Bacterial compositions and uses thereof

Abstract

The present disclosure relates to a bacterial composition, a method of producing a bacterial composition and various uses of the bacterial composition.

Description

[0001] BACTERIAL COMPOSITIONS AND USES THEREOF

[0002] BACKGROUND

[0003] Spontaneous Pre-term birth accounts for ~15 million births annually and is the major cause of neonatal mortality and morbidity worldwide even if occurring later in pregnancy. The aetiology of spontaneous pre-term birth is multifactorial and infection accounts for ~40% of causes. The vaginal and intestinal microbiota in pregnant women is essential in establishing and maintaining a healthy pregnancy including prevention of spontaneous preterm birth.

[0004] The vaginal microbiota differs in composition from that of the gut or skin and can vary due to the menstruation cycle, during pregnancy, contraceptive use, antibiotic use, and douching. In healthy women of reproductive age, Lactobacillus species are the dominant microorganisms in the vagina, with Lactobacillus crispatus, L. gasseri, L. iners or L. jensenii as the most common and abundant strains. A reduction in lactobacilli has been associated with bacterial vaginosis (BV), typically with an elevated relative abundance of Gardnerella vaginalis, Prevotella spp. and Atopobium vaginae. BV is in turn associated with adverse pregnancy outcomes, including spontaneous pre-term birth, even in mild or asymptomatic cases. Hence making it difficult to predict which pregnancies are most at risk.

[0005] Probiotics that can beneficially modify the vaginal microbiome may thus protect against BV and hereby reduce the risk of spontaneous pre-term birth in pregnant women. However, to date, no such probiotic has been identified.

[0006] There remains a need for a probiotic product that is easily consumed, appealing and which can beneficially modify the vaginal microbiome to prevent BV. Moreover, there is a need for a probiotic product that may be ingested orally, and which can modify the vaginal microbiome to prevent, ameliorate and / or treat BV. Finally, there is a need for such probiotic product, which is also appealing and ingestible by pregnant woman, who offer suffer from nausea.

[0007] SUMMARY

[0008] Against this background, the inventors have developed a probiotic bacterial composition which can be consumed orally and modify the vaginal microbiome to promote an increase in Lactobacillus and decrease the abundance of bacterial species generally associated with BV. The probiotic bacterial composition of the present disclosure may prevent, ameliorate or treat vaginal microbiome dysbiosis. Surprisingly, the bacteria of the composition can be administered orally and yet affect the vaginal microbiota. In being able to beneficially modify the vaginal microbiome, the bacterial composition of the disclosure could also prevent and / or ameliorate and / or treat BV, and in pregnant women, reduce the risk of preterm birth.

[0009] In a first aspect the present disclosure relates to a bacterial composition comprising at least two bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1.

[0010] In particular, the present disclosure relates to said bacterial composition further comprising at least one ingredient selected from the group consisting of a prebiotic, a protein, a carbohydrate having a glycaemic index below 50, and an omega-3 fatty acid. Optionally, such a bacterial composition may be formulated as a frozen confectionary product, such as an ice cream.

[0011] A further aspect of the present disclosure relates to a method of producing a bacterial composition as described in the first aspect of the disclosure. Particularly, the method may be for producing the bacterial composition of the first aspect of the disclosure formulated as an ice cream.

[0012] In another aspect of the disclosure, the bacterial compositions may be used to reduce the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a subject, the use comprising administering to the subject the bacterial composition.

[0013] Yet another aspect of the disclosure provides a method of treating or preventing bacterial vaginosis in a subject; and / or the bacterial composition of the first aspect of the disclosure for use in such method.

[0014] A further aspect of the disclosure provides a method of preventing spontaneous pre-term birth in a pregnant subject; and / or the bacterial composition of the first aspect of the disclosure for use in such method.

[0015] DETAILED DESCRIPTION

[0016] In the description of the disclosure various embodiments and / or individual components are disclosed. As will be apparent to the ordinarily skilled practitioner, all combinations of such embodiments and components taught in the disclosure are possible and can result in preferred embodiments of the present disclosure.

[0017] Any percentages and ratios are calculated by weight unless otherwise indicated. All percentages, parts and ratios are calculated based on the total composition unless otherwise indicated.

[0018] Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, concentrations, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ± 10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%

[0019] A first aspect of the present disclosure provides a bacterial composition comprising at least two bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1.

[0020] The at least two bacterial species may each be any viable and / or dead strain of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1. It will be appreciated by one skilled in the art that the bacterial strains in bacterial composition may be present at varying ratios. For example, one bacterial strain may be present at a ratio of 1: 1, 1: 1,5, 1 :2, 1 :3, 1:4, 1:5, 1 :5, 1 :6, 1:7, 1:8, 1:9 or 1 : 10 compared to one or more other bacterial strains present in the bacterial composition. In a preferred embodiment, the bacterial strains are present at a 1: 1 ratio.

[0021] In one embodiment of the first aspect of the disclosure, the bacterial composition comprises at least three bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus rhamnosus GR-1.

[0022] In a preferred embodiment, the bacterial composition comprises at least Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus rhamnosus GR-1. In a preferred embodiment, the bacterial composition comprises no other bacterial species than Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus rhamnosus GR-1 The number of viable bacteria of the bacterial composition, or of each bacterial strain, may be determined by plating the bacteria on a suitable medium (e.g. solidified agar in a standard sized Petri dish) and counting the number of colonies formed. The measure, colony forming unit (CFU) is well understood in the art and is used to quantify the amount of viable (live) bacteria in the composition.

[0023] In one embodiment, each one or more of the bacterial strains are present at a concentration of about IxlO9- IxlO5colony-forming units (CFU) per ml bacterial composition, such as about IxlO8- IxlO5, about IxlO7- IxlO5, about IxlO9- IxlO6, about IxlO9- IxlO7, or about IxlO8- IxlO6In a preferred embodiment, each one or more of the bacterial strains are present at a concentration of about IxlO7CFU / ml.

[0024] In another embodiment, each one or more of the bacterial strains are present at a concentration of about IxlO9- IxlO5colony-forming units (CFU) per g bacterial composition, such as about IxlO8- IxlO5, about IxlO7- IxlO5, about IxlO9- IxlO6, about IxlO9- IxlO7, or about IxlO8- IxlO6In a preferred embodiment, each one or more of the bacterial strains are present at a concentration of about IxlO7CFU / g.

[0025] In one embodiment, the bacterial composition further comprises at least one ingredient selected from the group consisting of a prebiotic, a protein, a carbohydrate having a glycaemic index below 50, and an omega-3 fatty acid. In some embodiments, the bacterial composition comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more ingredients selected from the group consisting of a prebiotic, a protein, a carbohydrate having a glycaemic index below 50, and an omega-3 fatty acid. In one embodiment, the bacterial composition comprises one or more prebiotic, one or more protein and one or more carbohydrates having a glycaemic index below 50.

[0026] The term "prebiotic" as used herein is intended to encompass food ingredients beneficially affecting the host by selectively stimulating growth and / or activity of at least one gastrointestinal and / or urogenital bacteria. It will be appreciated that dietary prebiotics are typically nondigestible fibre compounds that pass undigested through the upper part of the gastrointestinal tract.

[0027] It will be appreciated by one skilled in the art that prebiotics have been shown to improve survival and growth of bacterial strains in the gut (such as probiotics) when administered exogenously to a subject. For more information see Gibson, et al. Nat Rev Gastroenterol Hepatol 14, 491-502 (2017).

[0028] Accordingly, in one embodiment, the prebiotic is a non-digestible fibre. In some embodiments, the prebiotic is selected from the group consisting of pectin, cellulose, resistant starch, resistant dextrin, inulin, lignin, chitin, a beta-glucan, an oligofructose, Galacto-oligosaccharide(s) and any combination thereof. In a preferred embodiment, the prebiotic is pectin.

[0029] The term "protein" as used herein is intended to encompass food ingredients comprising one or more macromolecules comprising amino acid chains. The term is meant to encompass specific type of protein molecule with a unique sequence of amino acids and combinations of different protein molecules. It will be appreciated that the properties of protein mixtures are determined by the collective properties of the individual proteins present.

[0030] Accordingly, in some embodiments the protein is selected from the group consisting of whey, whey protein, casein, soy protein, pea protein, hemp protein, egg white protein, collagen and any combination thereof. In one embodiment, the protein comprises whey and casein. In some embodiments, the protein is a mixture of whey and casein at a ratio of about 50 / 50, about 60 / 40, about 70 / 30, about 75 / 25, about 80 / 20, about 85 / 15, about 90 / 10, where the ratio is the whey to casein (whey / casein) ratio expressed as weight concentration (w / w). In a preferred embodiment, the protein is a mixture of whey and casein at a ratio of about 80 / 20 (whey / casein) (w / w) as this ratio mimics the ratio of whey / casein in human breast milk.

[0031] As mentioned the 80 / 20 ratio of whey protein to casein protein closely mirrors the natural composition of human breast milk during early lactation. This specific protein profile is considered optimal for human nutrition due to the complementary physiological roles of whey and casein. Whey protein is rapidly digested and absorbed, providing an immediate supply of essential amino acids necessary for protein synthesis and metabolic function. In contrast, casein is digested more slowly, contributing to a sustained release of amino acids and prolonged satiety.

[0032] In the context of maternal nutrition, particularly during pregnancy, this 80 / 20 ratio is of increased relevance. Pregnant individuals have elevated protein requirements to support both maternal tissue expansion and fetal development. The rapid digestibility of whey can help meet acute nutritional demands, while casein supports prolonged amino acid availability, enhancing nitrogen retention and fetal growth. Moreover, the high biological value and immunoactive components of whey protein, including lactoferrin and immunoglobulins, may contribute to immune support for both mother and fetus. Accordingly, the 80 / 20 whey-to-casein ratio provides a balanced amino acid profile and temporal delivery of nutrients, aligning closely with the physiological needs of pregnant individuals and their developing fetus.

[0033] "Glycaemic index" (or GI) is a term known in the art used to quantify the relative rise in the blood glucose level two hours after consuming that food. The glycemic index is a number from 0 to 100 assigned to a food, with pure glucose arbitrarily given the value of 100. A food or food ingredient is considered to have a low GI if it is 55 or less; high GI if 70 or more; and mid-range GI if 56 to 69. The glycaemic index values of several foods are for example, disclosed in Jenkins, D et al., (2023) Glycemic index of foods: a physiological basis for carbohydrate exchange, The American Journal of Clinical Nutrition; Volume 34, Issue 3, pages 362-366.

[0034] In some embodiments the carbohydrate has a glycaemic index of about 50 or less, 45 or less, about 40 or less, about 35 or less, about 30 or less, about 25 or less, about 20 or less, about 15 or less, about 10 or less, about 5 or less. In some embodiments the carbohydrate is selected from the group consisting of Agave syrup, Palm sugar, Maltitol, Lactose, Barely Mai Syrup, Fructose, Galactose, Xylitol, Sorbitol, Mannitol, and any combination thereof. In a preferred embodiment, the carbohydrate is Agave syrup.

[0035] In some embodiments, the bacterial composition further comprises an oil. In some embodiments, the oil is a vegetable oil or a plant oil. As used herein, the term "vegetable oil" or "plant oil" refers to an oil extracted and / or obtained from a seed or other parts of a plant. In some embodiments, the vegetable oil or plant oil is selected from the group consisting of olive oil, sunflower oil, soybean oil, canola oil, corn oil, peanut oil, sesame oil, coconut oil, palm oil, grapeseed oil, avocado oil, safflower oil, flaxseed oil, walnut oil, hemp oil, rice bran oil, mustard oil, pumpkin seed oil, macadamia nut oil, hazelnut oil, apricot kernel oil, almond oil, argan oil, babassu oil, black seed oil, borage oil, camelina oil, chia seed oil, cottonseed oil, cranberry seed oil, and perilla oil.

[0036] In an alternative embodiment, the oil is an animal derived oil. In some embodiments, the animal derived oil is selected from the group consisting of lard, tallow, schmaltz, duck fat, goose fat and fish oil. In a preferred embodiment, the animal derived oil is fish oil. In some embodiments, the bacterial composition comprises an oil in a concentration (w / w) of about 0.1% to about 1%, such as about 0.1% to about 0.8%, about 0.2% to about 0.7%, about 0.3% to about 0.6%, or about 0.3% to about 0.5%. In a preferred embodiment, the bacterial composition comprises oil in a concentration (w / w) of about 0.3% to about 0.5%.

[0037] It will be appreciated by one skilled in the art that fish oil may contain one or more Omega- 3 fatty acid. Accordingly, in some embodiments the Omega-3 fatty acid is comprised in the fish oil.

[0038] "Omega-3 fatty acid" (or co-3 I n-3 fatty acid) are known to have a wealth of health benefits. The term "Omega-3 fatty acid" (or co-3 I n-3 fatty acid) is a term known in the art and encompasses unsaturated fatty acids with multiple double bonds, where the first double bond is between the third and fourth carbon atoms from the methyl end of the molecule. It will be appreciated that many omega-3 fatty acids are known in the art and may be used included in some embodiments of the present disclosure, including, but not limited to the group consisting of Hexadecatrienoic acid (HTA), o-Linolenic acid (ALA) , Stearidonic acid (SDA), Eicosatrienoic acid (ETE), Eicosatetraenoic acid (ETA) ,

[0039] Eicosapentaenoic acid (EPA), Heneicosapentaenoic acid (HPA), Docosapentaenoic acid (DPA), Clupanodonic acid, Docosahexaenoic acid (DHA), Tetracosapentaenoic acid, Tetracosahexaenoic acid (Nisinic acid) and any combination thereof. In one embodiment, the omega-3 fatty acid is selected from the group consisting of o-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0040] In one embodiment, the bacterial composition further comprises a flavouring agent. In a preferred embodiment, the flavouring agent is a natural flavouring agent.

[0041] The term "flavouring agent" is used herein to mean any substance that may be added to a food product to enhance or modify their taste or aroma. The term as used herein encompasses both single chemical compounds having smell or odour, and complex mixture of chemical compounds that grouped together have smell or odour. The term as used herein, also encompasses flavour enhancers, sweeteners, acidulants and salt substitutes. Preferably, a flavouring agent is safe for animal and / or human consumption. It will be appreciated that flavouring agents can be natural (i.e., of natural origin) or artificial / synthetic (i.e., chemically synthesized). Natural flavouring agents, include but are not limited to, those comprising plant or animal tissue, and / or flavouring agents extracted therefrom. Synthetic flavouring agents may be nature-identical synthetic flavouring agents (i.e., chemically identical to some substance that can be found in nature). In one embodiment, the bacterial composition comprises Sugar kelp (Saccharina latissimi) as a flavouring agent.

[0042] An embodiment of the disclosure relates to the bacterial composition further comprises an emulsifier.

[0043] The term "emulsifier" as used herein refers to any substance that is a surfactant having lipophilic and hydrophilic properties and which enables mixing of hydrophilic and hydrophobic liquids. Preferably, the emulsifier is safe for human consumption. The term as used herein encompasses both single chemical compounds and complex mixtures. For example, the emulsifier may be isolated lecithin or egg yolk, which comprises lecithin(s).

[0044] In some embodiments, the emulsifier is selected from the group consisting of egg yolk, lecithin, polysorbate, Carrageenan, Guar Gum, Monoglycerides, diglycerides and any combination thereof. In a preferred embodiment, the emulsifier is egg yolk. In a preferred embodiment, the bacterial composition comprises egg yolk as an emulsifier at a concentration (w / w) of about 5% to about 15%, preferably 11%.

[0045] In embodiments of the first aspect of the disclosure, the bacterial composition is a human and / or animal food. Accordingly, in some embodiments the bacterial composition is safe for consumption by animals and / or humans.

[0046] In one embodiment, the bacterial composition is a symbiotic composition. The symbiotic composition may be a complementary symbiotic composition or a synergistic symbiotic composition. A complementary symbiotic composition is to be understood as the combination of one (or more) probiotic with a one (or more) prebiotic. A complementary symbiotic composition combines a prebiotic and a probiotic that work independently to elicit one or more health benefits. In a synergistic symbiotic composition live microorganism(s) (such as the bacterial species of the present disclosure) is coadministered with a substrate that is utilized by microorganism(s), to enhance the microorganism(s) functionality. Synergistic symbiotic composition work together (not independently) to bring about the resulting health benefits.

[0047] The bacterial composition of the first aspect of the disclosure may be provided together with a suitable carrier, diluent or excipient. Accordingly, the bacterial composition may be provided as a liquid formulation or a solid formulation. The bacterial composition may be diluted and further solidified, for example by freezing of the diluent. Water or other aqueous solvents are examples of a suitable diluent, which may be frozen to yield a solid frozen composition.

[0048] In some embodiments, the bacterial composition is a liquid composition, a frozen composition or a dry composition. In a preferred embodiment, the bacterial composition is a frozen composition.

[0049] In will be appreciated that frozen compositions intended for human consumption may be formulated to appeal to the human subject, for example, by being formulated as desserts or other confectionary products. Accordingly, in a preferred embodiment the frozen composition is an ice cream.

[0050] The term "ice cream" is used herein to refer to a confectionary frozen dessert that is cooled below the freezing point of water and stirred to incorporate air and prevent the formation of large ice crystals from forming.

[0051] Probiotic bacterial compositions formulated as a confectionary product, and particularly as an ice cream, are particularly advantageous in prophylactic or treatment application in pregnant subjects. One advantage is that confectionary products, and in particular ice cream compositions are more appealable to a wider range of patients as compared to alternative probiotic compositions such as pills, capsules, drinks or shakes.

[0052] Further, probiotic ice cream compositions are especially advantageous in prophylactic or treatment applications in pregnant subjects as ice cream has been shown decrease the severity of nausea and vomiting in pregnant woman experiencing morning sickness or hyperemesis gravidarum (see Bhardwaj et al., 2020 and Badr et al., 2021). It is thus expected that a probiotic ice cream composition will lead to an increased consumption of the probiotic composition by pregnant woman and / or to increased compliance with a probiotic administration regime.

[0053] In some embodiments, the ice-cream may be based on dairy products, such as whey or milk. In an alternative embodiment, the ice-cream may be free from animal products.

[0054] The term "milk" is to be understood as the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk.

[0055] In a particular embodiment, the bacterial composition comprises in the range of 5% protein to about 25% protein (w / w), such as at about 5% protein to about 25% protein (w / w), about 6% protein to about 23% protein (w / w), about 7% protein to about 20% protein (w / w), about 8% protein to about 20% protein (w / w), about 9% protein to about 18% protein (w / w), or about 10% protein to about 15% protein (w / w). In a preferred embodiment, the bacterial composition comprises in the range of about 10% protein to about 15% protein (w / w). In a further preferred embodiment, the bacterial composition comprises about 12% protein (w / w).

[0056] In an additional or alternative embodiment, the bacterial composition comprises in the range of 5% carbohydrate to about 25% carbohydrate (w / w), such as at about 5% carbohydrate to about 25% carbohydrate (w / w), about 6% carbohydrate to about 23% carbohydrate (w / w), about 7% carbohydrate to about 20% carbohydrate (w / w), about 8% carbohydrate to about 20% carbohydrate (w / w), about 9% carbohydrate to about 18% carbohydrate (w / w), about 10% carbohydrate to about 15% carbohydrate (w / w) or about 12% carbohydrate to about 15% carbohydrate (w / w). In a preferred embodiment, the bacterial composition comprises in the range of about 12% carbohydrate to about 15% carbohydrate (w / w). In a further preferred embodiment, the bacterial composition comprises about 15% carbohydrate (w / w).

[0057] In an additional or alternative embodiment, the bacterial composition comprises in the range of 5% fat to about 25% fat (w / w), such as at about 5% fat to about 25% fat (w / w), about 6% fat to about 23% fat (w / w), about 7% fat to about 20% fat (w / w), about 8% fat to about 20% fat (w / w), about 9% fat to about 18% fat (w / w), about 10% fat to about 15% fat (w / w). In a preferred embodiment, the bacterial composition comprises in the range of about 10% fat to about 15% fat (w / w). In a further preferred embodiment, the bacterial composition comprises about 14% fat (w / w). In some embodiments, the bacterial composition comprises in the range of about 0.001% (w / w) to about 0.01% choline (w / w), preferably 0.05% (w / w).

[0058] A person skilled in the art will appreciate that the viability of the bacteria in the composition of the disclosure will be affected over time by the storage and use conditions of the composition, such as the temperature, humidity, water content, pH, water activity, or osmolarity of the composition. For example, the decrease in bacterial viability over time may be higher in a liquid formulation compared to a frozen formulation or a dry formulation. Such loss of viability can be measured by determining the CFU value (or the CFU value per gram composition) of a composition before and after exposure to specific conditions. The CFU value may be determined as detailed above. In some embodiments, the bacterial composition is a frozen composition and has a loss in viability of the at least to bacterial species as measured by CFU / g of less than about 3, about 2,5, less than about 2, less than about 1,5, less than about 1, or less than about 0,5 log units after storage for about 1 day to 56 weeks at about -5°C to -25°C. In some embodiments, the loss in viability is less than about 3, less than about 2,5, less than about 2, less than about 1,5, less than about 1, or less than about 0,5 log units after storage for about 1 to 10 weeks at about -5°C to -25°C. In some embodiments, the loss in viability is less than about 3 log units after storage for about 1 to 4 weeks at about -10°C to -20°C.

[0059] In a preferred embodiment, the bacterial composition is a frozen composition and has a loss in viability of the at least two bacterial species as measured by CFU / g of less than about 3 log units after storage for about 2 weeks at about -18°C.

[0060] In some embodiments the bacterial composition is a liquid composition. In further embodiments the liquid composition may be a ready-to-drink liquid composition. In some embodiments the liquid composition is a carbonated beverage. In an alternative embodiment, the liquid composition is a non-carbonated beverage.

[0061] In some embodiments, the liquid composition has a loss in viability of the at least to bacterial species as measured by CFU / g of less than about 3, about 2,5, less than about 2, less than about 1,5, less than about 1, or less than about 0,5 log units after storage for about 1 day to 4 weeks at about 1°C to 10°C. In some embodiments, the loss in viability is less than about 3, less than about 2,5, less than about 2, less than about 1,5, less than about 1, or less than about 0,5 log units after storage for about 2 to 14 days at about 1°C to 10°C. In some embodiments, the loss in viability is less than about 3 log units after storage for about 5 to 9 days at about 1°C to 5°C.

[0062] In a preferred embodiment, the liquid composition has a loss in viability of the at least two bacterial species as measured by CFU / g of less than about 3 log units after storage for about 1 week at about 4°C.

[0063] In some embodiments, the bacterial composition is a dry composition. The bacterial composition may be formulated with a suitable solid carrier. Examples of a suitable solid carrier include maltodextrin, inulin, potato starch, corn starch, other vegetable starch, microcrystalline cellulose and any combination thereof.

[0064] It will be appreciated that dry compositions may be prepared using a variety of techniques and methods known in the art. The choice of technique will depend on the bacteria of the composition, for example on their ability to form spores. A dry bacterial composition will usually be prepared by drying a liquid composition comprising the bacteria. Preferably, the method or technique of drying should not result in complete loss of bacterial viability.

[0065] In some embodiments, the dry composition is selected from the group consisting of a freeze-dried, spray dried, vacuum dried and air-dried composition. In some embodiments, the dry composition is formulated as a powder or granulate.

[0066] In some embodiments, the dry composition has a low water activity (Aw). It will be appreciated that products, particularly food and animal feed products, with low water activity inhibit growth of bacteria and / or fungi, which generally results in longer shelf life. Similarly, the bacterial composition formulated in a dry composition having low water activity will be shelf-stable for longer, and the number of bacteria will remain stable.

[0067] Accordingly, in some embodiments the dry composition has a water activity (Aw) in the range of about 0.01-0.8, for example about 0.02-0.77, about 0.03-0.74, about 0.04-0.71, about 0.05-0.68, about 0.05-0.65, about 0.05-0.62, about 0.05-0.6, about 0.01-0.77, about 0.01-0.74, about 0.01-0.71, about 0.01-0.68, about 0.01-0.65, about 0.01-0.62, about 0.01-0.59, about 0.01-0.56, about 0.01-0.53, about 0.01-0.5, about 0.01-0.47, about 0.01-0.44, or about 0.01-0.40 at 37°C. In one embodiment, the dry composition has a water activity in the range of about 0.05-0.6 at 37°C. In a preferred embodiment the dry composition has a water activity in the range of about 0.01-0.4 at 37°C.

[0068] It will be appreciated that water activity (aw) can be calculated following the formula: aw = p / ps, where p is the partial water vapor pressure in equilibrium with the analyte, and ps is the (partial) vapor pressure of pure water at the same temperature. Accordingly, in some embodiments, the water activity of the bacterial composition is determined following aw = p / ps, where p is the partial water vapor pressure in equilibrium with the bacterial composition and ps is the (partial) vapor pressure of pure water at the same temperature. Water activity and / or vapor pressure values for the bacterial composition and / or pure water can be measured by several methods, which are well known in the art. For example, these values can be obtained using Resistive electrolytic hygrometers, capacitance hygrometers, and / or dew point hygrometers. The water-activity may also be measured following the standard determination of water activity as set forth in ISO 18787 (International Organization for Standardization, ISO 18787:2017. "Determination of water activity"; 2017). Accordingly, in one embodiment water activity is determined using the standard determination of water activity as set forth in ISO 18787. In one embodiment of the first aspect of the disclosure, the bacterial composition is adapted for oral administration.

[0069] A person skilled in the art will appreciate that the bacterial composition of the disclosure may be adapted for oral administration to a subject, when it is safe to be swallowed and / eaten by the subject. Accordingly, in some embodiments the bacterial composition is formulated as a tablet, capsule, syrup, solution, suspension, powder, emulsion, or other means of oral administration.

[0070] A second aspect of the present disclosure provides a method for producing the bacterial composition of the first aspect of the disclosure comprising the steps of:

[0071] (i) Mixing whey with milk to obtain a first solution;

[0072] (ii) concentrating the first solution by reducing the volume of said first solution to approximately 1 / 15 - 1 / 20 of the original volume to obtain a concentrated solution;

[0073] (iii) subjecting the concentrated solution to pasteurization to obtain a pasteurized solution;

[0074] (iv) combining two or more bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 with a prebiotic and oil, to obtain a bacterial solution;

[0075] (v) Mixing said pasteurized solution with said bacterial solution to obtain the bacterial composition.

[0076] In some embodiments step (i) comprises mixing whey with milk at a whey / milk ratio of 95 / 5 (w / w), 90 / 10 (w / w), 85 / 15 (w / w), 80 / 20 (w / w), 75 / 25 (w / w), 70 / 30 (w / w), 65 / 35 (w / w), 60 / 40 (w / w), 55 / 45 (w / w), or 50 / 50 (w / w). In a preferred embodiment, the whey / milk ratio is of 80 / 20 (w / w).

[0077] In some embodiments step (iv) comprises admixing said bacterial species and said oil. In some embodiments, the bacterial species are encapsulated in said oil. Admixing and / or encapsulating said bacterial species with said oil is particularly advantageous in the production of bacterial compositions which are subsequently cooled and / or frozen, such as in the production of a probiotic ice cream. Particularly, said admixing or encapsulation in oil avoids formation of ice crystals in and / or around the bacterial cells during the freezing process, which can lead to cell damage and lower viability. Accordingly, said oil admixed with or encapsulating said bacterial strains acts as a cryoprotectant and increases cryopreservation and bacterial cell viability in a frozen bacterial composition.

[0078] In a preferred embodiment the method for producing the bacterial composition of the first aspect of the disclosure comprising the steps of:

[0079] (i) Mixing whey with milk at a whey / milk ratio of 80 / 20 (w / w) to obtain a first solution;

[0080] (ii) concentrating the first solution by reducing the volume of said first solution to approximately 1 / 15 - 1 / 20 of the original volume to obtain a concentrated solution;

[0081] (iii) subjecting the concentrated solution to pasteurization to obtain a pasteurized solution;

[0082] (iv) combining two or more bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 with a prebiotic and plant oil, to obtain a bacterial solution;

[0083] (v) Mixing said pasteurized solution with said bacterial solution and obtaining a bacterial composition.; and

[0084] (vi) lowering the temperature of said mixed solution to no more than 0°C, to obtain a frozen ice cream bacterial composition.

[0085] In one embodiment, the milk comprises less than 0.5% fat, such as less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%.

[0086] In a preferred embodiment the volume of the first solution is reduced to approximately 1 / 18 of the original volume. In some embodiments, the volume of the first solution is reduced by evaporation of the water content of the solution, for example by heating and / or boiling.

[0087] "Pasteurizing" as used herein means treatment of the milk substrate to reduce or eliminate the presence of live organisms, such as microorganisms. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow.

[0088] In one embodiment, the concentrated solution is pasteurized by low-temperature pasteurization. As used herein "low temperature pasteurization" encompasses pasteurization processes carried out at a temperature of 70°C or lower. Accordingly, in some embodiments the concentrated solution is pasteurized by heating to about 60°C- 70°C, for example about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, or about 70°C. In some embodiments, heating of the solution may be carried out for about 15-60 minutes, such about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, or about 60 minutes.

[0089] In one embodiment, the concentrated solution is pasteurized by heating the solution to about 60°C-70°C for about 15-60 minutes. In a preferred embodiment the solution is heated to about 65°C for about 30 minutes.

[0090] Low temperature pasteurization can advantageously be used in the method of the invention as it avoids protein denaturation which occurs in the more common high-temperature pasteurization (i.e., above 70°C). Low temperature pasteurization thus allows for the retention of non-denaturated protein in the product, which improves taste and consistency.

[0091] In one embodiment, the method further comprising a step of adding a flavouring agent to the pasteurized solution of step (iii). In one embodiment the flavouring agent comprises fruit or is isolated or extracted from fruit. In one embodiment, the fruit is pasteurized before being added to the pasteurized solution of step (iii). The fruit may be pasteurized, for example, as described above for the concentrated solution. In one embodiment, the method further comprises a step of adding an omega-3 fatty acid to the pasteurized solution of step (iii). In a preferred embodiment, the omega-3 fatty acid is first mixed with the flavouring agent, before being added to the pasteurized solution of step (iii). In a preferred embodiment the omega-3 fatty acid and the flavouring agent are mixed at a temperature above 60°C, such as about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C or about 80°C. In a preferred embodiment the omega-3 fatty acid and the flavouring agent are mixed at about 72°C. In one embodiment, the omega-3 fatty acid is first mixed with the flavouring agent immediately after pasteurization of the flavouring agent. It will be appreciated that the elevated temperature of the pasteurized solution after pasteurization may aid in mixing of the solution and the fatty acid and / or the flavouring agent.

[0092] In some embodiments, the flavouring agent and / or omega-3 fatty acid are added to the pasteurized solution of step (iii) at temperature below about 10°C. In one embodiment, at a temperature between about 0°C and about 10°C, such as about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C.

[0093] In some embodiments the bacterial solution of step (iv) is a liquid solution. In some embodiments, the bacterial solution further comprises one or more prebiotics. In one embodiment, the bacterial solution further comprises an oil, preferable a plant derived oil, such as almond oil, avocado seed oil, canola oil, cocoa butter, coconut oil, corn oil, cottonseed oil, flax seed oil, grapeseed oil, hemp oil, olive oil, palm kernel oil, peanut oil, pumpkin seed oil, rice bran oil, safflower seed oil, sesame seed oil, soybean oil, sunflower seed oil, walnut oil and any combination thereof.

[0094] In some embodiments, the temperature in step (vi) is lowered until the mixed solution is frozen. In some embodiments, the temperature is lowered below 0°C. In one embodiment, a gas is introduced to the mixed solution before or during freezing. In some embodiments, air is introduced by stirring and or mixing of the solution during freezing.

[0095] In one embodiment, the bacterial composition comprises in the range of 30% to 50% whey, such as at about 30% to about 50% whey (w / w), about 32% to about 48% whey (w / w), about 34% to about 46% whey (w / w), about 36% to about 47% whey (w / w), about 38% to about 46% whey (w / w) or about 40% to about 45% whey (w / w). In a preferred embodiment, the bacterial composition comprises in the range of about 40% to about 45% whey (w / w). In a further preferred embodiment, the bacterial composition comprises about 43% whey (w / w).

[0096] In an additional or alternative embodiment, the bacterial composition comprises in the range of about 10% to about 40% milk (w / w), such as at about 10% to about 40% milk (w / w), about 15% to about 35% milk (w / w), about 20% to about 33% milk (w / w) or about 25% to about 30% milk (w / w). In a preferred embodiment, the bacterial composition comprises in the range of about 25% to about 30% milk (w / w). In a further preferred embodiment, the bacterial composition comprises about 27% milk (w / w).

[0097] In an additional or alternative embodiment, the bacterial composition comprises in the range of about 1% to about 20% carbohydrate having a glycaemic index below 50 (w / w), such as at about 1% to about 20% carbohydrate having a glycaemic index below 50 (w / w), about 3% to about 18% carbohydrate having a glycaemic index below 50 (w / w), about 5% to about 16% carbohydrate having a glycaemic index below 50 (w / w), about 6% to about 14% carbohydrate having a glycaemic index below 50 (w / w), or about 8% to about 12% carbohydrate having a glycaemic index below 50 (w / w). In a preferred embodiment, the bacterial composition comprises in the range of about 8% to about 12% carbohydrate having a glycaemic index below 50 (w / w). In a further preferred embodiment, the bacterial composition comprises about 10% carbohydrate having a glycaemic index below 50 (w / w). In a preferred embodiment, the carbohydrate having a glycaemic index below 50 is agave syrup.

[0098] In an additional or alternative embodiment, the bacterial composition comprises in the range of about 0.01% to about 1% of a prebiotic (w / w), such as at about 0.04% to about 1% of a prebiotic (w / w), about 0.04% to about 0.8% of a prebiotic (w / w), about 0.05% to about 0.6% of a prebiotic (w / w), about 0.08% to about 0.4% of a prebiotic (w / w), or about 0.1% to about 0.15% of a prebiotic (w / w). In a preferred embodiment, the bacterial composition comprises in the range of about 0.1% to about 0.15% of a prebiotic (w / w). In a further preferred embodiment, the bacterial composition comprises about 0.125% of a prebiotic (w / w). In a preferred embodiment, the prebiotic is pectin.

[0099] In an additional or alternative embodiment, the bacterial composition comprises in the range of about 0.1% to about 1% omega-3 fatty acid containing oil (w / w), such as at about 0.1% to about 1% omega-3 fatty acid containing oil (w / w), about 0.15% to about 0.9% omega-3 fatty acid containing oil (w / w), about 0.2% to about 0.7% omega-3 fatty acid containing oil (w / w), or about 0.3% to about 0.5% omega-3 fatty acid containing oil (w / w). In a preferred embodiment, the bacterial composition comprises in the range of about 0.3% to about 0.5% omega-3 fatty acid containing oil (w / w). In a further preferred embodiment, the bacterial composition comprises about 0.4% omega-3 fatty acid containing oil (w / w). In a preferred embodiment, the omega-3 fatty acid containing oil is fish oil.

[0100] A third aspect of the disclosure provides a use of a bacterial composition according to the first aspect of the disclosure, for reducing the number of bacteria selected from the group consisting of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a subject, the use comprising administering to the subject the bacterial composition. A non-limiting example of an embodiment of the third aspect of the disclosure is provided in Example 1.

[0101] A fourth aspect of the disclosure provides a method of reducing the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a subject, comprising administering the bacterial composition according to the first aspect of the disclosure to the subject. A nonlimiting example of an embodiment of the third aspect of the disclosure is provided in Example 1.

[0102] It will be appreciated by the skilled person that Prevotella spp., Gardnerella spp., and Atopobium spp. are different genus of bacteria. As used herein, each bacterial use cited herein is to be understood as encompassing all species and / or strains of said bacterial genus individually or in combination. Accordingly, reduction of the number a genus in a subject should be understood as either the reduction of the average number of bacteria belonging to said genus or as the reduction of one or more bacterial species or strains belonging to said genus. In some embodiments Prevotella spp. is P. copri, P. bivia, P. amnii, and / or P. timonensis. In some embodiments, Gardnerella spp. is Gardnerella vaginalis. In some embodiments, Atopobium spp. is Atopobium vaginae.

[0103] In one embodiment of the third or fourth aspect of the disclosure, the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. are reduced in the vagina and / or the gastrointestinal tract of the subject. In a preferred embodiment the reduction in in the vagina of the subject.

[0104] In some embodiments, the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. are reduced when compared to the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp in a subject not having been administered the bacterial composition. In some embodiments, the reduction is a reduction of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% compared to the subject not having been administered the bacterial composition. In an additional or alternative embodiment, the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a sample obtained from a subject having been administered the bacterial composition are reduced when compared to the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp obtained from a subject not having been administered the bacterial composition. In some embodiments, the reduction is a reduction in the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% in a sample obtained from a subject having been administered the bacterial composition, compared to a sample obtained from a subject not having been administered the bacterial composition.

[0105] Methods and / or techniques to determine the number of individual bacteria of particular genus, species or strain will be known to the skilled person. In some embodiments determining the number of specific bacteria comprises obtaining a sample from the urogenital and / or gastrointestinal tract of the subject, preferably from the vagina. In some embodiments, the sample from the subject may be cultured in culture media, and the bacteria may be determined by morphological and / or phenotypic characterization. In a preferred embodiment, the number of specific bacteria is determined by isolating nucleic acid from said sample from the subject. In one embodiment the number of specific bacteria may be determined by analysing said nucleic acid, for example by PCR, quantitative PCR (qPCR), reverse-transcription qPCR (RT-qPCR), Northern blotting, Southern blotting, or sequencing. In a preferred embodiment the nucleic acid is sequenced to determine the number of specific bacteria in a sample of the subject. In some embodiments, sequencing of the nucleic acid comprises sequencing of the 16S ribosomal rRNA sequence. In an alternative embodiment, sequencing comprises whole genome sequencing. It will be appreciated that a person skilled in the art will know how to determine which sequences determined in the nucleic acid correspond to specific bacteria, for example by bioinformatic comparison to sequences present in nucleic acid databases and repositories, such as RefSeq, PDB, nr / nt, as maintained, for example, by the National Library of Medicine, National Institute of Health - USA.

[0106] As previously described, dysbiosis of the bacterial flora in the vagina is known to be correlated with onset of bacterial vaginosis (BV). Specifically, elevated numbers of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the vagina of a subject have been linked to BV. Similarly, reduced levels of Lactobacillus species in the vagina of the subject are linked to BV. It will therefore be appreciated that reducing the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the vagina of the subject may prevent and / or ameliorate and / or treat bacterial vaginosis in a subject. Similarly, increasing the number of Lactobacillus in the vagina of the subject may prevent and / or ameliorate and / or treat bacterial vaginosis in a subject.

[0107] A used herein "bacterial vaginosis" in line with the medical consensus as a dysbiosis of the naturally occurring bacterial composition in the vagina of a subject. Typically, BV is associated with a reduction in the number of lactobacilli and an increase in the relative abundance of Gardnerella vaginalis, Prevotella spp. and Atopobium vaginae.

[0108] Accordingly, a fifth aspect of the disclosure provides the bacterial composition according to the first aspect of the disclosure for use in preventing and / or ameliorating and / or treating bacterial vaginosis in a subject.

[0109] In one embodiment the use is for preventing bacterial vaginosis in a healthy subject. In one embodiment, the use is for treating BV in a subject experiencing BV.

[0110] A sixth aspect of the disclosure provides a method of preventing and / or ameliorating and / or treating bacterial vaginosis in a subject in need thereof, comprising administering to the subject a bacterial composition according to the first aspect of the disclosure, thereby preventing and / or ameliorating and / or treating bacterial vaginosis in the subject.

[0111] In one embodiment the method is a method of preventing bacterial vaginosis in a healthy subject. In one embodiment, the method is a method of ameliorating and / or treating BV in a subject experiencing BV.

[0112] Bacterial Vaginosis in pregnant women has been associated with spontaneous pre-term birth. By "spontaneous pre-term birth" we include any condition where a pregnant subject gives birth before 37 weeks of pregnancy are completed and where the birth occurs spontaneously and without external intervention. As such, the term excludes induction of labour and caesarean births. It is known that infections, and particularly bacterial vaginosis, in pregnant women can lead to spontaneous pre-term birth. It will thus be appreciated that treating and / or preventing bacterial vaginosis can prevent spontaneous pre-term births in pregnant subjects.

[0113] Accordingly, a seventh aspect of the disclosure provides the bacterial composition according to the first aspect of the disclosure for use in preventing spontaneous pre-term birth in a pregnant subject. An eighth aspect of the disclosure provides a method of preventing spontaneous pre-term birth in a pregnant subject, comprising administering to the subject a bacterial composition according to the first aspect of the disclosure.

[0114] In some embodiments of the seventh or eighth aspect of the disclosure, the pregnant subject is at high risk of spontaneous pre-term birth. By "high risk of spontaneous preterm birth" we mean patients who previously had spontaneous pre-term birth (before gestational age (GA) 37+0), and / or a late spontaneous miscarriage (from week GA 16+0), and / or had a history of cervical conization. Accordingly, in some embodiments the subject having high risk of spontaneous pre-term birth is a subject having had a spontaneous preterm birth before, and / or a late spontaneous miscarriage, and / or had a history of cervical conization.

[0115] In one embodiment, the bacterial composition is administered to the subject during some or all of the duration of the pregnancy. In some embodiments, the bacteria composition is administered at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months or at least 9 months before the due date for the pregnancy. In some embodiments the bacterial composition is further administered before conception.

[0116] In some embodiments of the fifth, sixth, seventh and / or eighth aspect of the disclosure the subject has dysbiosis of the microbial flora in the vagina.

[0117] A ninth aspect of the disclosure provides the bacterial composition according to the first aspect of the disclosure for use in preventing and / or ameliorating and / or treating bacterial dysbiosis in the vagina in a subject. In a specific embodiment the bacterial composition according to the first aspect of the disclosure is for use in preventing and / or ameliorating and / or treating bacterial dysbiosis in the vagina in a subject.

[0118] As used herein "dysbiosis" refers to a deleterious imbalance or disturbance in the normally occurring microbial flora in a subject. It will be appreciated that dysbiosis can occur due to an increased amount of certain bacterial species or strains, a reduced amount of certain bacterial species or strains or an overall increase or reduction in the number of bacteria. With respect to the present disclosure "dysbiosis in the vagina" involves an increase in the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp.

[0119] Preferably, the numbers of (i) Prevotella spp., Gardnerella spp., and / or Atopobium spp and / or (ii) Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 are detected in a relevant biological sample from a subject. For example, samples can be taken from the vagina or the gastrointestinal tract of the subject. Preferably the samples are obtained from the vagina of the subject.

[0120] The number of (i) Prevotella spp., Gardnerella spp., and / or Atopobium spp and / or (ii) Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in a test sample can be compared to a number of said (i) Prevotella spp., Gardnerella spp., and / or Atopobium spp or (ii) Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in a control sample. The control sample may advantageously be derived from a healthy subject or from a pool of samples from healthy subjects, and is preferably treated in the same way as is the test sample. Thus, preferably the control sample is sampled in the same way as is the test sample, if applicable, nucleic acid is isolated in the same way as is the test sample, and, if applicable, hybridization, quantitative amplification, and / or sequencing is performed under the same conditions to allow a fair comparison of the test sample and control sample. It is not necessary to determine the number of (i) Prevotella spp., Gardnerella spp., and / or Atopobium spp. or (ii) Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in a control sample each time a test sample is measured; once the number is reliably determined in a control sample, the number values may be stored, e.g., in a computer, and used for the comparative purposes.

[0121] As used herein a "healthy subject" is a subject free from any diagnosed diseases or disorders. The healthy subject may be a pregnant subject or a not pregnant subject. Preferably, the healthy subject is a human. As described previously, the vaginal and / or urogenital microbiota of a subject can vary due to the menstruation cycle, during pregnancy, contraceptive use, antibiotic use, and age of the subject. Hence, it may be advantageous to compare to the number of one or more bacteria present in the vagina of a subject to that present in a healthy subject that that is similar with regards to one or more of the parameters above.

[0122] The number of said Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a test sample can be compared to the same bacteria in a control sample. An increase number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. spp. in a test sample compared to a control sample is related to an indication or diagnosis that the test sample is from a subject suffering from a bacterial dysbiosis in the vagina. A bacterial dysbiosis in the vagina may be associated with BV. The number of said Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in a test sample can be compared to the same bacteria in a control sample. A decreased number of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in a test sample compared to a control sample is related to an indication or diagnosis that the test sample is from a subject suffering from a bacterial dysbiosis in the vagina.

[0123] In a specific embodiment an increase number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a test sample compared to a control sample and a decreased number of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in a test sample compared to a control sample is related to an indication or diagnosis that the test sample is from a subject suffering from a bacterial dysbiosis in the vagina.

[0124] As used herein, the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a test sample is increased when it is significantly higher than the level of said Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a control sample. It is also considered increased when the level of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the test sample is at least 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold or higher than the numbers of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the control sample. As used herein, the numbers or level of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a test sample is decreased when it is significantly lower than the level of said Oscillospira bacteria in a control sample. It is also considered decreased when the level of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the test sample is at least 1-fold, 2-fold, 5-fold, 10-fold, 100-foldor 1000-fold lower than the level of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the control sample.

[0125] As used herein, the number of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in a test sample is increased when it is significantly higher than the level of said Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in a control sample. It is also considered increased when the level of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in the test sample is at least 1- fold, 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold or higher than the numbers of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in the control sample. As used herein, the numbers or level of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in a test sample is decreased when it is significantly lower than the level of said Oscillospira bacteria in a control sample. It is also considered decreased when the level of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in the test sample is at least 1-fold, 2-fold, 5-fold, 10-fold, 100-fold or 1000-fold lower than the level of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 in the control sample.

[0126] In some embodiments of the fifth, sixth, seventh, eight, and / or ninth aspect of the disclosure, the subject has an increased number of at least one of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the vagina. In one embodiment, all of Prevotella spp., Gardnerella spp., and Atopobium spp. are increased in the vagina of the subject. In some embodiments the number is increased compared to the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the vagina of a healthy subject. In some embodiments, the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. is increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% compared to the number in a healthy subject.

[0127] In some embodiments, the subject has a lower number of Lactobacillus spp. in the vagina. In one embodiment, at least one of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1 is decreased in the vagina of the subject. In some embodiments, the number of Lactobacillus spp. and / or the number of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1 is lower compared to the number in the vagina of a healthy subject. In some embodiments, the number is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% lower compared to the number in a healthy subject.

[0128] In some embodiments, the number of at least one of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the vagina is reduced following administration of the bacterial composition. In one embodiment, the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. is reduced following administration of the bacterial composition when compared to the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp in a subject not having been administered the bacterial composition. In some embodiments, the reduction is a reduction of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% compared to the subject not having been administered the bacterial composition. In some embodiments all of Prevotella spp., Gardnerella spp., and Atopobium spp. in the vagina are reduced following administration of the bacterial composition. In one embodiment, the number of at least one of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1 is increased in the vagina of the subject following administration of the bacterial composition. In one embodiment, the number of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1 is increased in the vagina of the subject following administration of the bacterial composition when compared to the number in a subject not having been administered the bacterial composition. In some embodiments, the increase is an increase of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% compared to the subject not having been administered the bacterial composition. In some embodiments all of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 are increased following administration of the bacterial composition.

[0129] In one embodiment, the number of at least one of or all of Prevotella spp., Gardnerella spp., and / or Atopobium spp. is reduced and the number of at least one of or all of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1 is increased in the vagina of the subject following administration of the bacterial composition.

[0130] Administration may be on an as-needed or as-desired basis in order to (i) maintain or increase numbers of at least two bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 for example, once-monthly, once-weekly, or daily, including multiple times daily, to arrive at a total daily dose or amount of at least two bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1 whether administered every day, one day per week, one day per month, or on a given day as needed. The amount of bacterial composition utilized may be dependent on a variety of factors, including the health status of the subject, age, gender, or other like factors of ordinary consideration. Preferably, the subjected administered the composition in accordance with the disclosure is mammal, most preferably a human.

[0131] In some embodiments the bacterial composition is administered at a daily dose of about 2x10“ - 2xl07CFU, such as a daily dose of about 2xl07CFU, about 2xl08CFU, about 2xl09CFU, about 2xlO10CFU, or about 2xlOnCFU. In a preferred embodiment the bacterial composition is administered at a daily dose of about 2xl09CFU. It will be appreciated that the most effective daily dose to be administered may depend on the subject to which it is administered, for example may depend on the age, weight, BMI, or diet of the subject. Accordingly, the daily dose of the bacterial composition may be calculated by factoring the above parameters of the subject.

[0132] In one embodiment, the method and or use further comprises a step of monitoring the concentration of at least one of Prevotella spp., Gardnerella spp., and / or Atopobium spp. and / or at least one of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1 is increased in the vagina of the subject following administration of the bacterial composition. In some embodiments, the daily dose of the bacterial composition is adjusted based on said monitoring.

[0133] In some embodiments, the bacterial composition is administered to the subject by oral administration vaginal administration or rectal administration. In a preferred embodiment, the bacterial composition is administered to the subject by oral administration.

[0134] In some embodiments of the third, fourth, fifth, sixth, seventh, eighth and / or ninth aspects of the present invention, the bacterial composition is administered to the subject formulated as a frozen composition. In some embodiments, the frozen composition is an ice cream. In some embodiments, the bacterial composition is prepared according to the method of the second aspect of the present invention.

[0135] As previously detailed, ice cream compositions are particularly advantageous in prophylactic and / or treatment applications in pregnant subjects due to ice cream consumption decreasing morning sickness and / or hyperemesis gravidarum, or at least one symptom thereof, in the pregnant subjects. Accordingly, in some embodiments, the bacterial composition is administered to a pregnant subject experiencing one or more symptoms associated with morning sickness and / or hyperemesis gravidarum. In some embodiments, the pregnant subject is experiencing one or more symptoms selected from the group consisting of nausea, vomiting, and weight loss. In some embodiments, at least one symptom of morning sickness and / or hyperemesis gravidarum is decreased and / or alleviated in a pregnant subject following administration of the bacterial composition.

[0136] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the disclosure.

[0137] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. All publications, patents, patent applications or other disclosures in this specification are incorporated herein by reference in their entirety.

[0138] Preferences, options and embodiments for a given aspect, feature or parameter of the disclosure should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the disclosure.

[0139] Some embodiments of the present invention are also disclosed in the following numbered list of items:

[0140] 1. A bacterial composition comprising at least two bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1.

[0141] 2. The bacterial composition of item 1, comprising at least three bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus rhamnosus GR-1.

[0142] 3. The bacterial composition of any one of items 1-2, comprising at least Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus rhamnosus GR-1.

[0143] 4. The bacterial composition of any of the preceding items, wherein the bacterial strains are present at a concentration of about IxlO9- IxlO5colony-forming units (CFU) / ml, preferably wherein the bacterial strains are present at a concentration of about IxlO7CFU / ml.

[0144] 5. The bacterial composition of any of the preceding items, further comprising at least one ingredient selected from the group consisting of a prebiotic, a protein, a carbohydrate having a glycaemic index below 50, and an omega-3 fatty acid.

[0145] 6. The bacterial composition of item 5, wherein the prebiotic is a non-digestible fibre.

[0146] 7. The bacterial composition of any one of items 5-6, wherein the prebiotic is selected from the group consisting of pectin, cellulose, resistant starch, resistant dextrin, inulin, lignin, chitin, a beta-glucan, an oligofructose, and a Galacto-oligosaccharide, or a combination thereof, preferably wherein the prebiotic is pectin.

[0147] 8. The bacterial composition of any one of items 5-7, wherein the protein is selected from the group consisting of whey, whey protein, casein, soy protein, pea protein, hemp protein, egg white protein and collagen, or a combination thereof.

[0148] 9. The bacterial composition according to item 8, wherein the protein comprises whey protein and casein, preferably wherein the whey protein and casein are at an 80 / 20 ratio (w / w).

[0149] 10. The bacterial composition any one of items 5-9, wherein the carbohydrate having a glycaemic index below 50 is selected from the group consisting of Agave syrup, Palm sugar, Maltitol, Lactose, Barely Mai Syrup, Fructose, Galactose, Xylitol, Sorbitol and Mannitol, or a combination thereof, preferably wherein the carbohydrate is Agave syrup.

[0150] 11. The bacterial composition of any one of items 5-10, wherein the omega-3 fatty acid is selected from the group consisting of o-linolenic acid (ALA), eicosa pentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0151] 12. The bacterial composition of any of the preceding items further comprising an oil.

[0152] 13. The bacterial composition of item 12, wherein the oil is a plant oil or an animal derived oil. The bacterial composition of item 12 or 13, wherein the bacterial strains are admixed and / or encapsulated in said oil. The bacterial composition of any one of the preceding items, further comprising a flavouring agent. The bacterial composition of item 15, wherein the favouring agent is a non-synthetic flavouring agent. The bacterial composition of any of the preceding items, further comprising an emulsifier. The bacterial composition of item 17, wherein the emulsifier is selected from the group consisting of egg yolk, lecithin, polysorbate, Carrageenan, Guar Gum, Monoglycerides and diglycerides, or a combination thereof, preferably wherein the emulsifier is egg yolk. The bacterial composition of any one of the preceding items, wherein the composition is a human and / or animal food. The bacterial composition of any one of the preceding items, wherein the composition is a symbiotic composition. The bacterial composition of any one of the preceding items, wherein the composition is a liquid composition, a frozen composition, or a dry composition. The bacterial composition of item 21, wherein the frozen composition is an ice cream. The bacterial composition of item 22, wherein the frozen composition has a loss in viability of the at least two bacterial species as measured by CFU / g of less than 3 log units after storage for 2 weeks at -18°C. The bacterial composition of item 21, wherein the liquid composition is a ready-to- drink liquid composition, such a carbonated or non-carbonated beverage. 25. The bacterial composition of item 24, wherein liquid composition has a loss in viability of the at least two bacterial species as measured by CFU / g of less than 3 log units after storage for 1 week at 4°C.

[0153] 26. The bacterial composition according to item 21, wherein the dry composition is selected from the group consisting of a freeze-dried, spray dried, vacuum dried and air-dried composition.

[0154] 27. The bacterial composition according to item 26, wherein the dry composition has a loss in viability of the at least two bacterial species as measured by CFU / g of less than 3 log units after storage for 2 weeks at 37°C and water activity (Aw) < 0.4, preferably less than 2.5 log unit after storage for 2 weeks at 37°C and Aw < 0.4.

[0155] 28. The bacterial composition according any one of items 26-27, wherein the dry composition is in the form of a powder and / or a granulate.

[0156] 29. The bacterial composition according to any one of items 26-28, wherein the water activity (Aw) of the dry composition is in the range of about 0.01-0.8, preferably in the range of about 0.05-0.6, most preferably in the range of about 0.01-0.4.

[0157] 30. The bacterial composition of any one of the preceding items, wherein the composition is adapted for oral administration.

[0158] 31. A method for producing the bacterial composition according to any one of items 1- 20 comprising the steps of:

[0159] (i) Mixing whey with milk;

[0160] (ii) concentrating the first solution by reducing the volume of said first solution to approximately 1 / 15 - 1 / 20 of the original volume to obtain a concentrated solution, preferably wherein the volume of the first solution is reduced to approximately 1 / 18 of the original volume;

[0161] (iii) subjecting the concentrated solution to pasteurization to obtain a pasteurized solution;

[0162] (iv) combining two or more bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1, optionally with a prebiotic and oil, to obtain a bacterial solution;

[0163] (v) mixing said pasteurized solution with said bacterial solution to obtain the bacterial composition. The method of item 31, wherein the pasteurization of step (iii) comprises heating the concentrated solution to 60°C-70°C for 15-60 minutes, preferably 65°C for 30 minutes. The method of item 31 or 32, further comprising a step of adding a flavouring agent to the pasteurized solution of step (iii), optionally wherein the flavouring agent comprises fruit, further optionally wherein the fruit is pasteurized before being added to the pasteurized solution of step (iii). The method of any one of items 31-33, further comprising a step of adding an omega-3 fatty acid to the pasteurized solution of step (iii), optionally wherein the omega-3 fatty acid is first mixed with the flavouring agent of item 30, before being added to the pasteurized solution of step (iii), further optionally wherein the omega- 3 fatty acid and the flavouring agent are mixed at 72°C. The method of any one of items 31-34, wherein the oil is a plant oil. The method of any one of items 31-35, wherein step (v) is carried out at a temperature below 10°C, optionally wherein step (v) is carried out at a temperature between 10°C and 0°C. Use of a bacterial composition according to any one of items 1-30, for reducing the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a subject, the use comprising administering to the subject the bacterial composition. The use according to item 37, wherein the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. are reduced in the vagina and / or the gastrointestinal tract of the subject when compared to the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp in a subject not having been administered the bacterial composition. 39. A method of reducing the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a subject, comprising administering the bacterial composition according to any one of items 1-30 to the subject.

[0164] 40. The method of item 39, wherein method is for reducing the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the vagina and / or the gastrointestinal tract of the subject when compared to the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp in a subject not having been administered the bacterial composition.

[0165] 41. A bacterial composition according to any one of items 1-30 for use in preventing and / or ameliorating and / or treating bacterial vaginosis in a subject.

[0166] 42. A bacterial composition according to any one of items 1-30, for use in preventing spontaneous pre-term birth in a pregnant subject.

[0167] 43. A bacterial composition for use according to item 41 or 42, wherein the subject or pregnant subject is a subject in high risk of spontaneous pre-term birth.

[0168] 44. A method of preventing and / or ameliorating and / or treating bacterial vaginosis in a subject in need thereof, comprising administering to the subject a bacterial composition according to any one of items 1-230.

[0169] 45. A method of preventing spontaneous pre-term birth in a pregnant subject, comprising administering to the subject a bacterial composition according to any one of items 1-30.

[0170] 46. The method according to item 44 or 45, wherein the subject or pregnant subject is a subject in high risk of spontaneous pre-term birth.

[0171] 47. A method of preventing and / or ameliorating and / or treating bacterial dysbiosis in the vagina in a subject in need thereof, comprising administering to the subject a bacterial composition according to any one of items 1-30.

[0172] 48. The method according to item 47, wherein bacterial dysbiosis in the vagina is a bacterial dysbiosis in the vagina. 49. A bacterial composition according to any one of items 1-30 for use in preventing and / or ameliorating and / or treating bacterial dysbiosis in the vagina in a subject.

[0173] 50. The bacterial composition according to item 49, wherein bacterial dysbiosis in the vagina is bacterial dysbiosis in the vagina.

[0174] 51. The use or method of any one of items 37-50, wherein the bacterial composition is administered at a daily dose of about 2xlOn- 2xl07colony-forming units (CFU), preferably wherein the bacterial composition is administered at a daily dose of about 2xlO9CFU.

[0175] 52. The use or method of any of items 37-51, wherein the administration results in an increased number of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1 in the vagina and / or the gastrointestinal tract of the subject when compared to the number of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1 in a subject not having received the bacterial composition.

[0176] 53. The use or method of any of items 37-52, wherein the bacterial composition is administered orally.

[0177] 54. The use or method of any of items 37-53, wherein the subject is experiencing one or more symptoms associated with morning sickness and / or hyperemesis gravidarum.

[0178] 55. The use or method according to item 54, wherein the symptoms are selected from the group consisting of nausea, vomiting, and weight loss.

[0179] 56. The use or method of any of items 37-55, wherein at least one symptom of morning sickness and / or hyperemesis gravidarum is decreased and / or alleviated in a pregnant subject following administration of the bacterial composition.

[0180] Description of the Figures

[0181] Figure 1. - Univariate analysis of intervention and time effect on bacterial taxa. Linear mixed-effects model showing time and interaction (time point: intervention) effect on specific genera from 16s (a, b) and specific Lactobacillus species copy numbers from qPCR (c, d), stratified by time trajectory. Forest plots showing models beta estimate and 95% CI (x-axis) of taxa for the interaction term (control :time (bottom line), intervention :time (top line)) and time term (middle line). Solid lines indicating significant effects on the respective taxa (likelihood ratio test, FDR-BH), for the vaginal microbiota (a,c) and gut microbiota (b,d), respectively.

[0182] Figure 2. - Effect of intervention and time on the prevalence of specific Lactobacillus species detected using qPCR in the two swab sites. Upwards triangles show increase and downward triangles show a decrease. Size of triangles show effect size (Cliff's delta).

[0183] Stars show significance in the likelihood ratio test. Cliff's D effect size prevalence revealed a significant difference between the control and intervention group with good compliance for some of the tested Lactobacillus spp.

[0184] Embodiments and features of the present disclosure are also illustrated by the following non-limiting examples.

[0185] EXAMPLES

[0186] EXAMPLE 1

[0187] Aim of the study

[0188] The overall aim of this randomized controlled feasibility study among pregnant women with a high risk of spontaneous preterm birth, was to assess the feasibility and compliance of vaginal commensal probiotics, given as daily ice cream throughout pregnancy. The primary aim was to characterize gut and vaginal microbiota from swabs taken throughout pregnancy with respect to composition, compartmentalization, progression, and colonization of the probiotic strains within and between groups throughout pregnancy. The secondary aims were to examine uptake, participant experience and satisfaction, and to register pregnancy outcomes in both groups.

[0189] Materials and Methods

[0190] Study design and cohort

[0191] This was a randomized controlled feasibility study based at Odense University Hospital in Denmark between April 2019 and August 2020. Forty-five pregnant women with a high risk of SPONTANEOUS PRE-TERM BIRTH were recruited in the study during their first antenatal midwife session at Odense University Hospital (OUH), and 43 agreed to participate. Recruited women were in their first trimester and had previously a SPONTANEOUS PRE-TERM BIRTH (before gestational age (GA) 37+0), and / or a late spontaneous miscarriage (from week GA 16+0), and / or had a history of cervical conization. Exclusion criteria were women <18 years old, current or previous gestational diabetes, pre-gestational diabetes, twin pregnancy, allergy to milk protein, previous bariatric surgery, and non-Danish language skills. All participants provided written informed consent and the study was reviewed by the ethics committee (REC nr: S- 20180157). The women were randomized by a computer through OPEN RedCap into two groups at 2: 1, an intervention group (n=29), and a control group (n= 14). The intervention group followed the intervention protocol with daily ice cream and provided microbiome swab samples. The control group did not ingest the ice cream during their pregnancies but followed the usual care protocol and provided microbiome swab samples. All women were followed up from inclusion until after delivery.

[0192] Intervention

[0193] The intervention group was instructed to ingest two custom-designed ice creams of 100ml beakers from Skaro is (a manufacturer of organic ice cream and functional foods) daily from inclusion until labour. The ice cream contained the probiotic strains Lactobacillus crispatus, L. jensenii, L. gasseri, and L. rhamnosus GR-1. All strains were added in the same amounts: 0.000125 g / lOOg ice cream, IxlO7CFU / ml bacteria with a bacteria concentration of 100 billion CFU / g (CFU=colony-forming unit).

[0194] Prebiotics, protein, carbohydrates with low glycaemic index and omega-3 fatty acid from fish were also added. The ice cream varied in five flavours and the women could decide which flavours they wanted.

[0195] A representative simplified list of ingredients for the ice cream (excluding the probiotic bacterial strains and flavouring agents) can be found in Table A.

[0196] Table A - Simplified ingredient list of the ice cream (excluding the probiotic bacterial strains and certain flavouring agents).

[0197] Data collection

[0198] All 43 women attended three appointments at the antenatal care units during their pregnancies. The first appointment was an inclusion, randomization, and baseline meeting where all women completed a questionnaire and provided vaginal and rectal swabs. In addition, the intervention group also received forms to register their daily ice cream intake throughout their pregnancy. Second appointment (time point 2) and third appointment (time point 3) involved registration of pregnancy complications as well as vaginal and rectal swabbing for all women. Descriptive data (Table 1) were collected from all included women through interview together with electronic health records and documented in OPEN RedCap database at time point 1. Body Mass Index (BMI) was calculated. Based on compliance to ice cream ingestion, participants were divided into the following groups: excellent (ingested 67-100% of the ice cream), good (34-65%), poor (10-33%), no compliance (<10%) or no answer (N / A). Only women with excellent or good compliance were included in the metagenomic analysis.

[0199] Vaginal and rectal microbiome samples

[0200] The swabs were performed by self-sampling with standard swabbing kits containing 2ml liquid Amies medium, where the women would swab themselves from the vagina and rectum in the hospital bathroom. If the women were uncomfortable with self-sampling, the midwife would perform these instead. The women were given both oral and written information on how to swab correctly and were handed two separate swabbing kits marked V (vaginal) and R (rectal). The swabs were performed within the gestational time windows of week 9-18, week 16-32, and week 27-40. DNA isolation, protocol accuracy and 16S rRNA gene sequencing

[0201] Laboratory procedures were conducted under a hood with laminar flow (LabGarda ES Energy Sever Classe II Laminar Flow, NuAire Inc., Plymouth, MN, USA) to limit environmental contamination. Following the manufacturer's procedures, total DNA was extracted from all swab samples with the ZymoBIOMICS DNA Miniprep Kit (ZYMO Research Europe GmbH, Freiburg, Germany). 250 ml aliquots from the swab solution were processed. DNA extraction followed the manufacturer's recommendations with slight modifications for better mechanical disruption. To account for inter- person variations in laboratory workflow, the same person performed the DNA extraction on all swabs from the same subject on the same day. DNA concentrations were determined using a Qubit assay kit. V3-V4 hypervariable region of the 16S rRNA gene was PCR amplified using 16S rRNA-specific Primers:341F "Klindworth" - 785R "Klindworth". Bacterial 16S rRNA gene amplicons were sequenced targeting the V3-V4 (300bp paired-end sequencing) using Illumina MiSeq platform performed by LGC Genomics GmbH (Berlin, Germany).

[0202] Real-time qPCR

[0203] For all samples collected for each time point, qPCR was performed using an Applied Biosystems QuantStudio 3 system (Thermo Fisher Scientific, Darmstadt, Germany). Amplification and detection were performed in 96-well optical plates (Applied Biosystems) with SYBR-Green qPCR assay (Applied Biosystems). All amplifications were performed in duplicates in a final volume of 5 pL containing 13.8 pL of a 2xSYBR Green PCR Master Mix including ROX as a passive reference (Applied Biosystems), 500 nM of each Lactobacillus strain specific primers and 1.2 pL of template DNA (0.5 mg / mL). For amplification, the standard protocol of the Applied Biosystems QuantStudio 3 system was followed, i.e., an initial cycle at 95°C for lOmin, followed by 40 cycles at 95°C for 15 s, and 1 min at 60°C. PCR product specificity was tested by melting curve (Tm) analysis. Standard curves for quantification consisted of 10-fold serial dilutions in the range of lO^-lO^ copies of the 16S rRNA gene of the E. coli (Invitrogen, C404010) amplified with primers 27F and 1492R. The total amount of bacterial 16S in rectal swabs was quantified with universal primers, Univ337F and Univ518R.

[0204] Data manaqement and statistical analysis

[0205] Continuous variables were presented as means and standard deviations (normally distributed variables) or medians and ranges if not normally distributed. Categorical variables were presented as frequencies and proportions. The characteristics and outcomes between the two groups were compared using Mann-Whitney U test for continuous variables and Chi^-tests and Fisher's exact tests for categorical variables. A value of p<0.05 or a value of q<0.1 (Benjamini-Hochberg false discovery rate control (FDR) corrected p values) were considered significant for all analyses. Stata (v.17) was used for statistical data analysis and R was used for the bioinformatic data analysis. The raw sequences obtained were processed using LotuS (1.62). Poisson binomial model-based read filtering was applied. OTU clustering (UPARSE) was based on sequence similarity of 97%, while SILVA version 138 was used for taxonomic profiling. A total number of 4854 OTUs were detected. To account for the difference in sampling depth and avoid bias in alpha diversity metrics, rarefaction of the data was performed using the rtk package. OTU counts were rarefied to the smallest retained sample size (i.e., raw reads) to obtain relative microbiota abundances in each sample, accounting for read depth in each sample. Since the data were not normally distributed, nonparametric tests were used for all association tests. The Mann-Whitney-U test was used for discrete predictors. For pairs of continuous variables, a nonparametric Spearman correlation test was used. FDR was applied in all multiple testing situations to control the family-wise error rate at 10%. Hierarchical clustering was used to establish grouping patterns of the different study samples, including an updated adaptation of the approach used to define "enterotypes" in the human gut using the "Dirichlet multinomial" R package (v. 1.36.0). The Chi^-test implemented in base R was used to test for significant differences in the resulting community-type distribution between samples grouped by time point and intervention, respectively. Beta diversity was calculated as Bray-Curtis dissimilarities as implemented in the vegan R (v. 2.5-7) package. To determine the impact of intervention, time point, and swab site on the taxonomic composition of the microbiome, permutational multivariate analysis of variance (PERMANOVA) was performed. Bray-Curtis distances were used for all analyses. PERMANOVA test was performed using the "adonis" package in R (v. 0.4). Mantel test was performed using the "ape" package in R (v. 5.3).

[0206] Results

[0207] Characteristics of the study population

[0208] Of n=43 recruited women at high risk of PTB, n=6 withdrew from the intervention group within the first two weeks, leaving n=37 women to be followed throughout pregnancy. Accordingly, n=23 were randomized to ingest daily probiotic ice cream containing vaginally commensal lactobacilli (L. crispatus, L. gasseri, L. jensenii, L. rhamnosus GR- 1), prebiotic, omega-3 fatty acid, carbohydrates with low glycaemic index and protein, and n=14 were in the control group ingesting no such ice cream (Methods). Gut and vaginal microbiota composition were determined in all women through qPCR and 16S sequence analysis of swab samples taken at three time points during pregnancy, with trial satisfaction and outcomes assessed postpartum. Compliance was self-tracked throughout, and relevant demographic and clinical covariates were collected at enrolment (Methods).

[0209] Patient characteristics are displayed in Table 1. Controls had fewer previous pregnancies (p=0.015), and more conizations (p=0.015) than the intervention group, but no other significant differences were found in the registered parameters (all p>0.05) (Table 1). No significant differences were seen when comparing characteristics of the intervention group (n=23) with the women who withdrew from the study (n=6).

[0210] Table 1 *The P-value was calculated with the Mann-Whitney U-test / Wilcoxon rank sum test if the median was used and with the Chi2-test and

[0211] Fisher's exact in case of frequencies and proportions. PTB=Preterm birth, BMI=Body mass index, GA=Gestational age, IQR=lnterquartile range

[0212] Compliance with intervention

[0213] Each woman in the intervention group had variable compliance from time point to time point. Adherence to ice cream gradually decreased throughout the study, with 30.4% (n=7) displaying excellent compliance in period 1 and 4.35% (n= l) in period 3. A total of 13% (n=3) of the intervention group had good or excellent compliance throughout their pregnancy, while 43.5% (n=10) had good or excellent compliance in one or more periods. Additionally, 43% had no compliance during the last time period before birth. No participants in either the control or intervention group reported taking other probiotic supplements.

[0214] Diversity and composition of vaginal and gut microbiome during pregnancy

[0215] The following analysis was done with control group versus women in the intervention group with good or excellent adherence to the intervention. Those with no adherence were not included in the following analysis. As expected, the vaginal microbiome was significantly less diverse than and compositionally different from the gut microbiome (MWU test, q<0.001). There was no significant impact of time points or intervention on alpha diversity or multivariate measures of microbiome composition. Furthermore, microbiota from vaginal swabs was significantly different in microbial community composition than the microbiota from rectal swabs (Mantel statistic R: 0.5974, p<0.001). Comparison between the control and intervention group was based on their daily ice cream intake compliance. A PERMANOVA test at baseline showed no significant difference between groups in vaginal or gut microbiome composition before the intervention, implying no major discrepancies in host-microbiome interactions.

[0216] Bacterial body site distribution

[0217] To assess the basis for delivery of probiotic microbiota to the vaginal ecosystem by oral administration and subsequent gut passage, we investigated whether each 16S bacterial taxon was detectable in both vaginal and rectal swabs or only in one or the other as well as the impact of pregnancy progression and intervention status (Figures 1 and 2). In addition, to assess taxon sharing (and / or impact of cross- system compositional determinants) between both swab sites, we performed logistic regression on a presenceabsence matrix. Lactobacillus was found in all samples from both swab sites and subsequently removed from the analysis as uninformative.

[0218] Impact of pregnancy progression and synbiotic intervention on key vaginal microbiota

[0219] We next focused on a subset of bacterial taxa either linked previously to reproductive health such as BV or spontaneous pre-term birth, or that were vaginal commensals present within our tested probiotic ice cream (L. crispatus, L. gasseri, L. jensenii, L. rhamnosus GR-1). Using linear mixed-effects models we assessed whether their distribution and presence in our cohort was impacted by either the course of pregnancy or by the course of the intervention, representing the latter through interaction terms between intervention status and the passage of time (Figure 1). Figures 1 (a) and 1 (c) denotes that the ice cream intervention significantly increases vaginal carriage of L. jensenii (Likelihood ratio test (LRT) q<0.1) and significantly depletes a subset of Prevotella OTUs from baseline to time point 3.

[0220] Figure 1 (b) and 1 (d) displays the rectal distribution and presence over time and intervention of the specific lactobacilli species. There was a significant depletion of Lactobacillus from baseline to time point 3 in the intervention group. Furthermore, a significant enrichment in Prevotella was seen in the intervention group from time point 2 to time point 3 and an enrichment in L. crispatus from time point 2 to 3 in the control group.

[0221] Finally, a significant depletion of Atopobium and Gardnerella was evident in association with the treatment from baseline to time point 3.

[0222] Assessing probiotic penetrance and colonization through quantitative PCR

[0223] Going beyond the limitations of 16S amplicon sequencing, we used quantitative PCR (qPCR) to test for the presence of the probiotic strains specifically in each sample and L. iners. There was an upward shift of the specific Lactobacillus species in the entire cohort from baseline to time point 2 (Figure 2). In the control group the Lactobacillus enrichment then largely ceased before time point 2, whereas the enrichment continued in the intervention group until time point 3. Thus, all probands gained lactobacilli in the first half of pregnancy, but those who adhered to the intervention also continued to gain lactobacilli in the latter half of pregnancy. Women in the intervention group tended to have lower levels of lactobacilli at baseline than the control group, but this was not statistically significant. Figure 2 shows a significant increase in vaginal L. rhamnosus GR-1 in the intervention group from baseline to time point 3 (q<0.001). In addition, rectal amount increased significantly from baseline to time point 2 and between time point 2 and 3 (q<0.1). Moreover, both vaginal and rectal L. jensenii abundance increased significantly between time point 2 and 3 in the intervention group compared to the control group (q<0.01). L. gasseri abundance decreased significantly in rectal samples in the intervention group from baseline to time point 2 (q<0.001) but increased significantly from baseline to time point 3 compared to the control group (q<0.001). There was also an increasing trend of both vaginal and rectal L. crispatus and L. iners in the intervention group throughout the study, though neither was statistically significant (q>0.1). Since the different Lactobacillus species exist as a part of the normal vaginal microbiota, it was expected to also find these species in the control group vaginal swabs albeit a lower abundance.

[0224] Discussion

[0225] The inventors investigated the relationship between intake of ice cream containing vaginal commensal probiotics and the effect on the vaginal and gut microbiome in pregnant women at high risk of spontaneous pre-term birth. It was demonstrated that: i) while a small number of daily probiotics did not shift the overall diversity in either community, all women had an increase in lactobacilli during the first half of pregnancy, with a continued increase in the latter half of pregnancy in compliant women from the intervention group; ii) L. gasseri, L. jensenii and L. rhamnosus GR-1 from the ice cream could be recovered in both rectal and vaginal samples; iii) L. crispatus was found more often in the intervention group and iv) vaginal Prevotella, as well as gut Gardnerella and Atopobium (all unfavourable bacteria when present in the vagina), significantly decreased in association with the intervention.

[0226] A daily intake of the vaginal commensal strains (L. crispatus, L. jensenii, L. gasseri, L. rhamnosus GR-1) that promote a stable microbiota and carry out beneficial functions in the vagina, along with one or more dietary beneficial supplements, such as prebiotics, protein, carbohydrate or omega-3 fatty acids, may be a practical and efficient way to promote a healthy and stable microbiota composition throughout pregnancy. Several studies have demonstrated that probiotics are safe and effective for the treatment or prevention of inflammatory conditions such as necrotizing enterocolitis and inflammatory bowel disease.

[0227] Vaginal eubiosis is characterized by a significantly lower diversity than gut eubiosis. In the vagina, eubiosis is provided by a beneficial lactic-acid-producing microbiota, predominantly but not uniquely, from the genus Lactobacillus. Vaginal eubiosis is provided by such organisms through numerical dominance, prevention of biofilm formation through vaginal epithelial cell adhesion, by lactic acid production that decreases the vaginal pH, and hydrogen peroxide (H2O2) production, which is toxic to other microorganisms and pathogens. In contrast, vaginal dysbiosis constitutes a prolonged deviation from a low- diversity, Lactobac / 7 / us-abundant / -dominant vaginal microbiota to a microbiota that has a high diversity and high abundance of potentially pathogenic organisms. New information from molecular-based techniques demonstrates that worldwide, the eubiotic microbiota of the healthy vagina is dominated by one, or at the most two species of lactobacilli from a shortlist of four species: L. crispatus; L. gasseri; L. iners and L. jensenii.

[0228] The antimicrobial, antiviral and immunomodulatory properties of lactic acid, the major organic acid metabolite produced by lactobacilli has been previously demonstrated. Acidity levels as well as H2O2 production differ between Lactobacillus species. In women with a Lactobacillus dominated vaginal microbiota lactic acid concentrations are inversely proportional to vaginal pH, and lactic acid is primarily responsible for acidification of the vagina. The eubiotic vagina has a pH of no higher than 4.5 while the dysbiotic vagina has a pH of >4.5. Production of L- and D-isomers of lactic acid differs between lactobacilli species and emphasizes the D-isomer's possible protective role. Lactic acid also exists as a protonated (non-dissociated H+; neutrally charged ion) or a lactate anion (dissociated H+; negatively charged ion). The protonated form has antimicrobial and immunomodulatory properties compared to the lactate anion, and the protonated form of lactic acid predominates at a pH <3.9. Finally, the concentration of lactic acid in the eubiotic vagina is ~110mM compared to <20mM in vaginal dysbiosis. This contrasts with the concentrations of short chain fatty acids like acetate, succinate, butyrate, and propionate that barely reach a concentration of ImM in the eubiotic vagina but in vaginal dysbiosis, acetic acid concentrations may reach 120mM.

[0229] Approximately 95% of strains of L. crispatus and 94% of strains of L. jensenii produce H2O2, and 9% and 7% of such women respectively had BV. In contrast, approximately 71% of strains of L. gasseri and only 9% of strains of L. iners produced H2O2 of which 43% and 36% of such women respectively had BV in a previous study. A vaginal microbiota with L. iners dominance or low lactobacilli in general, especially in the first trimester of pregnancy, is associated with adverse pregnancy outcomes such as spontaneous pre-term birth or intra-uterine infection. L. iners co-exists well with unfavourable bacteria such as Gardnerella and Prevotella that significantly increase the risk of short cervix, spontaneous pre-term birth, late miscarriage through BV.

[0230] It has further been reported that the vaginal microbiota changes spatially throughout the vaginal tract, which may indicate that bacteria found in the lower part of the vagina might be in closer relation to the gut bacteria. These findings support our hypothesis that the vaginal microbiota can be improved through oral intake of probiotics.

[0231] Vaginal commensal Lactobacillus strains from the probiotic ice cream used in this example, as well as other embodiments of the disclosure, could provide a protective and / or ameliorating and / or curative mechanisms to the vaginal microbiota that is beneficial to pregnant women, and especially those at high risk of spontaneous pre-term birth. The hypothesis of a protective effect strengthens with the decrease in vaginal Prevotella, as well as of gut Gardnerella and Atopobium, in the intervention group, since all three bacteria are associated with BV and linked to spontaneous pre-term birth. Besides decreasing BV, the intervention could also prevent bacteria ascending from the vagina to the intrauterine space, which is a mechanism for spontaneous preterm labour leading to spontaneous preterm birth.

[0232] REFERENCES

[0233] 1. Gibson, G., Hutkins, R., Sanders, M. et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat Rev Gastroenterol Hepatol 14, 491-502 (2017). https: / / doi.org / 10.1038 / nrgastro.2017.75

[0234] 2. Jenkins, D et al., (2023) Glycemic index of foods: a physiological basis for carbohydrate exchange, The American Journal of Clinical Nutrition; Volume 34, Issue 3, pages 362-366.

[0235] 3. International Organization for Standardization, ISO 18787:2017. "Determination of water activity"; 2017

[0236] 4. Bhardwaj D, Chawla S, Bhardwaj B, Heer AK, Verma P, Kherav A. The Association of Vanilla Ice Cream in Symptomatic Relief of Nausea and Vomiting in Management of Hyperemesis Gravidarum: A Cohort Study. J Clin Obstet Gynecol Infertil. 2020; 4(1): 1048.

[0237] 5. Badr EA, El-Fattah NA, El-Gawad, Elsayed HN. "Effect of Ice food for Pregnant Women with Hyperemesis Gravidarum". Journals, I. OS R. 2021. PP 01-07.

Claims

CLAIMS1. A bacterial composition comprising Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus rhamnosus GR-1.

2. The bacterial composition of claim 1, wherein the bacterial strains are present at a concentration of about IxlO9- IxlO5colony-forming units (CFU) / ml, preferably wherein the bacterial strains are present at a concentration of about IxlO7CFU / ml.

3. The bacterial composition of any of the preceding claims, further comprising at least one ingredient selected from the group consisting of a prebiotic, a protein, a carbohydrate having a glycaemic index below 50, and an omega-3 fatty acid.

4. The bacterial composition of claim 3, wherein the prebiotic is a non-digestible fibre.

5. The bacterial composition of any one of claims 3-4, wherein the prebiotic is selected from the group consisting of pectin, cellulose, resistant starch, resistant dextrin, inulin, lignin, chitin, a beta-glucan, an oligofructose, and a Galacto-oligosaccharide, or a combination thereof, preferably wherein the prebiotic is pectin.

6. The bacterial composition of any one of claims 3-5, wherein the protein is selected from the group consisting of whey, whey protein, casein, soy protein, pea protein, hemp protein, egg white protein and collagen, or a combination thereof.

7. The bacterial composition according to claim 6, wherein the protein comprises whey protein and casein, preferably wherein the whey protein and casein are at an 80 / 20 ratio (w / w).

8. The bacterial composition any one of claims 3-7, wherein the carbohydrate having a glycaemic index below 50 is selected from the group consisting of Agave syrup, Palm sugar, Maltitol, Lactose, Barely Mai Syrup, Fructose, Galactose, Xylitol, Sorbitol and Mannitol, or a combination thereof, preferably wherein the carbohydrate is Agave syrup.

9. The bacterial composition of any one of claims 3-8, wherein the omega-3 fatty acid is selected from the group consisting of o-linolenic acid (ALA), eicosa pentaenoic acid (EPA) and docosahexaenoic acid (DHA).

10. The bacterial composition of any of the preceding claims further comprising an oil, optionally a plant oil or an animal derived oil.

11. The bacterial composition of claim 10, wherein the bacterial strains are admixed and / or encapsulated in said oil.

12. The bacterial composition of any one of the preceding claims, further comprising a flavouring agent.

13. The bacterial composition of claim 12, wherein the favouring agent is a nonsynthetic flavouring agent.

14. The bacterial composition of any of the preceding claims, further comprising an emulsifier.

15. The bacterial composition of claim 14, wherein the emulsifier is selected from the group consisting of egg yolk, lecithin, polysorbate, Carrageenan, Guar Gum, Monoglycerides and diglycerides, or a combination thereof, preferably wherein the emulsifier is egg yolk.

16. The bacterial composition of any one of the preceding claims, wherein the composition is a human and / or animal food.

17. The bacterial composition of any one of the preceding claims, wherein the composition is a liquid composition, a frozen composition, or a dry composition.

18. The bacterial composition of claim 17, wherein the frozen composition is an ice cream.

19. The bacterial composition of claim 18, wherein the frozen composition has a loss in viability of the at least two bacterial species as measured by CFU / g of less than 3 log units after storage for 2 weeks at -18°C.

20. A method for producing the bacterial composition according to any one of claims 1- 19 comprising the steps of:(i) Mixing whey with milk;(ii) concentrating the first solution by reducing the volume of said first solution to approximately 1 / 15 - 1 / 20 of the original volume to obtain a concentrated solution, preferably wherein the volume of the first solution is reduced to approximately 1 / 18 of the original volume;(iii) subjecting the concentrated solution to pasteurization to obtain a pasteurized solution;(iv) combining two or more bacterial species selected from the group consisting of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus rhamnosus GR-1, optionally with a prebiotic and oil, to obtain a bacterial solution;(v) mixing said pasteurized solution with said bacterial solution to obtain the bacterial composition.

21. The method of claim 20, wherein the pasteurization of step (iii) comprises heating the concentrated solution to 60°C-70°C for 15-60 minutes, preferably 65°C for 30 minutes.

22. The method of claim 20 or 21, further comprising a step of adding a flavouring agent to the pasteurized solution of step (iii), optionally wherein the flavouring agent comprises fruit, further optionally wherein the fruit is pasteurized before being added to the pasteurized solution of step (iii).

23. The method of any one of claims 20-22, further comprising a step of adding an omega-3 fatty acid to the pasteurized solution of step (iii), optionally wherein the omega-3 fatty acid is first mixed with the flavouring agent of claim 30, before being added to the pasteurized solution of step (iii), further optionally wherein the omega- 3 fatty acid and the flavouring agent are mixed at 72°C.

24. The method of any one of claims 20-23, wherein the oil is a plant oil.

25. The method of any one of claims 20-23, wherein step (v) is carried out at a temperature below 10°C, optionally wherein step (v) is carried out at a temperature between 10°C and 0°C.

26. A method of reducing the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in a subject, comprising administering the bacterial composition according to any one of claims 1-19 to the subject.

27. The method of claim 26, wherein method is for reducing the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp. in the vagina and / or the gastrointestinal tract of the subject when compared to the number of Prevotella spp., Gardnerella spp., and / or Atopobium spp in a subject not having been administered the bacterial composition.

28. A bacterial composition according to any one of claims 1-19 for use in preventing and / or ameliorating and / or treating bacterial vaginosis in a subject.

29. A bacterial composition according to any one of claims 1-19, for use in preventing spontaneous pre-term birth in a pregnant subject.

30. A bacterial composition for use according to claim 28 or 29, wherein the subject or pregnant subject is a subject in high risk of spontaneous pre-term birth.

31. A method of preventing and / or ameliorating and / or treating bacterial vaginosis in a subject in need thereof, comprising administering to the subject a bacterial composition according to any one of claims 1-19.

32. A method of preventing spontaneous pre-term birth in a pregnant subject, comprising administering to the subject a bacterial composition according to any one of claims 1-19.

33. The method according to claim 31 or 32, wherein the subject or pregnant subject is a subject in high risk of spontaneous pre-term birth.

34. A method of preventing and / or ameliorating and / or treating bacterial dysbiosis in the vagina in a subject in need thereof, comprising administering to the subject a bacterial composition according to any one of claims 1-19.

35. A bacterial composition according to any one of claims 1-19 for use in preventing and / or ameliorating and / or treating bacterial dysbiosis in the vagina in a subject.

36. The method or bacterial composition for use of any one of claims 26-35, wherein the bacterial composition is administered at a daily dose of about 2xlOn- 2xl07colony-forming units (CFU), preferably wherein the bacterial composition is administered at a daily dose of about 2xl09CFU.

37. The method or bacterial composition for use of any of claims 26-36, wherein the administration results in an increased number of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1 in the vagina and / or the gastrointestinal tract of the subject when compared to the number of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and / or Lactobacillus rhamnosus GR-1 in a subject not having received the bacterial composition.

38. The method or bacterial composition for use of any of claims 26-37, wherein the bacterial composition is administered orally.

39. The method or bacterial composition for use of any of claims 26-38, wherein the subject is experiencing one or more symptoms associated with morning sickness and / or hyperemesis gravidarum, optionally wherein the symptoms are selected from the group consisting of nausea, vomiting, and weight loss.

40. The method or bacterial composition for use of any of claims 26-39, wherein at least one symptom of morning sickness and / or hyperemesis gravidarum is decreased and / or alleviated in a pregnant subject following administration of the bacterial composition.

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