WO2023177875A1 - Methods and compositions for anaerobic contaminant testing - Google Patents

Abstract

Provided herein are methods and compositions related to detecting anaerobic contaminant microbes in a composition comprising a product microbe.

Description

METHODS AND COMPOSITIONS FOR ANAEROBIC CONTAMINANT TESTING

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Application No. 63/320,813, filed on March 17, 2022, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND

[2] Anaerobic bacteria are bacteria that grow poorly (or do not grow) in the presence of oxygen. Obligate (strict) anaerobic bacteria are particularly sensitive to even low levels of oxygen. In humans, many types of anaerobic bacteria are found in the gastrointestinal tract. As microbial culturing methods typically occur in atmospheric air (an aerobic environment), the culturing of anaerobic bacteria can be challenging and often requires specialized equipment and techniques.

[3] In addition, since most culturing methods involve atmospheric conditions, established methods of microbial contaminant testing do not fully capture potential contaminant microbes in anaerobic culturing conditions. Therefore, a method is needed to test for anaerobic contaminant microbes. A methodology is important, for example, to establish microbial purity, i.e., the absence (or acceptable levels) of extraneous or undesirable microorganisms, to ensure the purity and safety of an anaerobic bacteria product.

SUMMARY

[4] In certain aspects, the present disclosure provides a method of detecting anaerobic microbe contamination in a composition comprising a product microbe. The present disclosure includes that at least one characteristic of this method can be to suppress propagation of viable product microbe and allow for growth of other viable anaerobic microbes, so that the other viable anaerobic microbes can be detected. In some embodiments, the product microbe is anaerobic bacteria, e.g., grown under anaerobic conditions. The anaerobic growth conditions create the potential for growth of anaerobic contaminant microbes, in particular, viable anaerobic bacteria other than the product microbe. In some embodiments, the method comprises suppressing the growth of the product microbe (e.g., such that any growth is below the limit of detection) and permitting the growth of anaerobic contaminant microbes (non-product microbes) in a growth medium containing an antibiotic (e.g., an antibiotic at a concentration that suppresses (e.g., selectively suppresses) growth of the product microbe) under anaerobic conditions. This facilitates detection of lower-level contaminants relative to the product microbe. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 20 ppm or below about 10 ppm (parts per million). In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 5%, below about 4%, below about 3%, below about 2%, below about 1%, below about 0.5%, or below about 0.1%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.1%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.01%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.001%.

[5] In some embodiments, the product microbes are anaerobic bacteria of the genus Actinomyces, Bacteroides, Bifidobacterium, Clostridium, Foumierella, Fusobacterium, Harryflintia, Lactococcus, Megasphaera, Parabacteroides, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, or Veillonella. In some embodiments, the anaerobic bacteria are from the genus Prevotella. In some embodiments, the anaerobic bacteria are from a strain of Prevotella bacteria comprising one or more proteins listed in Table 1. In some embodiments, the anaerobic bacteria are from a strain of Prevotella substantially free of one or more of the proteins listed in Table 2. In some embodiments, the anaerobic bacteria are from a strain of Prevotella bacteria comprising one or more of the proteins listed in Table 1 and that is free or substantially free of one or more of the proteins listed in Table 2.

[6] In some embodiments, the Prevotella bacteria are of the species Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella jusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, or Prevotella veroralis. In some embodiments, the Prevotella bacteria are of the species Prevotella histicola.

[7] In some embodiments, the Prevotella is Prevotella Strain B 50329 (NRRL accession number B 50329 and herein after also referred to as “Prevotella Strain B”). In some embodiments, the Prevotella strain is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain B 50329.

[8] In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria comprising a protein listed in Table 1 and/or a gene encoding a protein listed in Table 1. In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria free or substantially free of a protein listed in Table 2 and/or a gene encoding a protein listed in Table 2. In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria comprising one or more proteins listed in Table 1 and/or one or more genes encoding one or more proteins listed in Table 1. In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria free or substantially free of one or more proteins listed in Table 2 and/or one or more genes encoding one or more proteins listed in Table 2. In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria comprising the proteins listed in Table 1 and/or genes encoding the proteins listed in Table 1. In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria free or substantially free of the proteins listed in Table 2 and/or genes encoding the proteins listed in Table 2.

[9] In some embodiments, the Prevotella histicola strain is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Prevotella histicola Strain C (ATCC Deposit Number PTA-126140).

[10] In some embodiments, the Prevotella histicola strain is Prevotella histicola Strain C (ATCC Deposit Number PTA-126140).

[11] In some embodiments, one or more antibiotics is used to suppress growth of the product microbe while allowing for growth of contaminant microbes, e.g., where each of the one or more antibiotics is used at a concentration that suppresses growth of the product microbe while allowing for growth of contaminant microbes. In some embodiments of the methods described herein, a sample of a composition comprising the product microbe (e.g., therapeutic composition or therapeutic agent) is incubated in a growth medium under anaerobic conditions to detect anaerobic contaminant microbes. In some embodiments, each one or more antibiotic is added to a growth medium (e.g., a separate growth medium is used for each of the one or more antibiotic) and is part of the growth medium used to grow (e.g., allow growth of) contaminant anaerobic microbes under anaerobic conditions. In some embodiments, the one or more antibiotics is used at a concentration to which the product microbe is sensitive (e.g., and growth of the product microbe is suppressed). In some embodiments, the one or more antibiotics is one or more antibiotics that the product microbe is sensitive to, preferably at low concentrations. In some embodiments, one or more contaminant anaerobic microbe is not sensitive to the concentration of the one or more antibiotics to which the product microbe is sensitive, and the one or more contaminant anaerobic microbe grows in the growth medium containing the one or more antibiotics (e.g., at the concentration to which the product microbe is sensitive). In some embodiments, one or more antibiotic is a broad-spectrum antibiotic. In some embodiments, one or more antibiotic is a narrow-spectrum antibiotic. In some embodiments, one antibiotic is used in the method to detect anaerobic contamination (i.e., contaminant microbes) in a composition (e.g., a therapeutic composition or therapeutic agent). In some embodiments, two antibiotics are used in the method to detect anaerobic contamination (i.e., contaminant microbes) in a composition (e.g., a therapeutic composition or therapeutic agent). In some embodiments, two or three antibiotics are used in the method to detect anaerobic contamination (i.e., contaminant microbes) in a composition (e.g., a therapeutic composition or therapeutic agent). In some embodiments, a first antibiotic is added to a first volume of growth medium, and, when used, a second antibiotic is added to a second volume of growth medium, and, when used, a third antibiotic is added to a third volume of growth medium. A sample of a composition comprising a product microbe can be added to the first growth medium (and, when used, to the second growth medium, and, when used, to the third growth medium) and incubated under anaerobic conditions that allow growth of anaerobic contaminant microbes and that suppress growth of the product microbe. In some embodiments, one or more antibiotic suppresses the growth of at least one gram-positive contaminant microbe. In some embodiments, one or more antibiotic suppresses the growth of at least one gram-negative contaminant microbe. In some embodiments, a first antibiotic suppresses the growth of at least one gram-positive contaminant microbe, and a second antibiotic suppresses the growth of at least one gram-negative contaminant microbe. In some embodiments, the growth medium is, or is comprised in, agar and/or a bacterial plate (e.g., agar plate). In some embodiments, the growth medium is a liquid growth media and/or is comprised in a liquid bacterial culture.

[12] In some embodiments, ampicillin is used as an antibiotic to suppress growth of the product microbe and allow growth of anaerobic contaminant microbes in the growth medium (e.g., ampicillin is used at a concentration that suppresses growth of the product microbe and allows growth of anaerobic contaminant microbes, e.g., under anaerobic conditions). In some embodiments, colistin is used as an antibiotic to suppress growth of the product microbe and allow growth of anaerobic contaminant microbes in the growth medium (e.g., colistin is used at a concentration that suppresses growth of the product microbe and allows growth of anaerobic contaminant microbes, e.g., under anaerobic conditions). In some embodiments, ampicillin and colistin are used (e.g., both are used in separate growth media) to suppress growth of the product microbe and allow growth of anaerobic contaminant microbes in the growth medium (e.g., both are used at concentrations that suppress growth of the product microbe and allow growth of anaerobic contaminant microbes e.g., under anaerobic conditions). In some embodiments, the anaerobic contaminant testing comprises growth of anaerobic contaminant microbes in separate ampicillin- and colistin- enriched growth media to suppress growth of the product microbe and allow growth of anaerobic contaminant microbes. In some embodiments, the ampicillin is at a concentration of about 20 mg/L to about 30 mg/L. In some embodiments, the ampicillin is at a concentration of about 25 mg/L. In some embodiments, the colistin is at a concentration of about 15 mg/L to about 25 mg/L. In some embodiments, the colistin is at a concentration of about 20 mg/L. [13] In some embodiments, the growth medium comprises Brain Heart Infusion (BHI). In some embodiments, the growth medium comprises about 32 g/ml to about 42 g/ml of the Brain Heart Infusion (BHI). In some embodiments the growth medium comprises about 37 g/ml of the Brain Heart Infusion (BHI). In some embodiments, the growth medium comprises Bacto Agar. In some embodiments, the growth medium comprises about 2.5 g/ml to about 12.5 g/ml of the Bacto Agar. In some embodiments, the growth medium comprises about 7.5 g/ml of the Bacto Agar. In some embodiments, the growth medium comprises a Vitamin K and Hemin Solution. In some embodiments, the growth medium comprises about 5 ml/L to about 15 ml/L of the Vitamin K and Hemin Solution. In some embodiments, the growth medium comprises about 10 ml/L of the Vitamin K and Hemin Solution. In some embodiments, the growth medium comprises a reducing agent. In some embodiments, the reducing agent comprises L-Cysteine-HCl. In some embodiments, the growth medium comprises about 0.25 g/ml to about 0.75 g/ml of L-Cysteine- HC1. In some embodiments, the growth medium comprises about 0.5 g/ml of L-Cysteine-HCl. In some embodiments, the growth medium comprises an antibiotic. In some embodiments, the antibiotic is used at a concentration that suppresses growth of the product microbe but allows growth (e.g., does not suppress growth) of an anaerobic contaminant microbe. In some embodiments, the antibiotic comprises ampicillin. In some embodiments, the growth medium comprises about 20 mg/L to about 30 mg/L of ampicillin. In some embodiments, the growth medium comprises about 25 mg/L of ampicillin. In some embodiments, the antibiotic comprises colistin. In some embodiments, the growth medium comprises about 15 mg/L to about 25 mg/L of colistin. In some embodiments, the growth medium comprises about 20 mg/L of colistin.

[14] In some embodiments, the growth medium comprises a Brain Heart Infusion (BHI), a Bacto Agar, a Vitamin K and Hemin Solution, and L-Cysteine-HCl. In some embodiments, the growth medium comprises or consists essentially of a Brain Heart Infusion (BHI), a Bacto Agar, a Vitamin K and Hemin Solution, L-Cysteine-HCl, and ampicillin. In some embodiments, the growth medium comprises or consists essentially of a Brain Heart Infusion (BHI), a Bacto Agar, a Vitamin K and Hemin Solution, L-Cysteine-HCl, and colistin.

[15] In some embodiments, one or more antibiotics (such as ampicillin and/or colistin) is used to suppress growth of a product microbe in a sample (e.g., a sample of a composition such as a therapeutic composition or therapeutic agent), e.g., the sample is tested separately in the presence of one or more antibiotics (such as ampicillin-containing and/or colistin-containing conditions, separately) (e.g., each antibiotic is present at a concentration that suppresses growth of the product microbe but does not suppress growth of an anaerobic contaminant microbe) to allow for growth of anaerobic contaminant microbes during incubation under anaerobic conditions. Anaerobic contaminant microbes (e.g., the presence, absence, type(s), and/or amounts of anaerobic contaminant microbes) can be assessed after incubation under anaerobic conditions for one or more days, such as 1-8 days, such as 5 days, at a temperature that allows growth, such as 32°C to 42°C, such as 37°C. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 20 ppm or below about 10 ppm (parts per million). In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 5%, below about 4%, below about 3%, below about 2%, below about 1%, below about 0.5%, or below about 0.1%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.1%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.01%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.001%. Anaerobic contaminant microbes can be assessed, e.g., after incubation, and measured, e.g., as total colony forming units per gram of sample (CFU/g) or as viable cell count per gram of sample (VCC/g). A composition (e.g., the source of a sample), such as a therapeutic composition or therapeutic agent, can be considered to contain anaerobic contaminant microbe (e.g., an unacceptable level thereof) if growth (as determined by the presence of colonies, e.g., after incubation) is detected (e.g., any CFUs detected) or if the CFU/g value is above an acceptable limit. In some embodiments, the acceptable limit is up to about 500 CFU/g. In some embodiments, the acceptable limit is up to about 1000 CFU/g. In some embodiments, the acceptable limit is up to about 2000 CFU/g. In some embodiments, the acceptable limit is up to about 3000 CFU/g. Anaerobic contaminant microbes can be assessed, e.g., after incubation, and measured, e.g., based on sequencing of nucleic acids present in growth media or a sample derived therefrom, e.g., by a method including high throughput sequencing and/or 16S rRNA gene sequence profiling.

[16] In some embodiments, one or more antibiotics (such as ampicillin and/or colistin) are used to suppress growth of Prevotella Strain B in a sample (e.g., a sample of a composition such as a therapeutic composition or therapeutic agent), e.g., the sample is tested separately in the presence of one or more antibiotics, such as ampicillin-containing and/or colistin-containing conditions separately) (e.g., each antibiotic is present at a concentration that suppresses growth of the product microbe but does not suppress growth of an anaerobic contaminant microbe) to allow for growth of anaerobic contaminant microbes during incubation under anaerobic conditions. Anaerobic contaminant microbes can be assessed (e.g., the presence, absence, type(s), and/or amounts of anaerobic contaminant microbes) after incubation under anaerobic conditions for one or more days, such as 1-8 days, such as 5 days, at a temperature that allows growth, such as 32°C to 42°C, such as 37°C. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 20 ppm or below about 10 ppm (parts per million). In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 5%, below about 4%, below about 3%, below about 2%, below about 1%, below about 0.5%, or below about 0.1%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.1%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.01%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.001%. Anaerobic contaminant microbes can be assessed, e.g., after incubation, and measured, e.g., as total colony forming units per gram of sample (CFU/g) or viable cell count per gram of sample (VCC/g). A composition (e.g., the source of a sample), such as a therapeutic composition or therapeutic agent (e.g., containing Prevotella Strain B), can be considered to contain anaerobic contaminant microbe (e.g., an unacceptable level thereof) if growth (as determined by the presence of colonies, e.g., after incubation) is detected (e.g., any CFUs detected) or if the CFU/g value is above an acceptable limit. In some embodiments, the acceptable limit is up to about 500 CFU/g. In some embodiments, the acceptable limit is up to about 1000 CFU/g. In some embodiments, the acceptable limit is up to about 2000 CFU/g. In some embodiments, the acceptable limit is up to about 3000 CFU/g. Anaerobic contaminant microbes can be assessed, e.g., after incubation, and measured, e.g., based on sequencing of nucleic acids present in growth media or a sample derived therefrom, e.g., by a method including high throughput sequencing and/or 16S rRNA gene sequence profiling.

[17] In some embodiments, growth of a control anaerobic microbe is assessed, e.g., in one or more separate plates containing the growth medium under anaerobic conditions. For example, the susceptibility of the control anaerobic microbe to one or more antibiotics (such as ampicillin and/or colistin) is known and evaluated. In some embodiments, growth in growth medium under anaerobic conditions with an antibiotic (one or more antibiotic) and without antibiotic is evaluated. In some embodiments, the control anaerobic microbe comprises one or more of: Prevotella veroralis ATCC 33779, Bacteroides ovatus ATCC 8483, and/or Bifidobacterium bifidum ATCC 15696.

[18] In some embodiments, the anaerobic contaminant microbe is gram-negative. In some embodiments, the anaerobic contaminant microbe is gram-positive. In some embodiments, the anaerobic contaminant microbe is from the genus Bifidobacterium, Lactobacillus, Lactococcus, Streptococcus, or Penicillium. In some embodiments, the anaerobic contaminant microbe is from the species Penicillium roqueforti. In some embodiments, the anaerobic contaminant microbe is from the genus Lactobacillus, Streptococcus, Blautia, or Akkermansia. In some embodiments, the anaerobic contaminant microbe is a spore-forming microbe contaminant. In some embodiments, the anaerobic contaminant microbe is from the genus Clostridium. In some embodiments, the anaerobic contaminant microbe is from the species Clostridium spp. In some embodiments, the anaerobic contaminant microbe is from the genus Bacillus. In some embodiments, the anaerobic contaminant microbe is from the species Bacillus spp. In some embodiments, the anaerobic contaminant microbe is from the genus Bacteroides. In some embodiments, the anaerobic contaminant microbe is a sewage leakage contaminant. In some embodiments, the anaerobic contaminant microbe is a soil contaminant. In some embodiments, the anaerobic contaminant microbe is an animal (e.g., human) fecal contaminant. In some embodiments, the anaerobic contaminant microbe is an air-tolerant (aerotolerant) microbe.

[19] In some embodiments, the anaerobic contaminant microbe (e.g., that grows in the presence of one or more antibiotics) (e.g., genus and/or species and/or strain of the anaerobic contaminant microbe) is identified (e.g., identified by 16S rRNA gene sequence profiling). In some embodiments, DNA is analyzed by 16S sequencing (e.g., 16S rRNA gene sequence profiling) to identify the contaminant microbe (e.g., genus and/or species and/or strain of the contaminant microbe). In some embodiments, DNA is extracted, PCR amplified, and analyzed by 16S sequencing (e.g., 16S rRNA gene sequence profiling) to identify the contaminant microbe (e.g., genus and/or species and/or strain of the contaminant microbe). BRIEF DESCRIPTION OF THE FIGURES

[20] Fig. 1 is a graph showing the propagation of product microbe Prevotella Strain B 50329 on anaerobic BHI agar; and its suppression on anaerobic BHI agar with ampicillin and on anaerobic BHI agar with colistin. LOD: limit of detection. VCC/g: viable cell count per gram. The limit of detection (LOD) is 100 VCC/g (l.E+02).

[21] Fig. 2 is a figure showing the composition of the mock community used to test for recovery of selected anaerobic microbes.

[22] Fig. 3 is a figure showing the composition of the fecal anaerobic mix community used to test for recovery of fecal anaerobic microbes.

[23] Figs. 4 A and 4B are graphs showing the recovery (as VCC/g) of the mock community (Fig. 4A) and fecal community (Fig. 4B) on anaerobic BHI control agar, anaerobic BHL ampicillin agar and anaerobic BHI-colistin agar. The limit of detection (LOD) is 100 VCC/g (l.E+02).

[24] Figs. 5A and 5B are diagrams showing that BHI-colistin agar testing (Fig. 5A) and BHL ampicillin agar testing (Fig. 5B) were able to detect 23 out of 30 strains in the mock community. The percentage values (%) provide the amount of growth under the given antibiotic condition as compared to the amount of growth on the BHI control agar.

[25] Figs. 6A-6C are graphs showing recovery of anaerobic microbes from the fecal community on BHI control agar (Fig. 6A), BHLampicillin agar (Fig. 6B), and BHI-colistin- agar (Fig. 6C). The percentage values (%) are the percentage of read counts from 16S profiling.

DETAILED DESCRIPTION

[26] As the field of microbe-based therapeutics grows, for example, to develop treatments for specific diseases or conditions, there will be more stringent requirements for product microbes. Requirements for product microbes include microbial safety and purity, e.g., the absence or low levels (e.g., below a set acceptable limit) of extraneous or undesirable microorganisms that are not the product microbe.

[27] For example, a product microbe composition can contain between 10e2 to 10e8 viable microbes per gram that will grow in anaerobic conditions, preventing potential contaminants at lower concentrations from being visualized and discovered using existing plating and enrichment methods. In order to allow detection of potential anaerobic contaminants, the propagation of product microbe has to be suppressed, while allowing other microbes to grow.

[28] Current product microbe contamination testing involves testing for total aerobic contaminant microbes and specific pathogens. However, there is no established method for testing for anaerobic contaminant microbes, even in a composition comprising anaerobic product microbes. Anaerobic contaminant microbes may potentially be present, or present at levels that exceed a threshold value, in anaerobic microbe-containing products, e.g., due to the anaerobic conditions under which microbes are grown. For example, during production, the rich media under strict anaerobic conditions allows growth of a product microbe but may also allow for the propagation of contaminating obligate or facultative anaerobes and/or aerotolerant microbes potentially present in the media or equipment, causing contamination (e.g., presence at a value that exceeds an acceptable limit) of the final composition.

[29] Furthermore, when product microbes are present in a composition it can be difficult to detect anaerobic contaminant microbes, particularly when they are present in low numbers. Thus, to detect anaerobic contaminant microbes in a sample comprising an anaerobic product microbe, an anaerobic contaminant testing procedure needs to suppress growth of the product microbe while allowing for growth of contaminant microbes. Described herein are methods and compositions for anaerobic contaminant testing of microbe product comprising anaerobic bacteria. The methods and compositions (such as growth media) can be used with samples of therapeutic agents and/or therapeutic compositions.

Definitions

[30] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a,” “an,” and “the” are understood to be singular or plural.

[31] The term “about” when used before a numerical value indicates that the value may vary within a reasonable range, such as within ± 10%, ± 5% or ± 1% of the stated value.

[32] As used herein, “anaerobic conditions” are conditions with reduced levels of oxygen compared to normal atmospheric conditions. For example, in some embodiments anaerobic conditions are conditions wherein the oxygen levels are partial pressure of oxygen (pCh) below about 8%. In some instances, anaerobic conditions are conditions wherein the pCh is below about 2%. In some instances, anaerobic conditions are conditions wherein the pCh is below about 0.5%. For example, in some embodiments, anaerobic conditions are conditions (e.g., for a liquid culture) wherein the redox potential (as measured with a redox sensor) is below about -100 mV, below about -200 mV, below about -300 mV, or below about -400 mV. For example, in some embodiments, anaerobic conditions are conditions (e.g., for agar plates) wherein the anaerobic conditions are in an anaerobic chamber. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 20 ppm or below about 10 ppm (parts per million). In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 5%, below about 4%, below about 3%, below about 2%, below about 1%, below about 0.5%, or below about 0.1%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.1%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.01%. In some embodiments, the anaerobic conditions comprise oxygen levels (e.g., in an anaerobic chamber) of below about 0.001%. In some embodiments, anaerobic conditions may be achieved by purging a bioreactor and/or a culture flask and/or a plate with a gas other than oxygen such as, for example, nitrogen and/or carbon dioxide (CO2). A liquid culture can be maintained in anaerobic conditions at a set redox potential; a culture flask and/or plate (e.g., agar plate) can be maintained in an anaerobic chamber.

[33] The term “decrease” or “deplete” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to a pretreatment state.

[34] As used herein, “engineered bacteria” are any bacteria that have been genetically altered from their natural state by human intervention and the progeny of any such bacteria. Engineered bacteria include, for example, the products of targeted genetic modification, the products of random mutagenesis screens and the products of directed evolution.

[35] The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. The term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence. [36] “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson etal. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., etal., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo etal. (1988) SIAM J Applied Math 48:1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)).

[37] The term “increase” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 10- fold, 100-fold, 10^A3 fold, 10^A4 fold, 10^A5 fold, 10^A6 fold, and/or 10^A7 fold greater after treatment when compared to a pre-treatment state.

[38] “Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, e.g., the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species. In some embodiments, the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence. In some embodiments, the entire genomes of two entities are sequenced and compared. In some embodiments, select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared. For 16S, OTUs that share > 97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU. See e.g., Claesson MJ, Wang Q, O’Sullivan O, Greene-Diniz R, Cole JR, Ross RP, and O’Toole PW. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. For complete genomes, MLSTs, specific genes, other than 16S, or sets of genes OTUs that share > 95% average nucleotide identity are considered the same OTU. See e.g., Achtman M, and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequently defined by comparing sequences between organisms.

Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof. Operational Taxonomic Units (OTUs) with taxonomic assignments made to, e.g., genus, species, and phylogenetic clade are provided herein.

[39] “Strain” refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species. The genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one nonnative plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic signatures between different strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.

[40] ‘ ‘Microbe” refers to any natural or engineered organism characterized as an archaeon, parasite, bacterium, fungus, microscopic alga, protozoan, and the stages of development or life cycle stages (e.g., vegetative, spore (including sporulation, dormancy, and germination), latent, biofilm) associated with the organism. Examples of gut microbes include: Actinomyces graevenitzii, Actinomyces odontolyticus, Akkermansia muciniphila, Bacteroides caccae, Bacteroides fragilis, Bacteroides putredinis, Bacteroides thetaiotaomicron, Bacteroides vultagus, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bilophila wadsworthia, Blautia, Butyrivibrio, Campylobacter gracilis, Clostridia cluster III, Clostridia cluster IV, Clostridia cluster IX (Acidaminococcaceae group), Clostridia cluster XI, Clostridia cluster XIII (Peptostreptococcus group), Clostridia cluster XIV, Clostridia cluster XV, Collinsella aerofaciens, Coprococcus, Corynebacterium sunsvallense, Desulfomonas pigra, Dorea formicigenerans, Dorea longicatena, Escherichia coli, Eubacterium hadrum, Eubacterium rectale, Faecalibacteria prausnitzii, Gemella, Lactococcus, Lanchnospira, Mollicutes cluster XVI, Mollicutes cluster XVIII, Prevotella, Rothia mucilaginosa, Ruminococcus callidus, Ruminococcus gnavus, Ruminococcus torques, and Streptococcus.

[41] As used herein a “product microbe” is a microbe that is intended to be in a composition.

[42] As used herein, a “contaminant microbe” is a microbe that is not intended to be present (e.g., not intended to be present at all or not intended to be present above an acceptable value) in a composition.

[43] “Therapeutic agent” includes a pharmaceutical agent and/or an agent for therapeutic use. In some embodiments, the therapeutic agent is, for example, a solution or dried form (for example, powder (such as a lyophilized powder or spray-dried powder) or lyophilate (for example, lyophilized powder or lyophilized cake)) that comprises bacteria (for example, as described herein). In some embodiments, the therapeutic agent is a pharmaceutical agent. In some embodiments, the therapeutic agent is (or is present in) a medicinal product, medical food, a food product, or a dietary supplement.

[44] “Therapeutic composition” includes a pharmaceutical composition. A therapeutic composition contains a therapeutic agent. In some embodiments, the therapeutic composition is a pharmaceutical composition. In some embodiments, the therapeutic composition is (or is present in) a medicinal product, medical food, a food product, or a dietary supplement. In some embodiments, a therapeutic composition provides a therapeutically effective amount of a therapeutic agent contained therein.

Product Microbes

[45] In certain aspects, provided herein are methods and compositions (such as growth media) for testing for anaerobic contaminant microbes in a composition (e.g., a therapeutic composition or therapeutic agent) comprising a product microbe. In some embodiments, the product microbe is anaerobic bacteria. In some embodiments, the product microbe comprises one strain of anaerobic bacteria. In some embodiments, the product microbe comprises more than one strain (e.g., 2, 3, 4, or 5 strains) of anaerobic bacteria. In some embodiments, the anaerobic bacteria are bacteria of the genus Actinomyces, Bacteroides, Bifidobacterium, Clostridium, Foumierella, Fusobacterium, Harryflintia, Lactococcus, Megasphaera, Parabacteroides, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, or Veillonella. In some embodiments, the anaerobic bacteria are bacteria of the genus Prevotella.

[46] In some embodiments, the anaerobic bacteria are Prevotella bacteria of the species Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella melanogenica, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella jusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, or Prevotella veroralis. In some embodiments, the anaerobic bacteria are Prevotella bacteria of the species Prevotella histicola.

[47] In some embodiments, the product microbe is a strain of Prevotella bacteria. In some embodiments, the strain is Prevotella histicola bacteria, for example, Prevotella Strain B 50329 (NRRL accession number B 50329). In some embodiments, the Prevotella strain is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain B 50329.

[48] In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more) proteins listed in Table 1 and/or genes encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more) proteins listed in Table 1. In some embodiments, the Prevotella bacteria comprise all of the proteins listed in Table 1 and/or genes encoding all of the proteins listed in Table 1.

Table 1 : Exemplary Prevotella proteins

T1

[49] In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria free or substantially free of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) proteins listed in Table 2 and/or genes encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) proteins listed in Table 2. In some embodiments, Prevotella bacteria are free of all of the proteins listed in Table 2 and/or genes encoding all of the proteins listed in Table 2.

Table 2: Other Prevotella proteins

[50] In some embodiments, the Prevotella bacteria are from a strain of Prevotella bacteria comprising one or more of the proteins listed in Table 1 and that is free or substantially free of one or more proteins listed in Table 2. In some embodiments, the Prevotella bacteria are from a strain of Prevotella bacteria that comprises all of the proteins listed in Table 1 and/or genes encoding all of the proteins listed in Table 1 and that is free of all of the proteins listed in Table 2 and/or genes encoding all of the proteins listed in Table 2.

[51] In some embodiments, the Prevotella histicola strain is a strain comprising at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Prevotella histicola Strain C (ATCC Deposit Number PTA-126140).

[52] In some embodiments, the Prevotella histicola strain is Prevotella histicola Strain C (ATCC Deposit Number PTA-126140).

Antibiotic Selection for Anaerobic Contaminant Testing

[53] Use of antibiotic selection provides a robust anaerobic contaminant microbe test. Anaerobic contaminant microbes in a sample (e.g., a sample of therapeutic composition or therapeutic agent) can be assessed, e.g., after incubation in anaerobic growth conditions that include one or more antibiotic. The conditions (including concentration of antibiotic) used are chosen to inhibit (suppress) the growth (e.g., propagation) of product microbe (e.g., live product microbe), e.g., of the therapeutic agent or of the therapeutic composition. The presence and/or level of anaerobic contaminant microbe can be evaluated, e.g., by detecting the presence of colonies and/or by determining colony forming units per gram (CFU/g) of sample (determining viable cell count per gram (VCC/g) of a sample is used interchangeably with CFU/g herein). A composition (e.g., the source of a sample), such as a therapeutic composition or therapeutic agent, can be considered to contain anaerobic contaminant microbe (e.g., an unacceptable level thereof) if growth, in the presence of an antibiotic (e.g., at a selected concentration e.g., that suppreses growth of product microbe), is detected (e.g., as determined by the presence of colonies, e.g., after incubation), e.g., if any CFUs are detected and/or if the CFU/g value is above an acceptable limit. In some embodiments, the acceptable limit is up to about 500 CFU/g. In some embodiments, the acceptable limit is up to about 1000 CFU/g. In some embodiments, the acceptable limit is up to about 2000 CFU/g. In some embodiments, the acceptable limit is up to about 3000 CFU/g. For example, if the acceptable limit is set at 1000 CFU/g, any amount of CFU/g up to 1000 CFU/g is below the acceptable limit, and any amount over 1000 CFU/g exceeds the acceptable limit.

[54] As described herein, to detect anaerobic contaminant microbes in a composition comprising an anaerobic product microbe, an anaerobic contaminant testing procedure suppresses growth of the product microbe while allowing for the growth of anaerobic contaminant microbes, if present. In some embodiments, use of antibiotic(s) (e.g., and concentration(s) thereof) that allow for recovery of multiple, or even most, anaerobic contaminant strains that may be present provide robust test results. In other words, in some embodiments, the selected antibiotic can be an antibiotic (e.g., and concentration) to which the product microbe is highly sensitive but that would not suppress multiple, or even most, other potential anaerobic contaminant microbes. The term “highly sensitive” can indicate an antibiotic concentration that is able to suppress growth of a given microbe (e.g., the product microbe) is lower (e.g., 10-, 25-, 50-, or 60-fold lower or even more-fold lower) than the concentration needed to suppress growth of another anaerobic strain (e.g., a contaminant strain). The term “highly sensitive” can, in various embodiments, indicate that an antibiotic concentration is able to suppress growth of a given microbe (e.g., the product microbe) by at least 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, or 10000-fold as compared to growth under control conditions that do not comprise the antibiotic. For example, with reference to Table 3 herein and minimum inhibitory concentration (MIC) testing, ampicillin at a concentration of 0.25 mg/L suppresses growth of P. histicola strain 1; yet ampicillin at a concentration of over 16 mg/L suppresses growth of P. histicola strain 2. In this example, P. histicola strain 1 is considered to be highly sensitive to ampicillin. [55] In some aspects of the anaerobic contaminant testing methods described herein, one or more (e.g., two) antibiotics are used to test for anaerobic contaminant microbes (e.g., in a sample). In some embodiments, the first antibiotic is an antibiotic (such as a broad-spectrum antibiotic) that suppresses the product microbe (e.g., that does not support growth of the product microbe and/or to which the product microbe is highly sensitive) but allows recovery of one or more gram-positive anaerobic contaminant microbes (e.g., allows growth of the gram-positive anaerobic contaminant microbes and/or is not an antibiotic to which the gram-positive anaerobic contaminant microbes are highly sensitive), e.g., under the anaerobic growth conditions. In some embodiments, the first antibiotic is an antibiotic (such as a narrow-spectrum antibiotic) that suppresses the product microbe (e.g., that does not support growth of the product microbe and/or to which the product microbe is highly sensitive) but allows recovery of one or more grampositive anaerobic contaminant microbes (e.g., allows growth of the gram-positive anaerobic contaminant microbes and/or is not an antibiotic to which the gram-positive anaerobic contaminant microbes are highly sensitive), e.g., under the anaerobic growth conditions. In some embodiments, the first antibiotic is an antibiotic (such as a broad- spectrum antibiotic) that suppresses the product microbe (e.g., that does not support growth of the product microbe and/or to which the product microbe is highly sensitive) but allows recovery of one or more gramnegative anaerobic contaminant microbes (e.g., allows growth of the gram-negative anaerobic contaminant microbes and/or t is not an antibiotic to which the gram-negative anaerobic contaminant microbes are highly sensitive), e.g, under the anaerobic growth conditions. In some embodiments, the first antibiotic is an antibiotic (such as a narrow-spectrum antibiotic) that suppresses the product microbe (e.g., that does not support growth of the product microbe and/or to which the product microbe is highly sensitive) but allows recovery of one or more gramnegative anaerobic contaminant microbes (e.g., allows growth of the gram-negative anaerobic contaminant microbes and/or is not an antibiotic to which the gram-negative anaerobic contaminant microbes are highly sensitive), e.g., under the anaerobic growth conditions.

[56] In some embodiments, if two or more antibiotics are used, the first antibiotic (such as a broad- spectrum antibiotic or narrow-spectrum antibiotic) suppresses the product microbe (e.g., that does not support growth of the product microbe and/or to which the product microbe is highly sensitive) but allows recovery of one or more gram-positive anaerobic contaminant microbes (e.g., allows growth of the gram-positive anaerobic contaminant microbes and/or is not an antibiotic to which the gram-positive anaerobic contaminant microbes are highly sensitive) and the second antibiotic is an antibiotic (such as a broad-spectrum antibiotic or narrowspectrum antibiotic) that suppresses the product microbe (e.g., that does not support growth of the product microbe and/or to which the product microbe is highly sensitive) but allows recovery of one or more gram-negative anaerobic contaminant microbes (e.g., allows growth of the gramnegative anaerobic contaminant microbes and/or is not an antibiotic to which the gram- negative anaerobic contaminant microbes are highly sensitive), e.g., under the anaerobic growth conditions. In some embodiments, the selected antibiotic is ampicillin. In some embodiments, the selected antibiotic is colistin. In some embodiments, the selected antibiotics are ampicillin and colistin. In some embodiments, the product microbe is highly sensitive to the selected antibiotic(s). In some embodiments, ampicillin is used at a concentration of about 25 mg/L (e.g., on an agar plate) and/or colistin is used at a concentration of about 20 mg/L (e.g., on an agar plate) for evaluating anaerobic contaminants in a sample containing Prevotella Strain B. Ampicillin is a broad spectrum antibiotic. Colistin is also known as polymyxin E and is a narrow spectrum antibiotic. Colistin is an antimicrobial agent with activity against most common gramnegative bacteria.

Anaerobic Contaminant Testing Growth Medium

[57] In some embodiments, the growth medium utilized in anaerobic contaminant testing supports the growth of anaerobic contaminant microbes over the growth of the product microbe. In some embodiments, the addition of one or more antibiotics (e.g., and at a selected concentration(s)) to which the product microbe is sensitive in the growth medium suppresses product microbe growth while allowing for growth of contaminant microbes when incubated under anaerobic conditions. The one or more antibiotics can be used in separate growth media.

[58] In some exemplary embodiments, growth medium is prepared as an agar plate under sterile conditions. Control growth medium without an antibiotic is prepared as an agar plate. Growth medium containing an antibiotic (such as ampicillin or colistin) at a selected concentration (e.g., that suppresses product microbe growth, such as 25 mg/L ampicillin or 20 mg/L colistin) is prepared as an agar plate. Under sterile anaerobic conditions, a known weight of sample is added to each of the plates. The sample can be of a therapeutic agent (such as in powder form) (that contains product microbe) or of a therapeutic composition (that contains product microbe). The plates are then incubated under anaerobic conditions at 37°C for five days. CFU/g is then determined.

[59] In some embodiments, the growth medium comprises Brain Heart Infusion (BHI). In some embodiments, the growth medium comprises about 37 g/ml of the Brain Heart Infusion (BHI).

[60] In some embodiments, the growth medium comprises Bacto Agar. In some embodiments, the growth medium comprises about 7.5 g/ml of the Bacto Agar.

[61] In some embodiments, the growth medium comprises a Vitamin K and Hemin Solution. In some embodiments, the growth medium comprises about 10 ml/L of the Vitamin K and Hemin Solution.

[62] In some embodiments, the growth medium comprises a reducing agent. In some embodiments, the reducing agent comprises L-Cysteine-HCl. In some embodiments, the growth medium comprises about 0.5 g/ml of L-Cysteine-HCl.

[63] In some embodiments, the growth medium comprises an antibiotic. In some embodiments, the antibiotic is ampicillin. In some embodiments, the growth medium comprises about 20 mg/L to about 30 mg/L of ampicillin. In some embodiments, the growth medium comprises about 25 mg/L of ampicillin. In some embodiments, the antibiotic is colistin. In some embodiments, the growth medium comprises about 15 mg/L to about 25 mg/L of colistin. In some embodiments, the growth medium comprises about 20 mg/L of colistin.

[64] In some embodiments, the growth medium comprises a Brain Heart Infusion (BHI), a Bacto Agar, a Vitamin K and Hemin Solution and L-Cysteine-HCl.

[65] In some embodiments, the growth medium consists essentially of a Brain Heart Infusion (BHI), a Bacto Agar, a Vitamin K and Hemin Solution, L-Cysteine-HCl and ampicillin. In some embodiments, the growth medium comprises about 20 mg/L to about 30 mg/L of ampicillin. In some embodiments, the growth medium comprises about 25 mg/L of ampicillin.

[66] In some embodiments, the growth medium consists essentially of a Brain Heart Infusion (BHI), a Bacto Agar, a Vitamin K and Hemin Solution, L-Cysteine-HCl and colistin. In some embodiments, the growth medium comprises about 15 mg/L to about 25 mg/L of colistin. In some embodiments, the growth medium comprises about 20 mg/L of colistin.

[67] In some embodiments, the growth medium comprising an antibiotic (e.g., at a selected concentration (e.g., that suppresses product microbe growth)) is used to suppress growth of product microbe in a sample and allow for growth and detection of anaerobic contaminant microbe during incubation under anaerobic conditions. In some embodiments, the growth medium comprising an antibiotic is used to suppress growth of bacteria of product microbe in a sample and allow for detection of anaerobic contaminant microbe during incubation under anaerobic conditions. Anaerobic contaminant microbe can be assessed after incubation for one or more days, such as 1 -7 days, such as 5 days, at a temperature that supports growth of the anaerobic contaminants, such as 32°C to 42°C, such as 37°C. Anaerobic contaminant microbe can be assessed, e.g., after incubation, and measured, e.g., as colony forming units per gram (CFU/g) or VCC/g of sample. A composition (e.g., the source of a sample), such as a therapeutic composition or therapeutic agent, can be considered to contain anaerobic contaminant microbe (e.g., an unacceptable level thereof) if growth (as determined by the presence of colonies, e.g., after incubation) is detected (e.g., any CFUs detected) or if the CFU/g value is above an acceptable limit. In some embodiments, the acceptable limit is about 500 CFU/g. In some embodiments, the acceptable limit is about 1000 CFU/g. In some embodiments, the acceptable limit is about 2000 CFU/g. In some embodiments, the acceptable limit is about 3000 CFU/g. [68] In some embodiments, the growth medium comprising an antibiotic (e.g., at a selected concentration (e.g., that suppresses product microbe growth)) is used to suppress growth of bacteria of the genus Prevotella or species Prevotella histicola in a sample and allow for growth and detection of anaerobic contaminant microbe during incubation under anaerobic conditions. In some embodiments, the growth medium comprising an antibiotic is used to suppress growth of bacteria of Prevotella Strain B in a sample and allow for detection of anaerobic contaminant microbe during incubation under anaerobic conditions. Anaerobic contaminant microbe can be assessed after incubation for one or more days, such as 1-7 days, such as 5 days, at a temperature that supports growth of the anaerobic contaminants, such as 32°C to 42°C, such as 37°C. Anaerobic contaminant microbe can be assessed, e.g., after incubation, and measured, e.g., as colony forming units per gram (CFU/g) or VCC/g of sample. A composition (e.g., the source of a sample), such as a therapeutic composition or therapeutic agent, can be considered to contain anaerobic contaminant microbe (e.g., an unacceptable level thereof) if growth (as determined by the presence of colonies, e.g., after incubation) is detected (e.g., any CFUs detected) or if the CFU/g value is above an acceptable limit. In some embodiments, the acceptable limit is about 500 CFU/g. In some embodiments, the acceptable limit is about 1000 CFU/g. In some embodiments, the acceptable limit is about 2000 CFU/g. In some embodiments, the acceptable limit is about 3000 CFU/g.

EXAMPLES

Example 1: Antibiotic Selection for Anaerobic Contaminant Microbes

[69] The goal of the presently exemplified anaerobic contaminant microbe test is to suppress propagation of Prevotella Strain B, and allow for growth of other viable anaerobic microbes, so that they can be detected. Prevotella Strain B is a gram-negative strict anaerobe. During production it is grown in rich media under strict anaerobic conditions, which may at the same time allow for the propagation of other obligate or facultative anaerobes and aerotolerant microbes if they are unintentionally present in the media or equipment, causing for contamination of the final product.

[70] Propagation of different anaerobes in the presence of Prevotella Strain B could be achieved by addition of specific antibiotics into the culture (growth) medium that suppress Prevotella Strain B propagation. To identify the antibiotic minimum inhibitory concentration (MIC), Prevotella Strain B and another Prevotella histicola strain were tested with Sensititre MIC commercial tests. To identify which antibiotic can suppress Prevotella Strain B (and the other Prevotella histicola strain), it was tested with in-house prepared agar media supplemented with various doses of antibiotics (both broad and narrow range antibiotics).

[71] Antibiotic sensitivity testing was done on a Prevotella histicola strain Prevotella Strain B 50329 (NRRL accession number B 50329 “Prevotella Strain B” or “P. histicola 1”) and another Prevotella histicola strain (“P. histicola 2”). The results are provided in Table 3, wherein the listed MIC values indicate the minimum concentration (in mg/L) of an antibiotic to inhibit growth each strain. As shown in Table 3, the minimum inhibitory concentration of ampicillin for P. histicola 1 is 0.25 mg/L, and the minimum inhibitory concentration of ampicillin for P. histicola 2 is above 16 mg/L. Antibiotic minimum inhibitory concentrations in Table 3 are in mg/L.

[72] Ampicillin inhibits (suppresses) growth of many gram-positive anaerobes, while allowing many gram-negative microbes to propagate. Colistin inhibits gram-negative bacteria and allows the propagation of gram-positive bacteria. The colistin culture is expected to detect grampositive anaerobes, and the ampicillin culture is expected to detect important gram-negative anaerobes, including members of the Bacteroides group, that are known to cause some human infections.

[73] 25 mg/L of ampicillin suppressed growth of Prevotella Strain B when grown on agar media. 20 mg/L of colistin suppressed growth of Prevotella Strain B. Data on the effects of ampicillin and colistin on Prevotella Strain B are shown in Fig. 1, which shows the suppression of Prevotella Strain B on anaerobic incubation on BHI-ampicillin agar and BHI-colistin agar, where growth, if any, was below the limit of detection for both antibiotic conditions . Growth, as measured by VCC/g, was detected on anaerobic BHI control agar. LOD: limit of detection. VCC/g: viable cell count per gram (VCC/g = CFU/g).

Table 3: Antibiotic minimum inhibitory concentration (MIC) for Prevotella Strain B 50329 (NRRL accession number B 50329 or “P. histicola I ”) compared to another Prevotella histicola strain (“P. histicola 2”)

[74] The effects of ampicillin and colistin were tested on a mock community and a fecal community of potential and likely anaerobic contaminant microbes. As shown in Fig. 2, the mock community comprised 30 strict anaerobic and air-tolerant species; 9 gram-negative (di derm) genera; 21 gram-positive (monoderm) genera; and included representative genera produced at manufacturing plants and spore-forming bacteria (for example environmental contaminant microbes). The pie chart area is proportional to the viable cell count of each individual strain in the mock community by VCC/mL (viable cell count/mL). Table 4 below shows the components and VCC/mL of the mock community recovered: 21 gram-positive and 9 gram-negative anaerobic and air-tolerant strains on BHI control agar, BHLcolistin agar, or BHL ampicillin agar. On BHI control agar, a total of 30 strains were recovered. On BHLcolistin agar, 18 gram-positive and 4 gram-negative strains were recovered. On BHLampicillin agar, 7 gramnegative strains were recovered. In Table 4, the limit of detection (LOD) is 100 CFU/g; <100 indicates no colonies were recovered as they were below the LOD.

Table 4: Mock community members

[75] Recovery of 30 individual strains that comprise mock community on ampicillin and colistin media is shown in Figs. 5 A and 5B. The method was able to detect 18 gram-positive and 8 gram-negative bacterial strains (Table 5). The detected 26 strains belong to 22 different taxonomic genera and 14 families from the human microbiome dominant phyla Firmicutes (Bacillota is a validly published synonym), Bacteroidetes (Bacteroidota is a validly published synonym) and Actinobacteria (Aclinomycelola is a validly published synonym) that make up a majority of the bacterial species of human gut microbiome (King CH et al., 2019, Turnbaugh PJ, et al. 2009).

Table 5: Bacterial species recovery on selective media with ampicillin and colistin

[76] As seen in Fig. 3, the fecal community was characterized by 16S rRNA gene sequence profiling (see also Table 6). Using 16S rRNA gene sequence analysis, representatives of five phyla were detected: Firmicutes (Bacillota is a validly published synonym), Bacteroidetes (Bacteroidota is a validly published synonym), Actinobacteria (Aclinomycelola is a validly published synonym), Pseudomonadota (synonym to Proteobacteria), Verrucomicrobiota, and 18 known genera shown in Table 6. Some of the recovered microorganisms were identified on family, class or phylum taxonomic level only.

Table 6: Relative abundance of bacteria of the fecal community members

[77] The graphs in Figs. 4A and 4B show the recovery (as VCC/g) of the mock community (Fig. 4A) and fecal community (Fig. 4B) on anaerobic BHI control agar, ampicillin anaerobic BHI agar and colistin anaerobic BHI agar. Colistin primarily selects for recovery of grampositive bacteria while ampicillin primarily selects for recovery of gram-negative bacteria. The mock community and fecal community test systems were plated in different dilutions on control and selective media with antibiotics (Figs. 4A and 4B). The limit of detection (LOD) was 100 VCC/g (l.E+02 VCC/g).

[78] The mock community colony number recovery on control medium was compared to the number of colonies propagated on ampicillin and colistin media (Fig. 4A, Table 7). About 60% of gram-positive colonies (VCC) were recovered on colistin and only 1% of gram-negative colonies were recovered on ampicillin. From the fecal community about 95% of gram-positive VCC on colistin and around 40% of gram-negative VCC on ampicillin selective media were recovered (Fig. 4B, Table 7).

Table 7, Recovered viable cell count for mock community and fecal community test systems

[79]

[80] To test for fecal community anaerobic contaminant recovery, BHI control agar, BHI- colistin agar, and BFH-ampicillin agar were processed. Fourteen dominant genera were identified on the BHI control agar and 12 genera were recovered on the two selective agar media (BHI- colistin and BHI-ampicillin). Six gram-negative genera were recovered on BFH-ampicillin agar;

6 gram-positive and 2 gram-negative genera were recovered on BHI-colistin agar. These data are shown in Figs. 6A-6C. Percentages in the figures relate to percent of the read count from 16S profiling. [81] The fecal community colonies that propagated on ampicillin or colistin agar media were washed out from the plates surface. Collected consortium biomass was analyzed by 16S rRNA profiling. The method detected a wide range of ampicillin and colistin resistant bacteria from four phyla (Table 8): Firmicutes (Bacillota is a validly published synonym), Bacteroidetes (Bacteroidota is a validly published synonym), Actinobacteria (Actinomycetota is a validly published synonym), and Pseudomonadota (synonym to Proteobacteria). Representatives of three detected phyla, Firmicutes, Bacteroidetes, and Actinobacteria, make up a majority of the bacterial species of human gut microbiome (King CH et al., (2019) Baseline human gut microbiota profile in healthy people and standard reporting template. PLoS ONE 14(9): e0206484; Turnbaugh PJ, et al. (2009) A core gut microbiome in obese and lean twins. Nature 457:480 - 484).

Table 8, Bacterial genera from fecal sample recovered on selective media with ampicillin and colistin by 16S rRNA gene sequence profiling

[82] Overall, the use of colistin and ampicillin to suppress Prevotella Strain B for anaerobic contaminant testing provided robust results. The use of these two antibiotics was able to detect 77% of strains in a mock community and 86% of genera in a fecal community. Testing with colistin was able to detect 80% of gram-positive strains and 70% of total strains in a mock community and 57% of genera in a fecal community. Testing with ampicillin was able to detect 17% of gram- negative strains in a mock community and 43% of gram-negative genera in a fecal community.

Example 2: Preparation of Plates and Media for Anaerobic Contaminant Microbe Testing

[83] BHI agar plates were prepared.

Table 9: Media general formula

[84] L-cysteine-HCl (100X) solution was prepared separately according to the following instructions:

• Weigh 5 g of L-Cysteine-HCl.

• Add 100 ml of distilled water, mix until dissolved (mildly heat solution if not dissolving) and immediately transfer to an anaerobic chamber.

• Allow to degas for 1 hour.

• Close with airtight cap and seal.

• Autoclave at 121 °C for 30 mins.

[85] One liter of media (see Table 9 above) was prepared according to the following instructions:

• Weigh and resuspend 37 g of BHI powder in water.

• Add 1.5% agar to the resuspension and mix.

• Autoclave media at 121 °C for 30 minutes.

• Once media is autoclaved, move it to 50-55°C incubator to cool. This is performed in non-anaerobic conditions (not in an anaerobic chamber). [As a variation, this step can be performed under anaerobic conditions, such as inside an anaerobic chamber]. • Add 1% of L-cysteine (100X) solution to the media (10 ml per liter).

• Add 1 % Hemin/Vitamin K solution to the media (10 ml per liter).

• Close caps tightly and move to a 42°C incubator to cool.

[86] BHI plates were prepared as follows: Pour the media into petri dishes (about 23 ml per plate) inside biosafety hood and allow to solidify before turning over and moving into anaerobic chamber. This pouring step is performed in non-anaerobic conditions (not in an anaerobic chamber). [As a variation, this step can be performed under anaerobic conditions, such as inside an anaerobic chamber],

[87] BHI plates with antibiotics (either ampicillin or colistin) were prepared according to the following instructions:

• Antibiotic is added to the BHI agar media when it reaches 42 - 45 °C, add antibiotics to reach the final working concentration required (see Table 10 below for ampicillin and colistin concentrations used for Prevotella Strain B studies). This is performed in non-anaerobic conditions (not in an anaerobic chamber). [As a variation, this step can be performed under anaerobe conditions, such as inside an anaerobic chamber],

• Media is mixed and immediately poured into petri plates (about 23 ml per plate) inside biosafety cabinet. This is performed in non-anaerobic conditions (not in an anaerobic chamber). [As a variation, this step can be performed under anaerobic conditions, such as inside an anaerobic chamber],

• Allow the agar to solidify in the biosafety cabinet and transfer into anaerobic chamber.

• Use within a week of preparation.

[88] The choice of an antibiotic(s) to add to BHI agar media for anaerobic contaminant testing depends on the product microbe. Antibiotic is chosen to suppress growth of product microbe but allow for growth of other viable anaerobic microbes, so that the other anaerobic microbes can be detected. The antibiotic is a broad-spectrum antibiotic or a combination of a broad-spectrum antibiotic and a narrow spectrum antibiotic. Here, the antibiotic(s) chosen suppresses growth of the product microbe at 25mg/L ampicillin and 20 mg/L colistin for Prevotella Strain B.

[89] Table 10 below provides the stock and working concentrations for ampicillin and colistin used in studies with Prevotella Strain B. Table 10: Antibiotic stock concentrations and final concentrations for BHI agar plates

Example 3: Exemplary Anaerobic Contaminant Testing of Therapeutic Agent

[90] Exemplary manufacturing processes of Prevotella Strain B are detailed in WO 2020/237009, herein incorporated by reference. Exemplary therapeutic agent and therapeutic composition are detailed in WO 2020/237009, WO 2022/061123, and WO 2022/061094, herein incorporated by reference.

[91] Anaerobic contaminant testing of samples of product microbe therapeutic agents, specifically samples of product microbe Prevotella Strain B, were conducted according to the instructions below:

• Weigh at least 1 gram of the sample to be tested. Record the weight.

• Process the sample in either an anaerobic chamber or in a biosafety cabinet. If processed in biosafety cabinet, work needs to be done promptly and plates incubated in anaerobic jars with sachet as quickly as possible. The plates used for the test are stored anaerobically and only removed from their anaerobic environment to be used for plating immediately.

• Resuspend the sample in anaerobic dilution blank to -1 dilution (multiply the weight of the sample by 9 and add that amount of dilution blank, for example for 1.24 grams of sample (1.24 g x 9 = 11.16 g) use 11.16 grams of dilution blank).

• Vortex thoroughly for 1 minute. Make sure there are no visible clumps left and the mixture is homogenous. Vortex more if required.

• Dilute the sample further to -2, -3, -4, -5 and -6 by making subsequent 1 :10 dilutions by adding 1 mL sample in 9 mL dilution blank. Vortex on high for 3 x 5 sec bursts in between each dilution. Change for a new sterile tip between dilutions.

• On BHI plates (BHI control), plate dilutions -4, -5, and -6. • On plates containing antibiotics (BHI-ampicillin; BHI-colistin) plate dilutions -1, -2, and -3.

• Plate the samples immediately on plates that are marked in advance with sample identifier and dilution.

• PlatelOO pl on each plate; use a minimum of 2 plates per dilution.

• Use a sterile disposable spreader to spread the bacteria as evenly as possible. Change spreaders and pipette tips between each dilution.

• Incubate the samples in anaerobic conditions (in an incubator inside anaerobic chamber or in an anaerobic jar with a gas generating sachet) at 37°C for 5 days before reading the results.

[92] To read the results, after the 5 days of anaerobic incubation, remove the plates from the incubator and allow them to cool to room temperature. Using a counter, count the colonies in the plates. To calculate the CFU/gram of powder, multiply the number of colonies by the dilution factor and divide by 0.1.

Example 4: Anaerobic Contaminant Testing of Prevotella Strain B Therapeutic Agent Samples

[93] Following the method in Example 3 above, Table 11 A below shows the total anaerobic contaminant microbe recovery of samples of preparations of therapeutic agent of Prevotella Strain B. The limit of detection was 100 CFU/g. The acceptance criteria were set at an acceptable limit of 2000 CFU/g for the plates containing an antibiotic. Therefore, the total number of anaerobic contaminant microbes exceeded the accepted level (2000 CFU/g) for the sample of therapeutic agent 3.

Table 11 A: Anaerobic contaminant microbe test results for Prevotella Strain B samples

[94] The total anaerobic contaminant microbe recovery of samples of four other preparations of therapeutic agent of Prevotella Strain B was determined. No anaerobic contaminants were detected in any of the four samples when grown with an antibiotic. Results are shown in Table 1 IB. The method limit of detection was 100 CFU/g.

Table 1 IB: Anaerobic contaminant microbe test results for Prevotella Strain B samples

Example 5: Anaerobic Contaminant Microbe Testing Controls

[95] Once the set of proposed media was established for anaerobic contaminant microbe testing of samples containing Prevotella Strain B, a range of ATCC strains were tested on the media with and without antibiotics to find robust positive and negative controls for the method. From the 16 ATCC strains tested, 3 were chosen to be used as controls: a) Bacteroides ovatus ATCC 8483 as positive control for ampicillin and colistin media, b) Prevotella veroralis ATCC 33779 as negative control for ampicillin and colistin media, and c) Bifidobacterium bifidum ATCC 15696 as positive control on colistin medium and negative control on ampicillin medium. These 3 strains were grown, diluted and stored with glycerol as stocks. Control stocks are used each time a sample is tested to confirm media performance. The control strains need to follow the expected pattern of growth on the selective and control plates, to verify that the media are working as expected (Table 12). Table 12 below shows control bacteria strains and their expected growth in the three different agars: BHI control, BHI-colistin (20 mg/L), and BHI-ampicillin (25 mg/L).

Table 12: Expected growth of control samples

[96] Prior to use in the anaerobic contaminant microbe assay, these bacteria strains were grown in anaerobic conditions in appropriate liquid media. Prevotella veroralis and Bacteroides ovatus were both grown in anaerobic Tryptic Soy Broth (TSB) with 0.5 g/L L-cysteine HC1 with Hemin and vitamin K added (see Table 13 below). The media final pH was 6.8 ± 0.3 at 25°C.

Table 13: Anaerobic tryptic soy broth (TSB) medium supplemented with hemin/vitamin K solution

[97] Bifidobacterium bifidum were grown in LMRS media (see Table 14 below). The media final pH was 6.5 ± 0.2 at 25°C.

Table 14: Anaerobic Lactobacillus MRS broth medium

[98] Control bacteria strains were prepared according to the instructions below:

• Inoculate the appropriate media with the control bacteria strain and incubated anaerobically for 18-20 hours, until the control sample looks dense (OD600 greater than 1).

• Dilute the control sample to -5 and -6 dilutions.

• Mix the diluted control sample 1 : 1 with Anaerobic Glycerol Solution or other cryoprotectant and make multiple aliquots.

• Freeze the aliquots and store at -80°C.

• Test the control samples after freeze-thaw cycle on BHI control plates, by plating 100 pls on 2 plates and incubating anaerobically.

• If the dilution is appropriate to be used for testing, the number of colonies on BHI control plates should be between 30 and 300.

[99] For use in the anaerobic contaminant microbe assay, these strains were plated every time a sample product was tested as follows: Plate 100 pl on each plate, using 2 plates of BHI control agar, 2 plates of BFH-ampicillin agar, and 2 plates of BHI-cohstin agar for each control strain. Use a spreader to evenly spread the samples on the plates, turn plates over and incubate in anaerobic chamber at 37°C for 5 days.

[100] All control strains propagated on control BHI agar, and performed as expected on selective antibiotics media (Table 15).

Table 15, Growth of control strains on anaerobic contaminant microbe method media

Incorporation by Reference

[101] All publications patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

[102] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS What is claimed is:

1. A method of detecting anaerobic contaminant microbes in a composition comprising a product microbe, the method comprising suppressing growth of the product microbe and permitting growth of anaerobic contaminant microbes of a sample of the composition in a medium under anaerobic conditions.

2. The method of claim 1 , wherein the product microbe is an anaerobic bacterium.

3. The method of claim 1, wherein the composition is a therapeutic agent or therapeutic composition.

4. The method of any one of claims 1-3, wherein the product microbe is an anaerobic bacterium of the genus Actinomyces, Bacteroides, Bifidobacterium, Clostridium, Foumierella, Fusobacterium, Harryflintia, Lactococcus, Megasphaera, Parabacteroides, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, or Veillonella.

5. The method of any one of claims 2-4, wherein the anaerobic bacterium is a Prevotella histicola bacterium.

6. The method of any one of claims 2-5, wherein the anaerobic bacterium is a Prevotella Strain B 50329 (NRRL accession number B 50329) bacterium.

7. The method of any one of claims 1 -6, wherein the method further comprises quantification of the anaerobic contaminant microbes, optionally wherein the anaerobic contaminant microbes are quantified by calculating CFU/gram.

8. The method of claim 7, wherein calculation of CFU/gram comprises multiplying the number of colonies in the medium by a dilution factor of the sample and dividing by 0.1.

9. The method of any one of claims 1-8, wherein the sample is diluted and subsequently incubated on the medium.

10. A growth medium for anaerobic contamination testing comprising a Brain Heart Infusion (BHI), a Bacto Agar, a Vitamin K and Hemin Solution, and a reducing agent, optionally wherein the reducing agent is L-Cysteine-HCl, and an antibiotic.

11. The growth medium of claim 10, wherein the antibiotic is ampicillin.

12. The growth medium of claim 10, wherein the antibiotic is colistin.

13. The growth medium of any one of claims 10-12, wherein the growth medium comprises or consists essentially of about 37 g/ml of the Brain Heart Infusion (BHI), about 7.5 g/ml of the Bacto Agar, about 10 ml/L of the Vitamin K and Hemin Solution, about 0.5 g/ml of L-Cysteine- HC1, and about 25 mg/L of ampicillin.

14. The growth medium of any one of claims 10-12, wherein the growth medium comprises or consists essentially of about 37 g/ml of the Brain Heart Infusion (BHI), about 7.5 g/ml of the Bacto Agar, about 10 ml/L of the Vitamin K and Hemin Solution, about 0.5 g/ml of L-Cysteine- HC1, and about 20 mg/L of colistin.