# Characterisation of potential anti-inflammatory next-generation probiotics resistant to bisphenol A

A. López-Moreno, C. Carbonne, C. Kropp, D. Rios-Covian, F. Pepke, P.

Langella, M. Aguilera, R. Martin

► To cite this version:

A. López-Moreno, C. Carbonne, C. Kropp, D. Rios-Covian, F. Pepke, et al.. Characterisation of potential anti-inflammatory next-generation probiotics resistant to bisphenol A. Beneficial Microbes, 2025, 16 (1), pp 91 - 107. 10.1163/18762891-bja00041. hal-05071306

HAL Id: hal-05071306

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Submitted on 16 May 2025

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RESEARCH ARTICLE

# Characterisation of potential anti-inflammatory next-generation probiotics resistant to bisphenol A

A. López-Moreno¹,² ID, C. Carbonne³, C. Kropp³, D. Rios-Covian³, F. Pepke³, P. Langella³ ID,
M. Aguilera¹,²,# ID and R. Martin³,‡,∗ ID

¹Department of Microbiology, Faculty of Pharmacy, University of Granada, 18071 Granada, Spain; ²Center of Biomedical Research, Institute of Nutrition and Food Technology 'José Mataix', University of Granada, 18016 Granada, Spain; ³INRAE, AgroParisTech, Micalis Institute, Université Paris-Saclay, 78350 Jouy-en-Josas, France; *rebeca.martin-rosique@inrae.fr

*These authors contributed equally.

Received 1 February 2024 | Accepted 20 August 2024 | Published online 27 September 2024 |
Published in issue 15 January 2025

## Abstract

The world is witnessing an increasing incidence of chronic non-communicable diseases (NCDs), such as inflammatory bowel disease (IBD), a group of complex gastrointestinal disorders characterised by inflammation. It is believed that environmental factors, such as exposure to pollutants and endocrine-disrupting chemicals (i.e. bisphenol A [BPA]), are playing a role in IBD pathophysiology. New research suggests a potential treatment solution: next-generation probiotic (NGP) strains isolated from human gut microbiota that can biodegrade xenobiotics and thus possibly modulate IBD triggered by these xenobiotics. In this study, we hypothesised that specific BPA-tolerant bacteria would exhibit beneficial, anti-inflammatory properties that could counter the effects of BPA exposure and concomitantly reduce colitis severity. We observed that two such strains, *Bacillus* sp. AM1 and *Paeniclostridium* sp., exhibited potential anti-inflammatory properties *in vitro* and *in vivo*. First, these bacteria were able to decrease the secretion of interleukin (IL)-8 cytokines by HT-29 cells that had been exposed to the proinflammatory cytokine tumour necrosis factor (TNF)-α. Second, when treated with *Bacillus* sp. AM1 and *Paeniclostridium* sp. (this latter had a stronger reducing effect on inflammatory markers), mice with chemically induced colitis displayed lower levels of colon damage, monocyte chemotactic protein 1 (MCP-1), lipocalin-2 (LCN-2), and proinflammatory cytokines (IL-1β and IL-6). Future research should clarify the underlying mechanisms at play and identify potential strategies for counteracting the systemic effects of IBD, including those exacerbated by BPA exposure. Our results suggest that one such strategy could be treatment with BPA-tolerant bacteria that possess anti-inflammatory properties.

## Keywords

probiotic – BPA – endocrine disruptors – colitis – DNBS
# 1 Introduction

Over recent decades, the world has been witnessing a steady increase in the incidence of chronic non-communicable diseases (NCDs), including cardiovascular diseases, cancer, obesity, diabetes, inflammatory bowel disease (IBD), and mental illnesses. These conditions all share a common underlying feature – inflammation – which could be contributing to disease pathogenesis and progression. The World Health Organization (WHO) estimates that NCDs are responsible for 74% of global mortality (WHO, 2023), underscoring their dramatic effect on public health.

IBD refers to a group of multifactorial gastrointestinal disorders that are characterised by inflammation. The underlying cause of IBD is not fully understood but is believed to be a combination of genetic factors and environmental triggers (Danese et al., 2004) that result in individuals displaying dysregulated immune responses to the gut microbiota they harbour (Sheehan et al., 2015). Proposed environmental risk factors for IBD include the Western diet, tobacco use, infections, gut microbiota dysbiosis, antibiotic misuse or abuse, oestrogen-containing medications, and exposure to endocrine-disrupting chemicals (EDCs) or pollutants (DeLuca et al., 2018; Legaki and Gazouli, 2016). It has been hypothesised that increased levels of these risk factors are driving increases in IBD worldwide and especially in industrialised countries (Ng et al., 2017).

Bisphenol A (BPA) is an EDC with oestrogen-mimicking activity that also has immunological and obesogenic effects. It is found in everyday products, including polycarbonate plastics, plastic toys, food packaging, and plastic bottles (Jalal et al., 2018). Exposure to BPA can lead to changes in gut microbial structure (Lear et al., 2021). It may also play an important role in the development of autoimmune diseases by heightening the immune response and may thus be a factor behind the increasing incidence of inflammatory diseases (Bansal et al., 2017; Sharif et al., 2022). Recent studies suggest that BPA exposure may also influence IBD development (Malaisé et al., 2020; Reddivari et al., 2017; Yin et al., 2022) and could worsen colitis pathophysiology in mice (DeLuca et al., 2018).

Imbalance or dysbiosis in the gut microbiota has been linked to the development of several inflammatory diseases, including IBD (West et al., 2015). Therefore, it has been proposed that modulating gut microbiota composition could be a promising approach for treating IBD. Probiotics, prebiotics, and faecal transplants are among the strategies that have been explored to date

(Boesmans et al., 2018; Frank et al., 2007). Wide use has been made of traditional probiotics, such as strains of lactobacilli and bifidobacteria. However, clinical trials have found that probiotic efficacy varies, as reflected in clinical parameters and outcomes, due to specific factors associated with different strains, diseases, doses, and individuals (Veiga et al., 2020). Additionally, classical probiotics persist in the gut for short periods and have a limited capacity to modulate microbiota composition. To overcome these constraints, researchers have designed next-generation probiotics (NGPs) that target specific diseases by using multi-omics to thoroughly characterise strain taxonomic traits and health benefits (López-Moreno et al., 2021). Members of the gut microbiota, NGPs are often *Bacteroides* or *Clostridia* species that produce beneficial biocompounds and short-chain fatty acids (SCFAs), such as butyrate and propionate (West et al., 2015).

By isolating potential NGPs from human gut microbiota, it may be possible to identify strains that can biodegrade xenobiotics and that could thus help modulate inflammatory diseases, including those potentially exacerbated by chemical contaminants. In previous research, we isolated bacteria from human gut microbiota that can tolerate and potentially degrade BPA (López-Moreno et al., 2022). Here, our aim was to determine whether two of these BPA-tolerant strains displayed anti-inflammatory effects *in vitro* and *in vivo*. Specifically, we hypothesised that the strains would help decrease colitis severity in a context of BPA exposure.

# 2 Materials and methods

## Bacteria isolation and growth conditions

In a previous study, we isolated *Bacillus* sp. AM1 and *Paeniclostridium* sp. using a BPA-directed culturing approach (López-Moreno et al., 2022); we obtained both strains from the gut microbiota of healthy, normal weight children. *Bacillus* sp. AM1 (GenBank CP047644.1) was cultured in brain-heart infusion (BHI) broth under aerobic conditions at 37 °C with shaking. *Paeniclostridium* sp. (GenBank MZ612832.1) was cultured in BHI broth supplemented with 0.05% cysteine (reducing agent) in an anaerobic chamber (5%/5%/90% CO₂, H₂, N₂) at 37 °C. Granulated agar (15 g/l, Difco, Becton Dickinson, Franklin Lakes, NJ, USA) was added when necessary.

For the bacteria used in the *in vitro* and *in vivo* experiments described below, cultures were centrifuged at 2,500×g at the end of the exponential phase. They were
then resuspended in IX Dulbecco's phosphate-buffered saline (DPBS, Gibco, ThermoFisher, Waltham, MA, USA) containing 16% glycerine and stored at -80 °C until their use in the experiments. Supernatants were collected by centrifuging the cultures at 4,000×g at 4 °C for 15 min and were stored at -20 °C until their use in SCFA quantification and *in vitro* testing.

### In vitro microbiological characterisation of the bisphenol A-tolerant strains

Characterising potential gastrointestinal resistance and shock tolerance

To evaluate their potential resistance to the gastrointestinal environment, the strains were exposed to conditions mimicking those of the digestive tract. The strains were grown in media with a pH of 3 or 6 and a bile salt concentration of 0.3% or 1% (bile salt source: Oxoid Sigma B3883, ThermoFisher). Strain growth curves were characterised by measuring optical density at 600 nm (OD₆₀₀).

Strain responses to pH and bile salt shock were also tested. Pre-culture aliquots of both strains were exposed to control conditions (PBS solution) or four shock conditions (PBS solution with bile salt concentration of 0.3%, 1%, pH 3 and 6). After being incubated for 1 h at 37 °C, the aliquots were centrifuged at 2,500×g, rinsed with 1 ml of PBS, added to the optimal medium (BHI), and incubated for 24 h at 37 °C. Colony-forming units (cfu/ml) were enumerated at 0, 30 min, 1 h, and 24 h post exposure.

### Determining antibiotic resistance

The strains' minimum inhibitory concentrations (MICs) were determined using European Food Safety Authority (EFSA) guidelines for testing antimicrobial susceptibility (EFSA FEEDAP, 2012) as well as EUCAST clinical breakpoint tables (v. 13.0) and criteria established by the National Committee for Clinical Laboratory Standards (CLSI) (Humphries *et al.*, 2018). For both strains, resistance to the following antibiotics was tested using E-test (BioMérieux, Marcy l'Étoile, France): clindamycin, chloramphenicol, erythromycin, gentamicin, kanamycin, tetracycline, and vancomycin. The resistance of *Bacillus* sp. AM1 to streptomycin was also tested, as was the resistance of *Paeniclostridium* sp. to ampicillin. Testing employed the agar disc-diffusion method and Wilkins-Chalgren agar (Difco) as recommended in E-test instructions. The results were recorded after 48 h of incubation.

### Measurement of short chain fatty acid production

Supernatants were obtained from bacteria in the exponential phase and were deproteinised at 4 °C by overnight exposure to phosphotungstic acid (10% [v/v]); Sigma Aldrich, St. Louis, MO, USA). The resulting samples were centrifuged at 12,000×g for 15 min. SCFA concentrations were analysed using gas-liquid chromatography (GLC; Nelson 1020, Perkin-Elmer, St Quentin en Yvelines, France) as described elsewhere (Lan *et al.*, 2008). The internal standard was 2-ethylbutyrate, and a panel of SCFA standards was employed. Three replicates were performed for each sample. The default software of the GLC apparatus (Agilent, Santa Clara, CA, USA) was used to collect the SCFA data and to integrate the peaks. To determine the final SCFA concentrations, the supernatants were weighed before and after protein precipitation to obtain the appropriate multiplication factor (i.e. the supernatant-to-sample-mass ratio).

### Characterising carbohydrate fermentation profiles

The strains' metabolic profiles of carbohydrate fermentation were determined using an API50 CHB/E Medium system (BioMérieux) in accordance with the manufacturer's instructions. Metabolic profiles were analysed after 24 and 48 h of growth at 37 °C under aerobic or anaerobic conditions, depending on each strain's optimal growth conditions.

### Assessing the strains' anti-inflammatory effects via immunomodulation assays

Anti-inflammatory assays were performed using the procedure described by Kechaou *et al.* (2013). On day 1, cells from human colon adenocarcinoma cell line HT-29 (American Type Culture Collection [ATCC]; Sigma Aldrich) were seeded in 24-well culture plates that contained DMEM (Lonza, Basel, Switzerland) supplemented with 10% (v/v) foetal bovine serum (foetal bovine serum (FBS); Gibco) and 1% (v/v) penicillin/streptomycin (P/S; Sigma Aldrich). They were kept at 37 °C under conditions of 5% CO₂ until 80% confluence was reached. On day 6, 24 h before bacterial co-culture, the initial culture medium was replaced by a culture medium containing 5% FBS. On day 7, the HT-29 cells were co-cultured with the strains at a multiplicity of infection (MOI) level of 40; they were simultaneously exposed to human tumour necrosis factor (TNF)-α (5 ng/ml; Peprotech, Cranbury, NJ, USA) for 6 h at 37 °C under conditions of 10% CO₂. Next, supernatants were collected from the cell samples and stored at -80 °C. Interleukin (IL)-8 concentrations in these samples were analysed using the Human IL-8 ELISA MAX Standard
Set (BioLegend, San Diego, CA, USA), in accordance with the manufacturer's instructions. Each strain and supernatant was tested three times using three independent assays. The results were expressed as IL-8/protein concentrations (pg/mg) and were normalised using the negative control as the reference value (i.e. IL-8 concentrations).

The strains' cytotoxicity levels were evaluated using HT-29 cells and a Lactate Dehydrogenase (LDH) CyQUANT™ Kit (ThermoFisher Scientific) in accordance with the manufacturer's instructions.

### In vivo effects of the strains in a mouse model with bisphenol A exposure and colitis

In the animal care facilities of the French National Institute for Agriculture, Food, and Environment (IERP, INRAE, Jouy-en-Josas, France), 126 specific pathogen free (SPF) male C57BL/6 mice (6-8 weeks old) (Janvier, Le Genest Saint Isle, France) were housed in cages in groups of 5. They were reared under controlled conditions (temperature: 20 ± 2 °C, light/dark cycle: 12 h/12 h) and had *ad libitum* access to food and water. The experiments described below were performed in accordance with European Community regulations for animal welfare and were approved by INRAE's animal research and ethics committee (Comethea; protocol number 16744-201807061805486 v. 2).

The mice experienced 1 week of acclimation prior to a 22-day experimental period during which there was (1) exposure to BPA, (2) treatment with the BPA-tolerant strains, (3) the chemical induction of colitis (Figure 1). First, the 126 mice were assigned to various combinations of the following experimental categories: (1) BPA exposure (BPA) vs no BPA exposure (CO); (2) treatment with *Bacillus* sp. AM1 (BAC) vs treatment with *Paenilostridium* sp. (PAE) vs no bacterial treatment (PBS); (3) induced colitis (DNBS) vs no colitis (Veh). The result was eight distinct experimental groups: Veh-CO-PBS (n = 18), Veh-BPA-PBS (n = 18), DNBS-CO-PBS (n = 18), DNBS-BPA-PBS (n = 18), DNBS-BPA-BAC (n = 18), DNBS-BPA-PAE (n = 18), DNBS-CO-BAC (n = 9), and DNBS-CO-PAE (n = 9). During the experiment, mouse mass was measured daily.

Each day for the first 5 days of the experiment, the mice were orally gavaged with 50 µg of BPA (category: BPA) or corn oil (category: CO) per kg of body mass. This dose of BPA was chosen because it is the compound's tolerable daily intake (TDI) as established by the US Environmental Protection Agency (US EPA, 2023). For the next 14 days, the mice experienced a daily double oral gavage with (1) the same dose of BPA or corn oil as

previously described and (2) either 10⁹ cfu/ml of *Bacillus* sp. AM1 or *Paenilostridium* sp. in PBS (category: BAC vs PAE) or PBS alone (category: PBS). On day 19 of the experiment, colitis was induced in most of the mice (n = 90) via the intrarectal injection of dinitrobenzene sulfonic acid (DNBS) (groups: DNBS-CO-PBS, DNBS-BPA-PBS, DNBS-BPA-BAC, DNBS-BPA-PAE, DNBS-CO-BAC, DNBS-CO-PAE). The methodology described in Martín *et al.* (2014) was employed, with some minor modifications. In brief, the mice received an intrarectal injection of DNBS (200 mg/kg; ICN Biomedical Inc., Santa Ana, CA, USA) that was dissolved in PBS-ethanol (70/30, v/v). The other mice (n = 36) received a control injection of PBS-ethanol (70/30, v/v) (groups: Veh-CO-PBS, Veh-BPA-PBS). The study was conducted in two separate cohorts, with half of the mice for each group tested in one batch and the other half in another batch, effectively constituting two independent experiments to validate the outcomes. Mice that died before the onset of colitis or due to the anaesthesia were excluded from the analysis (n = 2).

Three days after the intrarectal injections, blood samples were obtained from the submandibular veins of all the mice, which were then euthanised. The blood samples were centrifuged for 10 min at 12,000 rpm and 4 °C, and the resulting sera were stored at -80 °C until further analysis could occur.

### Assessing the strains' anti-inflammatory effects in the mice

We assessed the strains' effects in the different groups of mice by measuring the mice's macroscopic scores, microscopic scores, myeloperoxidase (MPO) activity, cytokine levels, and percentages of immune cell populations present in the spleen and mesenteric lymph nodes (MLNs).

### Macroscopic scores

Immediately after euthanasia, the mice's colons and small intestines were collected, and visible colon damage was assessed using a mouse-adapted Wallace scale.

<figure><img src="image_4.png" /><figcaption>FIGURE 1 Experimental timeline of bisphenol A (BPA) exposure, treatment with BPA-tolerant strains, and colitis induction.</figcaption></figure>
Macroscopic scoring was conducted as described in a previous study on DNBS-induced colitis (Martín *et al.*, 2014). Briefly, the macroscopic criteria (scale of 0-9) included macroscopic mucosal damage (e.g. presence of ulcers, thickening of the colon wall, adhesions between the colon and other intra-abdominal organs), stool viscosity (i.e. an indicator of diarrhoea), and the presence of hyperaemia.

### Microscopic scores

Histological characteristics were evaluated via haematoxylin-eosin-saffron (HES) staining and in accordance with the established protocols (Barone *et al.*, 2023; Martín *et al.*, 2014) of the histology facility associated with the @Bridge platform of UMR 1313 GABI. Colon damage was assessed using a scoring system that accounted for architectural distortions, goblet cell depletion, oedema/ulceration, and the degree of inflammatory cell infiltration (Ameho *et al.*, 1997). Image analysis was performed using Panoramic Viewer software.

### Myeloperoxidase activity and levels of lipocalin-2

MPO activity is used as a marker of neutrophil infiltration. It was measured in the mice's colon and ileum samples employing a modified version of the method described by Bradley *et al.* (1982). One centimetre of distal colon was recovered and homogenised (50 mg/ml) in cold 50-mM potassium phosphate buffer (pH 6) containing 5% hexadecyltrimethylammonium bromide (Sigma-Aldrich, Lyon, France) and hydrogen peroxide (H₂O₂; Sigma-Aldrich) in a Precellys® Evolution Homogeniser (VWR). Following the colorimetric reaction, absorbance was measured using a plate reader (Tecan, Lyon, France).

Lipocalin-2 (LCN-2) levels in the serum samples were determined utilising a Mouse Lipocalin-2/NGAL ELISA Kit (DuoSet; R&D systems, Inc., Minneapolis, MN, USA) in accordance with the manufacturer's instructions.

### Cytokine levels

Levels of 13 pre-selected cytokines (IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, monocyte chemotactic protein 1 (MCP-1), interferon (IFN)-β, IFN-γ, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF)) were determined in the serum and colon samples using a LEGENDplex™ Mouse Inflammation Panel (BioLegend) in accordance with the manufacturer's instructions.

Immune cell populations in the spleen and mesenteric lymph nodes

Mononuclear cells were isolated from the mice's spleens and MLNs via gentle extrusion of the tissue through a 50-µm-mesh Nylon cell strainer (BD Biosciences, San Jose, CA, USA); the resulting erythrocytes were then lysed using red blood cell lysing buffer (Sigma-Aldrich) as described elsewhere (Larauche *et al.*, 2012).

For the flow cytometry analysis, aliquots of 10⁶-10⁷ cells were taken from each sample and pre-incubated with purified anti-mouse CD16/CD32 monoclonal antibodies (eBioscience, San Diego, CA, USA). They were then labelled with anti-CD3-FITC, anti-CD4-PerCP, anti-Tbet-APC, and anti-Gata3-PE monoclonal antibodies (all from eBioscience) in accordance with the manufacturer's instructions. Intracellular staining was performed using a FoxP3 Staining Kit (eBioscience). Stained cells were analysed via flow cytometry (Accuri, Ann Arbor, MI, USA) and utilising CFlow Sampler software (BD Accuri, Ann Arbor, MI, USA).

### Statistical analysis

GraphPad software (v. 9.5.0; GraphPad Software, La Jolla, CA, USA) was used for the statistical analyses. All the results were expressed in whisker-boxes with individual points or as mean ± standard error of the mean (SEM). Non-parametric statistical analyses were performed: two-sided Mann Whitney U tests and Kruskal-Wallis tests, which were followed by Dunn's multiple comparison tests when there were more than two groups. A P-value of 0.05 was considered significant.

## 3 Results

### *In vitro* microbiological characteristics of the bisphenol A-tolerant strains

Both strains (*Bacillus* sp. and *Paeniclostridium* sp.) were able to grow in media with bile salt concentrations of 0.3 and 1% (Figure 2A, B). However, when exposed to bile salt shock, growth by both strains were affected at 30 min and 1 h. *Bacillus* sp. AM1, but not *Paeniclostridium* sp., had recovered after growing on optimal medium for 24 h (Supplementary Figure S1). Additionally, both species tolerated pH levels of 3 and 6, displaying no significant reduction in growth (Supplementary Figure S1).

Based on EFSA guidelines, *Bacillus* sp. AM1 was susceptible to all eight antibiotics tested (Supplementary Table S1). The susceptibility of *Paeniclostridium* sp. could not be formally assessed because no clinical
A

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /></figure>

C

<figure><img src="image_7.png" /></figure>

D

<figure><img src="image_8.png" /><figcaption>FIGURE 2 *In vitro* characteristics of the bisphenol A (BPA)-tolerant strains. Growth curve of (A) *Bacillus* sp. AM1 and (B) *Paeniclostridium* sp. in control media and media with bile salt concentrations of 0.3% and 1%. Short chain fatty acid (SCFA) production at the end of the exponential phase by (C) *Bacillus* sp. AM1 and (D) *Paeniclostridium* sp. For A-D, the mean ± standard error of the mean is shown.</figcaption></figure>

breakpoint tables exist for this genus (EUCAST, 2023),
but the strain's MICs were nonetheless determined
for eight commonly used antibiotics (Supplementary
Table S2).

*Bacillus* sp. AM1 and *Paeniclostridium* sp. differed in their SCFA production patterns (Figure 2C and D). *Bacillus* sp. generated high levels of acetate; moderate levels of isovalerate; and low levels of isobutyrate, propionate, and butyrate. In contrast, *Paeniclostridium* sp. generated high levels of acetate; moderate levels of isocaproate, isovalerate, and propionate; and low levels of isobutyrate.

*Bacillus* sp. AM1 was able to metabolise 24 target carbohydrates: glycerol, L-arabinose, ribose, D-xylose, D-glucose, D-fructose, D-mannose, inositol, mannitol, sorbitol, methyl-D-glucoside, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, D-raffinose, starch, and glycogen. While *Paeniclostridium* sp. was only able to metabolise 4 target carbohydrates: D-glucose, D-fructose, maltose, and L-fucose (Table 1).

*Immunomodulation assays assessing the strains'*
*anti-inflammatory effects*

The *in vitro* immunomodulation assay characterised the
strains' effects on IL-8 secretion by human HT-29 cells
exposed to TNF-α, a major proinflammatory cytokine
(Figure 3). Before conducting the assay, a cytotoxicity
test was performed to verify that potential cytotoxic
compounds had not been released by the bacteria,
which could have caused cell damage or death.
The results of the cytotoxicity test came out negative,
confirming that no cytotoxic compounds were present
(data not shown). *Bacillus* sp. AM1 displayed pro-
nounced anti-inflammatory activity, significantly reduc-
ing IL-8 production by approximately 30% (*P* < 0.05).
Although this figure was 20% for *Paeniclostridium* sp.,
this increase was not statistically significant (Figure 3A).
Next, tests using the supernatants from the exponential-
phase bacterial cultures confirmed that absence of any
anti-inflammatory effects, meaning that the bacteria
had not released anti-inflammatory molecules into the
medium (Figure 3B). Finally, to explore possible syn-
<table><caption>TABLE 1 Carbohydrate fermentation profiles of *Bacillus* sp. AM1 and *Paeniclostridium* sp.</caption><thead><tr><th>Target compound</th><th><i>Bacillus</i> sp.</th><th><i>Paeniclostridium</i> sp.</th></tr></thead><tbody><tr><td>Water</td><td>-</td><td>-</td></tr><tr><td>Glycerol</td><td>+</td><td>-</td></tr><tr><td>Erythritol</td><td>-</td><td>-</td></tr><tr><td>D-Arabinose</td><td>-</td><td>-</td></tr><tr><td>L-Arabinose</td><td>+</td><td>-</td></tr><tr><td>Ribose</td><td>+</td><td>-</td></tr><tr><td>D-Xylose</td><td>+</td><td>-</td></tr><tr><td>L-Xylose</td><td>-</td><td>-</td></tr><tr><td>Adonithol</td><td>-</td><td>-</td></tr><tr><td>Methyl xyloside</td><td>-</td><td>-</td></tr><tr><td>Galactose</td><td>-</td><td>-</td></tr><tr><td>D-Glucose</td><td>+</td><td>+</td></tr><tr><td>D-Fructose</td><td>+</td><td>+</td></tr><tr><td>D-Mannose</td><td>+</td><td>-</td></tr><tr><td>Sorbose</td><td>-</td><td>-</td></tr><tr><td>Rhamnose</td><td>-</td><td>-</td></tr><tr><td>Dulcitol</td><td>-</td><td>-</td></tr><tr><td>Inositol</td><td>+</td><td>-</td></tr><tr><td>Mannitol</td><td>+</td><td>-</td></tr><tr><td>Sorbitol</td><td>+</td><td>-</td></tr><tr><td>Methyl-D-mannoside</td><td>-</td><td>-</td></tr><tr><td>Methyl-D-glucoside</td><td>+</td><td>-</td></tr><tr><td>N-acetyl-glucosamine</td><td>-</td><td>-</td></tr><tr><td>Amygdalin</td><td>+</td><td>-</td></tr><tr><td>Arbutin</td><td>+</td><td>-</td></tr><tr><td>Esculine</td><td>+</td><td>-</td></tr><tr><td>Salicin</td><td>+</td><td>-</td></tr><tr><td>Cellobiose</td><td>+</td><td>-</td></tr><tr><td>Maltose</td><td>+</td><td>+</td></tr><tr><td>Lactose</td><td>+</td><td>-</td></tr><tr><td>Melibiose</td><td>+</td><td>-</td></tr><tr><td>Sucrose</td><td>+</td><td>-</td></tr><tr><td>Trehalose</td><td>+</td><td>-</td></tr><tr><td>Inulin</td><td>-</td><td>-</td></tr><tr><td>Melizitose</td><td>-</td><td>-</td></tr><tr><td>D-Raffinose</td><td>+</td><td>-</td></tr><tr><td>Starch</td><td>+</td><td>-</td></tr><tr><td>Glycogen</td><td>+</td><td>-</td></tr><tr><td>Xylitol</td><td>-</td><td>-</td></tr><tr><td>Gentibiose</td><td>-</td><td>-</td></tr><tr><td>Turanose</td><td>-</td><td>-</td></tr><tr><td>Lyxose</td><td>-</td><td>-</td></tr><tr><td>Tagatose</td><td>-</td><td>-</td></tr><tr><td>D-Fucose</td><td>-</td><td>-</td></tr><tr><td>L-Fucose</td><td>-</td><td>+</td></tr></tbody></table>

<table><caption>TABLE 1 (Continued)</caption><thead><tr><th>Target compound</th><th><i>Bacillus</i> sp.</th><th><i>Paeniclostridium</i> sp.</th></tr></thead><tbody><tr><td>D-Arabitol</td><td>-</td><td>-</td></tr><tr><td>L-Arabitol</td><td>-</td><td>-</td></tr><tr><td>Gluconate</td><td>-</td><td>-</td></tr><tr><td>2, Keto-gluconate</td><td>-</td><td>-</td></tr><tr><td>5, Keto-gluconate</td><td>-</td><td>-</td></tr></tbody></table>

ergies, three types of strain combinations were tested: 50% (v/v) of both strains (combination 1), 75% of *Paeniclostridium* sp. and 25% of *Bacillus* sp. AM1 (v/v) (combination 2), and 25% of *Paeniclostridium* sp. and 75% of *Bacillus* sp. AM1 (v/v) (combination 3). Indeed, combination 3 displayed synergistic anti-inflammatory activity, reducing IL-8 production by approximately 60% (Figure 3C).

### Effects of BPA exposure in mice with chemically induced colitis

The effects of BPA exposure in mice with and without DNBS-induced colitis were examined (Figure 4 and 5). Compared to their respective controls, mice that had been exposed to BPA (Veh-BPA-PBS and DNBS-BPA-PBS) showed a similar percent change in body mass during the post injection period.

Mice without DNBS-induced colitis that had been exposed to BPA (Veh-BPA-PBS) did not significantly differ in their physiological metrics, macroscopic scores, microscopic scores, or cytokine levels from mice that had not been exposed (Veh-CO-PBS) (Figure 4A-F). However, the BPA exposed mice (Veh-BPA-PBS) did have slightly higher levels of proinflammatory cytokines in their sera (IL-1α) (Figure 4D). Regarding the immune cell results a significant decrease in Th1 cells (CD3+ CD4+Tbet+ cells) in the MLNs and in Th2 cells (CD3+ CD4+GATA3+ cells) in the spleen was seen in the mice exposed to BPA (Veh-BPA-PBS) (Figure 4I,J).

In contrast, mice with DNBS-induced colitis that had been exposed to BPA (DNBS-BPA-PBS) had significantly higher microscopic scores than did their unexposed counterparts (DNBS-CO-PBS) (Figure 5C, E, F); no differences were observed in percent change in mass or macroscopic scores (Figure 5A, B). Furthermore, compared to the unexposed mice (DNBS-CO-PBS), the exposed mice (DNBS-BPA-PBS) had significantly higher levels of proinflammatory biomarkers (P = 0.03), such as levels of MPO activity (Figure 5D). No statistical differences were observed in the other cytokines (data not shown) or in the immune cell results.
<figure><img src="image_9.png" /><figcaption>FIGURE 3 Anti-inflammatory properties of the BPA-tolerant strains. Interleukin (IL-8) production by HT-29 cells exposed to tumour necrosis factor (TNF)-α in the presence of (A) *Bacillus* sp. AM1 and *Paeniclostridium* sp., (B) supernatant from *Bacillus* sp. AM1 and *Paeniclostridium* sp. cultures, and (C) combinations of *Bacillus* sp. AM1 and *Paeniclostridium* sp. in different proportions. For all graphs, the mean ± standard error of the mean is shown. Results of Kruskal-Wallis tests and Dunn's post-hoc tests; \* P < 0.05, \*\* P < 0.01.</figcaption></figure>

**In vivo effects of the BPA-tolerant strains**

The mouse experiment explored whether the anti-inflammatory properties of BPA-tolerant strains seen *in vitro* were also observed *in vivo*.

For mice with DNBS-induced colitis that had been exposed to BPA, the percent change in body mass was not affected by the treatment with the strains (treatment groups: DNBS-BPA-BAC and DNBS-BPA-PAE vs control: DNBS-BPA-PBS) (Figure 6A). There were also no significant differences in the macroscopic scores (Figure 6B), although the mice treated with the strains (DNBS-BPA-BAC, DNBS-BPA-PAE) tended to have lower scores than the untreated mice (DNBS-BPA-PBS). This difference was significant in the case of the microscopic scores for the mice treated with *Paeniclostridium* sp. (DNBS-BPA-PAE) (Figure 6C-E), which also had longer colons (Figure 6F). Similarly, serum levels of LCN-2 tended to be lower in the mice treated with the strains (DNBS-BPA-BAC, DNBS-BPA-PAE) than in the untreated mice (DNBS-BPA-PBS); again, this difference was only significant for the mice treated with *Paeniclostridium* sp. (Figure 6G). Serum levels of MCP-1 were significantly lower in mice treated with either strain (DNBS-BPA-BAC, DNBS-BPA-PAE) than in the untreated mice (DNBS-BPA-PBS) (Figure 6H). No statistical differences were found for any of the other cytokines (data not shown) or in the immune cell results (data not shown).

For the mice with DNBS-induced colitis that had not been exposed to BPA, the percent change in body mass was again unaffected by the treatment with the strains

(treatment groups: DNBS-CO-BAC, DNBS-CO-PAE vs control: DNBS-CO-PBS) (Figure 7A). That said, the mice treated with *Bacillus* sp. AM1 (DNBS-CO-BAC) tended to gain body mass faster than did untreated mice (DNBS-CO-PBS) in the post injection period. The mice treated with *Bacillus* sp. AM1 (DNBS-CO-BAC) tended to have lower macroscopic scores than did the untreated mice (DNBS-CO-PBS), although this difference was not significant (Figure 7B). Serum levels of LCN-2 (Figure 7C) were lower in the mice treated with the strains (DNBS-CO-BAC, DNBS-CO-PAE) than in the untreated mice (DNBS-CO-PBS), although this difference was not significant. There were also strain-specific effects. Compared to the untreated mice (DNBS-CO-PBS), the mice treated with *Bacillus* sp. AM1 (DNBS-CO-BAC) had significantly higher serum levels of IL-1α and MCP-1 (Figure 7D-E), while the mice treated with *Paeniclostridium* sp. (DNBS-CO-PBS) had significantly lower colon levels of IL-1β, IL-10, and IL-6 (Figure 7F-H). No statistical differences were found in other cytokines measured (data not shown) or in the immune cell results (data not shown).

4 Discussion

It has been hypothesised that chronic exposure to endocrine disruptors, such as BPA, could be a major contributor to the increasing incidence of obesity, immunological disorders, and intestinal diseases (e.g.
<figure><img src="image_10.png" /><figcaption>FIGURE 4 Effects of BPA exposure in mice without DNBS-induced colitis. The treatment group was Veh-BPA-PBS, and the control group was Veh-CO-PBS. (A) Percent change in body mass between the time of the intrarectal injection (day 19) and the end of the experiment (day 22). Colon (B) macroscopic scores and (C) microscopic scores. (D) Interleukin (IL)-1α levels in the sera. Photos showing the histological features seen in the colons of the (E) unexposed mice (Veh-CO-PBS) and (F) exposed mice (Veh-BPA-PBS). Levels of (G) IL-1β and (H) monocyte chemotactic protein 1 (MCP-1) in the colon. (I) Percentage of CD3+CD4+Tbet+ cells in the mesenteric lymph nodes (MLNs). (J) Percentage of CD3+CD4+GATA3+ cells in the MLNs. Results of Mann Whitney U tests; \* *P* < 0.05, \*\* *P* < 0.01.</figcaption></figure>

IBD, Crohn's disease, ulcerative colitis) (Bernstein and Shanahan, 2008; DeLuca et al., 2018; Peinado et al., 2023). IBD is a debilitating lifelong condition for which no cure is currently available (Mishra et al., 2020), but microbiome-based therapies could provide a novel approach for treating the disease's symptoms. Indeed, there is increasing evidence that certain commensal bacteria exhibit anti-inflammatory properties that could help in this regard (Frank et al., 2007; Peterson et al., 2008; Sokol et al., 2008). Against this backdrop, we investigated the capacity of BPA-tolerant bacteria to temper inflammation both in vitro and in vivo. Our study is the first to examine the efficacy of this intervention in an experimental clinical-like context.

There is debate as to whether BPA exposure can exacerbate colitis pathophysiology (DeLuca et al., 2018; Roy et al., 2013). Here, we dosed a group of mice with the tolerable daily intake (TDI) for BPA established by the US EPA (50 µg/kg/day) and then induced col-

itis using DNBS. We demonstrated that BPA exposure slightly worsened colitis disease pathogenesis (increasing microscopic colon damage and MPO activity), as has been found in previous studies (Malaisé et al., 2020). However, in the mice without colitis, BPA exposure did not trigger inflammation in and of itself, perhaps because of the relatively short period of exposure. Taken together, these findings suggest that BPA exposure can exacerbate colitis-related inflammation but may otherwise have minimal effects on baseline inflammation in the absence of colitis. To clarify how BPA exposure was affecting colitis-related inflammation, we assessed levels of proinflammatory biomarkers, such as cytokines. However, we had little foundational information since there have been few studies looking at cytokine expression in mice with DNBS-induced colitis that have been exposed to BPA. In our experiment, in the mice that had been exposed to BPA, we saw increased levels of microscopic damage in colon tissue, as well as increased
<figure><img src="image_11.png" /><figcaption>FIGURE 5 Effects of bisphenol A (BPA) exposure in mice with DNBS-induced colitis. The treatment group was DNBS-BPA-PBS, and the control group was DNBS-CO-PBS. (A) Percent change in body mass between the time of the intrarectal injection (day 19) and the end of the experiment (day 22). Colon (B) macroscopic scores and (C) microscopic scores. (D) Levels of myeloperoxidase (MPO) activity. Photos showing the histological features seen in the colons of the (E) unexposed mice (DNBS-CO-PBS) and (F) exposed mice (DNBS-BPA-PBS). (G) Serum levels of IL-1α. (H) Colon levels of monocyte chemotactic protein 1 (MCP-1). (I) Percentage of CD3+CD4+Tbet+ cells in the spleen. (J) Percentage of CD3+CD4+GATA3+ cells in the spleen. Results of Mann Whitney U tests; \* *P* < 0.05, \*\* *P* < 0.01.</figcaption></figure>

levels of MPO activity, a signal of neutrophil infiltration. Although no significant differences were found in other proinflammatory biomarkers, a trend of increased IL-1α was observed in the BPA group. This pattern existed in mice with and without colitis and indicates the presence of acute inflammation. In general, during the innate immune response, MPO and MCP-1 are released in larger amounts to boost leukocyte chemotaxis and phagocytosis (Fico *et al.*, 2018). Therefore, our results could suggest that innate immune responses are activated by BPA exposure. Our findings are in line with those of previous animal studies, in which a positive correlation was found between BPA exposure and increased levels of proinflammatory cytokines such as IL-6, IL-8, IL-1α, TNF-α, and MPO (DeLuca *et al.*, 2018;

Ogo *et al.*, 2018; Wang *et al.*, 2021). Moreover, research on humans has observed a positive correlation between levels of BPA in the serum or urine and increased levels of inflammatory markers such as MCP-1, IL-6, and TNF-α in the serum (Savastano *et al.*, 2015).

We were interested in exploring the anti-inflammatory properties of BPA-tolerant species (*Bacillus* sp. AM1 and *Paeniclostridium* sp.) because the One Health approach suggests that microbial bioremediation could be a potential strategy in the face of increasing exposure to EDCs, including BPA (Kyrila *et al.*, 2021). *Bacillus* species have long been used as probiotics in humans and animals with the objective of preventing infectious diarrhoea caused by bacteria (Maity and Gupta, 2019; Sulthana *et al.*, 2019; Zani *et al.*, 1998). Furthermore,
<figure><img src="image_12.png" /><figcaption>FIGURE 6 Effects of treatment with bisphenol A (BPA)-tolerant strains on mice with DNBS-induced colitis that had been exposed to BPA. The strains were *Bacillus* sp. AM1 (BAC) and *Paeniclostridium* sp. (PAE); the treatment groups were DNBS-BPA-BAC and DNBS-BPA-PAE; and the control groups were DNBS-BPA-PBS and Veh-BPA-PBS. (A) Percent change in body mass between the time of the intrarectal injection (day 19) and the end of the experiment (day 22). Colon (B) macroscopic scores. Photos showing the histological features seen in the colons of mice treated with (C) *Bacillus* sp. AM1 (DNBS-BPA-BAC) and (D) *Paeniclostridium* sp. (DNBS-BPA-PAE). Colon (E) microscopic scores. (F) Colon length. (G-H) Serum levels of LCN-2 and MCP-1. Results of Kruskal-Wallis tests and Dunn's post hoc tests; \* *P* < 0.05, \*\* *P* < 0.01, and \*\*\* *P* < 0.001.</figcaption></figure>

researchers have looked at the antimicrobial effects of *Bacillus* species isolated from human gut microbiota (Torres-Sánchez *et al.*, 2021). Finally, non-pathogenic *Clostridium* species have been used to treat human gastrointestinal disease (Seki *et al.*, 2003; Shimbo *et*

al., 2005), and they display potentially beneficial anti-inflammatory effects in animals (Okamoto *et al.*, 2000).

Advances in gut microbiota research have spurred interest in NGPs in general and in butyrate-producing species in particular. The latter group of probiotics is being targeted with hopes of increasing colon butyrate
<figure><img src="image_13.png" /><figcaption>FIGURE 7 Effects of treatment with bisphenol A (BPA)-tolerant strains on mice with DNBS-induced colitis. The strains were *Bacillus* sp. AM1 (BAC) and *Paeniclostridium* sp. (PAE); the treatment groups were DNBS-CO-BAC and DNBS-CO-PAE; and the control groups were DNBS-CO-PBS and Veh-CO-PBS. (A) Percent change in body mass between the time of the intrarectal injection (day 19) and the end of the experiment (day 22). (B) Colon macroscopic scores. (C-E) Serum levels of lipocalin-2 (LCN-2), interleukin (IL)-1α, and monocyte chemotactic protein 1 (MCP-1). (F-H) Colon levels of IL-1β, IL-10, and IL-6. Results of Kruskal-Wallis tests and Dunn's post hoc tests; \* *P* < 0.05, \*\* \*P* < 0.01.</figcaption></figure>

production and counteracting disease-related micro-
biota dysbiosis (Boesmans *et al.*, 2018). The use of
butyrate producers such as *Butyricicoccus pullicaeco-
rum* or *Faecalibacterium* species has been shown to
yield beneficial effects in preclinical IBD models (Sokol
*et al.*, 2008, Eeckhaut *et al.*, 2013; Markowiak-Kopeć
and Śliżewska, 2020). Cross-feeding between acetate-
utilising bacteria and butyrate-producing bacteria is an
important metabolic interaction that allows microor-
ganisms to indirectly participate in butyrate produc-
tion (Rios-Covian *et al.*, 2015). We have found that

BPA-tolerant strains primarily produce acetate, but past
research has shown that treatments utilising *Bacillus*
spp. often increase the concentrations of SCFAs, includ-
ing butyrate (Prieto *et al.*, 2014). Given that SCFAs
exert immunomodulatory properties, our study evalu-
ated whether our two target BPA-tolerant strains (*Bacil-
lus* sp. AM1 and *Paeniclostridium* sp.) had an influ-
ence on inflammation mechanisms. We discovered
that both exhibited potent anti-inflammatory proper-
ties: *in vitro*, their presence decreased secretion of IL-8
cytokines by HT-29 cells exposed to the proinflam-
matory cytokine TNF-α. Numerous bacteria taxa have been categorised as NGPs, including *Faecalibacterium* species (Martín *et al.*, 2017), certain strains of lactobacilli (Torres-Maravilla *et al.*, 2016), and *Akkermansia muciniphila* (Zhai *et al.*, 2019); these groups have also exhibited anti-inflammatory properties. Moreover, in our study, we observed that *Bacillus* sp. AM1 did not exhibit antimicrobial resistance. We were unable to determine whether such was also true for *Paeniclostridium* sp. because the EFSA has yet to establish specific cutoffs for this genus, a task to which our work will help contribute. Future studies should analyse whole genome sequences from the taxon to verify the absence of antimicrobial resistance.

In our *in vivo* experiment, data indicated that both *Bacillus* sp. AM1 or *Paeniclostridium* sp. might limit colon damage in the mice, especially after BPA exposure, as reflected in the mice's lower macroscopic and microscopic scores. However, the effect was only significant for *Paeniclostridium* sp., and had a stronger reducing effect on inflammatory markers. Although previous research has observed a positive correlation between BPA exposure and LCN-2 levels (Malaisé *et al.*, 2020), we saw no such pattern, perhaps because of the duration of BPA exposure in our experiment. Interestingly, the mice treated with *Paeniclostridium* sp. in particular displayed lower LCN-2 levels. This result implies that *Paeniclostridium* sp. may have the capacity to mitigate the effects of BPA exposure, if LCN-2 is a reliable biomarker of the latter. Other studies using mouse models of acute colitis have found comparable results utilising treatments based on *Christensenella minuta* (Kropp et al., 2021) and *A. muciniphila* (Zhai et al., 2019). Moreover, in our study, for the mice with colitis that had not been exposed to BPA, treatment with *Paeniclostridium* sp. led to significantly lower levels of IL-1β and IL-6 in the colon. This finding reveals that treatments employing this strain could help reduce cytokine levels, which may climb in association with BPA exposure (Wang et al., 2021). Other research conducted in mouse models has shown that BPA exposure also enhances MCP-1 production (Sugita-Konishi et al., 2003). Interestingly, we found that, for mice with colitis that had been exposed to BPA, treatment with either target strain significantly reduced MCP-1 levels, suggesting that the presence of the BPA-tolerant strains could temper the immunological effects provoked by BPA exposure.

Our study's limitations are mainly methodological. First, the duration of BPA exposure was relatively short, especially compared to the potentially unlimited exposure that human populations experience. Second, while

it was helpful to use a model of acute inflammation as a first step, future research should employ models of chronic models is necessary to ensure that the observed results closely mimic the chronic conditions seen in humans. These issues aside, our study represents a pioneering effort to explore the abilities of NGPs to potentially modulate the degree to which BPA exposure exacerbates colitis severity. We used bacterial strains that had been isolated from humans, which may hold greater therapeutic promise for translational relevance, as they likely influence similar pathways and mechanisms in mice and humans alike. However, further research is necessary to determine how well our results in mice translate to humans, including research that evaluates the efficacy and safety of these probiotics in human populations. While animal models can offer valuable insights, it is important to acknowledge the inherent limitations of animal models when it comes to replicating the complexity of human physiology.

## 5 Conclusions

We found that proinflammatory biomarkers occurred at higher levels in mice exposed to BPA, indicating the presence of an altered inflammation profile. These results suggest a potential association between BPA exposure and the activation of innate immune responses, although future studies are needed to fully elucidate the underlying mechanisms involved. We discovered that two BPA-tolerant bacterial strains – *Bacillus* sp. AM1 and *Paeniclostridium* sp. – had potent anti-inflammatory properties; *in vitro*, their presence decreased secretion of IL-8 cytokines by HT-29 cells exposed to TNF-α, a powerful proinflammatory cytokine. *In vivo*, mice treated with the BPA-tolerant strains experienced less colon damage and had lower levels of MCP-1, LCN-2, and proinflammatory cytokines (IL-1β and IL-6), the latter only with *Paeniclostridium* sp. treatment. Future work should elucidate the mechanisms by which BPA provokes immunopathological effects as well as potential modulation strategies. In these efforts, it will be important to determine the molecular pathways by which xenobiotics generate inflammation.

We found that our target BPA-tolerant strains displayed anti-inflammatory properties that, may have the potential to mitigate certain adverse immunological effects in contexts where colitis is aggravated by BPA exposure. Our findings lay the groundwork for new therapeutic approaches, where inflammatory conditions
(e.g. colitis) exacerbated by xenobiotics could be modulated by specific xenobiotic-tolerant bacterial species.

## Supplementary material

Supplementary material is available online at: https://doi.org/10.6084/m9.fishshare.26976805

**Figure S1.** Effects of bile salt and pH shock on the growth (cfu/ml) of *Bacillus* sp. AM1 and *Paeniclostridium* sp.

**Table S1.** Antibiotic resistance results for *Bacillus* sp. AM1

**Table S2.** Antibiotic resistance results for *Paeniclostridium* sp.

## Authors' contribution

Conceptualisation, MA and RM; methodology, RM, and PL; investigation and formal analysis, AL-M, CK, and FP; funding acquisition, MA, AL-M, PL, and RM; writing-original draft preparation, AL-M; writing-review and editing, RM, MA, PL, CC, CK, DR-C, and FP; supervision, RM, CC, and DR-C. All authors have read and approved the final version of the manuscript.

## Conflict of interest

The authors declare no conflict of interest.

## Funding

This study was part of Ana López-Moreno's doctoral thesis and was financially supported by EFSA's EU-FORA Fellowship Programme (Cycle 2022/2023) as well as by Projects of Excellence funding from Andalucía's Council for University Studies, Research, and Innovation (Consejería de Universidad, Investigación e Innovación; P21-00341 DiETOXµBio).

## References

* Ameho, C.K., Adjei, A.A., Harrison, E.K., Takeshita, K., Morioka, T., Arakaki, Y., Ito, E., Suzuki, I., Kulkarni, A.D., Kawajiri, A. and Yamamoto, S., 1997. Prophylactic effect of dietary glutamine supplementation on interleukin 8 and tumour

necrosis factor alpha production in trinitrobenzene sulphonic acid induced colitis. Gut 41: 487-493.

* Bansal, A., Rashid, C., Xin, F., Li, C., Polyak, E., Duemler, A., van der Meer, T., Stefaniak, M., Wajid, S., Doliba, N., Bartolomei, M.S. and Simmons, R.A., 2017. Sex- and dose-specific effects of maternal bisphenol a exposure on pancreatic islets of first- and second-generation adult mice offspring. Environmental Health Perspectives 125: 097022.

* Barone, M., Ramayo-Caldas, Y., Estellé, J., Tambosco, K., Chadi, S., Maillard, F., Gallopin, M., Planchais, J., Chain, F., Kropp, C., Rios-Covian, D., Sokol, H., Brigidi, P., Langella, P. and Martín, R., 2023. Gut barrier-microbiota imbalances in early life lead to higher sensitivity to inflammation in a murine model of C-section delivery. Microbiome 11: 140.

* Bernstein, C.N. and Shanahan, F., 2008. Disorders of a modern lifestyle: reconciling the epidemiology of inflammatory bowel diseases. Gut 57: 1185-1191.

* Boesmans, L., Valles-Colomer, M., Wang, J., Eeckhaut, V., Falony, G., Ducatelle, R., Van Immerseel, F., Raes, J. and Verbeke, K., 2018. Butyrate producers as potential next-generation probiotics: safety assessment of the administration of *Butyricicoccus pullicaecorum* to healthy volunteers. mSystems 3: e00094-18.

* Bradley, P.P., Priebat, D.A., Christensen, R.D. and Rothstein, G., 1982. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. Journal of Investigative Dermatology 78: 206-209.

* Danese, S., Sans, M. and Fiocchi, C., 2004. Inflammatory bowel disease: the role of environmental factors. Autoimmunity Reviews 3: 394-400.

* DeLuca, J.A., Allred, K.F., Menon, R., Riordan, R., Weeks, B.R., Jayaraman, A. and Allred, C.D., 2018. Bisphenol-A alters microbiota metabolites derived from aromatic amino acids and worsens disease activity during colitis. Experimental Biology and Medicine 243: 864-875.

* Eeckhaut, V., Machiels, K., Perrier, C., Romero, C., Maes, S., Flahou, B., Steppe, M., Haesebrouck, F., Sas, B., Ducatelle, R., Vermeire, S. and Van Immerseel, F., 2013. *Butyricicoccus pullicaecorum* in inflammatory bowel disease. Gut 62: 1745-1752.

* European Committee on Antimicrobial Susceptibility Testing (EUCAST), 2023. When there are no breakpoints? Available at: https://www.eucast.org/clinical_breakpoints_and_dosing/when_there_are_no_breakpoints

* European Food Safety Authority Panel on Additives and Products or Substances used in Animal Feed (EFSA FEEDAP), 2012. Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA Journal 10: 2740.

* Fico, B.G., Whitehurst, M., Slusher, A.L., Mock, J.T., Maharaj, A., Dodge, K.M. and Huang, C.-J., 2018. The comparison
of acute high-intensity interval exercise vs continuous
moderate-intensity exercise on plasma calprotectin and
associated inflammatory mediators. Physiology and Behav-
ior 183: 27-32.

Frank, D.N., St Amand, A.L., Feldman, R.A., Boedeker, E.C., Harpaz, N. and Pace, N.R., 2007. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proceedings of the National Academy of Sciences of the USA 104: 13780-13785.

Humphries, R.M., Ambler, J., Mitchell, S.L., Castanheira, M., Dingle, T., Hindler, J.A., Koeth, L. and Sei, K., 2018. CLSI methods development and standardization working group best practices for evaluation of antimicrobial susceptibility tests. Journal of Clinical Microbiology 56. https://doi.org/10.1128/JCM.01934-17

Jalal, N., Surendranath, A.R., Pathak, J.L., Yu, S. and Chung, C.Y., 2018. Bisphenol A (BPA) the mighty and the mutagenic. Toxicology Reports 5: 76-84.

Kechaou, N., Chain, F., Gratadoux, J.-J., Blugeon, S., Bertho, N., Chevalier, C., Le Goffic, R., Courau, S., Molimard, P., Chatel, J.M., Langella, P. and Bermúdez-Humarán, L.G., 2013. Identification of one novel candidate probiotic *Lactobacillus plantarum* strain active against influenza virus infection in mice by a large-scale screening. Applied and Environmental Microbiology 79: 1491-1499.

Kropp, C., Le Corf, K., Relizani, K., Tambosco, K., Martinez, C., Chain, F., Rawadi, G., Langella, P., Claus, S.P. and Martin, R., 2021. The keystone commensal bacterium *Christensenella minuta* DSM 22607 displays anti-inflammatory properties both *in vitro* and *in vivo*. Scientific Reports 11: 11494.

Kyrila, G., Katsoulas, A., Schoretsaniti, V., Rigopoulos, A., Rizou, E., Doulgeridou, S., Sarli, V., Samanidou, V. and Touraki, M., 2021. Bisphenol A removal and degradation pathways in microorganisms with probiotic properties. Journal of Hazardous Materials 413: 125363.

Lan, A., Bruneau, A., Bensaada, M., Philippe, C., Bellaud, P., Rabot, S. and Jan, G., 2008. Increased induction of apoptosis by *Propionibacterium freudenreichii* TL133 in colonic mucosal crypts of human microbiota-associated rats treated with 1,2-dimethylhydrazine. British Journal of Nutrition 100: 1251-1259.

Larauche, M., Mulak, A., Kim, Y.S., Labus, J., Million, M. and Taché, Y., 2012. Visceral analgesia induced by acute and repeated water avoidance stress in rats: sex difference in opioid involvement. Neurogastroenterology and Motility 24: 1031-1047.

Lear, G., Kingsbury, J.M., Franchini, S., Gambarini, V., Maday, S.D.M., Wallbank, J.A., Weaver, L. and Pantos, O., 2021. Plastics and the microbiome: impacts and solutions. Environmental Microbiome 16: 2.

Legaki, E. and Gazouli, M., 2016. Influence of environmental factors in the development of inflammatory bowel diseases. World Journal of Gastrointestinal Pharmacology and Therapeutics 7: 112-125.

López-Moreno, A., Acuña, I., Torres-Sánchez, A., Ruiz-Moreno, Á., Cerk, K., Rivas, A., Suárez, A., Monteoliva-Sánchez, M. and Aguilera, M., 2021. Next generation probiotics for neutralizing obesogenic effects: taxa culturing searching strategies. *Nutrients* 13: 1617.

López-Moreno, A., Ruiz-Moreno, Á., Pardo-Cacho, J., Cerk, K., Torres-Sánchez, A., Ortiz, P., Úbeda, M. and Aguilera, M., 2022. Culturing and molecular approaches for identifying microbiota taxa impacting children's obesogenic phenotypes related to xenobiotic dietary exposure. *Nutrients* 14: 241.

Maity, C. and Gupta, A.K., 2019. A prospective, interventional, randomized, double-blind, placebo-controlled clinical study to evaluate the efficacy and safety of *Bacillus coagulans* LBSC in the treatment of acute diarrhea with abdominal discomfort. European Journal of Clinical Pharmacology 75: 21-31.

Malaisé, Y., Lencina, C., Placide, F., Bacquié, V., Cartier, C., Olier, M., Buettner, M., Wallbrecht, M., Ménard, S. and Guzylack-Piriou, L., 2020. Oral exposure to bisphenols induced food intolerance and colitis *in vivo* by modulating immune response in adult mice. Food and Chemical Toxicology 146: 111773.

Markowiak-Kopeć, P. and Śliżewska, K., 2020. The effect of probiotics on the production of short-chain fatty acids by human intestinal microbiome. *Nutrients* 12: 1107.

Martín, R., Chain, F., Miquel, S., Lu, J., Gratadoux, J.-J., Sokol, H., Verdu, E.F., Bercik, P., Bermúdez-Humarán, L.G. and Langella, P., 2014. The commensal bacterium *Faecalibacterium prausnitzii* is protective in DNBS-induced chronic moderate and severe colitis models. Inflammatory Bowel Diseases 20: 417-430.

Martín, R., Miquel, S., Benevides, L., Bridonneau, C., Robert, V., Hudault, S., Chain, F., Berteau, O., Azevedo, V., Chatel, J.M., Sokol, H., Bermúdez-Humarán, L.G., Thomas, M. and Langella, P., 2017. Functional characterization of novel *Faecalibacterium prausnitzii* strains isolated from healthy volunteers: a step forward in the use of *F. prausnitzii* as a next-generation probiotic. Frontiers in Microbiology 8: 1226.

Mishra, R., Dhawan, P., Srivastava, A.S. and Singh, A.B., 2020. Inflammatory bowel disease: Therapeutic limitations and prospective of the stem cell therapy. World Journal of Stem Cells 12: 1050-1066.

Ng, S.C., Shi, H.Y., Hamidi, N., Underwood, F.E., Tang, W., Benchimol, E.I., Panaccione, R., Ghosh, S., Wu, J.C.Y., Chan, F.K.L., Sung, J.J.Y. and Kaplan, G.G., 2017. Worldwide inci-
dence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. The Lancet 390: 2769-2778.

Ogo, F.M., de Lion Siervo, G.E.M., Staurengo-Ferrari, L., de Oliveira Mendes, L., Luchetta, N.R., Vieira, H.R., Fattori, V., Verri, W.A., Scarano, W.R. and Fernandes, G.S.A., 2018. Bisphenol A exposure impairs epididymal development during the peripubertal period of rats: inflammatory profile and tissue changes. Basic and Clinical Pharmacology and Toxicology 122: 262-270.

Okamoto, T., Sasaki, M., Tsujikawa, T., Fujiyama, Y., Bamba, T. and Kusunoki, M., 2000. Preventive efficacy of butyrate enemas and oral administration of *Clostridium butyricum* M588 in dextran sodium sulfate-induced colitis in rats. Journal of Gastroenterology 35: 341-346.

Peinado, F.M., Iribarne-Durán, L.M. and Artacho-Cordón, F., 2023. Human exposure to bisphenols, parabens, and benzophenones, and its relationship with the inflammatory response: a systematic review. International Journal of Molecular Sciences 24: 7325.

Peterson, D.A., Frank, D.N., Pace, N.R. and Gordon, J.I., 2008. Metagenomic approaches for defining the pathogenesis of inflammatory bowel diseases. Cell Host and Microbe 3: 417-427.

Prieto, M.L., O'Sullivan, L., Tan, S.P., McLoughlin, P., Hughes, H., O'Donovan, O., Rea, M.C., Kent, R.M., Cassidy, J.P., Gardiner, G.E. and Lawlor, P.G., 2014. Evaluation of the efficacy and safety of a marine-derived *Bacillus* strain for use as an in-feed probiotic for newly weaned pigs. PLoS ONE 9: e88599.

Reddivari, L., Veeramachaneni, D.N.R., Walters, W.A., Lozupone, C., Palmer, J., Hewage, M.K.K., Bhatnagar, R., Amir, A., Kennett, M.J., Knight, R. and Vanamala, J.K.P., 2017. Perinatal bisphenol A exposure induces chronic inflammation in rabbit offspring via modulation of gut bacteria and their metabolites. mSystems 2: e00093-17.

Rios-Covian, D., Gueimonde, M., Duncan, S.H., Flint, H.J. and de los Reyes-Gavilan, C.G., 2015. Enhanced butyrate formation by cross-feeding between *Faecalibacterium prausnitzii* and *Bifidobacterium adolescentis*. FEMS Microbiology Letters 362: fnv176.

Roy, A., Gaylo, A., Cao, W., Saubermann, L.J. and Lawrence, B.P., 2013. Neither direct nor developmental exposure to bisphenol A alters the severity of experimental inflammatory colitis in mice. Journal of Immunotoxicology 10: 334-340.

Savastano, S., Tarantino, G., D'Esposito, V., Passaretti, F., Cabaro, S., Liotti, A., Liguoro, D., Perruolo, G., Ariemma, F., Finelli, C., Beguinot, F., Formisano, P. and Valentino, R., 2015. Bisphenol-A plasma levels are related to inflammatory markers, visceral obesity and insulin-resistance: a

cross-sectional study on adult male population. Journal of Translational Medicine 13: 169.

Seki, H., Shiohara, M., Matsumura, T., Miyagawa, N., Tanaka, M., Komiyama, A. and Kurata, S., 2003. Prevention of antibiotic-associated diarrhea in children by *Clostridium butyricum* MIYAIRI. Pediatrics International 45: 86-90.

Sharif, K., Kurnick, A., Coplan, L., Alexander, M., Watad, A., Amital, H. and Shoenfeld, Y., 2022. The putative adverse effects of bisphenol A on autoimmune diseases. Endocrine, Metabolic and Immune Disorders – Drug Targets 22: 665-676.

Sheehan, D., Moran, C. and Shanahan, F., 2015. The microbiota in inflammatory bowel disease. Journal of Gastroenterology 50: 495-507.

Shimbo, I., Yamaguchi, T., Odaka, T., Nakajima, K., Koide, A., Koyama, H. and Saisho, H., 2005. Effect of *Clostridium butyricum* on fecal flora in *Helicobacter pylori* eradication therapy. World Journal of Gastroenterology 11: 7520-7524.

Sokol, H., Pigneur, B., Waterlot, L., Lakhdari, O., Bermúdez-Humarán, L.G., Gratadoux, J.-J., Blugeon, S., Bridonneau, C., Furet, J.-P., Corthier, G., Grangette, C., Vasquez, N., Pochart, P., Trugnan, G., Thomas, G., Blottière, H.M., Doré, J., Marteau, P., Seksik, P. and Langella, P., 2008. *Faecalibacterium prausnitzii* is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proceedings of the National Academy of Sciences of the USA 105: 16731-16736.

Sugita-Konishi, Y., Shimura, S., Nishikawa, T., Sunaga, F., Naito, H. and Suzuki, Y., 2003. Effect of bisphenol A on non-specific immunodefenses against non-pathogenic *Escherichia coli*. Toxicology Letters 136: 217-227.

Sulthana, A., Lakshmi, S.G. and Madempudi, R.S., 2019. Genome sequencing and annotation of *Bacillus subtilis* UBBS-14 to ensure probiotic safety. Journal of Genomics 7: 14-17.

Torres-Maravilla, E., Lenoir, M., Mayorga-Reyes, L., Allain, T., Sokol, H., Langella, P., Sánchez-Pardo, M.E. and Bermúdez-Humarán, L.G., 2016. Identification of novel anti-inflammatory probiotic strains isolated from pulque. Applied Microbiology and Biotechnology 100: 385-396.

Torres-Sánchez, A., Pardo-Cacho, J., López-Moreno, A., Ruiz-Moreno, Á., Cerk, K. and Aguilera, M., 2021. Antimicrobial effects of potential probiotics of *Bacillus* spp. isolated from human microbiota: *in vitro* and *in silico* methods. Microorganisms 9: 1615.

US Environmental Protection Agency (US EPA), 2023. Bisphenol A CASRN 80-05-7 | DTXSID7020182 | IRIS | US EPA, ORD. Available at: https://cfpub.epa.gov/ncea/iris2/chemicalLanding.cfm?substanceNmbr=356
Veiga, P., Suez, J., Derrien, M. and Elinav, E., 2020. Moving from probiotics to precision probiotics. Nature Microbiology 5: 878-880.

Wang, K., Qiu, L., Zhu, J., Sun, Q., Qu, W., Yu, Y., Zhao, Z., Yu, Y. and Shao, G., 2021. Environmental contaminant BPA causes intestinal damage by disrupting cellular repair and injury homeostasis in vivo and in vitro. Biomedicine and Pharmacotherapy 137: 111270.

West, C.E., Renz, H., Jenmalm, M.C., Kozyrskyj, A.L., Allen, K.J., Vuillermin, P., Prescott, S.L., MacKay, C., Salminen, S., Wong, G., Sinn, J., Stokholm, J., Bisgaard, H., Pawankar, R., Noakes, P., Kesper, D. and Tulic, M., 2015. The gut microbiota and inflammatory noncommunicable diseases: Associations and potentials for gut microbiota therapies. Journal of Allergy and Clinical Immunology 135: 3-13.

World Health Organization (WHO), 2023. Non communicable diseases. WHO, Geneva, Switzerland. Available at:

https://www.who.int/news-room/fact-sheets/detail/noncommunicable-diseases

Yin, F., Huang, X., Lin, X., Chan, T.F., Lai, K.P. and Li, R., 2022. Analyzing the synergistic adverse effects of BPA and its substitute, BHPF, on ulcerative colitis through comparative metabolomics. Chemosphere 287: 132160.

Zani, J.L., da Cruz, F.W., dos Santos, A.F. and Gil-Turnes, C., 1998. Effect of probiotic CenBiot on the control of diarrhoea and feed efficiency in pigs. Journal of Applied Microbiology 84: 68-71.

Zhai, R., Xue, X., Zhang, L., Yang, X., Zhao, L. and Zhang, C., 2019. Strain-specific anti-inflammatory properties of two *Akkermansia muciniphila* strains on chronic colitis in mice. *Frontiers in Cellular and Infection Microbiology* 9: 239.