruvic acid (e.g., sodium pyruvate), magnesium (e.g.,magnesium sulfate), and yeast extract reduces growth times compared to other enrichment media, such as buffered peptone water (BPW). The use of magnesium sulfate in combination with yeast extract provides the unexpected benefit of reduced growth time while also reducing the cost of the enrichment media due to the lower cost of magnesium sulfate compared to yeast extract.

[0027] The enrichment medium may include a selectivity agent that inhibits the growth ofgram-positive bacteria. This selectivity is beneficial for faster growth of the target pathogen, i.e., gram-negative bacteria, such as Salmonella, E. coli, or Cronobacter. For example, when selectivity agents are not present, growth rate may be slowed due to nutrient exclusion as a result of proliferation of competitive bacteria.

[0028] In some embodiments, the selectivity agent is selected from the group consisting ofantibiotics, detergents, and combinations thereof. In some embodiments, the selectivity agent is selected from the group consisting of vancomycin or a salt thereof, sodium 3 59099806.1Attorney Docket No.: 234994-559211 dodecylbenzenesulfonate (SDBS), novobiocin or a salt thereof, cefsulodin or a salt thereof, sulfadiazine or a salt thereof, bile salts, sodium lauryl sulfate (SLS), sodium dodecylbenzesulfonate (SDBS), Tergitol 4, and combinations thereof.

[0029] In some embodiments, the selectivity agent is selected from the group consisting ofvancomycin or a salt thereof, SDBS, and combinations thereof. In some embodiments, the selectivity agent is a combination of vancomycin or a salt thereof and SDBS. In some embodiments, the selectivity agent is vancomycin or a salt thereof. In some embodiments, the selectivity agent is vancomycin. In some embodiments, the selectivity agent is SDBS. In some embodiments, SDBS is the only selectivity agent. Vancomycin and / or SDBS inhibit gram-positive bacteria without harming injured pathogen cells. This also contributes to rapid growth rate of bacteria using the enrichment medium of the present invention.

[0030] Pyruvic acid may be in the form of a salt. For example, the pyruvic acid salt may besodium pyruvate, potassium pyruvate, or a combination thereof. In some embodiments, the pyruvic acid salt is sodium pyruvate. In solution, the pyruvic acid and the sodium cation may be dissociated, such that the sodium pyruvate is in the form of separate sodium cations and pyruvic acid anions. Likewise, potassium pyruvate may be in the form of separate potassium cations and pyruvic acid anions.

[0031] In some embodiments, the sodium pyruvate is present in the enrichment medium ina concentration of from about 0.1 to about 5 grams per liter, e.g., from about 0.2 to about 3 grams per liter, from about 0.5 to about 2 grams per liter, or from about 1 to about 1.5 grams per liter. Sodium pyruvate was found to significantly increase growth rate of bacteria in the enrichment medium of the present invention. As described in Example 3 below, certain concentrations of sodium pyruvate resulted in particularly rapid growth rate.

[0032] In some embodiments, pyruvic acid is present in the enrichment medium in aconcentration of from about 0.08 to about 4 grams per liter, e.g., from about 0.16 to about 2.4 grams per liter, from about 0.4 to about 1.6 grams per liter, or from about 0.8 to about 1.2 grams per liter.

[0033] Magnesium may be in the form of a salt. For example, the magnesium may bemagnesium sulfate, magnesium chloride, or a combination thereof. In some embodiments, the magnesium salt is magnesium sulfate. In some embodiments, the magnesium sulfate in the enrichment medium is anhydrous magnesium sulfate.

[0034] In some embodiments, the concentration of yeast extract in the enrichment mediumis from about 1 to about 9 grams per liter (e.g., from about 2.5 to about 7.5 grams per liter or from about 4 to about 6 grams per liter) and the concentration of magnesium sulfate is from about 0.5 to about 10 millimolar (e.g., from about 1 to about 5 millimolar or from about 2 to 4 59099806.1Attorney Docket No.: 234994-559211 about 4 millimolar). As noted above, the combined use of pyruvic acid (e.g., sodium pyruvate), magnesium (e.g., magnesium sulfate), and yeast extract is a surprising synergistic combination that reduces growth times compared to other enrichment media, such as buffered peptone water (BPW). The concentrations of sodium pyruvate, magnesium sulfate, and yeast extract in the enrichment medium also contribute to the reduced growth times.

[0035] In some embodiments, the concentration of yeast extract in the enrichment mediumis from about 1 to about 9 grams per liter (e.g., from about 2.5 to about 7.5 grams per liter or from about 4 to about 6 grams per liter) and the concentration of magnesium is from about 0.5 to about 10 millimolar (e.g., from about 1 to about 5 millimolar or from about 2 to about 4 millimolar).

[0036] In some embodiments, the enrichment medium further comprises potassiumchloride. In some embodiments, the enrichment medium further comprises sodium chloride. In some embodiments, the sodium chloride is in a concentration of less than or equal to about 3 grams per liter, e.g., from about 1.5 to about 2.5 grams per liter. Sodium chloride levels are normally 5 grams per liter in traditional media like buffered peptone water. The enrichment medium of the present invention may have reduced levels of sodium chloride, such as less than 5 grams per liter, less than about 3 grams per liter, or from about 1.5 to about 2.5 grams per liter. In some embodiments, the enrichment medium comprises from about 1 gram per liter to about 5 grams per liter of potassium chloride, e.g., from about 1.5 grams per liter to about 3 grams per liter.

[0037] In some embodiments, the enrichment medium is substantially free of sodiumphosphate. In some embodiments, the enrichment medium further comprises potassium phosphate (e.g., monobasic and / or dibasic). In some embodiments, higher phosphate levels are utilized in the enrichment medium of the present invention, which reduces shifts in pH.

[0038] In some embodiments, the enrichment medium further comprises phenol red. Thepresence of phenol red may aid as a pH indicator in case the sample enrichment starts off with an unusually acidic starting pH. In such an instance, NaOH may be added to balance pH to neutral. In some embodiments, the pH of the enrichment medium is from about 6.5 to about 7.5, e.g., from about 6.8 to about 7.2, or about 7.

[0039] In some embodiments, the enrichment medium further comprises glucose. In someembodiments, the enrichment medium comprises dextrose. In some embodiments, the enrichment medium comprises from about 1 gram per liter to about 5 grams per liter of dextrose, e.g., from about 1 gram per liter to about 3 grams per liter.

[0040] In some embodiments, the enrichment medium further comprises casein peptone. Insome embodiments, the concentration of casein peptone in the enrichment medium is reduced 5 59099806.1Attorney Docket No.: 234994-559211 compared to traditional enrichment medium. For example, the concentration of casein peptone in the enrichment medium may be less than about 10 grams per liter, less than about 7.5 grams per liter, from about 2 grams per liter to about 10 grams per liter, from about 2.5 grams per liter to about 7.5 grams per liter, or from about 4 grams per liter to about 6 grams per liter.

[0041] In some embodiments, the enrichment medium further comprises trisodium citratedihydrate. In some embodiments, the concentration of trisodium citrate dihydrate in the enrichment medium is from about 0.1 to about 2 grams per liter, e.g. from about 0.2 to about 1 grams per liter.

[0042] In some embodiments, the enrichment medium further comprises polysorbate 80(Tween 80).

[0043] In some embodiments, the enrichment medium is substantially free of chicken fat,thioglycolate, and / or mannitol. In some embodiments, the enrichment medium is substantially free of iron. In some embodiments, the enrichment medium is substantially free of casamino acids and / or lactose.

[0044] The enrichment medium may be irradiated, autoclavable, and / or ready-to-use.

[0045] In some embodiments, the enrichment medium comprises:from about 0.2 to about 3 grams per liter of sodium pyruvate; from about 2.5 to about 7.5 grams per liter of yeast extract; from about 1 to about 5 millimolar of magnesium sulfate; and vancomycin, SDBS, or a combination thereof.

[0046] In some embodiments, the enrichment medium comprises:from about 0.2 to about 3 grams per liter of sodium pyruvate; from about 2.5 to about 7.5 grams per liter of yeast extract; from about 1 to about 5 millimolar of magnesium sulfate; vancomycin, SDBS, or a combination thereof; casein peptone; sodium chloride; and potassium phosphate.

[0047] In some embodiments, the enrichment medium is capable of growing a samplecontaining gram-negative bacteria at 1 to 5 CFU / ml to produce an enriched sample containing greater than or equal to 1,000 CFU / ml after 6 hours of incubation at 42^C. In some embodiments, the enrichment medium is capable of growing a sample containing gram- 6 59099806.1Attorney Docket No.: 234994-559211 negative bacteria at 1 to 5 CFU / ml to produce an enriched sample containing greater than or equal to 10,000 CFU / ml after 6 hours of incubation at 42^C.

[0048] In another aspect, provided herein is an enrichment medium formulation usefulfor mixing with water to prepare an enrichment medium, the enrichment medium formulation comprising a pyruvic acid salt, a magnesium salt, and yeast extract. In another aspect, provided herein is an enrichment medium formulation useful for mixing with water to prepare an enrichment medium, the enrichment medium formulation comprising a pyruvic acid salt, a magnesium salt, yeast extract, and a selectivity agent that inhibits the growth of gram-positive bacteria. In another aspect, provided herein is an enrichment medium formulation useful for mixing with water to prepare an enrichment medium, the enrichment medium formulation comprising sodium pyruvate, magnesium sulfate, and yeast extract. In another aspect, provided herein is an enrichment medium formulation useful for mixing with water to prepare an enrichment medium, the enrichment medium formulation comprising sodium pyruvate, magnesium sulfate, yeast extract, and a selectivity agent that inhibits the growth of gram-positive bacteria. Such enrichment medium formulations may be in the form of a powder. Thus, embodiments of the present invention include dry formulations, e.g., a powder, and liquid formulations, e.g., with the materials mixed or dissolved in water.

[0049] The selectivity agents for use in the enrichment medium formulation (e.g.,powder) may include any of the various selectivity agents as described above with regard to the enrichment medium. The magnesium sulfate in the enrichment medium formulation may be anhydrous. For dry formulations (e.g., a powder), the pyruvic acid is in the form of a salt. Suitable salts include sodium pyruvate and potassium pyruvate. In some embodiments, the pyruvic acid salt is sodium pyruvate. Magnesium is also in salt form in dry formulations. Suitable salts include magnesium sulfate and magnesium chloride. In some embodiments, the magnesium salt is magnesium sulfate.

[0050] In some embodiments, the enrichment medium formulation (e.g., powder)comprises the sodium pyruvate in an amount of from about 0.4 wt% to about 20 wt% by weight of the enrichment medium formulation. For example, the amount of sodium pyruvate may be from about 0.8 wt% to about 12 wt%, from about 2 wt% to about 8 wt%, or from about 3.5 wt% to about 6 wt%, by weight of the enrichment medium formulation. “wt%” refers to weight percent, also known as percent by weight or percentage by weight.

[0051] In some embodiments, the enrichment medium formulation (e.g., powder)comprises the pyruvic acid salt in an amount of from about 0.4 wt% to about 20 wt% by weight of the enrichment medium formulation. For example, the amount of pyruvic acid salt may be from about 0.8 wt% to about 12 wt%, from about 2 wt% to about 8 wt%, or from 7 59099806.1Attorney Docket No.: 234994-559211 about 3.5 wt% to about 6 wt%, by weight of the enrichment medium formulation. “wt%” refers to weight percent, also known as percent by weight or percentage by weight.

[0052] In some embodiments, the enrichment medium formulation (e.g., powder)comprises the yeast extract in an amount of from about 4 wt% to about 35 wt% by weight of the enrichment medium formulation, and comprises the magnesium sulfate in an amount of from about 0.05 wt% to about 2 wt% by weight of the enrichment medium formulation. For example, the amount of yeast extract may be from about 10 wt% to about 30 wt% by weight of the enrichment medium formulation, and the amount of magnesium sulfate may be from about 0.1 wt% to about 1 wt% by weight of the enrichment medium formulation; or the amount of yeast extract may be from about 15 wt% to about 25 wt% by weight of the enrichment medium formulation, and the amount of magnesium sulfate may be from about 0.2 wt% to about 0.6 wt% by weight of the enrichment medium formulation.

[0053] In some embodiments, the enrichment medium formulation (e.g., powder)comprises the yeast extract in an amount of from about 4 wt% to about 35 wt% by weight of the enrichment medium formulation, and comprises the magnesium salt in an amount of from about 0.05 wt% to about 2 wt% by weight of the enrichment medium formulation. For example, the amount of yeast extract may be from about 10 wt% to about 30 wt% by weight of the enrichment medium formulation, and the amount of magnesium salt may be from about 0.1 wt% to about 1 wt% by weight of the enrichment medium formulation; or the amount of yeast extract may be from about 15 wt% to about 25 wt% by weight of the enrichment medium formulation, and the amount of magnesium salt may be from about 0.2 wt% to about 0.6 wt% by weight of the enrichment medium formulation.

[0054] The enrichment medium formulation may comprise any of the ingredients asdescribed above with regard to various embodiments of the enrichment medium, including sodium chloride, potassium chloride, phenol red, glucose (e.g., dextrose), casein peptone, potassium phosphate (dibasic and / or monobasic), trisodium citrate dihydrate, polysorbate 80, and combinations thereof.

[0055] In some embodiments, the enrichment medium formulation (e.g., powder)comprises sodium chloride in an amount of less than or equal to about 12 wt% by weight of the enrichment medium formulation, e.g., from about 5 wt% to about 10 wt% by weight of the enrichment medium formulation.

[0056] In some embodiments, the enrichment medium formulation (e.g., powder)comprises potassium chloride in an amount of from about 4 wt% to about 20 wt% by weight of the enrichment medium formulation, e.g., from about 4 wt% to about 12 wt%. 8 59099806.1Attorney Docket No.: 234994-559211

[0057] In some embodiments, the enrichment medium formulation (e.g., powder)comprises dextrose in an amount of from about 4 wt% to about 20 wt% by weight of the enrichment medium formulation, e.g., from about 4 wt% to about 12 wt%.

[0058] In some embodiments, the enrichment medium formulation (e.g., powder)comprises casein peptone in an amount of less than about 40 wt%, less than about 30 wt%, from about 8 to about 40 wt%, from about 10 to about 30 wt%, or from about 15 to about 25 wt% by weight of the enrichment medium formulation.

[0059] In some embodiments, the enrichment medium formulation (e.g., powder)comprises trisodium citrate dihydrate in an amount from about 0.4 to about 8 wt%, e.g., from about 0.8 to about 4 wt% by weight of the enrichment medium formulation.

[0060] In some embodiments, the enrichment medium formulation is substantially freeof sodium phosphate. In some embodiments, the enrichment medium formulation (e.g., powder) comprises potassium phosphate (e.g., monobasic and / or dibasic).

[0061] In some embodiments, the enrichment medium formulation is substantially freeof chicken fat, thioglycolate, and / or mannitol. In some embodiments, the enrichment medium is substantially free of iron. In some embodiments, the enrichment medium is substantially free of casamino acids and / or lactose.

[0062] In some embodiments, the enrichment medium formulation comprisesfrom about 0.8 wt% to about 12 wt% of sodium pyruvate; from about 10 wt% to about 30 wt% of yeast extract; from about 0.1 wt% to about 1 wt% magnesium sulfate; and vancomycin, SDBS, or a combination thereof.

[0063] In some embodiments, the enrichment medium formulation comprisesfrom about 0.8 wt% to about 12 wt% of sodium pyruvate; from about 10 wt% to about 30 wt% of yeast extract; from about 0.1 wt% to about 1 wt% magnesium sulfate; vancomycin, SDBS, or a combination thereof; casein peptone; sodium chloride; and potassium phosphate.

[0064] In some embodiments, the ingredients of the enrichment formulation (e.g.,according to the various embodiments described herein) are blended or combined in the powder. For example, in some embodiments, each of the ingredients to be present in the enrichment medium are blended or combined into a ready to use powder. For example, the powder may be such that when added to water, thereby forming an enrichment medium, the 9 59099806.1Attorney Docket No.: 234994-559211 enrichment medium is used in the method of enrichment without further addition of ingredients – i.e., all of the ingredients for enrichment may be present in the powder or may be present in the enrichment media prior to initiation of its use for enrichment.

[0065] In some embodiments, the enrichment medium formulation (e.g., powder) isirradiated. The enrichment medium formulation may be autoclavable and / or ready-to-use. As described above the enrichment medium formulation (e.g., powder) can be mixed with (e.g., dissolved in) water to make enrichment media for growing gram-negative bacteria.

[0066] In one aspect, provided herein is a method of making enrichment media,comprising mixing an enrichment medium formulation as described herein with water. In one embodiment of this aspect, provided herein is a method of making enrichment media, comprising dissolving an enrichment medium formulation as described herein in water.

[0067] In another aspect, provided herein is a method of growing gram-negative bacteriain an enrichment medium, comprising providing a sample comprising gram-negative bacteria, providing an enrichment medium according to one or more embodiments described herein, and growing the gram-negative bacteria in the enrichment medium.

[0068] In some embodiments, the gram-negative bacteria comprises Salmonella, E. coli,Cronobacter, or any combination thereof.

[0069] In some embodiments, growing the gram-negative bacteria in the enrichmentmedium includes incubating the gram-negative bacteria in the enrichment medium. In some embodiments, the incubation temperature is from about 34 to about 43 °C, e.g., about 35 °C or about 42°C.

[0070] In some embodiments, the method further comprises mixing an enrichmentmedium formulation (e.g., a powder) as described herein with water to prepare the enrichment medium.

[0071] In some embodiments, the method further comprises autoclaving the enrichmentmedium. In some embodiments, the enrichment medium is autoclaved at a temperature of less than or equal to 120 °C, e.g., less than or equal to 115 °C, less than or equal to about 110 °C, from about 100 to about 120 °C, from about 105 to about 115°C, or about 110 °C. The enrichment medium may include vancomycin in the medium. Autoclaving at lower temperature allows for the inclusion of vancomycin in the medium without the need to add vancomycin after autoclaving, as would be required at higher temperatures. This allows vancomycin to be included in an autoclavable and / or ready to use product.

[0072] In some embodiments, the method comprises growing the gram-negative bacteriain the enrichment medium for about 1 to about 8 hours, e.g., from about 2 hours to about 8 hours, from about 4 hours to about 8 hours, from about 6 hours to about 8 hours, or from 10 59099806.1Attorney Docket No.: 234994-559211 about 4 hours to about 6 hours. In some embodiments, the gram-negative bacteria are used in a detection assay after about 6 hours or less of growth time in the enrichment medium. In some embodiments, the gram-negative bacteria are used in a detection assay after about 4 to about 6 hours of growth time in the enrichment medium. The rapid growth rate achieved according to the present invention may allow for detection of the target pathogen (i.e., gram- negative bacteria) on the same day that a sample is collected and / or during the same working shift that the sample is collected. After enrichment, a portion of the sample may be aliquoted for testing with a molecular assay.

[0073] In some embodiments, the method comprises growing a sample of gram-negativebacteria containing 1 to 5 CFU / ml to produce an enriched sample containing greater than or equal to 1,000 CFU / ml after 6 hours of incubation at 42^C. In some embodiments, the method comprises growing a sample of gram-negative bacteria containing 1 to 5 CFU / ml to produce an enriched sample containing greater than or equal to 10,000 CFU / ml after 6 hours of incubation at 42^C.

[0074] Also disclosed herein is an enrichment medium useful for repair and growth ofgram-negative bacteria, comprising: from about 0.5 to about 2 grams per liter of sodium pyruvate; from about 2.5 to about 7.5 grams per liter of yeast extract; from about 1 to about 5 millimolar of magnesium sulfate; and water. In some embodiments, the concentration of yeast extract is from about 4 to about 6 grams per liter, and the concentration of magnesium sulfate is from about 2 to about 4 millimolar. In some embodiments, the enrichment medium further comprises potassium chloride and less than or equal to about 3 grams per liter of sodium chloride.

[0075] Also disclosed herein is an enrichment medium useful for repair and growth ofgram-negative bacteria, comprising: from about 0.4 to about 1.6 grams per liter of pyruvic acid; from about 2.5 to about 7.5 grams per liter of yeast extract; from about 1 to about 5 millimolar of magnesium; and water. In some embodiments, the concentration of yeast extract is from about 4 to about 6 grams per liter, and the concentration of magnesium is from about 2 to about 4 millimolar. In some embodiments, the enrichment medium further comprises potassium chloride and less than or equal to about 3 grams per liter of sodium chloride.

[0076] Also disclosed herein is an enrichment medium formulation useful for mixingwith water to prepare an enrichment medium, the enrichment medium formulation comprising: from about 2 wt% to about 8 wt% of sodium pyruvate; from about 10 wt% to about 30 wt% of yeast extract; from about 0.1 wt% to about 1 wt% magnesium sulfate; and 11 59099806.1Attorney Docket No.: 234994-559211 water. In some embodiments, the amount of yeast extract is from about 15 wt% to about 25 wt% by weight of the enrichment medium formulation, and the amount of magnesium sulfate is from about 0.2 wt% to about 0.6 wt% by weight of the enrichment medium formulation. In some embodiments, the enrichment medium further comprises potassium chloride and less than or equal to about 12 wt% of sodium chloride.

[0077] Also disclosed herein is an enrichment medium formulation useful for mixingwith water to prepare an enrichment medium, the enrichment medium formulation comprising: from about 2 wt% to about 8 wt% of a pyruvic acid salt; from about 10 wt% to about 30 wt% of yeast extract; from about 0.1 wt% to about 1 wt% a magnesium salt; and water. In some embodiments, the amount of yeast extract is from about 15 wt% to about 25 wt% by weight of the enrichment medium formulation, and the amount of magnesium salt is from about 0.2 wt% to about 0.6 wt% by weight of the enrichment medium formulation. In some embodiments, the enrichment medium further comprises potassium chloride and less than or equal to about 12 wt% of sodium chloride.

[0078] EXAMPLES

[0079] Example 1 – Exemplary Formula

[0080] Table 1 shows an exemplary formulation for an enrichment medium and anenrichment medium powder. The enrichment medium contains the listed materials in their respective concentrations in water. The enrichment medium powder contains the listed materials in their respective weight percentage concentrations (exclusive of water). Table 1 Enrichment Medium Enrichment Medium Powder (exclusive of59099806.1Attorney Docket No.: 234994-559211 Phenol Red 0.050 0.2% Antifoam 0.050 0.2%Bioingredients, Doral, FL), ProCel 351 PW Yeast Extract (Procelys, Milwaukee, WI), Sodium Chloride (American International Foods, Ada, MI), Potassium Chloride (RPI Corp, Mt. Prospect, IL), Dextrose, Cerelose Anhydrous (American International Foods, Ada, MI), Sodium Pyruvate (Gojira Fine Chemicals, Bedford Heights, OH), Potassium Phosphate Dibasic Anhydrous (Gojira Fine Chemicals, Bedford Heights, OH), Potassium Phosphate Monobasic Anhydrous (Gojira Fine Chemicals, Bedford Heights, OH), Magnesium Sulfate Anhydrous (RPI Corp, Mt. Prospect, IL), Phenol Red (Curtiss Laboratories, Ivyland, PA), Antifoam, Powder, DF-093 (Anhui Sinograce Chemical Co., LTD, Anhui Province, China), Sodium dodecylbenzenesulfonate (TCI Chemicals, Portland OR), Vancomycin Hydrochloride (Chem-Impex International Inc., Wood Dale, IL), TWEEN 80 (RPI Corp, Mt. Prospect, IL).

[0082] Example 2 – Growth Results for Healthy and Heat-Injured Salmonella EnteritidisATCC 13076 (SE 13076) Relative to Buffered Peptone Water (BPW) with Various Levels of Growth Enhancers

[0083] Ranges of four different growth enhancers were evaluated by growth curveanalysis and the use of Bioscreen C Pro (Oy Growth Curves Ab Ltd., Finland). A blend of enrichment medium (“common base”) was created without the four growth enhancers being analyzed. The common base was as follows: Casein Peptone 5.0 g / L, Sodium Chloride 2.0 g / L, Potassium Chloride 2.0 g / L, Potassium Phosphate, Dibasic 5.675 g / L, Potassium Phosphate, Monobasic 2.40 g / L, Citric Acid, Trisodium Citrate, Dihydrate 0.50 g / L, Vancomycin 0.005 g / L, Sodium Dodecylbenzenesulfonate (SDBS) 0.020 g / L, and Phenol Red 0.050 g / L. Minitab 18 generated a table of combination of different amounts of these four growth enhancers to be tested. The common base was used to prepare 25 versions for testing the ranges of yeast extract (0 to 10g / L), magnesium (0 to 4mM), dextrose (0 to 4g / L), and sodium pyruvate (0 to 2g / L). Media were sterilized at 110°C for 15 minutes and cooled before use.

[0084] Test strains were first grown on tryptic soy agar (TSA) plates at 35°C overnightbefore being suspended in sterile phosphate buffered saline (PBS) tubes and serially diluted to produce the target of approximately 10 – 50 cfu per strain (10ul) into Bioscreen wells 13 59099806.1Attorney Docket No.: 234994-559211 containing each of the 25 DOE media. The Minitab 18 software calculated that there would be a total of 18 replicates for one set of the 4 components (yeast extract at 5g / L, magnesium at 2mM, dextrose at 2g / L, and pyruvate at 1g / L). The remainder of the 24 media version had triplicates for a total of 90 Bioscreen wells inoculated per strain. Check plates were inoculated and incubated at 35°C for 24 hours.

[0085] Healthy cells of Salmonella Typhimurium ATCC 13076 were inoculated with anaverage of 35.0 cfu per well. Salmonella Enteritidis ATCC 13076 was selected for heat injury prior to enrichment in versions of the broth. Heat injury was performed on an overnight TSB culture (10ml) that was heated at 55°C for 10 minutes. Dilutions of Salmonella Enteritidis ATCC 13076 were plated onto TSA in triplicate before and after the cells were heat injured. TSA plates were incubated at 35°C for 48 hours to demonstrate that heat injury produced 2.6 logs die-off. Heat-injured cells of Salmonella Enteritidis (10ul) inoculated into Bioscreen wells (0.3ml) were an average of 35.0 cfu per well.

[0086] Bioscreen experiments were run for 48 hours at 42°C, with readings every 15minutes without shaking. Absorbance was recorded at 600nm for each Bioscreen run. At the completion of the Bioscreen runs, the initial absorbance readings were normalized to 0.100 per sample. Growth performance was evaluated by determining how many hours were needed to have the absorbance reach or pass 0.300 per replicate. These time values to reach 0.3 absorbance were entered into Minitab 18.

[0087] Minitab 18 was used to analyze the response surface of this data. Models werebuilt using a stepwise technique with alpha to enter or leave the model set at a significance level of 0.05.

[0088] SE 13076 was grown overnight in tryptic soy broth (TSB) and tested as healthy, andafter heat injury at 50°C for 15 minutes. Optical density (OD) readings were taken and the time to reach 0.3 OD was recorded. The results are shown in Table 2. 14 59099806.1Attorney Docket No.: 234994-559211 Table 2 Growth Enhancer Levels Growth Results (avg of triplicate) (Added to Common Base) SE 13076, Healthy SE 13076, Heat- on thp g y gy y , g pyseen by the growth results for Examples 2F and 2G. 2F has very good recovery of healthy and heat-injured SE 13076, while 2G, without pyruvate, has poor recovery.

[0090] Buffered peptone water, which was used for comparison in these examples, has thefollowing formulation: Enzymatic Digest of Casein, 10.0 g / L; Sodium Chloride, 5.0 g / L; Disodium Hydrogen Phosphate (anhydrous), 3.6 g / L; and Potassium Dihydrogen Phosphate, 1.5 g / L.

[0091] Example 3 – Recovery enhancers of injured cells comprising antioxidants such aspyruvate

[0092] Cells exposed to sublethal injury require substantially longer to recover from theinjury. To decrease the recovery time, antioxidants such as pyruvate can be added to the formulation. In this and in additional examples below an experiment was run where the response surface was modeled relative to the response of the strain of interest in the comparator case of BPW. The response variable in these experiments was how much faster the culture reached turbidity (OD change=0.2) relative to BPW.

[0093] Test strains were first grown on tryptic soy agar (TSA) plates at 35°C overnightbefore being suspended in sterile PBS tubes and serially diluted to produce the target of approximately 10 – 50 cfu per strain (10ul) into Bioscreen wells containing each of the 25 15 59099806.1Attorney Docket No.: 234994-559211 DOE media. The Minitab 18 software calculated that there would be a total of 18 replicates for one set of the 4 components (yeast extract at 5g / L, magnesium at 2mM, dextrose at 2g / L, and pyruvate at 1g / L). The remainder of the 24 media version had triplicates for a total of 90 Bioscreen wells inoculated per strain. Check plates were inoculated and incubated at 35°C for 24 hours.

[0094] Healthy cells of Salmonella Typhimurium ATCC 13076 were inoculated with anaverage of 35.0 cfu per well. Salmonella Enteritidis ATCC 13076 was selected for heat injury prior to enrichment in versions of the broth. Heat injury was performed on an overnight TSB culture (10ml) that was heated at 55°C for 10 minutes. Dilutions of Salmonella Enteritidis ATCC 13076 were plated onto TSA in triplicate before and after the cells were heat injured. TSA plates were incubated at 35°C for 48 hours to demonstrate that heat injury produced 2.6 logs die-off. Heat-injured cells of Salmonella Enteritidis (10ul) inoculated into Bioscreen wells (0.3ml) were an average of 35.0 cfu per well.

[0095] Bioscreen experiments were run for 48 hours at 42°C, with readings every 15minutes without shaking. Absorbance was recorded at 600nm for each Bioscreen run. At the completion of the Bioscreen runs, the initial absorbance readings were normalized to 0.100 per sample. Growth performance was evaluated by determining how many hours were needed to have the absorbance reach or pass 0.300 per replicate. These time values to reach 0.3 absorbance were entered into Minitab 18.

[0096] Minitab 18 was used to analyze the response surface of this data. Models werebuilt using a stepwise technique with alpha to enter or leave the model set at a significance level of 0.05.

[0097] Table 3 shows statistical analysis using Minitab software (version 18.1) forhealthy S. Enteritidis ATCC 13076 recovery relative to pyruvate levels and BPW. Yeast Extract and Magnesium were fixed at nominal levels of 5g / L and 2mM respectively in Common Base with the varying levels of sodium pyruvate as shown in Table 3. 16 59099806.1Attorney Docket No.: 234994-559211 Table 3 Pyruvate Time (hrs.) % Reduction ( / L) to Reach 0.3 in Growth

[0098] Unexpectons was not linear as thepyruvate levels increased. Growth time reductions leveled off at 1 g / L and decreased as Pyruvate levels increased to 2 g / L.

[0099] Example 4 – Magnesium combined with yeast extract allows for a reduced use ofyeast extract

[0100] To measure the effect of nutrients with unstressed cells, Salmonella strain ATCC13076 was used. In this experiment, there were significant interactions and large main effects between yeast extract and magnesium to improve Salmonella growth rates relative to BPW. This interaction yields a cost savings opportunity. Yeast extract is relatively expensive while magnesium is relatively inexpensive. Thus, as shown, as more Magnesium is added less yeast extract is required in the enrichment medium to obtain the same time savings. This optimum ratio is approximately 1:2 where 1 mM of additional magnesium added results in the need to add 2 g / L less of yeast extract.

[0101] Test strains were first grown on tryptic soy agar (TSA) plates at 35°C overnightbefore being suspended in sterile PBS tubes and serially diluted to produce the target of approximately 10 – 50 cfu per strain (10ul) into Bioscreen wells containing each of the 25 DOE media. The Minitab 18 software calculated that there would be a total of 18 replicates for one set of the 4 components (yeast extract at 5g / L, magnesium at 2mM, dextrose at 2g / L, and pyruvate at 1g / L). The remainder of the 24 media version had triplicates for a total of 90 Bioscreen wells inoculated per strain. Check plates were inoculated and incubated at 35°C for 24 hours.

[0102] Healthy cells of Salmonella Typhimurium ATCC 13076 were inoculated with anaverage of 35.0 cfu per well. Salmonella Enteritidis ATCC 13076 was selected for heat injury prior to enrichment in versions of the broth. Heat injury was performed on an 17 59099806.1Attorney Docket No.: 234994-559211 overnight TSB culture (10ml) that was heated at 55°C for 10 minutes. Dilutions of Salmonella Enteritidis ATCC 13076 were plated onto TSA in triplicate before and after the cells were heat injured. TSA plates were incubated at 35°C for 48 hours to demonstrate that heat injury produced 2.6 logs die-off. Heat-injured cells of Salmonella Enteritidis (10ul) inoculated into Bioscreen wells (0.3ml) were an average of 35.0 cfu per well.

[0103] Bioscreen experiments were run for 48 hours at 42°C, with readings every 15minutes without shaking. Absorbance was recorded at 600nm for each Bioscreen run. At the completion of the Bioscreen runs, the initial absorbance readings were normalized to 0.100 per sample. Growth performance was evaluated by determining how many hours were needed to have the absorbance reach or pass 0.300 per replicate. These time values to reach 0.3 absorbance were entered into Minitab 18.

[0104] Minitab 18 was used to analyze the response surface of this data. Models werebuilt using a stepwise technique with alpha to enter or leave the model set at a significance level of 0.05.

[0105] Table 4 shows the statistical analysis using Minitab software (version 18.1)showing faster growth of healthy SE 13076 relative to BPW when Magnesium and Yeast Extract (YE) levels were adjusted. Pyruvate and Dextrose were fixed at nominal levels of 1g / L and 2g / L respectively. Table 4 Magnesium (mM) 0 1 2 3 4

[0106] Growth rate is slowest in BPW, followed by the situation when Yeast Extract andMagnesium are removed from the media.

[0107] Surprisingly, incorporation of Mg allowed for lower amounts of Yeast Extract,which helps use less Yeast Extract for cost savings and for less challenges with sterilization (Maillard Reactions from heat and sugar).

[0108] Example 5 - Selective agent to restrict the growth of gram-positive bacteria18 59099806.1Attorney Docket No.: 234994-559211

[0109] For evaluation of gram-positive exclusivity performance results, the followingsteps were performed. Buffered Peptone Water (BPW) (Neogen, Lansing, MI, USA) was prepared by dissolving 20 g / L enrichment medium in DI water and sterilized by autoclavingfor 15 minutes at 121°C. The Example 1 Broth was prepared by dissolving 25.75 g / Lenrichment medium (components described in Example 1, Table 1) in DI water and sterilizing by autoclaving for 15 minutes at 110°C. Three gram-positive bacteria of Bacillus cereus ATCC 27348, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 6538 were employed. Test strains were first grown on tryptic soy agar (TSA) plates at 35°C overnight before being suspended in sterile PBS tubes and serially diluted to produce the target of 104 cfu per strain into duplicate tubes (10ml each). Check plates were inoculated and incubated at 35°C for 24 hours.

[0110] Inoculated tubes were placed in a 42°C water bath for 5 minutes before beingplaced in a 42°C incubator for the remainder of 6 hours. Tubes were removed and mixed by vortexing after 6 hours of incubation; 1ml of each duplicate tube was respectively combined into a sterile tube and mixed. Aliquots (10ul) of the combined duplicate tubes were aseptically removed from each BPW and enrichment medium suspension and spread onto their respective sterile TSA plates. TSA plates were incubated at 35°C for 24 hours and cell counts were recorded.

[0111] Table 5 shows exclusive organisms and recovery (cfu / ml) at 6 hours anddemonstrates the ability of the enrichment medium to select for gram-negative pathogens (suppress gram-positive bacteria) and shows the effect of vancomycin at (a gram-positive specific antibiotic at 5 mg / L) on restricting the growth of gram-positive bacteria when compared to non-selective BPW. Table 5 Plate Counts(cfu / ml)8

[0112] The Example 1 broth has negligible recovery of these exclusivity strainscompared to the reference method cited in the art. This is in contrast to the broad growth benefit seen with gram-negative pathogens such as Cronobacter, pathogenic E. coli, and Salmonella. 19 59099806.1Attorney Docket No.: 234994-559211

[0113] Test strains were first grown on tryptic soy agar (TSA) plates at 35°C overnightbefore being suspended in sterile PBS tubes and serially diluted to produce the target of approximately 10 cfu per strain into triplicate tubes of BPW and enrichment medium tubes (10ml each). Innoculation check plates were inoculated and incubated at 35°C for 24 hours.

[0114] Inoculated tubes of BPW and enrichment media were placed in a 42°C waterbath for 5 minutes before being placed in a 42°C incubator for the remainder of 6 hours. Tubes were removed and mixed by vortexing after 6 hours of incubation. Replicate aliquots (10ul) were aseptically removed from each suspension and spread onto their respective sterile plates (VRBA, TSA, XLD, or CASE). Plates were incubated at 35°C for 24 hours and cell counts were recorded.

[0115] Example 1 Broth reduces lag time of gram-negative pathogens using asynergistic range of growth enhancers (Yeast Extract, Magnesium, Pyruvate, and Dextrose). Additionally, Example 1 Broth can greatly suppress gram-positive bacteria that might compete with the recovery of the target gram-negative pathogens. The Example 1 Broth formulation has a unique combination of media components for growth and selectivity to shorten lag time and improve rapid detection of pathogens. Example 1 Broth used in this testing had Yeast Extract (5g / L), Magnesium (2mM), Dextrose (2g / L), and Pyruvate (1g / L).

[0116] Table 6 shows the enhanced recovery of target gram-negative organisms (cfu / ml)in Example 1 Broth compared to reference BPW media at 6 Hours of incubation (42^C). Table 6 A. Non-Salmonella BPW Example 1 BrothE l 1 159099806.1

Claims

Attorney Docket No.: 234994-559211 CLAIMS WHAT IS CLAIMED IS:

1. An enrichment medium useful for repair and growth of gram-negative bacteria, comprising: pyruvic acid; magnesium; yeast extract; a selectivity agent that inhibits the growth of gram-positive bacteria; and water.

2. The enrichment medium of claim 1, wherein the pyruvic acid is in the form of sodium pyruvate.

3. The enrichment medium of claim 1, wherein the magnesium is in the form of magnesium sulfate.

4. An enrichment medium useful for repair and growth of gram-negative bacteria, comprising: sodium pyruvate; magnesium sulfate; yeast extract; a selectivity agent that inhibits the growth of gram-positive bacteria; and water.

5. The enrichment medium of any preceding claim, wherein the selectivity agent is selected from the group consisting of antibiotics, detergents, and combinations thereof.

6. The enrichment medium of any preceding claim, wherein the selectivity agent is selected from the group consisting of vancomycin or a salt thereof, sodium dodecylbenzenesulfonate (SDBS), novobiocin or a salt thereof, cefsulodin or a salt thereof, sulfadiazine or a salt thereof, bile salts, sodium lauryl sulfate (SLS), sodium dodecylbenzesulfonate (SDBS), Tergitol 4 , and combinations thereof. 21 59099806.1Attorney Docket No.: 234994-559211 7. The enrichment medium of any preceding claim, wherein the selectivity agent is selected from the group consisting of vancomycin or a salt thereof, SDBS, and combinations thereof.

8. The enrichment medium of claim 7, wherein the selectivity agent is a combination of vancomycin or a salt thereof and SDBS.

9. The enrichment medium of claim 7, wherein the selectivity agent is SDBS.

10. The enrichment medium of any preceding claim, wherein the sodium pyruvate is present in a concentration of from about 0.1 to about 5 grams per liter.

11. The enrichment medium of claim 10, wherein the sodium pyruvate is present in a concentration of from about 0.2 to about 3 grams per liter.

12. The enrichment medium of claim 11, wherein the sodium pyruvate is present in a concentration of from about 0.5 to about 2 grams per liter.

13. The enrichment medium of claim 12, wherein the sodium pyruvate is present in a concentration of from about 1 to about 1.5 grams per liter.

14. The enrichment medium of any preceding claim, wherein the magnesium sulfate is anhydrous.

15. The enrichment medium of any preceding claim, wherein the concentration of yeast extract is from about 1 to about 9 grams per liter, and the concentration of magnesium sulfate is from about 0.5 to about 10 millimolar.

16. The enrichment medium of claim 15, wherein the concentration of yeast extract is from about 2.5 to about 7.5 grams per liter, and the concentration of magnesium sulfate is from about 1 to about 5 millimolar. 22 59099806.1Attorney Docket No.: 234994-559211 17. The enrichment medium of claim 16, wherein the concentration of yeast extract is from about 4 to about 6 grams per liter, and the concentration of magnesium sulfate is from about 2 to about 4 millimolar.

18. The enrichment medium of any preceding claim, further comprising potassium chloride.

19. The enrichment medium of any preceding claim, further comprising phenol red.

20. The enrichment medium of any preceding claim, further comprising glucose.

21. The enrichment medium of claim 20, wherein the glucose is dextrose.

22. The enrichment medium of any preceding claim, further comprising casein peptone.

23. The enrichment medium of any preceding claim, further comprising one or more of sodium chloride, dibasic potassium phosphate, monobasic potassium phosphate, and trisodium citrate dihydrate.

24. The enrichment medium of any preceding claim, comprising sodium chloride in a concentration of less than or equal to about 3 grams per liter.

25. The enrichment medium of claim 20, comprising sodium chloride in a concentration of about 1.5 to about 2.5 grams per liter.

26. The enrichment medium of any preceding claim, further comprising polysorbate 80 (Tween 80).

27. The enrichment medium of any preceding claim, wherein the enrichment medium is substantially free of chicken fat, thioglycolate, and / or mannitol.

28. The enrichment medium of any preceding claim, comprising: from about 0.2 to about 3 grams per liter of sodium pyruvate; from about 2.5 to about 7.5 grams per liter of yeast extract; from about 1 to about 5 millimolar of magnesium sulfate; and 23 59099806.1Attorney Docket No.: 234994-559211 vancomycin, SDBS, or a combination thereof.

29. The enrichment medium of any preceding claim, comprising: from about 0.2 to about 3 grams per liter of sodium pyruvate; from about 2.5 to about 7.5 grams per liter of yeast extract; from about 1 to about 5 millimolar of magnesium sulfate; vancomycin, SDBS, or a combination thereof; casein peptone; sodium chloride; and potassium phosphate.

30. The enrichment medium of any preceding claim, wherein the enrichment medium is capable of growing a sample of gram-negative bacteria containing 1 to 5 CFU / ml to produce an enriched sample containing greater than or equal to 1,000 CFU / ml after 6 hours of incubation at 42^C.

31. The enrichment medium of any preceding claim, wherein the enrichment medium is capable of growing a sample of gram-negative bacteria containing 1 to 5 CFU / ml to produce an enriched sample containing greater than or equal to 10,000 CFU / ml after 6 hours of incubation at 42^C.

32. The enrichment medium of any preceding claim, wherein the enrichment medium is substantially free of sodium phosphate.

33. A method of growing gram-negative bacteria in an enrichment medium, comprising providing a sample comprising gram-negative bacteria; providing an enrichment medium according to any one of claims 1-32; and growing the gram-negative bacteria in the enrichment medium.

34. The method of claim 33, wherein the gram-negative bacteria comprises Salmonella, E. coli, Cronobacter, or any combination thereof.

35. The method of claim 33 or 34, wherein the gram-negative bacteria are used in a detection assay after about 6 hours or less of growth time in the enrichment medium. 24 59099806.1Attorney Docket No.: 234994-559211 36. The method of claim 35, wherein the gram-negative bacteria are used in a detection assay after about 4 to about 6 hours of growth time in the enrichment medium.

37. An enrichment medium formulation useful for mixing with water to prepare an enrichment medium, the enrichment medium formulation comprising: a pyruvic acid salt; a magnesium salt; yeast extract; and a selectivity agent that inhibits the growth of gram-positive bacteria.

38. The enrichment medium formulation of claim 37, wherein the pyruvic acid salt is sodium pyruvate.

39. The enrichment medium formulation of claim 37, wherein the magnesium salt is magnesium sulfate.

40. An enrichment medium formulation useful for mixing with water to prepare an enrichment medium, the enrichment medium formulation comprising: sodium pyruvate; magnesium sulfate; yeast extract; and a selectivity agent that inhibits the growth of gram-positive bacteria.

41. The enrichment medium formulation of any preceding claim, wherein the formulation is a powder.

42. The enrichment medium of any preceding claim, wherein the selectivity agent is selected from the group consisting of antibiotics, detergents, and combinations thereof.

43. The enrichment medium formulation of any preceding claim, wherein the selectivity agent is selected from the group consisting of vancomycin or a salt thereof, sodium dodecylbenzenesulfonate (SDBS), novobiocin or a salt thereof, cefsulodin or a salt thereof, sulfadiazine or a salt thereof, bile salts, sodium lauryl sulfate (SLS), sodium dodecylbenzesulfonate (SDBS), Tergitol 4, and combinations thereof. 25 59099806.1Attorney Docket No.: 234994-559211 44. The enrichment medium formulation of any preceding claim, wherein the selectivity agent is selected from the group consisting of vancomycin or a salt thereof, SDBS, and combinations thereof.

45. The enrichment medium formulation of claim 44, wherein the selectivity agent is a combination of vancomycin or a salt thereof and SDBS.

46. The enrichment medium formulation of claim 44, wherein the selectivity agent is SDBS.

47. The enrichment medium formulation of any preceding claim, wherein the sodium pyruvate is present in an amount of from about 0.4 wt% to about 20 wt% by weight of the enrichment medium formulation.

48. The enrichment medium formulation of claim 47, wherein the sodium pyruvate is present in an amount of from about 0.8 wt% to about 12 wt% by weight of the enrichment medium formulation.

49. The enrichment medium formulation of claim 48, wherein the sodium pyruvate is present in an amount of from about 2 wt% to about 8 wt% by weight of the enrichment medium formulation.

50. The enrichment medium formulation of claim 49, wherein the sodium pyruvate is present in an amount of from about 3.5 wt% to about 6 wt% by weight of the enrichment medium formulation.

51. The enrichment medium formulation of any preceding claim, wherein the magnesium sulfate is anhydrous.

52. The enrichment medium formulation of any preceding claim, wherein the amount of yeast extract is from about 4 wt% to about 35 wt% by weight of the enrichment medium formulation, and the amount of magnesium sulfate is from about 0.05 wt% to about 2 wt% by weight of the enrichment medium formulation. 26 59099806.1Attorney Docket No.: 234994-559211 53. The enrichment medium formulation of claim 43, wherein the amount of yeast extract is from about 10 wt% to about 30 wt% by weight of the enrichment medium formulation, and the amount of magnesium sulfate is from about 0.1 wt% to about 1 wt% by weight of the enrichment medium formulation.

54. The enrichment medium formulation of claim 44, wherein the amount of yeast extract is from about 15 wt% to about 25 wt% by weight of the enrichment medium formulation, and the amount of magnesium sulfate is from about 0.2 wt% to about 0.6 wt% by weight of the enrichment medium formulation.

55. The enrichment medium formulation of any preceding claim, further comprising potassium chloride.

56. The enrichment medium formulation of any preceding claim, further comprising phenol red.

57. The enrichment medium formulation of any preceding claim, further comprising glucose.

58. The enrichment medium formulation of claim 57, wherein the glucose is dextrose.

59. The enrichment medium formulation of any preceding claim, further comprising casein peptone.

60. The enrichment medium formulation of any preceding claim, further comprising one or more of sodium chloride, dibasic potassium phosphate, monobasic potassium phosphate, and trisodium citrate dihydrate.

61. The enrichment medium formulation of any preceding claim, comprising sodium chloride in an amount of less than or equal to about 12 wt% by weight of the enrichment medium formulation.

62. The enrichment medium formulation of claim 61, comprising sodium chloride in an amount of about 5 wt% to about 10 wt% by weight of the enrichment medium formulation. 27 59099806.1Attorney Docket No.: 234994-559211 63. The enrichment medium formulation of any preceding claim, further comprising polysorbate 80 (Tween 80).

64. The enrichment medium formulation of any preceding claim, wherein the enrichment medium formulation is substantially free of chicken fat, thioglycolate, and / or mannitol.

65. The enrichment medium formulation of any preceding claim, comprising: from about 0.8 wt% to about 12 wt% of sodium pyruvate; from about 10 wt% to about 30 wt% of yeast extract; from about 0.1 wt% to about 1 wt% magnesium sulfate; and vancomycin, SDBS, or a combination thereof.

66. The enrichment medium formulation of any preceding claim, comprising: from about 0.8 wt% to about 12 wt% of sodium pyruvate; from about 10 wt% to about 30 wt% of yeast extract; from about 0.1 wt% to about 1 wt% magnesium sulfate; vancomycin, SDBS, or a combination thereof; casein peptone; sodium chloride; and potassium phosphate.

67. The enrichment medium formulation of any preceding claim, wherein the enrichment medium formulation is substantially free of sodium phosphate.

68. The enrichment medium formulation of any preceding claim, wherein the ingredients are blended or combined into the powder.

69. An enrichment medium useful for repair and growth of gram-negative bacteria, comprising: from about 0.5 to about 2 grams per liter of sodium pyruvate; from about 2.5 to about 7.5 grams per liter of yeast extract; from about 1 to about 5 millimolar of magnesium sulfate; and water. 28 59099806.1Attorney Docket No.: 234994-559211 70. The enrichment medium of claim 69, wherein the concentration of yeast extract is from about 4 to about 6 grams per liter, and the concentration of magnesium sulfate is from about 2 to about 4 millimolar.

71. The enrichment medium of claim 69 or 70, further comprising potassium chloride and comprising less than or equal to about 3 grams per liter of sodium chloride.

72. An enrichment medium formulation useful for mixing with water to prepare an enrichment medium, the enrichment medium formulation comprising: from about 2 to about 8 wt% of sodium pyruvate; from about 10 to about 30 wt% of yeast extract; from about 0.1 to about 1 wt% magnesium sulfate; and water.

73. The enrichment medium formulation of claim 63, wherein the amount of yeast extract is from about 15 wt% to about 25 wt% by weight of the enrichment medium formulation, and the amount of magnesium sulfate is from about 0.2 wt% to about 0.6 wt% by weight of the enrichment medium formulation.

74. The enrichment medium formulation of claim 63 or 64, further comprising potassium chloride and comprising less than or equal to about 12 wt% of sodium chloride. 29 59099806.1

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