EP4689056A2

EP4689056A2 - Media and media supplement for the growth of fastidious microorganisms

Info

Publication number: EP4689056A2
Application number: EP24717136.6A
Authority: EP
Inventors: Julian MARCHESI; Lauren ROBERTS
Original assignee: Imperial College Innovations Ltd
Current assignee: Ip2ipo Innovations Ltd
Priority date: 2023-03-29
Filing date: 2024-03-28
Publication date: 2026-02-11
Also published as: WO2024200789A2; WO2024200789A3; WO2024200789A9; GB202304580D0

Abstract

The invention relates media, compositions, medium supplements and a method of making same for high-yield culture of fastidious bacteria.

Description

MEDIA AND MEDIA SUPPLEMENT FOR THE GROWTH OF FASTIDIOUS MICROORGANISMS

FIELD OF THE INVENTION

The present invention relates to media for the growth of fastidious microorganisms (e.g. bacteria) such as those found in the vaginal microbiome such as Lactobacillus iners, or the intestinal microbiome such as Alistipes spp.

BACKGROUND TO THE INVENTION

Lactobacillus spp. are Gram-positive, facultatively anaerobic rod-shaped bacteria which are known to produce lactic acid by fermentation and are the main colonizers of the human vaginal tract. It has long been known that healthy vaginal microbiomes are those dominated by Lactobacillus spp. Of the four main lactobacilli found in the vagina, L. iners is one of the most frequently found with an estimated 50% of women harbouring this species. However, the presence of L. iners in women with bacterial vaginosis (BV) suggests that this species may not be protective against the disease, furthermore it has been demonstrated that an L. iners dominated vaginal microbiome is more likely to shift to this community state type (CST). Gardnerella vaginalis can also dominate the vaginal microbiome in BV.

Historically L. iners can be difficult to identify on vaginal smears as it is not always clearly Gram-positive, and some isolates have a more coccobacillary morphology as opposed to the classic bacillary morphology of Lactobacillus species. L. iners is a vaginal bacterium that is cultured anaerobically and is known to be fastidious in its growth requirements. Lactobacilli are usually grown on MRS (de Man, Rogosa and Sharpe) media, however this medium poorly supports the growth of L. iners due to its more complex nutritional requirements (Petrova Ml, Reid G, Vaneechoutte M, Lebeer S. Lactobacillus iners: ‘Friend or Foe?’ Trends in Microbiology. 2017 Mar 1 ;25(3):182-91). Columbia agar supplemented with 5% v/v defibrinated horse blood is the recommended medium for culturing L. iners and G. vaginalis. Colonies grown on this medium are very small and usually require 48 hours incubation to obtain sufficient growth. The other major difficulty arises in liquid culture of pure isolates, as blood cannot be added to broth because during concentration of the bacterial growth by centrifugation, the blood cells are also concentrated and contaminate the bacterial fraction. However, without the blood supplement the bacteria grow poorly in Columbia broth, with L. iners often failing to grow at all. Early research identified the components of secretions from the female genital tract and proposed a Chemically Defined Medium (CDM) to support the growth of vaginal bacteria (Geshnizgani AM, Onderdonk AB. Defined Medium Simulating Genital Tract Secretions for Growth of Vaginal Microflora. Journal of Clinical Microbiology. 1992; 30 (5): 1323-6), however this medium does not support the growth of L. iners and poorly supports growth of G. vaginalis. A modified version of NYCIII broth from the American Type Culture Collection (ATCC) (Hertzberger R. REBLAB. 2017 [cited 2021 Nov 22] available from: http://www.reblab.org/uncategorized/nyciii-medium/) has been used by some laboratories to culture G. vaginalis. However, this medium does not provide robust support for growing L. iners. Research by Bloom and co-workers demonstrated that MRS supplemented with cysteine and glutamine supported growth of L. iners (Bloom SM, Mafunda NA, Woolston BM, Hayward MR, Frempong JF, Abai AB, et al. Cysteine dependence of Lactobacillus iners is a potential therapeutic target for vaginal microbiota modulation. Nature Microbiology. 2022 Mar 1 ; 7(3): 434-50). However, robust growth was only obtained after 35+ hours.

There is a paucity of information about these vaginal bacteria, particularly L. iners, due to the lack of appropriate medium to grow them. A medium is reguired that can support vigorous growth of fastidious bacteria like L. iners and G. vaginalis e.g. for isolation from clinical swabs or during transport of a swab to a medical laboratory for analysis.

SUMMARY OF THE INVENTION

The invention disclosed concerns a new and improved medium for the growth of fastidious organisms such as bacteria. The fastidious bacteria can be fastidious vaginal bacteria. The fastidious bacteria can be fastidious intestinal bacteria. The fastidious vaginal bacteria can include species such as Lactobacillus iners (L. iners) and G. vaginalis. The fastidious intestinal bacteria can include species such as Alistipes spp. Current growth methods are only able to achieve low densities of L. iners after culturing for 48 hours. In contrast, the unigue combination of reagents in the media in this invention provides improved growth for Lactobacillus iners (L. iners) and G. vaginalis, e.g. enabling fast growth of L. iners to high densities within 24 hours. An object of the present invention is to provide a medium supplement and a medium composition which can allow fastidious microorganisms (e.g. bacteria) such as those from the vaginal microbiome or intestinal microbiome to grow to high yields and more guickly than is currently possible. Another object of the present invention is to provide a culture method capable of economically culturing fastidious anaerobes to a high final optical density. Media, compositions and supplements are provided that consist of readily available laboratory chemicals and can be manufactured more economically and still provide an improved medium for culture of fastidious microorganisms.

A culture medium for growth of fastidious microorganisms is provided comprising (a) a base composition (b) a second composition and (c) fetal bovine serum. The base composition comprises Polysorbate 80 (polyoxyethylene sorbitan monooleate) and/or cysteine hydrochloride. The second composition can comprise Menadione (Vitamin K3).

A medium supplement for culture of fastidious microorganisms (e.g. bacteria, such as those of the vaginal tract or intestinal tract) is provided, the medium supplement including Polyethylene glycol sorbitan monooleate (also known as Polysorbate 80, and TWEEN 80™), Cysteine- hydrochloride and/or menadione (vitamin K3). The medium supplement may include Polyethylene glycol sorbitan monooleate (also known as Polysorbate 80, and TWEEN 80™), Cysteine- hydrochloride and glycogen. The medium supplement may include a carbon source, such as glucose.

A medium composition for culture of fastidious microorganisms (e.g. bacteria, such as those of the vaginal tract or intestinal tract) is provided. The medium composition can comprise the base composition and second composition of the culture medium described herein, or the medium supplement described herein, optionally further comprising one or more from proteose peptone; yeast extract; fetal bovine serum; preferably comprising proteose peptone and yeast extract. A medium composition for culture of fastidious microorganisms (e.g. bacteria, such as those of the vaginal tract or intestinal tract) is provided, the medium composition comprising one or more from: peptone; yeast extract; fetal bovine serum; and a medium supplement as described herein. Preferably the medium composition comprises peptone and yeast extract.

A method of culturing fastidious microorganisms (e.g. bacteria, such as those of the vaginal tract or intestinal tract) is provided, the method including inoculating fastidious microorganisms into (i) the above described medium or (ii) a medium comprising the medium supplement, or (iii) a medium comprising the medium composition, and culturing the inoculated fastidious microorganisms.

A method of preparing a medium for culture of fastidious microorganisms (e.g. bacteria, such as those of the vaginal tract or intestinal tract) is provided comprising (a) making a part (I) by solubilizing the components of the base composition in water, and sterilizing the solution by autoclaving, preferably at 110°C to 130°C; (b) making a part (II) by solubilizing the components of the second composition in water, and sterilizing the solution by filtration; (c) mixing part (I) with part (II) in a 1 :1 ratio.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Growth curve of G. vaginalis in different media formulations. Optical Density (ODeoo, y-axis) plotted against time (hours, x-axis). (i) Black continuous line: NYCIII Glucose, (ii) Black dashed line: NYCIII Glycogen (instead of glucose), (iii) Black dotted line: NYCIII + Glycogen + Glucose, (iv) Black dash-dot-dash line: NYCIII + Glycogen + Urea, (v) Grey continuous line: NYCIII + Glycogen + Urea + Cysteine, (vi) Grey dashed line: NYCIII + Glycogen + Urea + Cysteine + Albumin + Mucin, (vii) Grey dotted line: Vaginal Microbe Media (VMM), (viii) Grey dash-dot-dash line: Chemically Defined Medium (CDM).

Figure 2. Growth curve of L. iners in different media formulations. Optical Density (ODeoo, y-axis) plotted against time (hours, x-axis). (i) Black continuous line: NYCIII Glucose, (ii) Black dashed line: NYCIII Glycogen (instead of glucose), (iii) Black dotted line: NYCIII + Glycogen + Glucose, (iv) Black dash-dot-dash line: NYCIII + Glycogen + Urea, (v) Grey continuous line: NYCIII + Glycogen + Urea + Cysteine, (vi) Grey dashed line: NYCIII + Glycogen + Urea + Cysteine + Albumin + Mucin, (vii) Grey dotted line: Vaginal Microbe Media (VMM), (viii) Grey dash-dot-dash line: Chemically Defined Medium (CDM).

Figure 3. Growth curve of L. iners in Vaginal Microbe Media (VMM) broth, for two L. iners isolates (DSM 13335 and DSM 13336). Optical Density (ODeoo, y-axis) of cultures grown in VMM broth plotted overa 32-hour time period, (i) Black continuous line: L. iners DSM 13336. (ii) Black dashed line: L. iners DSM 13335.

Figure 4. Growth curves of L. iners isolates in supplemented MRS (de Man, Rogosa and Sharpe) broth, shown on same scale as Figure 3. Adapted from data published by Bloom et al. Optical Density (ODeoo, y-axis) plotted against time (hours, x-axis). (i) Black continuous line: L. iners F3. (ii) Black dashed line: L. iners F1. (iii) Black dotted line: L. iners F2. (iv) Black dash-dot-dash line: L. iners M . (v) Grey continuous line: L. iners F4. (vi) Grey dashed line: L. iners A3, (vii) Grey dotted line: L. iners A4. (viii) Grey dash-dot-dash: L. iners A2.

Figure 5. Bar chart showing the number of Colony Forming Units (CFU) of L. iners isolates (DSM 13335 and DSM 13336) present when grown in Columbia broth versus Vaginal Microbe Media (VMM) broth. CFU was determined by serial dilution of 1 mL of culture, after pelleting and washing with PBS, followed by plating onto Columbia Blood Agar (CBA) plates and incubation in an anaerobic chamber for 48 hr. CFU is shown on a logarithmic scale on the y-axis, and bars are shown for Columbia broth (left) and VMM (right). For each broth, a bar is shown for (i) L. iners DSM 13335 (grey) and (ii) L. iners DSM 13336 (black).

Figure 6. Bar chart showing the number of Colony Forming Units (CFU) of L. iners commercial isolates (DSM 13335 and DSM 13336) and isolates from patients (L. iners 21 N1 and L. iners 24C1) present when grown in different media. CFU is shown on a logarithmic scale on the y-axis, and media are shown from left to right as follows: Columbia, MRS, MRS +, and VMM. For each medium, bars denoting different L. iners isolates are shown from left to right as follows: (i) L. iners DSM 13335 (grey), (ii) L. iners DSM 13336 (black), (iii) L. iners 21 N1 (diagonal grey stripes), and (iv) L. iners 24C1 (diagonal black stripes).

Figure 7. Growth curve of L. iners isolates comparing VMM to VMM base, for two L. iners isolates (DSM 13335 and DSM 13336). Optical Density (ODeoo, y-axis) of cultures grown in VMM or VMM base broth plotted over a 55-hour time period, (i) Black continuous line: VMM L. iners DSM 13335. (ii) Black dashed line: VMM L. iners DSM 13336. (iii) Black dotted line: VMM base L. iners DSM 13335. (iv) Black dash-dot-dash line: VMM base L. iners DSM 13336.

Figure 8. Growth curve of L. iners isolates with various compositions of VMM. A) L. iners DSM 13335. B) L. iners DSM 13336. Optical Density (ODeoo, y-axis) of cultures grown in VMM compositions plotted over a 40-hour time period, (i) Black continuous line: VMM. (ii) Black dashed line: VMM base, (iii) Black dotted line: VMM base + Glycogen, (iv) Black dash- dot-dash line: VMM base + Albumin, (v) Grey continuous line: VMM base + Urea, (vi) Grey dashed line: VMM base + MgSO4. (vii) Grey dotted line: VMM base + Vitamins, (viii) Grey dash-dot-dash line: VMM base + FBS.

Figure 9. Bar graph of the 24 hour time point comparing the impact of different formulations of VMM on the growth of L. iners isolates. A) L. iners DSM 13335. B) L. iners DSM 13336. Optical Density (ODeoo) of cultures is shown on the y-axis and media are shown from left to right as follows: VMM, VMM base, VMM base + Glycogen, VMM base + Albumin, VMM base + Urea, VMM base + MgSO4, VMM base + Vitamins, and VMM base + FBS.

Figure 10. Growth curves of L. iners isolates with VMM, VMM base and VMM base with FBS, glycogen and albumin. Optical Density (ODeoo, y-axis) of cultures plotted over a 24- hour time period. A) L. iners DSM 13335. B) L. iners DSM 13336. C) L. iners 21 . D) L. iners 24. (i) Black continuous line: VMM. (ii) Black dashed line: VMM base, (iii) Black dotted line: VMM base + FBS + Albumin + Glycogen (VMM2).

Figure 11. Bar graph of the 24 hour time point comparing the impact of the 3 formulations of VMM on the growth of L. iners isolates. Optical Density (ODeoo, y-axis) plotted for the following strains from left to right: L. iners DSM 13335, L. iners DSM 13336, L. iners 21 , and L. iners 24. For each L. iners isolate, the following formulations are plotted (from left to right): Solid black bar: VMM. Grey bar with dots: VMM base. Solid grey bar: VMM base + FBS + Albumin + Glycogen (VMM2).

Figure 12. Bar chart showing the number of Colony Forming Units (CFU) of L. iners isolate DSM 13335 present when grown in Columbia, MRS, supplemented MRS (MRS+), New York City III (NYC III), Vaginal Microbe Media (VMM), and VMM base supplemented with FBS, Albumin and Glycogen (VMM 2) broths. CFU was determined by plating serial dilutions of the culture onto Columbia Blood Agar (CBA) plates and incubated in an anaerobic chamber for 48 hrs. The inoculum was prepared by resuspending L. iners culture from a CBA plate in Columbia broth to an OD = 0.1. CFU is shown on a logarithmic scale on the y-axis, the inoculum is on the far left (dark grey) and the results for the 6 media are groups into the 24 hour and 48 hour readings. White: Columbia, White with black dots: MRS, White with black diagonal lines: MRS +, light grey: NYCIII, light grey with black dots: VMM, light grey with black diagonal lines: VMM2.

Figure 13. Bar chart showing the number of CFU of L. iners isolate DSM 13335 present when grown in Columbia, MRS, MRS+, NYC III, VMM, and VMM 2 broths. CFU was determined as described previously. The inoculum was prepared by resuspending L. iners culture from a CBA plate in Columbia broth to an OD = 0.1 , and then diluting the culture by serial dilution. CFU is shown on a logarithmic scale on the y-axis, the inoculum is on the far left (dark grey) and the results for the 6 media are groups into the 24 hour and 48 hour readings. White: Columbia, White with black dots: MRS, White with black diagonal lines: MRS +, light grey: NYCIII, light grey with black dots: VMM, light grey with black diagonal lines: VMM2

DETAILED DESCRIPTION

Even though L. iners is one of the most common bacteria in the vaginal microbiome, isolates have been difficult to grow from patient samples. The inventors have cultured bacteria from 25 swabs vaginal swabs yet only isolated L. iners from two. In contrast, L. iners can be found in 50% of vaginal swabs when analysed using DNA based assays (where no culturing is required). Bloom and colleagues found that L. iners does not require a reduced environment when it has access to enough bioavailable sources of cysteine (5). This observation may indicate that the transport media used for vaginal swabs is inadequate leading to fastidious vaginal bacteria such as L. iners not being able to survive on the swab from hospital to lab. Using this VMM as a transport medium with vaginal swabs may allow bacteria to persist long enough to be cultured in the lab.

Definitions

For the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

Fastidious microorganisms, such as bacteria, are microorganisms with complicated nutritional requirements, that will only grow in the presence of specific nutrients and/or specific conditions. Fastidious bacteria are difficult to culture. A practical consideration for fastidious bacteria is that a negative culture result for a fastidious bacterial could be a false negative. Fastidious bacteria of the vaginal tract that grow well using the methods, media and media supplements of the invention include Atopobium vaginae / Fannyhesse vaginae, Bifidobacterium dentium, Escherichia coli, Finegoldia magna, Fusobacterium nucleatum, Gardnerella vaginalis, Lacticaseibacillus rhamnosus, Lactobacilli us gasseri, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus vaginalis / Limosilactobacillus vaginalis, Prevotella amnii. Fastidious bacteria of the vaginal tract that grow well using the methods, media and media supplements of the invention also include add Dialister micraerophilus, Sneathia vaginalis and Megasphaera lornae. The fastidious bacteria of the vaginal tract may be selected from L. iners and G. vaginalis. Preferably the fastidious bacterium of the vaginal tract is L. iners.

The vaginal tract (VT) is also known as the female genital tract. The microbiota composition of the VT is linked to important women’s health and reproductive outcomes including preterm birth (miscarriage) human immunodeficiency virus (HIV) risk, and human papillomavirus (HPV) infection. The microorganisms that are found within the vaginal tract are collectively known as the vaginal microbiome. Other fastidious microorganisms such as bacteria of the intestinal tract have similar growth requirements. Fastidious bacteria of the intestinal tract suitable for growth using the methods, media and media supplements of the invention include Alistipes finegoldii, Alistipes onderdonkii, Alistipes putredins, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bifidobacterium longum, Citrobacter freundii, Clostridium difficile, Clostridium perfringes, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Roseburia hominis, and Ruminococcus gnavus. The fastidious bacteria of the intestinal tract may be selected from Alistipes spp., Fusobacterium nucleatum, Roseburia hominis and Ruminococcus gnavus. Preferably the fastidious bacteria of the intestinal tract are Alistipes spp and/or Roseburia spp.

Bacterial species which can be cultured using the media described herein include one or more or all of the bacterial species listed in Table 11 and Table 12.

As used herein, the term "medium" or "culture medium" refers to a solid, semisolid or liquid medium containing all of the nutrients and necessary physical growth parameters necessary for microbial growth or proliferation.

As used herein, the term "culture" or "growth" of microorganisms means multiplying microbial organisms by letting them reproduce in a predetermined culture medium under conditions conducive for their growth.

As used herein, the term "supplement" of medium refers to an additive consisting of selected components to promote the growth, proliferation or other characteristics of one or more desired anaerobes.

The ingredients in any of the tables herein may be provided as supplements, compositions or in the form of a kit comprising a dry composition.

As used herein, the term "includes", "comprises", "contains" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

As used herein, where a concentration is provided in g/L, that refers to the dry weight of compound added to the corresponding volume of water (w/v).

As used herein, where the amount of a component comprised in a culture medium is expressed as a weight % in w/v, based on the total volume of the culture medium. This may be calculated as follows:

A culture medium for growth of fastidious microorganisms (e.g. bacteria, such as those from the vaginal or intestinal tract) is provided which comprises (a) a base composition (b) a second composition and (c) fetal bovine serum. The base composition comprises Polysorbate 80 (polyoxyethylene sorbitan monooleate) and/or cysteine. The base composition can include one or more of sodium chloride, Polysorbate 80, D-glucose, Potassium phosphate dibasic, Potassium phosphate monobasic and cysteine (e.g. cysteine- hydrochloride). The base composition can further include proteose peptone and/or yeast extract. Where present, the components of the base composition can be used in the following amounts, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%. Where provided as a dry supplement, the components are provided in g, in corresponding proportions to the concentrations given below, e.g. for preparation of 500 mL or 1 L of medium. The base composition can comprise water.

Table 1

The base composition can comprise components according to recipe 1 or recipe 2, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%.

The second composition can comprise Menadione (Vitamin K3). The second composition can comprise water, preferably distilled water. The second composition can comprise one or more of the components in the following table, optionally present in the following amounts (where present), further optionally +/- 2% +/- 5%, +/- 10%, +/- 15%:

Table 2

The second composition can comprise components according to recipe 3 or recipe 4, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%. The culture medium can further comprise one or more components selected from the following table, optionally in the following amounts (where present), further optionally +/- 2% +/- 5%, +/- 10%, +/- 15%:

Table 3 A medium supplement for high yield culture of fastidious microorganisms (e.g. bacteria such as those from the vaginal or intestinal tract) is provided. The medium supplement includes cysteine (e.g. Cysteine- hydrochloride), more preferably 0.2 to 1 g/L cysteine hydrochloride; optionally 0.5 g/L (+/ 10%), and/or Menadione (Vitamin K3) and Tween80, more preferably 0.1 to 0.3 g/L TWEEN 80; optionally 0.2 g/L (+/- 10%). The medium supplement can comprise a carbon source e.g. glucose, preferably D-glucose, more preferably 4 g/L to 8 g/L D-glucose; optionally 5 g/L (+/- 10%). The medium supplement can one or more of: Sodium chloride, D-glucose, Potassium phosphate dibasic, Potassium phosphate monobasic, Polyethylene glycol sorbitan monooleate and Cysteine- hydrochloride. The medium supplement can further include one or more of :Glycogen, Bovine Serum Albumin, Mucin, Urea, Magnesium sulfate, Sodium bicarbonate, Hemin (5g/L), Menadione (Vitamin K3)(1g/L), Myo-inositol, Thiamine HCI, Pyridoxine HCI, Folic acid, D-Biotin, L-ascorbic acid, D-calcium pantothenate, Choline chloride, Nicotinamide (niacinamide), riboflavin, Cyancobalamin/Vitamin B12, Vitamin A, p-amino benzoic acid and Vitamin D (cholecalciferol). The medium supplement can comprise one or more selected from: Glycogen, Mucin, Urea, Magnesium sulfate, Hemin, Menadione, Vitamin mix and Bovine Serum Albumin. The medium supplement of the invention may include one or more of Sodium chloride, D-glucose, Potassium phosphate dibasic, Potassium phosphate monobasic, Polyethylene glycol sorbitan monooleate and Cysteine- hydrochloride. The medium supplement of the invention may comprise or consist of Sodium chloride, D- glucose, Potassium phosphate dibasic, Potassium phosphate monobasic, Polyethylene glycol sorbitan monooleate and Cysteine- hydrochloride. The medium supplement can further comprise proteose peptone and/or yeast extract. The medium supplement can further comprise additional buffers or salts, such as one or more selected from HEPES, potassium chloride and sodium bicarbonate. The components may be present in the any of the amounts provided in Table 1 , table 2 or table 3, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%.

A medium composition for culture of fastidious microorganisms (e.g. bacteria, such as those of the vaginal tract or intestinal tract) is provided comprising (i) the base composition and the second composition, or the medium supplement described herein, optionally further comprising one or more from proteose peptone; yeast extract; fetal bovine serum; preferably comprising proteose peptone and yeast extract. The medium composition can comprise one or more from: peptone; yeast extract; fetal bovine serum; and a medium supplement as described herein. Preferably the medium composition comprises peptone and yeast extract. The medium composition for culture of fastidious microorganisms (e.g. bacteria, such as those of the vaginal tract or the intestinal tract) may also contain one or more of :Glycogen, Bovine Serum Albumin, Mucin, Urea, Magnesium sulfate, Sodium bicarbonate, Hemin (5g/L), Menadione (Vitamin K3)(1g/L), Myo-inositol, Thiamine HCI, Pyridoxine HCI, Folic acid, D-Biotin, L-ascorbic acid, D- calcium pantothenate, Choline chloride, Nicotinamide (niacinamide), riboflavin, Cyancobalamin/Vitamin B12, Vitamin A, p-amino benzoic acid and Vitamin D (cholecalciferol). The components may be present in the any of the amounts provided in Table 1 , table 2 or table 3, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%.

The medium supplement of the present invention is used mainly for culture of anaerobes, and these anaerobes are vaginal or intestinal tract anaerobic microorganisms. The fastidious microorganisms which may be cultured using the medium supplement, compositions and methods may also be capable of growth in aerobic conditions.

The medium supplement or composition may include Cysteine, e.g. L-cysteine. Cysteine may include Cysteine HCI.

The medium supplement or composition may include a buffer such as HEPES (4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid)).

The medium supplement or composition may include a detergent. The detergent is preferably polyoxyethylene sorbitan monooleate (also known as polysorbate 80, TWEEN 80™), of chemical formula: where the sum of w + x + y + z = 20.

The medium supplement or composition may include a carbon source, such as glucose (e.g. D-glucose, also known as dextrose) or another suitable carbon source. The carbon source may be present at a concentration of 4g/L to 12 g/L; 4.5g/L to 11 g/L; 4.5g/L to 5.5 g/L; 5g/L to 5.5 g/L. Preferably the carbon source includes D-glucose at a concentration of about 5 g/L. The carbon source can be provided as a hydrate, e.g. D-glucose monohydrate. The weight given refers to the weight of D-glucose itself. The formula weight of D-glucose monohydrate is about 10% greater than the formula weight of D-glucose. Thus, the amount of D-glucose monohydrate needed to provide the equivalent amount of D-glucose is about 10% greater. Thus, when the carbon source is D-glucose monohydrate, it can be present at concentration of about 5.5 g/L. The carbon source may also include a polysaccharide of glucose, such as glycogen. The polysaccharide can be present at a concentration of 0.5g/L to 3 g/L; 0.5g/L to 2 g/L. Preferably the polysaccharide is present at about 1 g/L.

The medium supplement or composition may include albumin. The albumin is preferably serum albumin, most preferably bovine serum albumin, such as Sigma Aldrich cat # A4053. Albumin can be present at 1-3 g/L, e.g. 2 g/l +/- 10%.

The medium supplement of composition may include glycogen. Glycogen can be present at 0.5-2 g/L, e.g. at 1 g/L +/- 10%.

Preferably the medium supplement of the invention comprises albumin and glycogen.

The medium supplement or composition may include a vitamin mix comprising one or more of Thiamine HCI, Pyridoxine HCI, Folic acid, D-Biotin, L-ascorbic acid, D-calcium pantothenate, Choline chloride, Nicotinamide (niacinamide), riboflavin, Cyancobalamin/Vitamin B12, Vitamin A, p-amino benzoic acid, Vitamin K1 , Myo-inositol, Vitamin D (cholecalciferol). The one or more components can be present at the concentration provided in the table below.

Table 4

The medium supplement or composition may be solubilized in water, e.g. sterilised water, distilled water, double distilled water, deionized water. The water is preferably distilled water.

A culture medium for growth of fastidious microorganisms (e.g. bacteria from the vaginal or intestinal tract) is provided which comprises (a) a base composition (b) a second composition. The base composition can include one or more of polyoxyethylene sorbitan monooleate (also known as polysorbate 80, TWEEN 80™), sodium chloride, D-glucose, Potassium phosphate dibasic, Potassium phosphate monobasic, cysteine (e.g. cysteinehydrochloride), proteose peptone and/or yeast extract. The base composition can comprise or consist of proteose peptone, yeast extract, sodium chloride, D-glucose, dipotassium phosphate, monopotassium phosphate, Tween-80 and cysteine (e.g. cysteinehydrochloride). Where present, the components of the base composition can be used in the following amounts, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%. Where provided as a dry supplement, the components are provided in g, in corresponding proportions to the concentrations given below, e.g. for preparation of 500 mL or 1 L of medium. The base composition can comprise water.

Table 5.

The base composition can comprise components according to recipe 1 or recipe 2 in table 5, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%. A base composition supplement is also provided comprising the components of the base compositions described herein, such as in table 5, in the amounts described above, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%, except for proteose peptone and/or yeast extract. The base composition supplement can be combined with the second compositions described here, and used to prepare culture medium by the methods described herein, including incorporating proteose peptone, yeast and fetal bovine serum in the amounts described herein.

The base composition can comprise components according to recipe 1 or recipe 2 in table 5, further comprising 450 mL distilled water per 1 L total medium, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%.

The second composition can comprise water, preferably distilled water. The second composition can include one or more of glycogen, albumin and/or fetal bovine serum (FBS). The second composition can comprise one or more of the components in the following table, optionally present in the following amounts (where present), further optionally +/- 2% +/- 5%, +/- 10%, +/- 15%. The second composition can comprise or consist of albumin and glycogen, optionally present in the following amounts, further optionally +/- 2% +/- 5%, +/- 10%, +/- 15%. The second composition can comprise or consist of albumin and glycogen and FBS, optionally present in the following amounts, further optionally +/- 2% +/- 5%, +/- 10%, +/- 15%.

Table 6

The second composition can comprise components according to recipe 5 or recipe 6, further comprising 450 mL distilled water per 1 L total medium, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%.

The second composition can comprise components according to recipe 5 or recipe 6, further comprising fetal bovine serum. The second composition can comprise components according to recipe 5 or recipe 6, further comprising 100 mL fetal bovine serum per 1 L total medium, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%. A culture medium for expression of fastidious microorganisms (e.g. bacteria from the vaginal or intestinal tract) is provided comprising (a) a base composition as described herein and (b) a second composition as described herein. A culture medium for expression of fastidious microorganisms (e.g. bacteria from the vaginal or intestinal tract) is provided comprising (a) a base composition comprising components according to recipe 1 or recipe 2, optionally +/- 2% +/- 5%, +/- 10%, +/- 15% (b) a second composition comprising components according to recipe 5 or recipe 6, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%, and (c) fetal bovine serum, optionally comprising 100 mL fetal bovine serum per 1 L total medium, optionally +/- 2% +/- 5%, +/- 10%, +/- 15%. The culture medium can comprise or consist of Proteose peptone, Yeast extract, Sodium chloride, D-glucose, Dipotassium phosphate, Monopotassium phosphate, Tween 80, L-Cysteine hydrochloride, Albumin, Glycogen and FBS.

A method of culturing fastidious microorganisms (e.g. bacteria of the vaginal tract or intestinal tract) is provided, the method including inoculating fastidious microorganisms (e.g. bacteria of the vaginal or intestinal tract) into the above described medium and culturing the inoculated fastidious microorganisms (e.g. bacteria of the vaginal or intestinal tract). The culturing may be performed under anaerobic conditions.

The culturing of the inoculated anaerobes may be performed under conditions of a pH of 4 to 8 (e.g. about pH 7). The culture temperature can be 30-38°C (e.g. 37°C). The agitation speed of 0 to 200 rpm, preferably 0 rpm (no agitation). Typically culturing is performed at a nitrogen saturation of 80%, a hydrogen saturation 10% , and/or a carbon dioxide saturation of 10%.

A method of preparing a medium for culture of fastidious microorganisms (e.g. bacteria of the vaginal or intestinal tract) is provided comprising (a) making a part (I) by solubilizing the components of the base composition in water and sterilizing the solution (b) making a part (II) by solubilizing the components of the second composition and sterilizing the solution and c) mixing part (I) with part (II) in a 1 :1 ratio. The solution for part (I) can be sterilized by autoclaving, preferably at 110°C to 130°C, or boiling. Typically a solution can be sterilized by autoclaving or boiling for 10 to 60 minutes (e.g. 15 minutes). The solutions can be sterilized by filter sterilization, e.g. part (II) can be sterilized by filtration before combining it with a sterile part (I), or the solutions can be sterilized in a single step after combining part (I) and part (II). Preferably the components of part II are sterilized without heating the solution to above about 50°C, or to above about 100°C. VMM may be packaged in several configurations, wherein all components are preferably sterile in their packaging and use. An illustration of alternative packaging solution configurations are as follows:

1. All components of base composition and the second composition are freeze dried together and sterile water is used to reconstitute the components.

2. All components of the base composition are freeze dried and sterile water is used to reconstitute the components. All components of the second composition are freeze dried and sterile water is used to reconstitute the components.

3. All components of VMM/VMM 2/VMM base in solution are stored frozen or refrigerated.

4. All components of VMM/VMM 2/VMM base are in aqueous solution.

5. All components of base composition are freeze dried and sterile water is used to reconstitute the components. Components of part II (“additions”) are provided as a concentrated liquid which can be added aseptically to the sterilised base components.

6. A swab collection tube containing VMM/VMM2/VMM base agar (e.g. about 0.4% agar) and a swab.

7. A swab collection tube containing VMM/VMM2/VMM base broth (e.g. configuration 4) and a swab.

The following numbered paragraphs describe embodiments of the invention.

A1. A culture medium for expression of fastidious microorganisms comprising (a) a base composition (b) a second composition and (c) fetal bovine serum, wherein the base composition comprises Polysorbate 80 (polyoxyethylene sorbitan monooleate) and cysteine hydrochloride and/or wherein the second composition comprises Menadione (Vitamin K3).

A2. A culture medium according to embodiment A1 wherein the base composition comprises sodium chloride, Polysorbate 80, D-glucose, Potassium phosphate dibasic, Potassium phosphate monobasic and Cysteine- hydrochloride, and/or wherein the second composition comprises Menadione (Vitamin K3), optionally wherein the base composition further comprises proteose peptone and yeast extract. A3. A culture medium according to embodiment A1 or embodiment A2 wherein the base composition comprises the following components in the following amounts ( weight % in w/v) :

A4. A culture medium according to embodiment A3 wherein the base composition comprises the following components present in the following approximate weight percentages: A5. A culture medium according to any of embodiments A1 to A4 wherein the second composition comprises water (e.g. distilled water) and one or more of the following components in the following amounts:

A6. A culture medium according to any one of embodiments A1 to A5 wherein the second composition comprises water, preferably distilled water, and one or more of the following components in the following amounts:

A7. A culture medium according to any one of embodiments A1 to A6 wherein:

(a) the base comprises water, preferably distilled water, and the following components present in the following approximate weight percentages:

(b) the second composition comprises water (e.g. distilled water) and the following components in the following approximate weight percentages:

A8. The culture medium according to any one of embodiments A1 to A7 further comprising one or more components selected from : A9. The culture medium according to any one of embodiments A1 to A8 wherein the fastidious microorganisms are fastidious bacteria.

A10. The culture medium according to any one of embodiments A1 to A9 wherein the fastidious microorganisms are fastidious bacteria from the vaginal tract.

A11. The culture medium of embodiment A10 wherein the fastidious microorganisms are one or more selected from Atopobium vaginae / Fannyhesse vaginae, Bifidobacterium dentium, Escherichia coli, Finegoldia magna, Fusobacterium nucleatum, Gardnerella vaginalis, Lacticaseibacillus rhamnosus, Lactobacilli us gasseri, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus vaginalis / Limosilactobacillus vaginalis, Prevotella amnii, preferably wherein the fastidious microorganisms are Lactobacillus iners (L iners) or Gardnerella vaginalis (G. vaginalis).

A12. The culture medium according to any one of embodiments A1 to A9, wherein the fastidious microorganisms are fastidious bacteria from the intestinal tract.

A13. The culture medium of embodiment A12, wherein the fastidious microorganisms are one or more selected from Alistipes finegoldii, Alistipes onderdonkii, Alistipes putredins, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bifidobacterium longum, Citrobacter freundii, Clostridium difficile, Clostridium perfringes, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Roseburia hominis, Ruminococcus gnavus, preferably wherein the fastidious microorganisms are one or more selected from Alistipes spp., Fusobacterium nucleatum, Roseburia hominis and Ruminococcus gnavus.

A14. A medium supplement for high yield culture of fastidious microorganisms, the medium supplement comprising Cysteine- hydrochloride and/or Menadione (Vitamin K3) and Tween80.

A15. A medium supplement according to embodiment A14, further comprising glucose, preferably D-glucose.

A16. A medium supplement according to embodiment A15, comprising:

(a) 4 g/L to 8 g/L D-glucose; optionally 5.5 g/L (+/- 10%);

(b) 0.1 to 0.3 g/L TWEEN 80; optionally 0.2 g/L (+/- 10%);

(c) 0.2 to 1 g/L cysteine hydrochloride; optionally 0.5 g/L (+/ 10%).

A17. A medium supplement according to any one of embodiments A1 to A16, comprising one or more of: Sodium chloride, D-glucose, Potassium phosphate dibasic, Potassium phosphate monobasic, Polyethylene glycol sorbitan monooleate and Cysteinehydrochloride, optionally in the following amounts:

A18. A medium supplement according to embodiment A17, further comprising one or more of: Glycogen, Bovine Serum Albumin, Mucin, Urea, Magnesium sulfate, Sodium bicarbonate, Hemin (5g/L), Menadione (Vitamin K3)(1g/L), Myo-inositol, Thiamine HCI, Pyridoxine HCI, Folic acid, D-Biotin, L-ascorbic acid, D-calcium pantothenate, Choline chloride, Nicotinamide (niacinamide), riboflavin, Cyancobalamin/Vitamin B12, Vitamin A, p- amino benzoic acid and Vitamin D (cholecalciferol), optionally in the following amounts:

A19. A medium composition for culture of fastidious bacteria of the vaginal tract or intestinal tract comprising (i) the base composition and second composition of any one of embodiments A1 to A8, or

(ii) the medium supplement of any one of embodiments A1 to A18, optionally further comprising one or more from proteose peptone; yeast extract; fetal bovine serum; preferably comprising proteose peptone and yeast extract.

A20. The medium composition according to embodiment A19, wherein the fastidious bacteria are selected from the group Atopobium vaginae / Fannyhesse vaginae, Bifidobacterium dentium, Escherichia coli, Finegoldia magna, Fusobacterium nucleatum, Gardnerella vaginalis, Lacticaseibacillus rhamnosus, Lactobacilli us gasseri, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus vaginalis / Limosilactobacillus vaginalis, Prevotella amnii, preferably wherein the fastidious bacteria are Lactobacillus iners (L. iners) or Gardnerella vaginalis (G. vaginalis).

A21. The medium composition according to embodiment A19, wherein the fastidious bacteria are selected from the group Alistipes finegoldii, Alistipes onderdonkii, Alistipes putredins, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bifidobacterium longum, Citrobacter freundii, Clostridium difficile, Clostridium perfringes, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Roseburia hominis, Ruminococcus gnavus, preferably wherein the fastidious bacteria are one or more of Alistipes spp., Fusobacterium nucleatum, Roseburia hominis and Ruminococcus gnavus.

A22. A method of culturing fastidious bacteria comprising inoculating fastidious bacteria into (i) the culture medium of any one of embodiments A1 to A13 or (ii) a medium comprising the medium supplement of any one of embodiments A1 to A18 or (iii) a medium comprising the medium composition of any one of embodiments A1 to A21 and culturing the inoculated fastidious bacteria.

A23. A method of preparing a medium for culture of fastidious bacteria comprising

(a) making a part (I) by solubilizing the components of the base composition according to any one of embodiments A1 to A13 in water, and sterilizing the solution, optionally by autoclaving, preferably at 110°C to 130°C;

(b) making a part (II) by solubilizing the components of the second composition according to any one of embodiments A1 to A13 in water, and sterilizing the solution, optionally by filtration;

(c) mixing part (I) with part (II) in a 1 :1 ratio. A24. A method of preparing a medium for culture of fastidious bacteria comprising

(c) mixing part (I) with part (II) in a 1 :1 ratio.

EXAMPLES

The materials can suitably be purchased from trusted chemical producers and traders such as Sigma Aldrich, Merck, Corning, Gibco and Acros Organic e.g. as detailed in Table 1.

Example 1. Components of vaginal microbe medium (VMM)

The composition of the vaginal microbe medium (VMM) is listed in Table 7. To make the VMM agar the components of part I are dissolved in 450 ml deionised water and autoclaved at 121 °C for 15 minutes. The mixture is kept in a water bath set at 50°C. The components of part II are dissolved in 450 ml water and are stirred for 15 minutes. Part II is sterilized by filtration (pore size 0.22 pM). Part II is heated to 50°C to in a water bath. Part II is added to Part I and the agar is poured into petri dishes (approx. 30 ml per dish).

To make the VMM broth the components of part I (except the agar) are dissolved in 450 ml deionised water and autoclaved at 121°C for 15 minutes. The mixture is allowed to cool to at least 50°C. The components of part II are dissolved in 450 ml water and are stirred for 15 minutes. Part II is sterilized by filtration (pore size 0.22 pM). Part II is added to Part I and the media is stored at 4°C.

Table 7. Components of VMM.

Example 2. Experiments to test VMM

To demonstrate the improved rate at which high densities of L iners cultures can be achieved using VMM, a growth curve experiment was done. L. iners isolates (DSM 13335 and DSM 13336) were plated onto degassed VMM agar and incubated for 48 hours in an anaerobic chamber. The resulting culture was used to inoculate 10 mL of degassed VMM broth which was incubated in an anaerobic chamber for 24 hours. ODeoo of the culture was measured. The culture was used to inoculate degassed VMM broth to an ODeoo = 0.1. The cultures were incubated in an anaerobic chamber. The ODeoo of the cultures was measured over 32 hours.

The ODeoo measurements of the cultures grown in VMM broth were plotted over a 50-hour time period (Figure 1). The lag phase was around 4 hours and the exponential phase occurred between 5 and 10 hours. The performance of VMM was compared to two media (with and without additional supplements) identified from the art. The first medium was a modified version of NYCIII broth from the American Type Culture Collection (ATCC). The second was Chemically Defined Medium (CDM).

For 500 mL NYCIII modified medium: Table 8

*Optional - omit if using alternative sugar source e.g. Glycogen

For 1 litre of CDM:

Table 9 Further experiments were undertaken with NYCIII to which components of CDM were added:

NYCIII with Glycogen (e.g. 1 gram) instead of glucose.

NYCIII + Glycogen + Glucose

NYCIII + Glycogen + Urea

NYCIII + Glycogen + Urea + Cysteine

NYCIII + Glycogen + Urea + Cysteine + Albumin + Mucin

Growth assays were performed with modified NYCIII and CDM media as broths and using pure cultures of both L. iners and G. vaginalis. The bacteria showed slightly improved growth in NYCIII. When grown in CDM G. vaginalis showed little growth whereas L. iners showed almost none (Fig. 1 and 2). The addition of glycogen and urea to NYCIII resulted in good growth of G. vaginalis, but the additional components of CDM did not show marked improvement of growth (Fig. 1). For L. iners, the highest density of bacterial culture was in VMM (Fig.2).

Example 3. Comparison of Columbia and VMM broth for L. Iners growth

L. iners isolates (DSM 13335 and DSM 13336) were plated onto degassed Columbia agar supplemented with 5% v/v horse blood (CBA) from freezing media (12% v/w skim milk powder, 1% v/v DMSO, 1 % v/v 100% glycerol). Plates were incubated for 48 hours in an anaerobic chamber (Don Whitley Scientific) (10% CO2, 10% H2, 80% N2) at 37°C. The resulting culture was used to inoculate 10 mL of degassed Columbia broth which was incubated in an anaerobic chamber for 48 hours. The optical density (ODeoo) of the culture was measured. An aliquot (1 mL) of the culture was used to inoculate 10 mL of degassed Columbia broth or VMM broth. The cultures were incubated in the anaerobic chamber for 48 hours. Growth was monitored by measuring the ODeoo of the cultures (Fig. 3) and the number of Colony Forming Units (CFU) was determined (Fig. 5).

Example 4. Further comparison to other media.

Bloom and colleagues demonstrated adequate growth of L. iners strains in MRS broth supplemented with L-cysteine and L-glutamine, however the cultures only obtained an ODeoo « 1 after 35 hours whereas with VMM broth this optical density was achieved in 7 hours (Fig. 6).

Example 5. Experiments to develop pared-down VMM media. The aim of these experiments was to investigate which ingredients were most important for robust growth of L. iners.

Performance of ‘Part 1’ alone (VMM base)

In the following experiments, an amended version of VMM Part I was used, consisting of the components set out in Table 7 except for HEPES, potassium chloride and agar (VMM base). First, growth of L. iners isolates in VMM base was investigated for two commercial L. iners strains (DSM 13335 and DSM 13336), in comparison to the full VMM media (VMM base + Part II of Table 7). As shown in Fig. 7, the VMM base components were able to support growth, however, not to the same degree as the full recipe. Therefore, it was evident that some component/s of the Part II ‘additions’ were responsible for enabling the level of growth seen in VMM.

Performance of VMM base with individual components of Part II

Growth of L. iners DSM13335 and DSM13336 was tested with VMM, VMM base, and VMM base with the addition of one ingredient from Part II at a time. Growth curves (Fig. 8) and a bar chart plotting ODeoo at the 24 hour time point (Fig. 9) demonstrate that FBS is very important to L. iners growth. For L. iners DSM 13335 there was no significant difference in growth between VMM and VMM base with FBS. Albumin and Glycogen increased growth for L. iners 13335 and L. iners 13336 respectively.

A formulation with fewer components and comparable growth

VMM, VMM base and VMM base + FBS + glycogen + albumin were tested using two commercial isolates (L iners DSM 13335 and DSM 13336) and two isolates from patient swabs (L iners 21 and 24). Growth curves (Fig. 10) and a bar chart plotting ODeoo at the 24 hour time point (Fig. 11) demonstrate that the new formulation (VMM base + FBS + glycogen + albumin, ‘VMM2’) resulted in comparable growth results compared to the original VMM recipe. For some strains the new formulation VMM2 resulted in improved growth.

Summary

Based on these results we can further order the ingredients by importance:

Most important: Proteose peptone, Yeast extract, Sodium chloride, Glucose, Dipotassium phosphate, Monopotassium phosphate, TWEEN 80, L-Cysteine hydrochloride, FBS

High importance: Glycogen, Albumin

Medium importance: Mucin, Urea, Magnesium sulfate, Hemin, Menadione, Vitamin mix Least important: HEPES, Potassium chloride, Sodium bicarbonate

These experiments demonstrate that a pared-down VMM recipe according to Table 10 below is a viable option for a L. iners medium.

Table 10 - VMM2

For 1 litre:

VMM2 is prepared as described in Example 1 for VMM, with Part 1.1 and Part II.2 each dissolved in 450 ml water.

Example 6. Bacterial species supported by VMM and VMM2

To investigate which bacterial species could be supported by VMM and VMM2, the bacterial species listed in table 11 were cultured from freezing stocks onto degassed agar known to sustain the growth of the specific species. The cultures were incubated for 48 hours in an anaerobic chamber at 37°C, 70% humidity and gas composition: 10% carbon dioxide, 10% hydrogen, 80% nitrogen. Using an inoculation loop, ± 1 pl of culture was used to inoculate degassed VMM broth, VMM2 broth, VMM agar, and VMM2 agar. The cultures were incubated anaerobically as above for 48 hours. Growth of the bacterial species was confirmed by visual inspection of the agar plates for colonies and broth for turbidity. Any broth that was deemed turbid as compared to the control un-inoculated broth was noted down as sustaining bacterial growth. The growth of all bacteria was supported by VMM and the pared down version, VMM2. All bacterial species that were supported by VMM2 were also supported by VMM. Table 11.

In a further experiment, vaginal swabs were plated onto degassed VMM2 agar and incubated anaerobically (as above) for 72 hours. All bacterial growth on the plate was scraped and DNA was extracted from the culture. Metaxonomic profiling using 16S rRNA gene sequencing was performed to determine which species from the vaginal swabs were cultured using the agar. The unique bacterial species (i.e. those not found in the first list of 57 species) are listed in the table 12.

Table 112: Bacterial species cultured using VMM2 media.

This example demonstrates that VMM and VMM2 can support the growth of a range of bacteria from different phyla. The bacteria cultured using these media are common members of the skin, oral, gut and vaginal microbiomes and many of which are deemed fastidious bacteria.

Example 7.

Various media were used to culture L. iners using the same starting inoculum. Figure 12 is a bar chart showing the number of Colony Forming Units (CFU) of L. iners isolate DSM 13335 present when grown in Columbia, MRS, supplemented MRS (MRS+), New York City III (NYC III), Vaginal Microbe Media (VMM), and VMM base supplemented with FBS, Albumin and Glycogen (VMM 2) broths. CFU was determined by plating serial dilutions of the culture onto Columbia Blood Agar (CBA) plates and incubating in an anaerobic chamber for 48 hrs. The inoculum was prepared by resuspending L. iners culture from a CBA plate in Columbia broth to an OD = 0.1. CFU is shown on a logarithmic scale on the y-axis, the inoculum is on the far left (dark grey) and the results for the 6 media are groups into the 24 hour and 48 hour readings. White: Columbia, White with black dots: MRS, White with black diagonal lines: MRS +, light grey: NYCIII, light grey with black dots: VMM, light grey with black diagonal lines: VMM2. After some testing it was found that the starting inoculum was high, and it was thought that some broths may be slowing the death rate of the bacterium as opposed to supporting its growth. The starting inoculum of L. iners was reduced to 10¹ CFU to determine which media enable the growth of the bacterium. Figure 13 is a bar chart showing the number of CFU of L. iners isolate DSM 13335 present when grown in Columbia, MRS, MRS+, NYC III, VMM, and VMM 2 broths at 24 hours and 48 hours. CFU was determined as described previously. The inoculum was prepared by resuspending L. iners culture from a CBA plate in Columbia broth to an OD = 0.1 , and then diluting the culture by serial dilution. CFU is shown on a logarithmic scale on the y-axis, the inoculum is on the far left (dark grey) and the results for the 6 media are groups into the 24 hour and 48 hour readings. White: Columbia, White with black dots: MRS, White with black diagonal lines: MRS +, light grey: NYCIII, light grey with black dots: VMM, light grey with black diagonal lines: VMM2. It was found that Columbia, MRS and supplemented MRS broth do not support the growth or preserve the bacterium in any way. NYCIII broth supported the growth slightly, but this was only seen in one of the two replicates and only at 24 hours. VMM and VMM 2 supported the growth of L. iners and enabled the cultures to grow to a density of between 10⁴- 10⁵ and 10⁸ colony forming units after 24 and 48 hours respectively.

It will be appreciated that the above description is made by way of example and not limitation of the scope of the appended claims, including any equivalents as included within the scope of the claims. Various modifications are possible and will be readily apparent to the skilled person in the art. Likewise, features of the described embodiments can be combined with any appropriate aspect described above and optional features of any one aspect can be combined with any other appropriate aspect.

Claims

1. A culture medium for expression of fastidious microorganisms comprising (a) a base composition (b) a second composition and (c) fetal bovine serum, wherein the base composition comprises Polysorbate 80 (polyoxyethylene sorbitan monooleate) and cysteine hydrochloride and wherein the second composition comprises glycogen.

2. A culture medium according to claim 1 wherein the base composition comprises sodium chloride, Polysorbate 80, D-glucose, Potassium phosphate dibasic, Potassium phosphate monobasic and Cysteine- hydrochloride, and/or wherein the second composition comprises Menadione (Vitamin K3), optionally wherein the base composition further comprises proteose peptone and yeast extract.

3. A culture medium according to claim 1 or claim 2 wherein the base composition comprises the following components in the following amounts ( weight % in w/v) :

4. A culture medium according to claim 3 wherein the base composition comprises the following components present in the following approximate weight percentages:

5. A culture medium according to any previous claim wherein the second composition comprises water (e.g. distilled water) and one or more of the following components in the following amounts:

6. A culture medium according to any previous claim wherein the second composition comprises water, preferably distilled water, and one or more of the following components in the following amounts:

7. A culture medium according to claim 6 wherein the second composition comprises water, preferably distilled water, and the following components in the following amounts: 8. A culture medium according to any previous claim wherein:

(b) the second composition comprises water (e.g. distilled water) and the following components in the following approximate weight percentages: 9. The culture medium according to any previous claim further comprising one or more components selected from :

10. The culture medium of any previous claim wherein the fastidious microorganisms are fastidious bacteria.

11. The culture medium of any previous claim wherein the fastidious microorganisms are fastidious bacteria from the vaginal tract.

12. The culture medium of claim 11 wherein the fastidious microorganisms are one or more selected from Atopobium vaginae / Fannyhesse vaginae, Bifidobacterium dentium, Escherichia coli, Finegoldia magna, Fusobacterium nucleatum, Gardnerella vaginalis, Lacticaseibacillus rhamnosus, Lactobacilllus gasseri, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus vaginalis / Limosilactobacillus vaginalis, Prevotella amnii, preferably wherein the fastidious microorganisms are Lactobacillus iners (L. iners) or Gardnerella vaginalis (G. vaginalis).

13. The culture medium of any of claims 1-10, wherein the fastidious microorganisms are fastidious bacteria from the intestinal tract.

14. The culture medium of claim 13, wherein the fastidious microorganisms are one or more selected from Alistipes finegoldii, Alistipes onderdonkii, Alistipes putredins, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bifidobacterium longum, Citrobacter freundii, Clostridium difficile, Clostridium perfringes, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Roseburia hominis, Ruminococcus gnavus, preferably wherein the fastidious microorganisms are one or more selected from Alistipes spp., Fusobacterium nucleatum, Roseburia hominis and Ruminococcus gnavus.

15. A medium supplement for high yield culture of fastidious microorganisms, the medium supplement comprising Cysteine- hydrochloride, glycogen and Tween80.

16. A medium supplement according to claim 15, further comprising glucose, preferably D-glucose.

17. A medium supplement according to claim 16, comprising:

(a) 4 g/L to 8 g/L D-glucose; optionally 5.5 g/L (+/- 10%); (b) 0.1 to 0.3 g/L TWEEN 80; optionally 0.2 g/L (+/- 10%);

(c) 0.2 to 1 g/L cysteine hydrochloride; optionally 0.5 g/L (+/ 10%).

18. A medium supplement according to any of claims 15 to 17, comprising one or more of: Sodium chloride, D-glucose, Potassium phosphate dibasic, Potassium phosphate monobasic, Polyethylene glycol sorbitan monooleate and Cysteine- hydrochloride, optionally in the following amounts:

19. A medium supplement according to claim 18, further comprising one or more of: Glycogen, Bovine Serum Albumin, Mucin, Urea, Magnesium sulfate, Sodium bicarbonate, Hemin (5g/L), Menadione (Vitamin K3)(1g/L), Myo-inositol, Thiamine HCI, Pyridoxine HCI, Folic acid, D-Biotin, L-ascorbic acid, D-calcium pantothenate, Choline chloride, Nicotinamide (niacinamide), riboflavin, Cyancobalamin/Vitamin B12, Vitamin A, p-amino benzoic acid and Vitamin D (cholecalciferol), optionally in the following amounts:

20. A medium composition for culture of fastidious bacteria of the vaginal tract or intestinal tract comprising

(i) the base composition and second composition of any one of claims 1-9, or

(ii) the medium supplement of any one of claims 15-19, optionally further comprising one or more from proteose peptone; yeast extract; fetal bovine serum; preferably comprising proteose peptone and yeast extract.

21 . The medium composition according to claim 20, wherein the fastidious bacteria are selected from the group Atopobium vaginae / Fannyhesse vaginae, Bifidobacterium dentium, Escherichia coli, Finegoldia magna, Fusobacterium nucleatum, Gardnerella vaginalis, Lacticaseibacillus rhamnosus, Lactobacilli us gasseri, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus vaginalis / Limosilactobacillus vaginalis, Prevotella amnii, Dialister micraerophilus, Sneathia vaginalis and Megasphaera lornae preferably wherein the fastidious bacteria are Lactobacillus iners (L. iners) or Gardnerella vaginalis (G. vaginalis).

22. The medium composition according to claim 20, wherein the fastidious bacteria are selected from the group Alistipes finegoldii, Alistipes onderdonkii, Alistipes putredins, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bifidobacterium longum, Citrobacter freundii, Clostridium difficile, Clostridium perfringes, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Roseburia hominis, Ruminococcus gnavus, preferably wherein the fastidious bacteria are one or more of Alistipes spp., Fusobacterium nucleatum, Roseburia hominis and Ruminococcus gnavus.

23. A method of culturing fastidious bacteria comprising inoculating fastidious bacteria into (i) the culture medium of any one of claims 1-14 or (ii) a medium comprising the medium supplement of any one of claims 15-19 or (iii) a medium comprising the medium composition of any one of claims 20-22 and culturing the inoculated fastidious bacteria.

24. A method of preparing a medium for culture of fastidious bacteria comprising

(a) making a part (I) by solubilizing the components of the base composition according to any one of claims 1-14 in water, and sterilizing the solution, optionally by autoclaving, preferably at 110°C to 130°C;

(b) making a part (II) by solubilizing the components of the second composition according to any one of claims 1-14 in water, and sterilizing the solution, optionally by filtration;

(c) mixing part (I) with part (II) in a 1 :1 ratio.

25. A method of preparing a medium for culture of fastidious bacteria comprising

(c) mixing part (I) with part (II) in a 1 :1 ratio.