Methods for reducing levels of xenobiotics in the environment

Abstract

The present invention relates to methods for reducing the level of xenobiotics in an environment by contacting the environment with a composition comprising one or more bacterial strains. The present invention also relates to bacterial strains for use in the methods for reducing the level of xenobiotics in a subject. The present invention also relates to compositions comprising one or more bacterial strains.

Description

[Technical Field]

[0001] The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research innovation programme (grant agreement no. 866028).

[0002] The present invention relates to methods for reducing the level of xenobiotics in an environment by contacting the environment with a composition comprising one or more bacterial strains. The present invention also relates to bacterial strains for use in the methods for reducing the level of xenobiotics in a subject. The present invention also relates to compositions comprising one or more bacterial strains. [Background technology]

[0003] Xenobiotics are compounds that are foreign to the human body, such as environmental pollutants, food contact materials, pesticides, drugs, natural toxins, and processing contaminants. It is estimated that an individual human is exposed to up to 100,000 different xenobiotic compounds over the course of a lifetime. This includes man-made chemicals that resist normal environmental degradation processes, many of which are widely used in industrial, agricultural, and domestic applications. As a result of their resistance to degradation, they accumulate in the environment, humans, and animals. The environmental accumulation of xenobiotics is particularly evident in water systems, where xenobiotic levels often reach undesirable and even unsafe levels; water treatment plants are poorly equipped to deal with these types of contaminants.

[0004] Once ingested, degradation-resistant xenobiotics can persist for long periods of time (a process known as bioaccumulation). When xenobiotics reach toxic concentrations in the body, they can have a variety of negative physiological effects.

[0005] Per- and polyfluoroalkyl substances (PFAS) are among the most well-known xenobiotics, commonly known as "forever chemicals" because they persist in the environment. After their development in the 1940s and 1950s, these synthetic chemicals were widely used in industrial manufacturing and quickly became ubiquitous in the environment. PFAS bioaccumulation and associated toxic effects have been documented in animals and humans. Summary of the Invention [Problem to be solved by the invention]

[0006] There is an urgent unmet need for methods to reduce the levels of these types of xenobiotics in the environment, humans, and animals. [Means for solving the problem]

[0007] The present inventors have discovered numerous bacterial strains that can bioaccumulate and / or biotransform xenobiotics. Preferably, these bacterial strains are commonly found in the human microbiome and therefore do not pose an immediate safety risk to humans and animals. By bioaccumulating and / or biotransforming xenobiotics, the bacterial strains of the present invention reduce the bioavailability of xenobiotics, thereby preventing or reducing their accumulation in the environment, thereby improving environmental quality and reducing bioaccumulation in animals and humans (e.g., through ingestion or absorption). When administered to a subject, the bacterial strains of the present invention also help reduce the levels of xenobiotics already present in the subject; and also help prevent future bioaccumulation of xenobiotics.

[0008] The present invention provides a method for reducing the level of xenobiotics in an environment, comprising contacting the environment with a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia. The present invention also provides a method for reducing the level of xenobiotics in an environment, comprising contacting the environment with a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacte, Dorea, and Streptococcus.

[0009] The present invention also provides the use of a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus in a method for reducing the level of xenobiotics in the environment.

[0010] In some embodiments, the environment is an aqueous environment, and optionally the aqueous environment is drinking water or wastewater.

[0011] In some embodiments, the method includes detecting the presence and / or determining the abundance of one or more bacterial strains in the environment prior to contacting the environment with the composition.

[0012] In some embodiments, the method comprises: (i) contacting an environment with one or more bacterial strains; and then (ii) removing the one or more bacterial strains to provide a treated environment.

[0013] The present invention also provides a composition for use in a method for reducing xenobiotic levels in a subject, the composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia. The present invention also provides a composition for use in a method for reducing xenobiotic levels in a subject, the composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus.

[0014] In some embodiments, the methods include preventing or treating xenobiotic poisoning in a subject.

[0015] In some embodiments, the subject has ingested, is suspected of ingesting, or is at risk of ingesting a xenobiotic.

[0016] In some embodiments, the methods involve detecting the presence and / or measuring the abundance of one or more bacterial strains in the subject prior to administering the composition to the subject.

[0017] In some embodiments, the subject is a human. In some embodiments, the subject is an animal, optionally the animal is a cow, sheep, pig, poultry, cat or dog.

[0018] The present invention also provides a composition for reducing the level of xenobiotics, the composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia.The present invention also provides a composition for reducing the level of xenobiotics, the composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus.

[0019] In some embodiments, the one or more bacterial strains are selected from Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Collinsella aerofaciens, Coprococcus comes, Eubacterium rectale, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides merdae, and Roseburia intestinalis.In some embodiments, the one or more bacterial strains are Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Collinsella aerofaciens, Coprococcus comes, Eubacterium rectale, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides merdae, Roseburia intestinalis, Escherichia coli, Phocaeicola coprocola, Prevotella copri, Bacteroides eggerthii, Prevotella melaninogenica, Bacteroides fragilis, Bacteroides xylanisolvens, Butyrivibrio crossotus, Bacteroides coprocola, Roseburia hominis, Lacrimispora saccharolytica, Clostridium scindens, Fusobacterium nucleatum subsp. Nucleatum, Clostridium difficile, Phocaeicola vulgatus, Agathobacter rectalis, Roseburia inulinivorans, Dorea formicigenerans, Streptococcus salivarius, Fusobacterium nucleatum subsp. Animalis, Fusobacterium nucleatum subsp. Vincentii, Clostridium hylemonae, and Clostridium Selected from sporogenes.

[0020] In some embodiments, the composition comprises Bacteroides, and optionally, the composition comprises Bacteroides uniformis.

[0021] In some embodiments, one or more of the bacterial strains are genetically modified. In some embodiments, one or more of the bacterial strains comprises a genetic modification that results in the reduction or elimination of xenobiotic excretion from the one or more bacterial strains. In some embodiments, the genetic modification comprises the deletion or inactivation of at least one gene required for the activity of an efflux pump.

[0022] In some embodiments, the xenobiotic comprises one or more of a PFA, a bisphenol, and a pesticide.

[0023] In some embodiments, the xenobiotic comprises one or more of PFNA, PFOA, PFDeA, bisphenol AF, boscalid, propiconazole, pyrimethanil, tributyl-PO4, and triphenyl-PO4.

[0024] In some embodiments, the xenobiotic comprises a PFA, optionally the PFA comprises PFOA, PFNA, and / or PFDeA.

[0025] In some embodiments, the composition comprises a Bacteroides and the xenobiotic comprises PFA.

[0026] In some embodiments, the composition comprises Bacteroides uniformis and the PFA comprises PFNA, PFOA, and / or PFDeA.

[0027] In some embodiments, the composition is formulated for enteral delivery, and optionally, the composition is formulated for oral, nasal and / or rectal delivery.

[0028] In some embodiments, the composition further comprises one or more prebiotics to promote the growth of one or more bacterial strains.

[0029] In some embodiments, the one or more prebiotics are selected from arabinoxylan, xylose, fibrous dextran, corn fiber, polydextrose, lactose, N-acetyl-lactosamine, glucose, galactose, fructose, rhamnose, mannose, uronic acid, arabinose, fructose, fucose, lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose, xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose, xylooligosaccharides, fructooligosaccharides, galactooligosaccharides, lactosucrose, and soybean oligosaccharides.

[0030] In some embodiments, the one or more bacterial strains are lyophilized. In some embodiments, the composition further comprises a carrier, excipient, and / or diluent. In some embodiments, the composition comprises a gastro-resistant coating. In some embodiments, the composition is a sustained-release formulation.

[0031] The present invention also provides a dietary supplement comprising the composition of the present invention. [Brief explanation of the drawings]

[0032] [Figure 1] Strain bioaccumulation and biotransformation screen results. [Figure 2] Bioaccumulation of PFNA and biotransformation of propiconazole by Bacteroides dorei and Bacteroides uniformis. [Figure 3] PFA accumulation by B. uniformis (recovery of PFOA and PFNA in cell pellets). [Figure 4] PFA accumulation by B. uniformis at a range of different concentrations. The proportion of the accumulated compound remains constant for different concentrations. [Figure 5] PFA accumulation by B. uniformis for PFAs of various chain lengths. Accumulation increases with increasing chain length. [Figure 6] PFNA accumulation by B. uniformis over a 7-day time course. [Figure 7] Abundant gut bacterial species bioaccumulate and biotransform common chemical contaminants, accumulating and tolerating PFASs across a wide concentration range. a. Specificity of human gut bacteria in sequestering (bioaccumulating / biotransforming) chemical contaminants, as determined using mass spectrometry. Links between bacterial species and contaminants show >20% depletion and p<0.05 (Student's t-test). Link thickness is proportional to median depletion from six replicates (three biological, two technical). b, c. Examples showing bioaccumulation of the PFAS family compound perfluorononanoic acid (PFNA) (b) and biodegradation of the fungicide propiconazole (c) by B. uniformis. n=6 (three biological, two technical). d. Bioaccumulation of PFAS compounds of various chain lengths by B. uniformis (initial concentration for all compounds = 20 μM). n=3 technical replicates. e. Kinetics of PFNA depletion during growth of B. uniformis initiated from low cell density (OD600 = 0.05; initial PFNA concentration = 20 μM). n = 3 biological replicates. f. Kinetics of PFNA depletion by B. uniformis when initiated from high cell density (OD600 = 4). Approximately 50% of PFNA bioaccumulation occurs within the sample collection time frame (approximately 5 minutes). n = 2 biological replicates. g. PFNA bioaccumulate by B. uniformis grown at initial concentrations ranging from 0.01 to 100 μM. n = 4 technical replicates. h. Growth sensitivity of gut bacteria to PFAS is independent of bioaccumulation (n = 3 technical replicates); *bioaccumulating bacteria. [Figure 8]Genetic and morphological data support the intracellular accumulation of PFAS. E. coli efflux pump mutants show increased PFAS bioaccumulation potential, and PFAS-bioaccumulating bacteria display unique morphological characteristics under transmission electron microscopy (TEM). a. PFNA bioaccumulation by live, dead (heat-inactivated), and lysed (heat-inactivated + freeze-thaw + sonication) B. uniformis, O. splanchnicus, and E. coli cultures (OD600 = 3.75) in PBS buffer. n = 3 technical replicates. b. Schematic diagram of the AcrAB-TolC efflux pump. The resting state of the pump is shown on the left side of the image. Upon encountering a xenobiotic, the pump changes conformation and expels the xenobiotic outside the cell (right). c. Accumulation of PFDeA and PFNA by wild-type E. coli strains and corresponding efflux mutants (E. coli BW25113ΔtolC, E. coli C43(DE3)ΔacrAB-tolC). n = 3 technical replicates. d. Efflux mutants show increased PFNA and PFOA sensitivity. n=3 technical replicates. e-h. TEM of B. uniformis grown for 24 hours in mGAM+DMSO (e), 50 μM PFNA (f), 250 μM PFNA (g), or 125 μM PFDeA (h). i, j. TEM of E. coli BW25113 wild-type cells grown for 24 hours in mGAM+DMSO (i) or 250 μM PFNA (j). k, l. TEM of E. coli BW25113ΔtolC grown for 24 hours in mGAM+DMSO (k) or 250 μM PFNA (l). m, n. TEM of O. splanchnicus grown for 24 hours in mGAM+DMSO (m) or 250 μM PFNA (n). Arrows indicate changes in nucleoid appearance in PFAS-treated bacteria. [Figure 9]PFAS tolerance and bioaccumulation after adaptive laboratory evolution. a. Five enteric bacterial species were evolved through serial passage over 20 days in growth medium containing one of four PFAS compounds (500 μM perfluoroheptanoic acid (PFHpA), 500 μM PFOA, 250 μM PFNA, 125 μM PFDeA). b. Improved growth of adapted B. uniformis populations in the presence of 125 μM PFDeA and 250 μM PFNA. c, d. Adapted populations retain PFDeA (c) and PFNA (d) bioaccumulation ability. n = 4 independent populations per compound. [Figure 10] Increased PFNA shedding in mouse feces and GI tract after PFNA exposure. a) Experimental setup. b, c) Mice colonized with a community of 20 human gut bacterial strains (Com20) show higher PFNA shedding after exposure compared to germ-free (GF) controls. [Figure 11]Method setup and results of the community screen. a. Method workflow for the artificial community experiment. b. Method workflow for the single-strain experiment. c. Of the 42 tested contaminants, 18 were isolated by at least one synthetic gut bacterial community. Each community consisted of 10 bacteria (Community 1: Bacteroides caccae, Bacteroides dorei, Bacteroides thetaiomicron, Bacteroides uniformis, Bacteroides vulgatus, Colinsella aerofaciens, Coprococcus comes, Eubacterium rectale, Parabacteroides merdae, Roseburia intestinalis; Community 2: Akkermansia muciniphila, Bacteroides clarus, Bacteroides stercoris, Clostridium difficile, Eggerthella lenta, Eubacterium eligens, Fusobacterium nucleatum subsp. animalis, Odoribacter splanchnicus, Parabacteroides distastonis, Ruminococcus bromii). Colored squares indicate sequestration, i.e., a >25% reduction from the sterile supernatant. PFAS are shown in blue. n=6 (3 biological and 2 technical replicates). [Figure 12] Intrastrain variability for PFNA and PFOA bioaccumulation potential. a. Accumulation potential of eight different B. uniformis and two E. coli strains grown in mGAM containing 20 μM PFOA or PFNA for 24 h starting from an OD of 0.05. No interstrain differences were observed. b. Accumulation potential of eight different B. uniformis and two E. coli strains (OD600=3.75) incubated in PBS containing 20 μM PFOA or PFNA for 4 h. No interstrain differences were observed. n=3 technical replicates. [Figure 13]Intrastrain variability in PFNA and PFOA bioaccumulation potential. PFNA or PFOA accumulation by B. uniformis (OD600 = 3.75) in PBS containing different concentrations of PFNA ranging from 0.01 to 100 μM over a 4-hour period. n = 3 technical replicates. [Figure 14] Bioaccumulation of PFAS in live and dead bacterial biomass. Bioaccumulation of PFDeA, PFNA, and PFOA by live, dead (heat inactivated), and lysed (heat inactivated + freeze-thaw + sonication) B. uniformis, O. splanchnicus, and E. coli cultures (OD600 = 3.75) in PBS buffer (n = 3 technical replicates). [Figure 15] Effect of ALE on growth in the presence of high PFAS concentrations. Growth of bacterial strains in the presence of high PFAS concentrations over the course of adaptive laboratory evolution. Evolutionary lineages per compound, n=4. [Figure 16] Evolved strains retain PFAS bioaccumulation potential. a. Strains evolved in the presence of PFDeA show no alterations to PFDeA bioaccumulation potential. b. Strains evolved in the presence of PFNA show no alterations to PFNA bioaccumulation potential. n=4 evolutionary lineages. [Figure 17] Results showing differentially expressed proteins between B. uniformis treated with PFNA (20 μM) compared to DMSO. Green and blue dots indicate proteins with a log abundance ratio of >1 or <-1 (i.e., a 2-fold increase or decrease) and a multiple testing-corrected p-value of <0.05. [Figure 18] Results from a transposon library screen of P. merdae showing reduced fitness for mutants in genes encoding homologs of the three most upregulated proteins in B. uniformis , all of which are efflux pumps. [Figure 19] A. PFNA accumulation shows a bimodal distribution. B. Gram-positive and Gram-negative strains show differences in PFNA accumulation. C. Bacterial phyla show differences in PFNA accumulation. D. Correlation of PFNA accumulation between growth (mGAM) and resting (PBS) assays. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is based on the surprising discovery of bacterial strains capable of reducing the levels of a wide variety of xenobiotics. Preferably, the bacterial strains of the present invention are commonly found in the human microbiota and can therefore be used in a variety of settings without imposing immediate health risks to humans or animals. The inventors have discovered that the bacterial strains of the present invention possess the highly desirable ability to bioaccumulate and / or biotransform xenobiotics. Surprisingly, the inventors have found that several bacterial strains commonly found in the gut bioaccumulate "persistent chemical" PFA substances, including PFNA (perfluorononanoic acid) and PFOA (perfluorooctanoic acid). The inventors have made the particularly surprising discovery that the gut bacterial strains of the present invention bioaccumulate PFAS to a much higher degree than bacteria (e.g., Pseudomonas species) that have been isolated from PFAS-contaminated sites. The bacterial strains of the present invention preferably reduce the levels of xenobiotics in the environment, thereby improving environmental quality and reducing the risk of ingestion and / or absorption by subjects (e.g., humans or animals).

[0034] The present invention provides a method for reducing the level of xenobiotics in an environment, comprising contacting the environment with a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia. The present invention also provides a method for reducing the level of xenobiotics in an environment, comprising contacting the environment with a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus. As shown herein, bacterial strains from each of these genera can bioaccumulate and / or biotransform xenobiotics, such as PFAS.

[0035] As used herein, "biotransformation" of a xenobiotic involves the enzymatic conversion of a xenobiotic compound to a metabolite, and "bioaccumulation" of a xenobiotic by a bacterial strain of the invention involves the uptake and sequestration of the xenobiotic within the bacterial cell.

[0036] In some embodiments, the reduction in levels of a xenobiotic comprises bioaccumulation of the xenobiotic by one or more bacterial strains, hi some embodiments, the reduction in levels of a xenobiotic comprises biotransformation of the xenobiotic by one or more bacterial strains.

[0037] Contacting the composition with the environment may be by any suitable means, hi some embodiments, contacting the composition with the environment comprises applying the composition to the environment using one or more methods selected from spraying, fertigation, and / or injection into the environment.

[0038] The environment is typically an ex vivo environment. In some embodiments, the environment is an aqueous environment. In some embodiments, the aqueous environment is a water treatment facility, such as a drinking water treatment facility and / or a wastewater treatment facility. In some embodiments, the aqueous environment is industrial wastewater. In some embodiments, the aqueous environment is drinking water or water intended for drinking.

[0039] Suitably, the bacterial strain of the invention is common in the human microbiota, and therefore its presence in, for example, drinking water, does not pose a significant health risk to humans or animals.

[0040] In some embodiments, the method for reducing the level of xenobiotics in an environment is performed in situ.

[0041] In some embodiments, the method is a bioremediation method. In some embodiments, the method is a bioremediation method performed in situ.

[0042] In some embodiments, the environment is land that is contaminated or suspected to be contaminated with xenobiotics. In some embodiments, the land is agricultural land. In some embodiments, the land is residential or domestic land. In some embodiments, the land is forest.

[0043] In some embodiments, the environment is within a bioreactor (eg, growth medium).

[0044] In some instances, for example, when bacterial cells bioaccumulate xenobiotics from an environment, it is preferable to remove bacterial strains after treating the environment. In some embodiments, a method includes: (a) contacting an environment containing or suspected of containing a xenobiotic with a composition that reduces the level of the xenobiotic in the environment, and then (b) removing one or more bacterial strains to provide a treated environment. For example, the environment may be filtered to remove the bacterial strains. In some embodiments, a method includes: (a) contacting an aqueous environment containing or suspected of containing a xenobiotic with a composition that reduces the level of the xenobiotic in the aqueous environment, and then (b) removing one or more bacterial strains to provide a treated aqueous environment. For example, the aqueous environment may be filtered to remove the bacterial strains. An appropriate filter size can be easily determined by the user.

[0045] In some embodiments, the method comprises contacting an aqueous environment containing or suspected of containing a xenobiotic with one or more bacterial strains of the present invention, wherein the one or more bacterial strains are immobilized on a support. In this embodiment, the aqueous environment may be continuously flowed over the support, or the support may be added to the aqueous environment for a predetermined period of time. The bacteria may be immobilized on a range of supports, such as glass or polymer beads, sand or gravel-type materials, polysaccharide-based matrices, and membranes.

[0046] The present invention also provides a composition for use in a method for reducing xenobiotic levels in a subject, the composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia. The present invention also provides a method for reducing xenobiotic levels in a subject, the method comprising administering to the subject a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia. The present invention also provides a composition for use in a method for preventing or treating xenobiotic intoxication in a subject, the composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia. The present invention also provides a method of preventing or treating xenobiotic poisoning in a subject, comprising administering to the subject a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia.

[0047] The present invention also provides a composition for use in a method for reducing the level of xenobiotics in a subject, the composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus. The present invention also provides a method of reducing xenobiotic levels in a subject, comprising administering to the subject a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus. The present invention also provides a composition for use in a method of preventing or treating xenobiotic poisoning in a subject, the composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus.The present invention also provides a method of preventing or treating xenobiotic poisoning in a subject, comprising administering to the subject a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus.

[0048] As mentioned above, the bacterial strains of the present invention are highly prevalent in the human microbiome, indicating that they can be safely administered to humans and animals.

[0049] As used herein, "xenobiotic intoxication" includes any adverse physiological effects caused by the ingestion of a xenobiotic. Xenobiotics may be ingested through a variety of routes, for example, in drinking water and / or food, and then absorbed from the intestine by active and / or passive mechanisms. Adverse physiological effects associated with the absorption and / or bioaccumulation of xenobiotics by a subject include, but are not limited to, inflammation, increased oxidative stress, and increased cytotoxicity. In some embodiments, a decrease in xenobiotic levels in a subject includes a decrease in ingested xenobiotic levels.

[0050] In some embodiments, reducing xenobiotic levels in a subject includes reducing the bioavailability of the xenobiotic due to bioaccumulation and / or biotransformation. Bioaccumulation results in the sequestration of ingested xenobiotics within bacterial cells, thereby reducing the bioavailability of the xenobiotic in the subject. Biotransformation results in the metabolism of the xenobiotic, thereby reducing the bioavailability of the xenobiotic in the subject. As used herein, xenobiotic bioavailability is the proportion of the xenobiotic available for absorption into the subject, typically from the intestine. Advantageously, the inventors have demonstrated that mice colonized with the human microbiota bacterial strains of the present invention excrete higher levels of xenobiotics than germ-free controls.

[0051] In some embodiments, the subject has ingested a xenobiotic, is suspected of ingesting a xenobiotic, or is at risk of ingesting a xenobiotic. In some embodiments, the subject is at risk of ingesting drinking water or food contaminated with a xenobiotic. In some embodiments, the subject is suspected of ingesting drinking water or food contaminated with a xenobiotic. In some embodiments (e.g., when the subject is at risk of exposure to a xenobiotic), the composition is for use as a prophylactic or defensive measure. In some embodiments, the subject is at risk of ingesting a xenobiotic during work or recreational activities. For example, the subject may work in close contact with a xenobiotic or visit an environment contaminated with a xenobiotic.

[0052] In some embodiments, the method includes detecting the presence and / or measuring the abundance of one or more bacterial strains in the environment prior to contact with the composition. The presence and / or abundance of bacterial strains in the environment can be determined using methods known in the art, such as cell-based methods and / or molecular methods (e.g., qPCR).

[0053] In some embodiments, the method includes detecting the presence and / or measuring the abundance of one or more bacterial strains in a subject prior to administering the composition to the subject. The bacterial strains of the present invention are common to the human microbiota, but the composition of the microbiota varies between individuals. Some individuals' microbiota will contain each of the bacterial strains of the present invention, while other individuals' microbiota may contain only a subset (or potentially even none) of the bacterial strains of the present invention. The presence and / or abundance of bacterial strains in a subject can be determined using methods known in the art, such as cell-based and / or molecular methods (e.g., qPCR). The amount of bacterial strain administered to a subject can vary based on the presence and / or abundance of the bacterial strain in the subject. For example, if a subject has a high abundance of a bacterial strain, a lower amount may be administered compared to the amount administered to a subject in which the bacterial strain is absent or low in abundance, and vice versa.

[0054] In some embodiments, the subject is a human. In some embodiments, the subject is an animal, optionally selected from cows, sheep, pigs, poultry (e.g., chickens, turkeys), cats, or dogs.

[0055] The present invention also provides a composition for reducing the level of xenobiotics in the environment, the composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia. The present invention also provides a composition for reducing the level of xenobiotics in the environment, the composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus. As shown herein, bacterial strains from each of these genera can bioaccumulate and / or biotransform xenobiotics, such as PFAs.

[0056] References herein to a "composition" will be understood to include compositions of the invention, compositions for use in methods of the invention, and compositions for use in therapeutic methods of the invention.

[0057] In some embodiments, the composition comprises two or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia, hi some embodiments, the composition comprises three or more (e.g., four or more, five or more, six or more, or seven) bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia.

[0058] In some embodiments, the composition comprises two or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus. In some embodiments, a composition comprises three or more (e.g., four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, or seventeen or seventeen) bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus. As shown herein, bacterial strains from each of these genera can bioaccumulate and / or biotransform xenobiotics.

[0059] In some embodiments, the composition comprises one or more of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Collinsella aerofaciens, Coprococcus comes, Eubacterium rectale, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides merdae, and Roseburia intestinalis.

[0060] In some embodiments, the composition comprises Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Collinsella aerofaciens, Coprococcus comes, Eubacterium rectale, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides merdae, Roseburia intestinalis, Escherichia coli, Phocaeicola coprocola, Prevotella copri, Bacteroides eggerthii, Prevotella melaninogenica, Bacteroides fragilis, Bacteroides xylanisolvens, Butyrivibrio crossotus, Bacteroides coprocola, Roseburia hominis, Lacrimispora saccharolytica, Clostridium scindens, Fusobacterium nucleatum subsp. Nucleatum, Clostridium difficile, Phocaeicola vulgatus, Agathobacter rectalis, Roseburia inulinivorans, Dorea formicigenerans, Streptococcus salivarius, Fusobacterium nucleatum subsp. Animalis, Fusobacterium nucleatum subsp. Vincentii, Clostridium hylemonae, and Clostridium sporogenes. As demonstrated herein, bacterial strains from each of these genera are capable of bioaccumulating and / or biotransforming xenobiotics.

[0061] In some embodiments, the composition comprises two or more of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Collinsella aerofaciens, Coprococcus comes, Eubacterium rectale, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides merdae, and Roseburia intestinalis. In some embodiments, the composition comprises three or more (e.g., four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, or thirteen) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Collinsella aerofaciens, Coprococcus comes, Eubacterium rectale, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides merdae, and Roseburia intestinalis.

[0062] These include Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, and Bacteroides thetaiotaomicron、Collinsella aerofaciens、Coprococcus comes、Eubacterium rectale、Odoribacter splanchnicus、Parabacteroides distasonis、Parabacteroides merdae、Roseburia intestinalis、およびEscherichia coli, 2 of which were identified as Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, and Bacteroides dorei、Bacteroides stercoris、Bacteroides thetaiotaomicron、Collinsella aerofaciens、Coprococcus comes、Eubacterium rectale、Odoribacter splanchnicus、Parabacteroides distasonis、Parabacteroides merdae、Roseburia intestinalis、Escherichia coli、Phocaeicola coprocola、Prevotella copri、Bacteroides eggerthii、Prevotella melaninogenica、Bacteroides fragilis、Bacteroides xylanisolvens、Butyrivibrio crossotus、Bacteroides coprocola、Roseburia hominis、Lacrimispora saccharolytica、Clostridium scindens、Fusobacterium nucleatum subsp.Nucleatum, Clostridium difficile, Phocaeicola vulgatus, Agathobacter rectalis, Roseburia inulinivorans, Dorea formicigenerans, Streptococcus salivarius, Fusobacterium nucleatum subsp. Animalis, Fusobacterium nucleatum subsp. Vincentii, Clostridium hylemonae, and Clostridium sporogenes (e.g., 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, or 36).

[0063] In some embodiments, one or more bacterial strains are genetically modified. In some embodiments, one or more of the bacterial strains comprises a genetic modification that results in the reduction or elimination of xenobiotic excretion from the one or more bacterial strains. In some embodiments, the genetic modification comprises the deletion or inactivation of at least one gene required for the activity of an efflux pump. In some embodiments, the genetic modification comprises the deletion or inactivation of at least one gene encoding an efflux pump or a component thereof. Efflux pumps are a common mechanism used by several bacterial species to reduce the intracellular concentration of toxic compounds. Disrupting or disrupting the activity of efflux pumps increases bioaccumulation by the bacterial strain. Gene deletion or inactivation can be achieved using any suitable method known in the art, such as site-directed mutagenesis, homologous recombination, CRISPR-Cas9-based methods, or transcription activator-like effector nuclease (TALEN)-based methods.

[0064] In some embodiments, the genetic modification comprises the deletion or inactivation of at least one gene required for an RND (resistance-nodulation-division) family transporter or a homolog thereof. RND family transporters are widely distributed, particularly among Gram-negative bacteria.

[0065] In some embodiments, the RND family transporter is an AcrAB-TolC complex or a homolog thereof. In some embodiments, the genetic modification comprises deletion or inactivation of at least one gene encoding a TolC family protein or a homolog thereof, such as tolC or a homolog thereof. In some embodiments, the genetic modification comprises deletion or inactivation of one or more (e.g., one, two, or all three) of arcA or a homolog thereof, acrB or a homolog thereof, and tolC or a homolog thereof.

[0066] In some embodiments, the genetic modification comprises the deletion or inactivation of a gene encoding a membrane fusion protein (MFP) subunit of an RND family efflux transporter (e.g., UniProt Accession Number: R9I2M9) or a homolog thereof.

[0067] In some embodiments, the genetic modification comprises the deletion or inactivation of a gene encoding a hydrophobe / amphiphile efflux-1 (HAE1) family RND transporter (e.g., UniProt Accession No. R9I2L8) or a homolog thereof.

[0068] In some embodiments, the genetic modification comprises the deletion or inactivation of the gene encoding the outer membrane factor (OMF) lipoprotein of the NodT family efflux transporter (e.g., UniProt Accession Number: R9I2R1) or a homolog thereof.

[0069] As used herein, homologues include functional and structural homologues, identifiable using methods known in the art, for example, by sequence and / or structural homology.

[0070] In some embodiments, the composition comprises Bacteroides. In some embodiments, the composition comprises one or more (e.g., two or more, three or more, four or more, five or more, or six) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, and Bacteroides thetaiotaomicron. In some embodiments, the composition comprises Bacteroides. In some embodiments, the composition comprises one or more (e.g., 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) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, and Bacteroides thetaiotaomicron, Bacteroides eggerthii, Bacteroides fragilis, Bacteroides xylanisolvens, and Bacteroides coprocola.

[0071] In some embodiments, the composition comprises Bacteroides uniformis. In some embodiments, the composition comprises one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, or eight) of Bacteroides uniformis DSM110080, Bacteroides uniformis DSM108148, Bacteroides uniformis DSM108147, Bacteroides uniformis DSM108145, Bacteroides uniformis DSM108146, Bacteroides uniformis HM-716, Bacteroides uniformis DSM6597, and Bacteroides uniformis HM-715. The inventors have found that these eight different strains of B. uniformis exhibit comparable levels of PFNA and PFOA bioaccumulation, indicating that bioaccumulation of these compounds is a species-level characteristic of B. uniformis.

[0072] In some embodiments, the composition comprises a genetically modified Bacteroides. In some embodiments, the Bacteroides comprises a genetic modification that results in reduced or eliminated xenobiotic efflux. In some embodiments, the genetic modification comprises the deletion or inactivation of at least one gene required for the activity of an efflux pump. In some embodiments, the genetic modification comprises the deletion or inactivation of at least one gene required for the activity of an R9I2M9 efflux transporter RND family, an R9I2L8 hydrophobe / amphiphile efflux-1 (HAE1) family RND transporter, and / or an R9I2R1 NodT family efflux transporter.

[0073] In some embodiments, the composition comprises Parabacteroides. In some embodiments, the composition comprises one or more (e.g., both) of Parabacteroides distasonis and Parabacteroides merdae.

[0074] In some embodiments, the composition comprises a genetically modified Parabacteroides. In some embodiments, the Parabacteroides comprises a genetic modification that results in reduced or eliminated xenobiotic efflux. In some embodiments, the genetic modification comprises the deletion or inactivation of at least one gene required for the activity of an efflux pump. In some embodiments, the genetic modification comprises the deletion or inactivation of at least one gene required for the activity of a homolog of the Bacteroides R9I2M9 efflux transporter RND family, the R9I2L8 hydrophobe / amphiphile efflux-1 (HAE1) family RND transporter, and / or the R9I2R1 NodT family efflux transporter.

[0075] In some embodiments, the composition comprises Bacteroides and / or Parabacteroides. In some embodiments, the composition comprises one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, or eight) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Parabacteroides distasonis, and Parabacteroides merdae. In some embodiments, the composition comprises Bacteroides and / or Parabacteroides. In some embodiments, the composition comprises one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Parabacteroides distasonis, Parabacteroides merdae, Bacteroides eggerthii, Bacteroides fragilis, Bacteroides xylanisolvens, and Bacteroides coprocola.

[0076] In some embodiments, the composition comprises Collinsella. In some embodiments, the composition comprises Collinsella aerofaciens.

[0077] In some embodiments, the composition comprises Coprococcus. In some embodiments, the composition comprises Coprococcus comest.

[0078] In some embodiments, the composition comprises a Eubacterium. In some embodiments, the composition comprises a Eubacterium rectale.

[0079] In some embodiments, the composition comprises Odoribacter. In some embodiments, the composition comprises Odoribacter splanchnicus.

[0080] In some embodiments, the composition comprises Roseburia. In some embodiments, the composition comprises Roseburia intestinalis. In some embodiments, the composition comprises one or more (e.g., two or more, or three) of Roseburia intestinalis, Roseburia hominis, and Roseburia inulinivorans.

[0081] In some embodiments, the composition comprises Escherichia. In some embodiments, the composition comprises E. coli.

[0082] In some embodiments, the composition comprises an E. coli strain comprising a genetic modification that results in the reduction or elimination of xenobiotic efflux. In some embodiments, the E. coli has been genetically modified to delete or inactivate one or more genes encoding the AcrAB-TolC efflux pump. In some embodiments, the E. coli has been genetically modified to delete or inactivate tolC. In some embodiments, the E. coli is E. coli BW25113 delta-tolC. In some embodiments, the E. coli has been genetically modified to delete or inactivate tolC, acrA, and / or acrB. In some embodiments, the E. coli is E. coli C43(DE3) delta acrAB-tolC.

[0083] In some embodiments, the composition comprises Phocaeicola. In some embodiments, the composition comprises Phocaeicola coprocola and / or Phocaeicola vulgatus.

[0084] In some embodiments, the composition comprises Prevotella. In some embodiments, the composition comprises Prevotella copri and / or Prevotella melaninogenica.

[0085] In some embodiments, the composition comprises Butyrivibrio. In some embodiments, the composition comprises Butyrivibrio crossotus.

[0086] In some embodiments, the composition comprises Lacrimispora. In some embodiments, the composition comprises Lacrimispora saccharolytica.

[0087] In some embodiments, the composition comprises Clostridium. In some embodiments, the composition comprises one or more (e.g., two or more, three or more, or four) of Clostridium scindens, Clostridium difficile, Clostridium hylemonae, and Clostridium sporogenes.

[0088] In some embodiments, the composition comprises Fusobacterium. In some embodiments, the composition comprises Fusobacterium nucleatum. In some embodiments, the composition comprises one or more (e.g., two or more, or three) of Fusobacterium nucleatum subsp. Nucleatum, Fusobacterium nucleatum subsp. Animalis, and Fusobacterium nucleatum subsp. Vincentii.

[0089] In some embodiments, the composition comprises Agathobacter. In some embodiments, the composition comprises Agathobacter rectalis.

[0090] In some embodiments, the composition comprises Dorea. In some embodiments, the composition comprises Dorea formicigenerans.

[0091] In some embodiments, the composition comprises Streptococcus. In some embodiments, the composition comprises Streptococcus salivarius.

[0092] In some embodiments, the composition comprises Phocaeicola vulgatus, Bacteroides uniformis, Bacteroides fragilis, Bacteroides thetaiotaomicron, Erysipelatoclostridium ramosum, Agathobacter rectalis, Roseburia intestinalis, Veillonella parvula, Eggerthella lenta, Fusobacterium nucleatum, Enterocloster bolteae, Clostridium perfringens, Lacrimispora saccharolytica, Streptococcus salivarius, Ruminococcus gnavus, Bariatricus comes, Parabacteroides merdae, Streptococcus parasanguinis, Collinsella aerofaciens, and Dorea formicigenerans (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, one or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty).

[0093] In some embodiments, the composition comprises Erysipelatoclostridium ramosum. In some embodiments, the composition comprises Veillonella parvula. In some embodiments, the composition comprises Eggerthella lenta. In some embodiments, the composition comprises Enterocloster bolteae. In some embodiments, the composition comprises Clostridium perfringens. In some embodiments, the composition comprises Lacrimispora saccharolytica. In some embodiments, the composition comprises Ruminococcus gnavus. In some embodiments, the composition comprises Bariatricus comedii. In some embodiments, the composition comprises Streptococcus parasanguinis.

[0094] In some embodiments, the composition comprises one or more (e.g., 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) of Bacteroides caccae, Bacteroides dorei, Bacteroides thetaiomicron, Bacteroides uniformis, Bacteroides vulgatus, Colinsella aerofaciens, Coprococcus comes, Eubacterium rectale, Parabacteroides merdae, and Roseburia intestinalis. In some embodiments, the composition comprises one or more (e.g., 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) of Akkermansia muciniphila, Bacteroides clarus, Bacteroides stercoris, Clostridium difficile, Eggerthella lenta, Eubacterium eligens, Fusobacterium nucleatum subsp. animalis, Odoribacter splanchnicus, Parabacteroides distastonis, and Ruminococcus bromii.

[0095] In some embodiments, the composition comprises Collinsella aerofaciens. In some embodiments, the composition comprises Akkermansia muciniphila. In some embodiments, the composition comprises Eggerthella lenta. In some embodiments, the composition comprises Eubacterium eligens. In some embodiments, the composition comprises Ruminococcus bromii.

[0096] In some embodiments, the xenobiotics include one or more of PFAs (per- and polyfluoroalkyl substances), bisphenols, and pesticides.

[0097] The terms "PFA" and "PFAS" are used interchangeably herein. Both terms refer to per- and polyfluoroalkyl substances.

[0098] In some embodiments, the xenobiotic comprises one or more of PFNA, PFOA, PFDeA, bisphenol AF, boscalid, propiconazole, pyrimethanil, tributyl-PO4, and triphenyl-PO4.

[0099] In some embodiments, the xenobiotic is PFA. In some embodiments, the xenobiotic comprises one or more (e.g., two or more, or three) of PFNA (perfluorononanoic acid), PFOA (perfluorooctanoic acid), and PFDeA (perfluorodecanoic acid).

[0100] In some embodiments, the xenobiotic is a bisphenol. In some embodiments, the xenobiotic comprises bisphenol AF.

[0101] In some embodiments, the xenobiotic is an insecticide. In some embodiments, the xenobiotic comprises one or more (e.g., two or more, or three) of boscalid, propiconazole, and pyrimethanil.

[0102] In some embodiments, the xenobiotic is a PFA and the composition comprises one or more (e.g., two or more, or three) of Bacteroides, Odoribacter, and Parabacteroides. In some embodiments, the xenobiotic is a PFA and the composition comprises one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Odoribacter splanchnicus, Parabacteroides distasonis, and Parabacteroides merdae. In some embodiments, the PFA comprises PFOA and / or PFNA.

[0103] In some embodiments, the xenobiotic is a PFA and the composition comprises one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or fourteen) of Bacteroides, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus.In some embodiments, the xenobiotic is PFA and the composition is Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides merdae, Phocaeicola coprocola, Prevotella copri, Bacteroides eggerthii, Prevotella melaninogenica, Bacteroides fragilis, Bacteroides xylanisolvens, Butyrivibrio crossotus, Bacteroides coprocola, Roseburia hominis, Parabacteroides distasonis, Lacrimispora saccharolytica, Clostridium scindens, Fusobacterium nucleatum subsp. Nucleatum, Clostridium difficile, Phocaeicola vulgatus, Agathobacter rectalis, Roseburia inulinivorans, Dorea formicigenerans, Streptococcus salivarius, Fusobacterium nucleatum subsp. Animalis, Fusobacterium nucleatum subsp. Vincentii, Clostridium hylemonae, and Clostridium sporogenes (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty-one or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more, twenty-six or more, twenty-seven or more, twenty-eight or more, twenty-nine or more, thirty or more, thirty-one or more, or thirty-two or more).In some embodiments, the PFA comprises PFOA and / or PFNA. As shown herein, each of these bacterial strains can efficiently bioaccumulate PFAs, such as PFNA.

[0104] In some embodiments, the xenobiotic is PFA and the composition comprises Bacteroides. In some embodiments, the xenobiotic is PFA and the composition comprises one or more (e.g., two or more, three or more, four or more, five or more, or six) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, and Bacteroides thetaiotaomicron. In some embodiments, the xenobiotic is a PFA and the composition comprises one or more (e.g., 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) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, and Bacteroides thetaiotaomicron, Bacteroides eggerthii, Bacteroides fragilis, Bacteroides xylanisolvens, and Bacteroides coprocola. In some cases, the xenobiotic is a PFA and the composition comprises Bacteroides uniformis. In some embodiments, the PFA comprises PFNA, PFOA, and / or PFDeA.

[0105] In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises an E. coli strain comprising a genetic modification that results in reduced or eliminated xenobiotic efflux. In some embodiments, the E. coli has been genetically modified to delete or inactivate one or more genes encoding the AcrAB-TolC efflux pump. In some embodiments, the E. coli has been genetically modified to delete or inactivate tolC. In some embodiments, the E. coli is E. coli BW25113 delta-tolC. In some embodiments, the E. coli has been genetically modified to delete or inactivate tolC, acrA, and / or acrB. In some embodiments, the E. coli is E. coli C43(DE3) deltaAcrA-AcrB-TolC. As demonstrated herein, E. coli strains comprising genetic modifications that result in reduced or eliminated xenobiotic efflux exhibit increased PFNA accumulation.

[0106] In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Phocaeicola. In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Phocaeicola coprocola and / or Phocaeicola vulgatus.

[0107] In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Prevotella. In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Prevotella copri and / or Prevotella melaninogenica.

[0108] In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Butyrivibrio. In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Butyrivibrio crossotus.

[0109] In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Lacrimispora. In some embodiments, the xenobiotic is a PFAS, optionally PFNA, and the composition comprises Lacrimispora saccharolytica.

[0110] In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Clostridium. In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises one or more (e.g., two or more, three or more, or four) of Clostridium scindens, Clostridium difficile, Clostridium hylemonae, and Clostridium sporogenes.

[0111] In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Fusobacterium. In some embodiments, the composition comprises Fusobacterium nucleatum. In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises one or more (e.g., two or more, or three) of Fusobacterium nucleatum subsp. Nucleatum, Fusobacterium nucleatum subsp. Animalis, and Fusobacterium nucleatum subsp. Vincentii.

[0112] In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Agathobacter. In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Agathobacter rectalis.

[0113] In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Dorea. In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Dorea formicigenerans.

[0114] In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Streptococcus. In some embodiments, the xenobiotic is a PFAS, optionally PFNA, PFOA, and / or PFDeA, and the composition comprises Streptococcus salivarius.

[0115] In some embodiments, the xenobiotic is a bisphenol and the composition comprises one or more (e.g., two or more, three or more, four or more, five or more, or six) of Bacteroides, Collinsella, Eubacterium, Odoribacter, Parabacteroides, and Roseburia. In some embodiments, the xenobiotic is a bisphenol and the composition comprises one or more (e.g., 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) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides thetaiotaomicron, Collinsella aerofaciens, Eubacterium rectale, Odoribacter splanchnicus, Parabacteroides merdae, and Roseburia intestinalis. In some embodiments, the bisphenol comprises bisphenol AF.

[0116] In some embodiments, the xenobiotic is an insecticide and the composition includes one or more (e.g., two or more, three or more, four or more, five or more, or six) of Bacteroides, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, and Roseburia. In some embodiments, the xenobiotic is an insecticide and the composition comprises one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Coprococcus comes, Eubacterium rectale, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides merdae, and Roseburia intestinalis. In some embodiments, the insecticide comprises boscalid, propiconazole, and / or pyrimethanil.

[0117] In some embodiments, the xenobiotic is tributyl-PO4 and the composition comprises one or more (e.g., two or more, three or more, four or more, or five) of Bacteroides, Coprococcus, Odoribacter, Parabacteroides, and Roseburia. In some embodiments, the xenobiotic is tributyl-PO4 and the composition comprises one or more (e.g., 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) of Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Coprococcus comes, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides merdae, and Roseburia intestinalis.

[0118] In some embodiments, the xenobiotic is triphenyl-PO4 and the composition includes one or more (e.g., two or more, or three) of Bacteroides, Coprococcus, and Odoribacter. In some embodiments, the xenobiotic is triphenyl-PO4 and the composition includes one or more (e.g., two or more, or three) of Bacteroides dorei, Coprococcus comest, and Odoribacter splanchnicus.

[0119] It will be appreciated that xenobiotics may adopt different forms depending on the surrounding environment. For example, a xenobiotic in solution may be deprotonated. The present invention is not limited to any particular xenobiotic form and includes, for example, salts, hydrates, solvates, crystalline forms, amorphous forms, and mixtures thereof of the xenobiotics described herein.

[0120] The bacterial strain may be wild-type or genetically modified. The composition typically comprises viable cells. In some embodiments, the composition comprises at least 10 4 Bacterial cells, optionally at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , or at least 10 15 Contains bacterial cells.

[0121] In some embodiments, the composition comprises dividing cells, e.g., log phase cells. In some embodiments, the composition comprises non-dividing cells, e.g., quiescent or inactive cells.

[0122] In some embodiments, the composition comprises one or more bacterial strains adapted for bioaccumulation and / or biotransformation of a xenobiotic. In some embodiments, the one or more bacterial strains are adapted for bioaccumulation and / or biotransformation of a xenobiotic by adaptive evolution. Adaptive evolution can include exposing one or more bacterial strains to a medium containing the xenobiotic, dividing the bacterial strains for a predetermined period of time or until a predetermined cell density is reached, and then transferring a subset of the bacterial strain population to a new medium containing the xenobiotic. Typically, multiple serial transfers (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 serial transfers) are performed to provide an adapted bacterial cell population. In some embodiments, the xenobiotic concentration can increase after several serial transfers, e.g., after five serial transfers. The bioaccumulation and / or biotransformation activity of the adapted bacterial cell population may be compared to the bioaccumulation and / or biotransformation activity of the starting bacterial cell population to identify adapted bacterial cell populations with improved activity.

[0123] In some embodiments, the xenobiotic level is reduced by at least 5%, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, when compared to the level of the xenobiotic in the contact area of ​​the environment prior to contact with the composition. In some embodiments, the level of reduction is assessed less than 6 hours after contact with the composition, e.g., less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, or less than 30 minutes after contact with the composition. In some embodiments, the level of reduction is assessed more than 6 hours after contact with the composition, e.g., more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, or more than 96 hours, more than 1 week, or more than 1 month after contact with the composition.

[0124] In some embodiments, the level of the xenobiotic is reduced by at least 5%, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, when compared to the level of the xenobiotic in the subject before administering the composition to the subject. In some embodiments, the level of the xenobiotic in the subject refers to the level of the xenobiotic in the intestine of the subject. In some embodiments, the level of reduction is assessed less than 6 hours after administration of the composition, e.g., less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, or less than 30 minutes after administration of the composition. In some embodiments, the level of reduction is assessed more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, or more than 96 hours, more than 1 week, or more than 1 month after administration of the composition.

[0125] In some embodiments, the composition is formulated for oral, rectal, and / or nasal delivery (e.g., via a nasoduodenal tube). In some embodiments, the composition is formulated for delivery to the intestine of a subject.

[0126] In some embodiments, the composition is in the form of a capsule, tablet, powder, or liquid. In some embodiments, the one or more bacterial strains are lyophilized.

[0127] In some embodiments, the composition includes one or more prebiotics to promote the growth of one or more bacterial strains. In some embodiments, the method includes administering one or more prebiotics to a subject prior to, concurrently with, or following administration of the composition.

[0128] The present invention provides a dietary supplement comprising the composition of the present invention.In some embodiments, the dietary supplement comprises one or more prebiotics.In some embodiments, the dietary supplement is a food or beverage.In some embodiments, the dietary supplement is a tablet or capsule.

[0129] In some embodiments, the one or more prebiotics are selected from arabinoxylan, xylose, fibrous dextran, corn fiber, polydextrose, lactose, N-acetyl-lactosamine, glucose, galactose, fructose, rhamnose, mannose, uronic acid, arabinose, fructose, fucose, lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose, xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose, xylooligosaccharides, fructooligosaccharides, galactooligosaccharides, lactosucrose, and soybean oligosaccharides.

[0130] In some embodiments, the composition comprises a sweetener, a flavoring agent, and / or a coloring agent. Sweeteners include, but are not limited to, glucose, dextrose, fructose, and saccharin. Flavoring agents include, but are not limited to, synthetic and natural oils and plant extracts. Suitable coloring agents include food coloring agents.

[0131] In some embodiments, the composition further comprises a carrier, excipient, and / or diluent. Typically, the carrier is a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate buffered saline. Non-limiting examples of excipients include mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Non-limiting examples of diluents include sugars (e.g., monosaccharides, disaccharides, and oligosaccharides), calcium sulfate dihydrate, dextrin, starch, and glycine.

[0132] In some embodiments, the composition comprises a preservative, non-limiting examples of which include sucrose, sodium ascorbate, glutathione, and cryoprotectants (e.g., disaccharides, polyols, or polysaccharides).

[0133] In some embodiments, the composition comprises a gastroresistant coating. In some embodiments, the composition is in the form of a capsule or tablet comprising a gastroresistant coating. Preferably, a gastroresistant coating can be used to aid in targeting the composition of the present invention, for example, to the intestine. Preferably, this helps increase the abundance of viable bacterial strains in the intestine.

[0134] Gastric-resistant coatings are typically selected from fatty acids, waxes, shellac, plastics, and vegetable fibers, such as hydroxypropylmethylcellulose phthalate, methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, cellulose acetate trimellitate, sodium alginate, hydroxypropylmethylcellulose acetate succinate, polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (CAP), zein, methyl methacrylate-methacrylic acid copolymer, and aqueous enteric coating solutions (e.g., ethyl cellulose, medium chain triglycerides, oleic acid, sodium alginate, stearic acid).

[0135] In some embodiments, the composition is a sustained-release formulation. In some embodiments, the composition is in the form of a sustained-release capsule or tablet. Sustained-release formulations include sustained-release formulations (when extended release is intended), pulsed-release formulations, and delayed-release formulations (e.g., to target different regions of the digestive tract, such as the intestine). In some embodiments, the sustained-release formulations of the present invention are formulated to allow the composition to be gradually released in the digestive tract (e.g., distributing the bacterial strain throughout the intestine) or released in a delayed manner (e.g., delaying release of the bacterial strain until it reaches the intestine). Sustained-release formulations are known in the art and typically include, for example, polymer-based components or coatings. Typically, sustained-release formulations can be formulated to release within 1 to 10 hours, optionally within 2 to 8 hours, or 3 to 6 hours after ingestion. [Example]

[0136] The present invention will be further clarified by the following examples, which are intended to be purely illustrative of the present invention and not limiting in any way.

[0137] Example 1 A number of bacterial strains prevalent in the human microbiome were investigated for their ability to biotransform and / or bioaccumulate xenobiotics.

[0138] Experimental setup For each xenobiotic, a set concentration of 20 μM was screened. Each xenobiotic-strain interaction was screened in three biological replicates with two technical replicates each. Screens were performed under anaerobic conditions in 96-well plates in a 500 μl volume. Plates containing 250 μl modified Gifu anaerobic medium (mGAM) at a 40 μM xenobiotic concentration were prepared the night before and placed in an anaerobic chamber overnight to ensure anaerobic conditions for inoculation. On the day of the screen, each well was inoculated with 250 μl of the second subculture to obtain a starting OD of 0.05. 600 For compound control wells, sterile mGAM was added to each well.

[0139] The plates were incubated at 37°C for 24 hours and then removed from the anaerobic chamber for further processing. 100 μl of the entire culture was transferred to a fresh 96-well plate and stored at -80°C until extraction. The remainder of the culture was centrifuged at 4000 rpm at 25°C for 15 minutes. 100 μl of the supernatant was transferred to a fresh 96-well plate and stored at -80°C until extraction.

[0140] 50 μl samples were extracted with 200 μl ice-cold MeOH:ACN (1:1) containing 60 μM internal standard and incubated at 4° C. for 30 min. The plate was then centrifuged at 4000 rpm for 10 min at 4° C. The supernatant was transferred to a 96-well plate for LC-MS analysis. Samples for concentration calibration and sterile compound controls were processed in the same manner.

[0141] Data Analysis: The concentration of the target compound in each sample was estimated using a standard curve. Statistical analysis was performed in RStudio version 1.3.1093 using a t-test. The false discovery rate (FDR) method was used to correct p-values ​​for the number of compounds tested. Additionally, the median value for each strain was compared to the median value of the compound control. The cutoff values ​​for significant reduction were set at p-corrected <0.05 and a median reduction >20%. Bioaccumulation was defined as a significant reduction in the supernatant concentration but not in the total lysate sample, and biotransformation was defined as a significant reduction in the concentration in both the supernatant and lysate samples.

[0142] result: Levels of exemplary xenobiotics, PFNA, PFOA, bisphenol AF, boscalid, propiconazole, pyrimethanil, tributyl-PO4, and triphenyl-PO4, were found to be significantly reduced by at least one of the bacterial strains tested. Bisphenol AF (BPAF), PFOA, PFNA, and triphenyl-PO4 were primarily bioaccumulated, while boscalid, propiconazole, pyrimethanil, and tributyl-PO4 were primarily biotransformed (Figure 1). Representative data showing the bioaccumulation of PFNA and the biotransformation of propiconazole by Bacteroides dorei and Bacteroides uniformis are provided in Figure 2.

[0143] Consideration: The present inventors have discovered that various intestinal bacterial strains can reduce the concentrations of xenobiotics through bioaccumulation and / or biotransformation. Bisphenols (exemplified by bisphenol AF) were found to be accumulated by up to 60% by some bacterial strains. PFAs (exemplified by PFOA and PFNA) were also shown to be accumulated by up to 60% and 75%, respectively. While not wishing to be bound by theory, the present inventors believe that the results showing biotransformation of PFOA and PFNA may be the result of incomplete extraction from cells, and that strains showing biotransformation (i.e., reduced levels of PFOA and PFNA in the supernatant) are more likely to bioaccumulate these compounds. Pesticides (e.g., boscalid, propiconazole, and pyrimethanil) are typically biotransformed by bacterial strains. The results presented herein demonstrate that intestinal bacterial strains can be used to reduce the levels of a range of xenobiotics through bioaccumulation and biotransformation.

[0144] Example 2 To validate the experimental setup and extraction method, we used PFA and B. uniformis as a model system. PFOA and PFNA bioaccumulation assays were repeated in B. uniformis using glass vials. Ultrasonication enhanced the extraction and improved the extraction of xenobiotic compounds from the cells.

[0145] Experimental setup: PFOA and PFNA were tested at a concentration of 20 μM in B. uniformis in three technical replicates. Screens were performed in glass vials in 3 ml volumes under anaerobic conditions. Glass vials containing 1.5 ml mGAM at a 40 μM xenobiotic concentration were prepared the night before and placed in an anaerobic chamber overnight to ensure anaerobic conditions for inoculation. On the day of the screen, each well was inoculated with 1.5 ml of the second subculture to achieve a starting OD of 0.05. 600 For compound controls, sterile mGAM was added to each glass vial.

[0146] The vials were incubated at 37°C for 24 hours and then removed from the anaerobic chamber for further processing. 1 ml of the entire culture was transferred to a fresh glass vial. The remainder of the culture was centrifuged at 4000 rpm at 25°C for 15 minutes, and 1 ml of the supernatant was transferred to a fresh glass vial. The remainder of the supernatant was removed from the cell pellet, and the pellet was resuspended in 2 ml of water, of which 1 ml was transferred to a fresh glass vial. All samples were stored at -80°C until extraction.

[0147] For extraction, samples were thawed and sonicated for 3 minutes. Each 1 ml sample was then extracted with 4 ml ice-cold MeOH:ACN (1:1) containing 60 μM internal standard, sonicated again for 3 minutes, and incubated at 4°C for 10 minutes. The vials were then centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was transferred to LC-MS vials for analysis. Samples for concentration calibration and sterile compound controls were processed in the same manner.

[0148] Data Analysis: The median value for each sample was compared to the median value for the compound control.

[0149] result: PFOA was reduced to 32% by B. uniformis, with 20% recovered from the cell pellet, whereas PFNA was reduced to 42% by B. uniformis, with 48% recovered from the cell pellet, confirming the accumulation of PFOA and PFNA by B. uniformis (Figure 3).

[0150] Example 3 B. uniformis response in accumulating PFA at various doses, accumulation of PFA at various chain lengths, and PFNA accumulation over time To determine the ability of B. uniformis to accumulate PFNA, concentrations ranging from 0.78 to 500 μM were tested in a resting cell assay. Furthermore, the accumulation ability of B. uniformis was tested for two PFAs of different chain lengths. Finally, PFNA accumulation was measured over a 7-day period to determine how rapidly PFNA accumulates and whether it is released again over time.

[0151] Experimental setup: PFNA was tested in B. uniformis at concentrations ranging from 0.78 to 500 μM in three technical replicates. Additionally, two different PFAs (PFNA and PFDeA) of varying chain lengths were tested in three technical replicates at a set concentration of 20 μM. The screen was performed under anaerobic conditions in a 96-well plate in a 400 μl volume. Plates containing 200 μl PBS at 2x the xenobiotic concentration were prepared the night before and placed in an anaerobic chamber overnight to ensure anaerobic conditions for inoculation. On the day of the assay, the bacterial culture was centrifuged, the supernatant removed, and the cell pellet resuspended in PBS to an OD of 7.5. 600 Each well was inoculated with 200 μl of culture in PBS to a starting OD of 3.75. 600 For compound control wells, sterile PBS was added to each well.

[0152] The plates were incubated at 37°C for 4 hours and then removed from the anaerobic chamber for further processing. 50 μl of the entire culture was transferred to a fresh 96-well plate and stored at -80°C until extraction. The remainder of the culture was centrifuged at 4000 rpm for 10 minutes at 4°C. 50 μl of the supernatant was transferred to a fresh 96-well plate and also stored at -80°C until extraction.

[0153] 50 μl of sample was extracted with 200 μl ice-cold MeOH:ACN (1:1) containing 60 μM internal standard and incubated for 10 min at 4° C. The plate was then centrifuged at 4000 rpm for 15 min at 4° C. The supernatant was transferred to a 96-well plate for LC-MS analysis. Samples for concentration calibration and sterile compound controls were processed in the same manner.

[0154] Data Analysis: The standard curve was used to estimate the concentration of the target compound in each sample. Statistical analysis was performed in RStudio version 1.3.1093 using a t-test. The FDR method was used to correct p-values ​​for the number of concentrations tested. Furthermore, the median value of each sample was compared to the median value of the compound control. The cutoff value for significant reduction was set at p-correction < 0.05 and median reduction > 20%.

[0155] result: PFNA accumulated significantly at all concentrations tested, at a constant rate of 25–40% (Figure 4).

[0156] We found that B. uniformis accumulated PFNA up to 25% and PFDeA up to 60% (Figure 5). These results suggest that the accumulation ability of B. uniformis increases with increasing PFA lipophilicity.

[0157] Over the 7-day time course, the percentage of PFNA that accumulated remained constant. Even at time 0, 30% of PFNA had accumulated. This suggests that within the time it took to collect samples after adding PFNA, B. uniformis had already accumulated a significant amount of the compound, and this did not change over the course of another 7 days (Figure 6), indicating that once bioaccumulated, the xenobiotic is not released back into the environment.

[0158] LCMS methods used in the examples A total of two quadrupole time-of-flight (QTOF) methods were set up to detect xenobiotics.

[0159] QTOF method in positive scanning mode An Agilent method (Aimei Zou, SP, et al., Agilent Technologies, Inc. Comprehensive LC / MS / MS Workflow of Pesticide Residues in Food Using the Agilent 6470 Triple Quadrupole LC / MS System, (2020)) was adapted to enable the analysis of a wide range of xenobiotics. Briefly, LC-MS analysis was performed on an Agilent 1290 Infinity II LC system coupled to an Agilent 6546 LC / Q-TOF (Agilent). Separation was performed using a ZORBAX RRHD Eclipse Plus column (C18, 2.1 x 100 mm, 1.8 μm; Agilent) with a ZORBAX Eclipse Plus (C18, 2.1 x 5 mm, 1.8 μm; Agilent) guard column at 40 °C. The multisampler was maintained at a temperature of 5 °C. The injection volume was 1 μL, and the flow rate was 0.4 mL / min. The mobile phase consisted of A: water + 0.1% formic acid + 5 mM ammonium formate; B: methanol + 0.1% formic acid + 5 mM ammonium formate. A 10-min gradient started with 5% solvent B, increased to 30% in 1 min, then increased to 100% over 7 min and held for an additional 3 min, followed by equilibration to starting conditions (5% mobile phase B) for 5 min. QTOF MS scans were operated in positive scanning mode (m / z 30–1500). Source parameters were as follows: gas temperature: 200 °C, drying gas: 9 L / min, nebulizer: 20 psi, sheath gas temperature: 400 °C, sheath gas flow: 12 L / min, VCap: 3000 V, nozzle voltage: 0 V, fragmentor: 110 V, skimmer: 45 V, and Octa RF Vpp: 750 V. Online mass calibration was performed using reference solutions (121.05 and 922.01 m / z). Compounds were identified based on their retention time, accurate mass and fragmentation pattern.

[0160] QTOF in negative scanning mode A QTOF method was designed in negative scanning mode to detect compounds that were undetectable in positive scanning mode (Jurek, A. & Leitner, E. Food Additives & Contaminants: Part A 35, 2256-2269, (2018)). Briefly, LC-MS analysis was performed on an Agilent 1290 Infinity II LC system coupled to an Agilent 6546 LC / Q-TOF (Agilent). Separation was performed using a ZORBAX RRHD Eclipse Plus column (C18, 2.1 x 100 mm, 1.8 μm; Agilent) with a ZORBAX Eclipse Plus (C18, 2.1 x 5 mm, 1.8 μm; Agilent) guard column at 40 °C. The multisampler was maintained at a temperature of 5 °C. The injection volume was 1 μL, and the flow rate was 0.4 mL / min. The mobile phase consisted of A: water + 5 mM ammonium acetate + 0.03% acetic acid; B: methanol + 5 mM ammonium acetate + 0.03% acetic acid. A 10-min gradient started at 35% solvent B and increased to 100% in 9 min, followed by a 5-min equilibration to the starting conditions (35% mobile phase B). QTOF MS scans were operated in negative scanning mode (m / z 30–1500). Source parameters were as follows: gas temperature: 200 °C, drying gas: 9 L / min, nebulizer: 20 psi, sheath gas temperature: 400 °C, sheath gas flow: 12 L / min, VCap: 3000 V, nozzle voltage: 0 V, fragmentor: 110 V, skimmer: 45 V, and Octa RF Vpp: 750 V. Online mass calibration was performed using reference solutions (112.99 and 1033.99 m / z). Compounds were identified based on their retention time, accurate mass, and fragmentation pattern. Some compounds that were undetectable with these mobile phases were run in the same manner but with mobile phases A:water; B:methanol.

[0161] Data Analysis: Selected xenobiotic standards were qualified using Agilent MassHunter Qualitative Analysis 10.0 software. TIC, EIC, and EIC fragment graphs were extracted for each compound. Xenobiotic compounds in each sample were quantified using Agilent MassHunter TQF Quantitative Analysis (Quant-My-Way) software.

[0162] Example 4 We performed a community-based screening approach, evaluating the ability of a mixture of gut bacterial strains to sequester contaminant compounds during a 4-hour exposure (Figure 11a). Eighteen contaminants were found to be sequestered by more than 25% by one or both synthetic communities (Figure 11c). Ten of these compounds were then tested for sequestration by individual strains over a 24-hour incubation period (Figure 11b); seven contaminants were found to be sequestered by at least one of the bacterial strains (Figure 7a). By comparing whole culture and supernatant concentrations to compound controls, we were able to distinguish between observed sequestration from bioaccumulation and biodegradation. In the context of this example, bioaccumulation is defined as compound sequestration that is at least 20% from the supernatant but is completely recovered in the whole culture sample, while biodegradation is defined as both the supernatant and whole culture samples showing greater than 20% sequestration (Figure 7b, c). These interactions provide a starting point for a mechanistic understanding of the role of the microbiota in contaminant xenobiotics.

[0163] The extent of bioaccumulation over 24 h of 20 μM PFNA exposure varied from 25% (P. merdae) to 74% (O. splanchnicus), and for PFOA, from 23% (P. merdae) to 58% (O. splanchnicus). Growth of B. uniformis and PFNA concentration over an 11-h period showed that growth correlated with sequestration from the medium (Figure 7e). When 20 μM PFNA was spiked into already grown B. uniformis, 50% of the PFNA bioaccumulated within minutes (Figure 7f). This was surprising, as previous reports from environmental bacteria, Pseudomonas species, isolated from a PFAS-contaminated site, showed much lower efficiency, with ~40% bioaccumulation of perfluorohexane sulfate (PFHxS) over a 5-day period followed by solvent preconditioning to promote sequestration. B. uniformis accumulated 60–70% of PFNA at submicromolar concentrations and approximately 55% at concentrations ranging from 1 to 100 μM PFNA (Figure 7g, Figure 13). Furthermore, B. uniformis and other abundant gut bacterial strains showed consistent growth up to high micromolar concentrations of PFAS (Figure 7h). In experiments with 250 μM PFNA exposure, B. uniformis accumulated approximately 40% of the chemical, corresponding to an apparent intracellular concentration of approximately 18 mM, which was much higher than that of most natural metabolites.

[0164] Previous studies have shown that accumulation in lipid bilayers may be the primary mechanism of PFAS bioaccumulation; therefore, we considered bioaccumulation specificity. While all PFAS-bioaccumulating bacteria are Gram-negative species, not all tested Gram-negative species, including Escherichia coli, accumulated PFAS to the same extent (Figure 12a, b). This specificity is inconsistent with membrane interactions being the primary mechanism underlying PFAS bioaccumulation. To examine underlying differences between species, we tested whether inactive cell mass (i.e., dead, lysed cells) could bioaccumulate PFAS. In resting cell assays in PBS buffer (OD = 3.75), B. uniformis and O. splanchnicus bioaccumulated PFOA, PFNA, and PFDeA by ~20%, ~55%, and ~85%, respectively, with dead and lysed cultures accumulating similar amounts. On the other hand, viable E. coli bioaccumulated much lower levels of PFOA, PFNA, and PFDeA (~5%, ~25%, and ~40%), whereas dead and dissolved E. coli bioaccumulated similar amounts to B. uniformis and O. splanchnicus (Figures 8a and 14). Therefore, transport across membranes appears to distinguish E. coli from other bioaccumulating Gram-negative species. Furthermore, the observed degree of bioaccumulation does not appear to be membrane-only, as this would be physiologically impossible, since the bioaccumulating bacteria exhibit robust growth and the ability to accumulate over a significant range of PFNA concentrations, even though they exhibit up to one PFNA molecule per two lipid molecules in the cytoplasmic membrane. This data suggests that PFAS bioaccumulation is intracellular and not a passive phenomenon driven by attachment to membrane lipid bilayers.

[0165] We next examined how cells cope with high mM intracellular levels of highly effective surfactants like PFAS and maintain their proliferation. To gain insight into the morphology of bioaccumulating cells, we used transmission electron microscopy (TEM) to compare control, DMSO-treated, and cells bioaccumulating PFNA or PFDeA. PFAS-treated cells were characterized by significant changes in nucleoid appearance in TEM (Figures 9e–n). This suggests interactions between PFNA and PFDeA and intracellular proteins and other macromolecules. Therefore, the incorporation of xenobiotics in / around these granular structures appears to be an effective mechanism by which cells maintain their survival and proliferation.

[0166] The intracellular storage of PFAS supports the existence of a transport mechanism capable of importing and / or effluxing these organofluorine surfactants without affecting the integrity of the cell membrane. To test this, we measured the bioaccumulation of E. coli gene knockout mutants lacking one or more genes encoding efflux pump proteins (Figure 8b). Efflux pumps are a common mechanism used by several bacterial species to reduce the intracellular concentration of toxic compounds. We reason that because E. coli can pump PFASs, at least to some extent, it does not bioaccumulate PFNA to the same extent as other Gram-negative bacteria tested. Supporting this hypothesis, two efflux pump mutants showed appreciable bioaccumulation of up to 30–40% PFNA and 60–70% PFDeA from the medium, whereas the wild-type strain accumulated only 10–20% PFNA and 30–40% PFDeA (Figure 8c). Furthermore, these mutants showed increased sensitivity to high concentrations of PFNA, but not PFDeA (Figure 8d). These results further confirm that PFAS bioaccumulation is not a passive but a membrane-interaction-driven process, and that bacterial cells may regulate bioaccumulation through transport mechanisms.

[0167] We next used another strategy to confirm the biological nature of PFAS bioaccumulation: adaptive laboratory evolution (Figure 9a). We reasoned that if cells possess mechanisms to regulate bioaccumulation and / or cope with high intracellular levels, tolerance to high PFAS exposure would rapidly evolve under natural selection. Furthermore, we sought to determine whether evolution at high PFAS concentrations would lead to strains with altered bioaccumulation potential. Therefore, we evolved B. uniformis, B. thetaiomicron, P. merdae, C. difficile, and E. coli BWΔTolC in media containing 500 μM PFHpA, 500 μM PFOA, 250 μM PFNA, or 125 μM PFDeA through serial transfer. Within 20 transfers (20 days), corresponding to approximately 100 generations, growth of B. uniformis improved from 2% to 76% for PFDeA and from 7% to 48% for PFNA compared to the untreated control population (Figure 9b). This rapid adaptation confirmed the evolvability of bacteria-PFAS interactions. No significant adaptation in growth was observed for the other strains and compounds tested (Figure 15). No differences were observed when comparing the bioaccumulation potential of the parental versus evolved populations for PFNA and PFDeA (Figures 9c, d; Figures 16a, b). Because the results described herein suggest that bioaccumulation potential is retained while bacterial growth can change, this is promising for the use of bacterial strains for PFAS removal under high and / or long-term exposure conditions.

[0168] To determine the in vivo relevance of gut bacterial accumulation of PFAS, germ-free C57BL / 6 mice or C57BL / 6 mice colonized with a community of 20 human gut bacterial strains (Com20) were administered a single oral dose of PFNA (10 mg / kg body weight) via gavage. Fecal samples were collected over the following two days, and on day 3, colon and small intestinal content samples were collected after euthanasia (Figure 10a). Colonized mice exhibited substantially higher excretion of PFNA at all follow-up time points (3 h, p = 0.009, fold change = 9; 1 day, p = 0.001, fold change = 2.9; 2 days, p < 0.001, fold change = 2.9; 3 day colon, p < 0.001, fold change = 3.4; 3 day small intestine, p = 0.007, fold change = 2) (Figure 10b). Increased clearance through fecal material in colonized mice indicates that PFNA accumulation by gut bacteria also occurs in vivo. Combined with in vitro results showing variable degrees of PFNA accumulation by different gut bacterial species, our mouse data indicate that gut microbiota composition is a critical factor determining PFAS toxicokinetics.

[0169] In summary, we have discovered bacterial species with a remarkably high capacity for intracellular accumulation of PFAS contaminants by gut bacteria. The specificity and genetic traceability of PFAS bioaccumulation offers the possibility of using commensal bacterial strains to remove PFAS from, for example, the human body.

[0170] Using 16S sequencing, mouse fecal samples were analyzed for their microbiota. Seventeen of 20 human gut bacterial strains colonized the intestines of Com20-inoculated germ-free mice. No differences in composition were observed between control (DMSO) and PFNA (10 mg / kg body weight)-treated Com20-colonized mice (Table 1). In both DMSO- and PFNA-treated groups, the proportion of bacteria classified as high PFNA-accumulating was approximately 70% (Table 2). [Table 1] TIFF2025535288000002.tif198164 [Table 2]

[0171] method Bacterial strains and cultures Strains were selected to represent abundant members prevalent in the healthy human gut microbiota. E. coli mutants were obtained from the Typas laboratory (EMBL Heidelberg, BW25113 wild-type, BW25113 ΔtolC) and the Luisi laboratory (University of Cambridge, C43 (DE3) wild-type, C43 (DE3) ΔacrAB-tolC). All bacterial experiments were performed in an anaerobic chamber (Coy Laboratory Products) filled with 2% hydrogen and 12% carbon dioxide in nitrogen. The chamber was equipped with a palladium catalyst system for oxygen removal, a dehumidifier, and a hydrogen sulfide removal system. Bacteria were grown at 37°C in modified Gifu Anaerobic Medium (mGAM, HyServe, Germany, manufactured by Nissui Pharmaceutical), prepared according to the manufacturer's instructions and sterilized by autoclaving. Bacteria for starting cultures were inoculated directly from frozen glycerol stocks and grown in 10 ml of medium in 15 ml plastic tubes for one or two days (depending on growth rate). The cultures were then diluted 100-fold and again incubated for the same period before the experiment began. Unless otherwise specified, screening plates / tubes containing medium were prepared the day before at 2x compound concentration (2% DMSO) and placed in a chamber overnight to ensure anaerobic conditions for inoculation. Inoculation was performed 1:1 with bacterial culture, and plates were sealed with AlumaSeal II film (A2350-100EA) to avoid evaporation during incubation.

[0172] Community-based screening approach (Fig. 11a, c) On the day of the screen, communities were combined by pooling the second passages of individual strains according to OD600 values. The combined communities were then centrifuged at 4000 rpm at 25°C for 15 minutes, and the pellets were resuspended in PBS buffer to generate communities with an OD600 of 7.5. Each well was inoculated 1:1 with the community in PBS to a starting OD600 of 3.75. For compound control wells, sterile PBS was added to each well. Plates were incubated at 37°C for 4 hours, after which they were centrifuged at 4000 rpm at 21°C for 15 minutes. The supernatants and compound controls were transferred to fresh 96-well plates and stored at -80°C until extraction.

[0173] Single-strain xenobiotic screen (Figure 7a, c, b) Ten compounds identified through the "community-based screening approach" were tested for sequestration by individual strains. On the day of the screen, each well was inoculated 1:1 with a second subculture to a starting OD600 of 0.05. For compound control wells, sterile mGAM was added to each well. Plates were incubated at 37°C for 24 hours, after which they were removed from the anaerobic chamber for sample collection. All cultures, supernatants, and compound control samples were collected and stored at -80°C until extraction.

[0174] PFAS bioaccumulation analysis Resting cell assay (Figures 7d, 2c; Figures 12b, 3) On the day of the screen, each well was inoculated 1:1 with culture in PBS to a starting OD600 of 3.75. For compound control wells, sterile PBS was added to each well. Samples were incubated at 37°C for 4 hours, after which they were removed from the anaerobic chamber for sample collection. All cultures, supernatants, and compound control samples were collected and stored at -80°C until extraction.

[0175] Proliferation assay (Figure 7g; Figure 12a) On the day of the screen, each well was inoculated 1:1 with the second subculture to a starting OD600 of 0.05. For compound control wells, sterile mGAM was added to each well. Samples were incubated at 37°C for 24 hours, after which they were removed from the anaerobic chamber for sample collection. All cultures, supernatants, and compound control samples were collected and stored at -80°C until extraction.

[0176] PFAS time course experiment PFAS time course experiment in growing B. uniformis cultures (Figure 7e). On the day of the screen, each tube was inoculated 1:1 with the second subculture to a starting OD600 of 0.05. Samples were incubated at 37°C for 11 hours. OD600 was measured every hour and supernatant samples were also collected every hour for PFNA analysis and stored at -80°C until extraction.

[0177] PFAS time course experiment in stationary-phase B. uniformis cultures (Figure 7f) A 1.5 ml stationary second-subculture of B. uniformis or pure mGAM was spiked with 15 μl of 2 mM PFNA in DMSO. Whole culture, supernatant, and compound control samples were collected at 0, 15, 30, and 60 min and stored at -80°C until extraction.

[0178] PFAS accumulation in viable, heat-inactivated, and lysed bacterial cultures (Figure 8a, Figure 14) On the day of the screen, each well was inoculated 1:1 with viable, heat-inactivated, or lysed culture in PBS to a starting OD600 of 3.75. The second subculture was spun down, and the pellet was resuspended in PBS to an OD600 of 7.5. Each culture was divided into three aliquots: viable, heat-inactivated, and lysed culture. The viable culture was used directly. Bacteria were heat-inactivated at 70°C for 40 minutes, and the lysed culture was further freeze-thawed three times and sonicated for 3 minutes. After adding the respective culture or sterile PBS to each well, the plate was sealed and incubated at 37°C. After 4 hours, all cultures, supernatants, and compound control samples were collected and stored at -80°C until extraction.

[0179] Sample extraction for LC / MS / MS Bacterial samples For the "community-based screening approach," 70 μl of supernatant was extracted with 140 μl of ice-cold methanol:acetonitrile (1:1) containing internal standards (caffeine, ibuprofen) and incubated at 4°C for 30 minutes. For the "single-strain xenobiotic screen" and all other PFAS bioaccumulation screens, 50 μl of sample was extracted with 200 μl of ice-cold methanol:acetonitrile (1:1) containing internal standards (caffeine, ibuprofen) and incubated at 4°C for 15 minutes. The plate was then centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was transferred to a 96-well plate for LC-MS analysis. Samples for concentration calibration and sterile compound controls were processed in the same manner.

[0180] Mouse fecal sample Frozen fecal samples were weighed into beaded tubes, and 250 μl of extraction buffer (methanol + 0.05% KOH + 15 μM caffeine) was added. The tubes were then homogenized at 1500 rpm for 10 minutes, followed by centrifugation at 14,000 rpm for 5 minutes at 4°C. 20 μl of supernatant was added to 80 μl of water + 0.1% formic acid, vortexed, incubated at 4°C for 15 minutes, and centrifuged at 14,000 rpm for 5 minutes at 4°C. The supernatant was transferred to an LCMS vial containing an insert. Samples for concentration calibration were processed in the same manner.

[0181] LC-MS / MS (QTOF) xenobiotic measurement QTOF Parameters Briefly, LC-MS analysis was performed on an Agilent 1290 Infinity II LC system coupled with an Agilent 6546 LC / Q-TOF (Agilent). Depending on the xenobiotic being measured, the QTOF MS scans were operated in positive or negative scanning mode (m / z 30–1500). Source parameters were as follows: gas temperature: 200 °C, drying gas: 9 L / min, nebulizer: 20 psi, sheath gas temperature: 400 °C, sheath gas flow: 12 L / min, VCap: 3000 V, nozzle voltage: 0 V, fragmentor voltage: 110 V, skimmer voltage: 45 V, and Octa-RF voltage: 750 V. Online mass calibration was performed using reference solutions (positive: 121.05 and 922.01 m / z; negative: 112.99 and 1033.99 m / z). The collision energies used were 0, 10, 20, and 40 V. Compounds were identified based on their retention time, accurate mass, and fragmentation pattern. For all measured compounds, pure standards were obtained from Sigma Aldrich (Merck KGaA, Darmstadt, Germany) and used for method development, compound identification, and calibration.

[0182] Five different LC methods were applied: A 15-minute reversed-phase LC method used with a QTOF in positive ionization mode (Figure 11c). Separation was performed using a ZORBAX RRHD Eclipse Plus column (C18, 2.1 x 100 mm, 1.8 µm; Agilent) with a ZORBAX Eclipse Plus (C18, 2.1 x 5 mm, 1.8 µm; Agilent) guard column at 40 °C. The multisampler was maintained at a temperature of 4 °C. The injection volume was 1 µL, and the flow rate was 0.4 mL / min. The mobile phase consisted of A: water + 0.1% formic acid + 5 mM ammonium formate; B: methanol + 0.1% formic acid + 5 mM ammonium formate. A 15-min gradient started at 5% solvent B, increased to 30% in 1 min, then increased to 100% in 7 min, held for an additional 3 min, and then returned to 5% solvent B for 5 min for re-equilibration.

[0183] A 10-min reversed-phase dual-pump LC method used with a QTOF in positive ionization mode (Figure 7a, c). Separation was performed using two ZORBAX RRHD Eclipse Plus columns (C18, 2.1 x 5 mm, 1.8 µm; Agilent) with ZORBAX Eclipse Plus (C18, 2.1 x 100 mm, 1.8 µm; Agilent) guard columns at 40 °C. The multisampler was maintained at a temperature of 4 °C. The injection volume was 1 µL, and the flow rate was 0.4 mL / min. The mobile phase consisted of A: water + 0.1% formic acid + 5 mM ammonium formate; B: methanol + 0.1% formic acid + 5 mM ammonium formate. A 10-min gradient started at 5% solvent B, increased to 30% in 1 min, then increased to 100% in 7 min, held for 1.7 min, and then returned to 5% solvent B in 8.8 min and held for 10 min. The re-equilibration gradient started at 5% solvent B, then ramped to 100% solvent B in 0.1 min and held for 4 min before returning to the starting conditions of 5% solvent in 4.1 min.

[0184] A 13-minute reversed-phase LC method used with a QTOF in negative ionization mode ("community-based screening approach" (Figure 11c)) Separation was performed using a ZORBAX RRHD Eclipse Plus column (C18, 2.1 x 100 mm, 1.8 µm; Agilent) with a ZORBAX Eclipse Plus (C18, 2.1 x 5 mm, 1.8 µm; Agilent) guard column at 40 °C. The multisampler was maintained at a temperature of 4 °C. The injection volume was 1 µL, and the flow rate was 0.4 mL / min. The mobile phase consisted of A: water; B: methanol. A 13-minute gradient started at 35% solvent B, increased to 100% in 9 minutes, held for 1 minute, and then returned to 35% solvent B for 3 minutes for re-equilibration.

[0185] A 10-minute reversed-phase dual-pump LC method used with a QTOF in negative ionization mode (Figures 7a, b, d; Figure 8a, c; Figure 12; Figure 13; Figure 14; Figure 16). Separation was performed using two ZORBAX RRHD Eclipse Plus columns (C18, 2.1 x 100 mm, 1.8 µm; Agilent) with ZORBAX Eclipse Plus (C18, 2.1 x 5 mm, 1.8 µm; Agilent) guard columns at 40 °C. The multisampler was maintained at a temperature of 4 °C. The injection volume was 1 µL, and the flow rate was 0.4 mL / min. The mobile phase consisted of A: water + 5 mM ammonium acetate + 0.03% acetic acid; B: methanol + 5 mM ammonium acetate + 0.03% acetic acid. A 10-min gradient started at 35% solvent B, increased to 100% in 7 min, held for 1.7 min, then returned to 35% solvent B in 8.8 min and maintained until 10 min. The re-equilibration gradient started at 35% solvent B, then ramped to 95% solvent B in 0.1 min and held for 4 min before returning to the starting conditions of 35% solvent B in 4.1 min.

[0186] A 2-minute reversed-phase LC method used with a QTOF in negative ionization mode (Figure 9c, d). Separation was performed using a ZORBAX RRHD Eclipse Plus column (C18, 3.0 x 50 mm, 1.8 μm; Agilent) with a ZORBAX Eclipse Plus (C18, 2.1 x 5 mm, 1.8 μm; Agilent) guard column at 40 °C. The multisampler was maintained at a temperature of 4 °C. The injection volume was 1 μL, and the flow rate was 0.8 mL / min. The mobile phase consisted of A: water + 5 mM ammonium acetate + 0.03% acetic acid; B: methanol + 5 mM ammonium acetate + 0.03% acetic acid. A 2-minute gradient started at 30% solvent B, increased to 100% in 0.5 min, held for 1 min, then returned to 30% solvent B in 1.1 min and held for 2 min.

[0187] LC-MS / MS (QQQ) PFNA measurement QQQ parameters Briefly, LC-MS / MS analysis was performed on an Agilent 1290 Infinity II LC system coupled to an Agilent 6570 LC / TQ (Agilent). The QQQ was operated in Dynamic MRM mode. Source parameters were as follows: gas temperature: 300 °C, gas flow: 910 L / min, nebulizer: 50 psi, sheath gas temperature: 300 °C, sheath gas flow: 11 L / min, VCap: 3500 V (positive mode) or 3000 V (negative mode), nozzle voltage: 2000 V (positive mode) or 500 V (negative mode). For PFNA detection, the scan segments were as follows: precursor ion: 463; product ions: 418.9 and 294.1; fragmentor voltages: 64 and 80 V; collision energy: 8 V. Pure standards were obtained from Sigma Aldrich (Merck KGaA, Darmstadt, Germany) and used for method development, compound identification and calibration.

[0188] 2-min reversed-phase LC method used in QQQ (Figure 7e, f, g) Separation was performed using a ZORBAX RRHD Eclipse Plus column (C18, 3x50mm, 1.8µm; Agilent) with a ZORBAX Eclipse Plus (C18, 2.1x5mm, 1.8µm; Agilent) guard column at 40°C. The multisampler was maintained at a temperature of 4°C. The injection volume was 1µL, and the flow rate was 0.8mL / min. The mobile phase consisted of A: water + 0.1% formic acid; B: methanol + 0.1% formic acid. A 2-min gradient started at 30% solvent B, increased to 100% in 0.5 min, held for 1 min, then returned to 30% solvent B at 1.05 min and held for 2 min.

[0189] A 10-minute reversed-phase LC method used with QQQ for mouse fecal samples (Figure 10). Separation was performed using a ZORBAX RRHD Eclipse Plus column (C18, 2.1 x 100 mm, 1.8 µm; Agilent) with a ZORBAX Eclipse Plus (C18, 2.1 x 5 mm, 1.8 µm; Agilent) guard column at 40 °C. The multisampler was maintained at a temperature of 4 °C. The injection volume was 2 µL, and the flow rate was 0.4 mL / min. The mobile phase consisted of A: water + 0.1% formic acid; B: methanol + 0.1% formic acid. A 10-min gradient started at 5% solvent B, increased to 90% in 5 min, further increased to 100% in 7 min, then returned to 5% solvent B in 7.1 min and held until 10 min.

[0190] LC-MS / MS data analysis Selected xenobiotic standards were qualified using Agilent MassHunter Qualitative Analysis 10.0 software. TIC, EIC, and EIC fragment graphs were extracted for each compound. Xenobiotic compounds in each sample were quantified using Agilent MassHunter TQF Quantitative Analysis (version 10.1) or Agilent MassHunter QQQ Quantitative Analysis software (version 10.1).

[0191] Concentrations of target compounds were estimated using calibration curves based on pure compound standards. Data analysis was performed in RStudio version 1.3.1093. The median value of each sample group was compared to the median value of the compound control, and an appropriate reduction was selected as a cutoff to ensure adequate reduction relative to the compound control distribution (median reduction >25% for "Community-Based Screening Approach"; >20% for "Single-Strain Xenobiotic Screen"; all other PFAS bioaccumulation screens >20%). Further statistical comparisons were performed using t-tests (two-tailed), with p-values ​​FDR-corrected for the number of compounds tested ("Single-Strain Xenobiotic Screen"). (When t-tests were performed, corrected p-values ​​are provided in the respective figures.) A corrected p-value of <0.05 was considered significant.

[0192] "Single-strain xenobiotic screen" bioaccumulation was defined as compound sequestration of at least 20% from the supernatant but not from the whole culture sample and corrected p<0.05, while biodegradation was defined as both supernatant and whole culture samples showing greater than 20% compound sequestration and corrected p<0.05.

[0193] PFAS bacterial growth screen (Figures 7h, 8d) Plates (Corning 3795) containing 50 μl mGAM containing 2x PFAS (2% DMSO) concentrations were prepared the night before and placed in an anaerobic chamber overnight to ensure anaerobic conditions for inoculation. On the day of the screen, each well was inoculated with 50 μl of the second subculture to achieve a starting OD of 0.05. The plates were sealed with a gas-permeable membrane (Breath-Easy, Merck, catalog number Z380059), which was further punctured with a syringe to prevent gas accumulation. The plates were stacked without lids and incubated at 37°C for 24 hours in a stacker-incubator system (Biostack 4, Agilent BioTek) connected to a plate reader (Epoch 2, Agilent BioTek), with OD recorded every hour.

[0194] Growth curve analysis was performed in RStudio version 1.3.1093. First, the lowest OD value for each growth curve was set to 0. Then, the raw AUC was calculated for each well using the "bayestestR" package and the area_under_curve() function. Further processing of the growth curves was performed by plate. AUC values ​​were normalized by the median AUC of all control wells (DMSO controls) on each plate to determine percent growth inhibition.

[0195] Conventional ultrathin section transmission electron microscopy (Figure 8e-n) On the day of the experiment, each tube containing the 2x concentration of PFAS was inoculated 1:1 with a second subculture to a starting OD600 of 0.05. After incubating the samples at 37°C for 24 hours, the bacterial culture was spun down and the supernatant removed. The bacteria were then fixed with half-Karnovsky's fixative as 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4 with NaOH) for several hours at room temperature. For conventional transmission electron microscopy, post-fixation was performed with a mixture of 1% osmium tetroxide and 1% potassium ferrocyanide in cacodylate buffer. After en bloc staining with 5% aqueous uranyl acetate, dehydration through an ethanol series and resin infiltration were completed for plastic embedding in TER (TAAB Epoxy Resin). After polymerization at 65°C for several days, ultrathin sections (~60 nm) were cut using an ultramicrotome (Leica EM UCT / UC7 / Artos-3D, Vienna, Austria), mounted on formvar-carbon film on EM copper grids, and stained with lead citrate. Bacterial ultrastructure was observed using an FEI Talos F200C 200 kV transmission electron microscope (Thermo Fischer Scientific, Einthoven, Netherlands) equipped with a Ceta-16M CMOS-based camera (4k x 4k pixels, 16-bit dynamic range) and a JEM-1400 Flash TMP (JEOL Ltd., Tokyo, Japan) and a TVIPS TemCam-XF416 CMOS (Tietz Video and Image Processing Systems GmbH, Germany), as described in Amelio, I. et al. P Natl Acad Sci USA 117, 15694-15701, (2020).

[0196] Adaptive laboratory evolution (Figure 9; Figure 15) B. uniformis, B. thetaiomicron, O. splanchnicus, P. merdae, and E. coli BW25113 ΔtolC were evolved for 20 days in 500 μM PFHpA, 500 μM PFOA, 250 μM PFNA, and 125 μM PFDeA. DMSO was used as a control. Four replicate lineages for each PFAS compound and eight for the DMSO control were evolved in parallel. 2 ml deep-well stock plates containing 100x stocks of each PFAS in DMSO were prepared prior to the experiment and stored at -80°C until use. On day 0, each well was inoculated with a second subculture to a starting OD600 of 0.05. The following day, 50 μl of the grown culture was transferred to a fresh compound plate containing PFAS / DMSO in mGAM. Every 5 days, growth of strains in the presence of PFAS was measured by transferring 100 μl of starting culture to clear-bottom plates, which were measured and analyzed as described in the section "PFAS-Bacterial Growth Screen." On day 20, glycerol stocks were prepared from each lineage and stored at -80°C.

[0197] Mouse experiment (Figure 10) Animal experiments were approved by the local authorities (Regierungspraesidium Tuebingen, H 02 / 20 G). Germ-free C57BL / 6J mice were bred in-house (Gnotobiotic Mouse Facility, Tuebingen). Mice were housed under sterile conditions in flexible film isolators (Zoonlab) and transferred to the Isocage P system (Tecniplast) for experiments. Mice were provided ad libitum with autoclaved drinking water and γ-irradiated maintenance diet (Altromin). Five- to six-week-old female (n = 3) and male (n = 15) mice were used, and animals were randomly assigned to experimental groups. Mice were maintained in groups of three per cage throughout the experiment. All animals were scored daily for health status.

[0198] Preparation and inoculation of Com20 bacterial community The Com20 community included Phocaeicola vulgatus, Bacteroides uniformis, Bacteroides fragilis, Bacteroides thetaiotaomicron, Erysipelatoclostridium ramosum, Agathobacter rectalis, Roseburia intestinalis, Veillonella parvula, Eggerthella lenta, Fusobacterium nucleatum, Enterocloster bolteae, Clostridium perfringens, Lacrimispora saccharolytica, Streptococcus salivarius, Ruminococcus gnavus, Bariatricus comes, Parabacteroides merdae, Streptococcus parasanguinis, Collinsella aerofaciens, and Dorea formicigenerans. The Com20 community was prepared under anaerobic conditions (Coy Laboratory Products Inc., 2% H2, 12% CO2, balance N2). Consumables, glassware, and media were pre-reduced for at least two days before bacterial inoculation. Each strain was grown in monoculture overnight at 37°C in 5 ml of its respective growth medium. The following day, bacteria were subcultured (1:100) into 5 ml of fresh medium and incubated at 37°C for 16 hours, except for Eggerthella lenta, which was grown for two days. The optical density (OD) at 578 nm was determined, and equal proportions of bacteria were mixed together to a total OD of 0.5 in a final volume of 10 ml (OD of 0.025 for each of the 20 strains). After adding 2.5 ml of 50% glycerol (with a few crystals of palladium black (Sigma-Aldrich)), 200 μl aliquots were prepared in glass vials (2 ml, Supelco, ref. 29056-U) and frozen directly at -80°C. The frozen vials were used within three months.

[0199] For inoculation of germ-free mice, cages were transferred to the ISOcage Biosafety Station (IBS) (Tecniplast) via a 2% Virkon S disinfectant solution (Lanxess) dipping bath. Frozen Com20 community glycerol stocks (one per mouse) were kept on dry ice and then thawed during transfer to the IBS. After thawing, the mixture was used immediately with a maximum of 3 minutes of minimal oxygen exposure. Mice were inoculated by oral gavage (50 μl), and inoculation was repeated 48 hours later using the same protocol. A germ-free control group was left untreated. The IBS was sterilized with 3% perchloracetic acid (Wofasteril, Kesla Hygiene AG).

[0200] Ten days after the second inoculation of Com20, mice were orally gavaged with either PFNA (10 mg / kg in 25% DMSO) in a volume of 50 μl. Fresh fecal samples were collected in sterile, weighed 1.5 ml Eppendorf tubes before treatment, 3 hours, 1 day, and 2 days after treatment and immediately frozen at -80°C. Three days after treatment, mice were euthanized by CO2, cervically dislocated, and dissected. Intestinal contents were collected from the colon and small intestine and collected in the same manner.

[0201] Data analysis and replication All data analysis was performed using open-source packages accessed through Rstudio (version 1.3.1093). Xenobiotic compounds in each sample were quantified using Agilent MassHunter TQF Quantitative Analysis (version 10.1) or Agilent MassHunter QQQ Quantitative Analysis software (version 10.1). All t-tests were two-tailed. Biological replicates refer to different inoculum cultures, while technical replicates refer to experiments starting with the same inoculum culture. None of the data points correspond to repeated measurements of the same sample. Fold changes refer to median ratios.

[0202] Example 5 We performed proteomic analysis in Bacteroides uniformis after treatment with PFNA (20 μM) and DMSO (control) to determine differences in the proteome induced in response to perfluorononanoic acid (PFNA). Figure 17 and Table 3 show proteins differentially expressed in PFNA-treated cells compared to DMSO.

[0203] In B. uniformis, 37 proteins were significantly up-regulated and 19 were significantly down-regulated after PFNA treatment. More than half of the up-regulated proteins were related to plasma membrane and transporter function, with the top three up-regulated proteins being efflux pumps (R9I2M9 efflux transporter RND family: 5.39-fold change; R9I2L8 hydrophobe / amphiphile efflux-1 (HAE1) family RND transporter: 5.03-fold change; R9I2R1 NodT family efflux transporter: 4.79-fold change). These results support the involvement of efflux pumps in PFNA bioaccumulation.

[0204] The involvement of transporters was further supported by genetic analysis in another bioaccumulating gut bacterium, Parabacteroides merdae. We used a pool of transposon mutants of P. merdae (hereafter referred to as the "transposon library") to identify mutants with increased or decreased fitness upon PFNA exposure. The composition of the transposon library was determined after treatment with PFNA (500 μM) and DMSO (control). Homologs of the three most upregulated proteins (efflux pumps) in B. uniformis showed decreased fitness upon PFNA exposure compared to DMSO (Figure 18, Table 4). This suggests that the absence of these efflux pumps rendered the strain less tolerant to PFNA exposure, supporting their role as PFNA efflux factors.

[0205] Taken together, proteomic and genetic analyses indicate the involvement of efflux pumps in the extracellular export of PFNA, which influences the amount of PFNA bioaccumulation. Therefore, knockout of genes encoding efflux pumps is expected to increase the ability of bacterial cells to accumulate PFNA. [Table 3] TIFF2025535288000005.tif249165TIFF2025535288000006.tif66164 [Table 4]

[0206] Example 6 Several bacterial and yeast strains were tested for their ability to accumulate perfluorononanoic acid (PFNA). To allow for interspecies comparison, a resting cell assay, i.e., an OD of approximately 3.75, was used. 600 The study was performed using bacterial cells suspended in PBS buffer at a density of 100 μg / mL. The percentage of total PFNA removed from the supernatant, i.e., cell-free buffer, is shown in Figure 19A. The distribution of PFNA bioaccumulation showed a bimodal distribution (Figure 19B). When plotted as a group by Gram staining, Gram-negative bacteria showed higher PFNA accumulation compared to Gram-positive strains (Figure 19C). PFNA accumulation was further correlated with phylum. Most bacteria belonging to Actinomycetota, Bacillota, and Pseudomonadota showed lower accumulation (<20%), while all bacteria belonging to Fusobacteriota and Bacteroidota accumulated PFNA at greater than 20%. Using a two-compartment Gaussian mixture model, strains were classified as low- or high-PFNA accumulators with a cutoff of 25% PFNA accumulation, resulting in 40 high-accumulator strains (Table 5).

[0207] Comparing these results with those shown for 14 bacterial strains grown in modified Gifu anaerobic medium (mGAM) revealed a strong positive correlation (Pearson rank correlation: r = 0.76, p = 0.002; Spearman rank correlation: ρ = 0.64, p = 0.015) (Figure 19E). Although there was some variability due to differences in experimental setup, this comparison demonstrates the applicability of both screening approaches and highlights that PFNA accumulation can occur in very different environmental settings (PBS = nutrient-free environment; mGAM = nutrient-rich environment). [Table 5]

[0208] method Bacterial strains and in vitro incubation Microbial strains included three yeasts, spanning all major phyla of gut bacteria, including intraspecific divergences, several probiotic strains, and strains isolated from kefir. Experiments with kefir strains were performed under aerobic conditions, while all other bacterial experiments were conducted in an anaerobic chamber (Coy Laboratory Products) filled with 2% hydrogen and 12% carbon dioxide in nitrogen. The chamber was equipped with a palladium catalyst system for oxygen removal, a dehumidifier, and a hydrogen sulfide removal system. Bacteria were grown at 37°C in either mGAM, MRS, or YPD medium. Starting bacteria / yeast cultures were inoculated directly from frozen glycerol stocks and grown in 10 ml of growth medium in 15 ml plastic tubes for one or two days (depending on growth rate). The cultures were then diluted 100-fold and incubated again for the same period before starting the experiment. Screening plates containing 40 μM PFNA in PBS (2% DMSO) were placed in the chamber overnight to ensure anaerobic conditions for inoculation. Inoculation was performed 1:1 with bacterial culture in PBS to an OD of 3.75 and a PFNA concentration of 20 μM. Plates were sealed with AlumaSeal II film (A2350-100EA) to avoid evaporation during incubation. Plates were incubated at 37°C for 4 hours before being removed from the anaerobic chamber for sample collection. Total culture, supernatant, and compound control samples were collected and stored at -80°C until extraction.

[0209] Sample extraction 50 μl of sample was extracted with 200 μl ice-cold methanol:acetonitrile (1:1) containing internal standards (caffeine, ibuprofen) and incubated for 15 min at 4°C. The plate was then centrifuged at 4000 rpm for 10 min at 4°C. The supernatant was transferred to a 96-well plate for LC-MS analysis. Samples for concentration calibration and sterile compound controls were processed in the same manner.

[0210] QTOF parameters (Batch 1-2) Briefly, LC-MS analysis was performed on an Agilent 1290 Infinity II LC system coupled with an Agilent 6546 LC / Q-TOF (Agilent). The QTOF MS scan was operated in negative scanning mode (m / z 30–1500). Source parameters were as follows: gas temperature: 200 °C, drying gas: 9 L / min, nebulizer: 20 psi, sheath gas temperature: 400 °C, sheath gas flow: 12 L / min, VCap: 3000 V, nozzle voltage: 0 V, fragmentor: 110 V, skimmer: 45 V, and Octa RF Vpp: 750 V. Online mass calibration was performed using reference solutions (112.99 and 1033.99 m / z). Collision energies used were 0 V, 10 V, 20 V, and 40 V. Compounds were identified based on their retention time, accurate mass, and fragmentation pattern. For all measured compounds, pure standards were obtained from Sigma Aldrich (Merck KGaA, Darmstadt, Germany) and used for method development, compound identification and calibration.

[0211] 10-minute reversed-phase dual-pump LC method used with QTOF (batches 1-2) Separation was performed using two ZORBAX RRHD Eclipse Plus columns (C18, 2.1 x 100 mm, 1.8 µm; Agilent) with ZORBAX Eclipse Plus (C18, 2.1 x 5 mm, 1.8 µm; Agilent) guard columns at 40 °C. The multisampler was maintained at a temperature of 4 °C. The injection volume was 1 µL, and the flow rate was 0.4 mL / min. The mobile phase consisted of A: water + 5 mM ammonium acetate + 0.03% acetic acid; B: methanol + 5 mM ammonium acetate + 0.03% acetic acid. A 10-min gradient started at 35% solvent B, increased to 100% in 7 min, held for 1.7 min, then returned to 35% solvent B in 8.8 min and maintained until 10 min. The re-equilibration gradient started at 35% solvent B, then ramped to 95% solvent B in 0.1 min and held for 4 min before returning to the starting conditions of 35% solvent B in 4.1 min.

[0212] QQQ parameters (batches 3 to 8) Briefly, LC-MS / MS analysis was performed on an Agilent 1290 Infinity II LC system coupled to an Agilent 6570 LC / TQ (Agilent). The QQQ was operated in Dynamic MRM mode. Source parameters were as follows: gas temperature: 300 °C, gas flow: 910 L / min, nebulizer: 50 psi, sheath gas temperature: 300 °C, sheath gas flow: 11 L / min, VCap: 3500 V (positive mode) or 3000 V (negative mode), nozzle voltage: 2000 V (positive mode) or 500 V (negative mode). For PFNA detection, the scan segments were as follows: precursor ion: 463; product ions: 418.9 and 294.1; fragmentor voltages: 64 and 80 V; collision energy: 8 V. Pure standards were obtained from Sigma Aldrich (Merck KGaA, Darmstadt, Germany) and used for method development, compound identification and calibration.

[0213] 2-minute reversed-phase LC method used in QQQ (batches 3–8) Separation was performed using a ZORBAX RRHD Eclipse Plus column (C18, 3x50mm, 1.8µm; Agilent) with a ZORBAX Eclipse Plus (C18, 2.1x5mm, 1.8µm; Agilent) guard column at 40°C. The multisampler was maintained at a temperature of 4°C. The injection volume was 1µL, and the flow rate was 0.8mL / min. The mobile phase consisted of A: water + 0.1% formic acid; B: methanol + 0.1% formic acid. A 2-min gradient started at 30% solvent B, increased to 100% in 0.5 min, held for 1 min, then returned to 30% solvent B at 1.05 min and held for 2 min.

[0214] LC-MS / MS data analysis Selected xenobiotic standards were qualified using Agilent MassHunter Qualitative Analysis 10.0 software. TIC, EIC, and EIC fragment graphs were extracted for each compound. Xenobiotic compounds in each sample were quantified using Agilent MassHunter TQF Quantitative Analysis (version 10.1) or Agilent MassHunter QQQ Quantitative Analysis software (version 10.1).

[0215] A calibration curve based on pure compound standards was used to estimate the linearity of target compound concentrations within each batch. Data analysis was performed in RStudio version 1.3.1093. The median value of each sample group was compared to the median value of the compound control within each batch to calculate the percent of PFNA accumulation. Further statistical comparisons were performed using t-tests (two-tailed), and p-values ​​were FDR-corrected for the number of strains tested. A corrected p-value of <0.05 was considered significant. A two-compartment Gaussian mixture model was used to determine whether the strain had high or low accumulation.

Claims

1. 1. A method for reducing the level of xenobiotics in an environment, comprising contacting the environment with a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus.

2. Use of a composition comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus in a method for reducing the level of xenobiotics in the environment.

3. 3. The method of claim 1 or the use of claim 2, wherein the environment is an aqueous environment, optionally the aqueous environment is drinking water or wastewater.

4. 4. The method of claim 1 or claim 3, or the use of claim 1 or claim 2, wherein the method comprises detecting the presence and / or measuring the abundance of the one or more bacterial strains in the environment prior to contacting the environment with the composition.

5. 5. The method of any one of claims 1, 3 or 4, or the use of any one of claims 2 to 4, wherein the method comprises: (i) contacting the environment with the one or more bacterial strains; and then (ii) removing the one or more bacterial strains to provide a treated environment.

6. 1. A composition for use in a method for reducing xenobiotic levels in a subject, comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus.

7. 7. The composition for use of claim 6, wherein the method comprises preventing or treating xenobiotic intoxication in the subject.

8. 8. The composition for use of claim 6 or claim 7, wherein the subject has ingested, is suspected of ingesting, or is at risk of ingesting the xenobiotic.

9. 9. The composition for use according to any one of claims 6 to 8, wherein the method comprises detecting the presence of and / or measuring the abundance of said one or more bacterial strains in said subject prior to administering said composition to said subject.

10. The composition for use according to any one of claims 6 to 9, wherein the subject is a human.

11. The composition for use according to any one of claims 6 to 10, wherein the subject is an animal, optionally wherein the animal is a cow, sheep, pig, poultry, cat or dog.

12. 1. A composition for reducing levels of xenobiotics, comprising one or more bacterial strains selected from Bacteroides, Collinsella, Coprococcus, Eubacterium, Odoribacter, Parabacteroides, Roseburia, Escherichia, Phocaeicola, Prevotella, Butyrivibrio, Lacrimispora, Clostridium, Fusobacterium, Agathobacter, Dorea, and Streptococcus.

13. The one or more bacterial strains are Bacteroides uniformis, Bacteroides caccae, Bacteroides clarus, Bacteroides dorei, Bacteroides stercoris, Bacteroides thetaiotaomicron, Collinsella aerofaciens, Coprococcus comes, Eubacterium rectale, Odoribacter splanchnicus, Parabacteroides distasonis, Parabacteroides merdae, Roseburia intestinalis, Escherichia coli, Phocaeicola coprocola, Prevotella copri, Bacteroides eggerthii, Prevotella melaninogenica, Bacteroides fragilis, Bacteroides xylanisolvens, Butyrivibrio crossotus, Bacteroides coprocola, Roseburia hominis, Lacrimispora saccharolytica, Clostridium scindens, Fusobacterium The method according to any one of claims 1 or 3 to 5, the use according to any one of claims 2 to 5, the composition for the use according to any one of claims 6 to 11, or the composition according to claim 12, wherein the bacterial strain selected from Fusobacterium nucleatum subsp. Nucleatum, Clostridium difficile, Phocaeicola vulgatus, Agathobacter rectalis, Roseburia inulinivorans, Dorea formicigenerans, Streptococcus salivarius, Fusobacterium nucleatum subsp. Animalis, Fusobacterium nucleatum subsp. Vincentii, Clostridium hylemonae, and Clostridium sporogenes.

14. 14. The method of any one of claims 1, 3 to 5 or 13, the use of any one of claims 2 to 5 or 13, the composition for use of any one of claims 6 to 11 or 13, or the composition of claim 12 or 13, wherein the composition comprises Bacteroides, and optionally the composition comprises Bacteroides uniformis.

15. 15. The method of any one of claims 1, 3 to 5, 13 or 14, the use of any one of claims 2 to 5, 13 or 14, the composition for use of any one of claims 6 to 11, 13 or 14, or the composition of any one of claims 12 to 14, wherein one or more of the bacterial strains comprises a genetic modification that results in the reduction or elimination of xenobiotic excretion from the one or more bacterial strains.

16. 16. The method of claim 15, the use of claim 15, the composition for the use of claim 15, or the composition of claim 15, wherein the composition comprises Bacteroides having a genetic modification that results in the reduction or elimination of xenobiotic efflux from Bacteroides, and optionally the genetic modification comprises the deletion or inactivation of at least one gene required for the activity of the R9I2M9 efflux transporter RND family, the R9I2L8 hydrophobe / amphiphile efflux-1 (HAE1) family RND transporter, and / or the R9I2R1 NodT family efflux transporter.

17. 17. The method of claim 15 or claim 16, the use of claim 15 or claim 16, the composition for the use of claim 15 or claim 16, or the composition of claim 15 or claim 16, wherein the composition comprises E. coli having a genetic modification that results in the reduction or elimination of xenobiotic export from E. coli, optionally the genetic modification comprising the deletion or inactivation of at least one gene required for the activity of the AcrAB-TolC efflux pump, and optionally the genetic modification comprising the deletion or inactivation of tolC, AcrA, and / or AcrB.

18. 18. The method of any one of claims 1, 3 to 5 or 13 to 17, the use of any one of claims 2 to 5 or 13 to 17, the composition for use of any one of claims 6 to 11 or 13 to 17, or the composition of any one of claims 12 to 17, wherein the xenobiotics comprise one or more of PFAS, bisphenols, and pesticides.

19. The xenobiotics are PFNA, PFOA, PFDeA, bisphenol AF, boscalid, propiconazole, pyrimethanil, tributyl-PO 4 , and triphenyl-PO 4 19. The method of any one of claims 1, 3 to 5 or 13 to 18, the use of any one of claims 2 to 5 or 13 to 18, the composition for the use of any one of claims 6 to 11 or 13 to 18, or the composition of any one of claims 12 to 18, comprising one or more of:

20. 20. The method of any one of claims 1, 3 to 5 or 13 to 19, the use of any one of claims 2 to 5 or 13 to 19, the composition for the use of any one of claims 6 to 11 or 13 to 19, or the composition of any one of claims 12 to 19, wherein the xenobiotic comprises a PFAS, optionally wherein the PFAS comprises PFOA, PFNA, and / or PFDeA.

21. 20. The method of any one of claims 1, 3 to 5 or 13 to 20, the use of any one of claims 2 to 5 or 13 to 20, the composition for use of any one of claims 6 to 11 or 13 to 20, or the composition of any one of claims 12 to 20, wherein the composition comprises Bacteroides and the xenobiotic comprises a PFAS.

22. 22. The method of claim 21, the use of claim 21, the composition for the use of claim 21, or the composition of claim 21, wherein the composition comprises Bacteroides uniformis and the PFAS comprises PFNA, PFOA, and / or PFDeA.

23. 23. The method of any one of claims 1, 3 to 5 or 13 to 22, the use of any one of claims 2 to 5 or 13 to 22, the composition for use of any one of claims 6 to 11 or 13 to 22, or the composition of any one of claims 12 to 22, wherein the composition is formulated for intestinal delivery, and optionally the composition is formulated for oral, nasal and / or rectal delivery.

24. 24. The method of any one of claims 1, 3 to 5 or 13 to 23, the use of any one of claims 2 to 5 or 13 to 23, the composition for use of any one of claims 6 to 11 or 13 to 23, or the composition of any one of claims 12 to 23, wherein the composition further comprises one or more prebiotics for promoting the growth of the one or more bacterial strains.

25. 25. The method of claim 24, the use of claim 24, the composition for the use of claim 24, or the composition of claim 24, wherein the one or more prebiotics are selected from arabinoxylan, xylose, fibrous dextran, corn fiber, polydextrose, lactose, N-acetyl-lactosamine, glucose, galactose, fructose, rhamnose, mannose, uronic acid, arabinose, fructose, fucose, lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose, xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose, xylooligosaccharides, fructooligosaccharides, galactooligosaccharides, lactosucrose, and soybean oligosaccharides.

26. The method of any one of claims 1, 3 to 5 or 13 to 25, the use of any one of claims 2 to 5 or 13 to 25, the composition for the use of any one of claims 6 to 11 or 13 to 25, or the composition of any one of claims 12 to 25, wherein the one or more bacterial strains are freeze-dried.

27. 26. The method of any one of claims 1, 3 to 5 or 13 to 26, the use of any one of claims 2 to 5 or 13 to 26, the composition for use of any one of claims 6 to 11 or 13 to 26, or the composition of any one of claims 12 to 26, wherein the composition further comprises a carrier, excipient and / or diluent.

28. 27. The method of any one of claims 1, 3 to 5 or 13 to 27, the use of any one of claims 2 to 5 or 13 to 27, the composition for use of any one of claims 6 to 11 or 13 to 27, or the composition of any one of claims 12 to 27, wherein the composition comprises a gastroresistant coating.

29. 28. The method of any one of claims 1, 3 to 5 or 13 to 28, the use of any one of claims 2 to 5 or 13 to 28, the composition for the use of any one of claims 6 to 11 or 13 to 28, or the composition of any one of claims 12 to 28, wherein the composition is a sustained release formulation.

30. 30. A dietary supplement comprising the composition of any one of claims 12 to 29.

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