# Postbiotic Effect of Escherichia coli CEC15 and Escherichia coli Nissle 1917 on a Murine Model of 5-FU-induced Intestinal Mucositis

Rafael de Assis Glória, Tales Fernando Da Silva, Tomás Andrade Magalhães

Gomes, Kátia Duarte Vital, Simone Odília Antunes Fernandes, Valbert

Nascimento Cardoso, Ênio Ferreira, Jean-Marc Chatel, Philippe Langella,

Claire Cherbuy, et al.

► To cite this version:

Rafael de Assis Glória, Tales Fernando Da Silva, Tomás Andrade Magalhães Gomes, Kátia Duarte Vital, Simone Odília Antunes Fernandes, et al. Postbiotic Effect of Escherichia coli CEC15 and Escherichia coli Nissle 1917 on a Murine Model of 5-FU-induced Intestinal Mucositis. Probiotics and Antimicrobial Proteins, 2024, 10.1007/s12602-024-10414-0. hal-05156875

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# Postbiotic effect of Escherichia coli CEC15 and Escherichia coli Nissle 1917 on amurine model of 5-FU-induced intestinal mucositis

**Rafael de Assis Glória**

Institute of Biological Sciences, Department of Genetics, Ecology, and Evolution, Federal University of Minas Gerais

**Tales Fernando da Silva**

Institute of Biological Sciences, Department of Genetics, Ecology, and Evolution, Federal University of Minas Gerais

**Tomás Andrade Magalhães Gomes**

Institute of Biological Sciences, Department of Genetics, Ecology, and Evolution, Federal University of Minas Gerais

**Kátia Duarte Vital**

Department of clinical and toxicological analysis, Faculty of Pharmacy, Federal University of Minas Gerais

**Simone Odília Antunes Fernandes**

Department of clinical and toxicological analysis, Faculty of Pharmacy, Federal University of Minas Gerais

**Valbert Nascimento Cardoso**

Department of clinical and toxicological analysis, Faculty of Pharmacy, Federal University of Minas Gerais

**Ênio Ferreira**

Department of general pathology, Federal University of Minas Gerais

**Jean-Marc Chatel**

Université Paris Saclay, INRAE, AgroParisTech, UMR1319, MICALIS

**Philippe Langella**

Université Paris Saclay, INRAE, AgroParisTech, UMR1319, MICALIS

**Claire Cherbuy**

Université Paris Saclay, INRAE, AgroParisTech, UMR1319, MICALIS

**Yves Le Loir**

INRAE, Institut Agro, STLO, 35042

**Gwénaël Jan**

INRAE, Institut Agro, STLO, 35042

**Éric Guédon**
Institute of Biological Sciences, Department of Genetics, Ecology, and Evolution, Federal University of Minas Gerais

## Research Article

**Keywords:** Escherichia coli, Postbiotics, mucositis, 5-Fluorouracyl, CEC15, Nissle 1917

**Posted Date:** November 15th, 2023

**DOI:** https://doi.org/10.21203/rs.3.rs-3592871/v1

**License:** © Ⓟ This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** No competing interests reported.

**Version of Record:** A version of this preprint was published at Probiotics and Antimicrobial Proteins on November 26th, 2024. See the published version at https://doi.org/10.1007/s12602-024-10414-0.
# Abstract

Postbiotics are preparations of inanimate microorganisms and/or their components that confers a health benefit on the host. The *Escherichia coli* strains Nissle 1917 (EcN) and CEC15 have shown beneficial effects, in their live form, in murine models of intestinal inflammation, such as colitis and mucositis. However, the use of live bacteria, in special by immunocompromised individuals, had raised concerns. Based on this, the present study evaluated the effects as postbiotic of heat-inactivated, and cell-free supernatant preparations of EcN and CEC15 in attenuating 5-fluorouracil (5-FU)-induced intestinal mucositis in mice and compared them with the probiotic effects of the live preparations.
BALB/c mice were fed daily, by gavage, with 10¹⁰ CFU of live or inactivated bacteria, or with 300 µL of cell-free supernatant for 12 days. On the 10th day, all animals, except for the control group, received an intraperitoneal injection of 5-FU (300 mg/kg). After 72 h of 5-FU administration, animals were euthanized, and the ileum and blood were collected for analysis. Treatments with live and heat-inactivated CEC15 mitigated weight loss, preserved intestinal length, reduced histological damage, maintained goblet cells, decreased neutrophil infiltration, and modulated expression of inflammatory and barrier genes, when compared to 5-FU mucositis controls. EcN showed more limited effects. CEC15 upregulated mRNA expression of the mucin *MUC2* and tight junction protein *TJP1*. Overall, CEC15 demonstrated protective effects against 5-FU-induced mucositis, whatever the way it was administered: live bacteria, heat-inactivated bacteria, or cell-free supernatant. This suggests CEC15 mediates a protective response via secreted metabolites and does not require viability.

# 1. INTRODUCTION

Probiotics are live microorganisms that, when administered in adequate amounts, can potentially bring health benefits to the host [1]. Lactic acid bacteria (LAB), such as members of the *Lactobacillaceae* family, *Lactococcus* spp., and *Streptococcus* spp., represent most probiotics found on the market today, together with *Bifidobacterium* spp., *Propionibacterium* spp., and the yeast *Saccharomyces* spp. Most of these organisms are found naturally in the human gastrointestinal tract. Nonetheless, some Gram-negative bacteria have also been identified with beneficial effects on the host [2].

One of the most studied Gram-negative probiotic bacteria is *Escherichia coli* Nissle 1917 (EcN). EcN is considered safe and effective and has been approved by the FDA for treating acute diarrhea in children. Studies have shown that EcN can produce various bioactive compounds that may have beneficial effects on human health. For example, EcN can produce bacteriocins, small peptides with antimicrobial activity against many pathogenic bacteria [3]. In addition, EcN can produce short-chain fatty acids, such as acetic acid, that promote a stimulatory effect on intestinal motility [4], through the modulation of the gut microbiota, by colonizing the human gastrointestinal tract (GIT), compete with other bacteria, and interact with host cells [5-7], and by modulating the diversity of the gut microbiota by reducing the number of pathogenic bacteria, which may lead to improved gut function and overall health [8-14].
Although EcN is generally considered safe, some concerns exist regarding its use in immunocompromised patients. These concerns relate to characteristics suggested by EcN genome sequence and which may negatively affect the health of these individuals, such as the toxin called colibactin, a potential genotoxic agent that can lead to DNA damage in intestinal cells and increase the risk of colorectal cancer [15, 16]. Finally, some studies have suggested that EcN may increase the production of pro-inflammatory cytokines in patients with inflammatory bowel disease (IBD). Although the production of pro-inflammatory cytokines is a normal immune system response to infection, this response can be exacerbated in IBD patients, leading to damage to intestinal tissue [17]. Therefore, there are concerns regarding the use of EcN in IBD patients.

In 2013, *E. coli* CEC15 (CEC15) was isolated from the feces of a newborn rat during a study that sought to identify bacteria with probiotic potential [18]. This study showed that early colonization by CEC15 triggered an adaptive response in the colonic epithelium of the mice, leading to the remodeling of the epithelial tissue, thickening of the mucus layer, and affecting ion and water transport. Colonization by *E. coli* CEC15 had a long-term effect on the colonic epithelium, leading to a shift in intestinal homeostasis toward a new equilibrium state [18].

Since then, new studies have sought to characterize this strain by evaluating its beneficial potential in *vivo* in murine models of intestinal inflammation. One work using gnotobiotic mice evaluated the effects of CEC15 colonization of the gastrointestinal tract, in comparison with the commercial EcN strain. CEC15 colonization increased expression of genes related to immunity and to defense against pathogens, mainly due to an increased production of mucin, a protein that coats the intestinal epithelium and helps preventing pathogen entry, evidencing the potential of this strain in modulating gut defense mechanisms [19]. More recently we evaluated and compared the protective effect of both strains, EcN and CEC15, in a model of intestinal mucositis induced by the drug 5-Fluorouracil (5-FU) in mice [20]. This model presents high severity in the inflammatory process and allows a better evaluation of probiotics' anti-inflammatory effects [21]. We demonstrated the preventive beneficial issue of CEC15 in this acute inflammatory model [20].

Recent research emphasizes the effect of various bacterial components, suggesting that live microorganisms are not required for beneficial action [22]. Furthermore, the use of killed microorganisms (also named paraprobiotics or ghost probiotics) and of their secreted products (also named metabiotics) may offer safety advantages, reducing the risk of infection and microbial translocation observed in immunocompromised individuals after probiotic administration [22, 23]. Therefore, an alternative to the possible unwanted effects caused by probiotics is the use of postbiotics [24]. A postbiotic is a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host. [25]. Because they are inactivated or inert components, postbiotics are considered safer than probiotics given the concerns related to the use of live microorganisms in specific patient populations, including immunosuppressed individuals [26]. These preparations can significantly reduce the risk of microbial translocation and infections, are more stable than probiotics, and have a lower risk of associated antimicrobial resistance.
Some of the mechanisms associated with the efficacy of postbiotics include epithelial barrier enhancement, immunomodulatory effects, and antimicrobial effects [27–29]. Currently, postbiotics are applied as a promising treatment strategy for health conditions, especially in gastrointestinal disorders [30]. Therefore, their application would be an efficient complement to probiotics and a driving force for developing a comprehensive health industry. Here we evaluated the potential beneficial role of postbiotic preparations from *E. coli* CEC15 and Nissle 1917 strains in the context of an acute intestinal inflammation, an in vivo intestinal mucositis induced by the cancer chemotherapy drug 5-FU. For that, we compared the effect of live bacteria (as control), heat-inactivated bacteria, and cell-free supernatant of both strains on the intestinal barrier and on inflammatory markers.

## 2. MATERIALS AND METHODS

### 2.1. Animals

Male BALB/c mice aged 6 weeks (n = 8/group), acquired from the Central Animal Facility of the Federal University of Minas Gerais (UFMG), were used. During the experiments, the mice were kept in mini-isolators in the animal house of the Department of Genetics, Ecology and Evolution of the Institute of Biological Sciences (ICB), UFMG, fed with standard mouse chow, water *ad libitum*, and a 12-hour light cycle. The experimental protocols used in this study were performed according to the guidelines approved by the Ethics Committee on Animal Use (CEUA) - UFMG (Protocol: 210/2022).

### 2.2. Bacterial cultures and sample preparation

For this experiment, both *E. coli* strains (Nissle 1917 and CEC15) were grown in LB medium with shaking (150 rpm) for 18 h at 37°C. To collect the supernatant, 30 mL of culture was centrifuged at 6000 x g for 15 min. The supernatant was subsequently filtered using a 0.22 µm syringe filter and aliquoted into 300 µL samples, kept at -80°C until use for the inactivated bacteria. The overnight culture was diluted to an optical density of 0.6 (λ = 600 nm) (OD<sub>600</sub>), equivalent to 2 x 10<sup>8</sup> CFU/mL, and centrifuged at 6000 x g for 15 minutes. The supernatant was discarded, the pellet was washed three times with sterile PBS, and finally resuspended in 3 mL of PBS. The wash was then incubated at 65°C for 1 hour to inactivate the bacteria, and after incubation, 300 µL samples were aliquoted. The inactivated bacteria and supernatant were seeded on LB agar and incubated for 48 hours at 37°C to confirm sterility. The live bacteria doses were prepared daily throughout the experiment. For that, overnight culture was diluted to an OD<sub>600</sub> of 0.2, to a final volume of 30 mL, and grown until reach an OD<sub>600</sub> of 0.6 and centrifuged at 6000 x g for 15 minutes. The supernatant was discarded, the pellet was washed three times with sterile PBS, and resuspended in 3 mL of PBS. The doses were aliquoted into microtubes in 300 µL volumes.

### 2.3. Experimental model and animal groups

The animals were randomly divided into eight experimental groups as described below: Ct- (negative control), MUC (induction of mucositis by 5-FU without treatment), EcN (treated with live EcN and induction of mucositis by 5-FU), EcNi (treated with heat-inactivated EcN and induction of mucositis by 5-FU).
FU), EcNs (treated with supernatant of EcN culture and induction of mucositis by 5-FU), CEC (treated with live CEC15 and induction of mucositis by 5-FU), CECi (treated with heat-inactivated CEC15 and induction of mucositis by 5-FU), CECs (treated with culture supernatant of CEC15 and induction of mucositis by 5-FU). Over 12 days, all animals received daily by gavage 300 µL of live, inactivated bacteria or supernatant according to the corresponding group, except for the Ct- and MUC-groups that received PBS. The experimental design is shown in Fig. 1.

The induction of intestinal mucositis in mice was based on the model previously described by Carvalho and colleagues (Carvalho et al., 2017). The animals received an intraperitoneal injection containing 300 mg/kg of 5-FU in a single dose for induction of inflammation 10 days after the beginning of the experimental period. Sterile PBS administered by the same route was used as a control. After 72 hours after the induction of mucositis, the animals were euthanized by deep anesthesia with ketamine + xylazine (270 and 90 mg/kg, respectively) to perform the analyses.

## 2.4. Food/water consumption and weight change

The feed was weighed on a semi-analytical scale during all the days of the experiment, and the water volume was measured daily with the aid of a volumetric measuring cylinder to evaluate the food and water consumption. The results were presented in two periods: before the induction of the disease (days 0–10) and after the induction of inflammation (days 11–13), to verify if the proposed treatments can improve the clinical picture of low food and water consumption caused by intestinal mucositis. The animals were weighed on semi-analytical scales every day of the experiment to assess weight variation in grams (weight gain or loss before and after the induction of mucositis) in the animals.

## 2.5. Evaluation of intestinal permeability

To evaluate the intestinal permeability of the animals, on the 13th experimental day, all the mice received by gavage 100 µL of a solution containing 18.5 MBq of diethylenetriaminepentaacetic acid radiolabeled with technetium-99m (⁹⁹mTc-DTPA) with a radiochemical purity of 99.4% performed by chromatography on Wattman paper. After 4 hours, all mice were euthanized, and blood was collected by axillary puncture, weighed, and placed in appropriate tubes for radioactivity determination. The levels of radioactivity in the blood were determined using an automated gamma counter (PerkinElmer Wallac Wizard 1470-020 Gamma Counter; PerkinElmer Inc.) The results are presented as the percentage of radiation dose, which was calculated using the following equation:

$$ \%DTPA/g = \frac{cpmbloodtreatment}{cpmbloodcontrol} \times 100 $$

Where cpm represents counts of radioactivity per minute.

## 2.6. Histological analysis

After the euthanasia procedure, samples of the ileum from the animals in each group were collected for histological analysis. The intestines were cut longitudinally, stretched with the serosal layer in contact
with the filter paper, and opened by the anti-mesenteric edge, removing all its contents without damage to the mucosa. The organs were then placed on a flat surface, and they were rolled in a spiral with the mucosa facing inward to form rolls from the distal to the proximal portion. Subsequently, the rolls were fixed by immersion in a 10% buffered formalin solution, where they were kept until processed. The material was processed for paraffin embedding, and 4 µm sections of each sample were stained with hematoxylin/eosin (HE), and periodic acid-Schiff (PAS). The pathologist examined microscopic slides, and a histopathological grading system evaluated mucosal and muscular lesions. Photographic images were obtained using an Olympus BX43 microscope and a Q-Color 5 digital camera captured with Q-Capture Pro 7 software (QImaging, BC, Canada).

**2.6.1. Histological score**

Histological score analyses were performed using the histological sections described above. The score proposed by Howarth and collaborators was used [31] to evaluate the association of mucositis with histological changes of the ileal mucosa so that the microscopic findings evaluated were villous atrophy, rupture of the surface enterocyte borders, depletion of calyceal cells, loss of crypt architecture, destruction of crypt cells, abscess formation in the crypts, infiltration of lymphocytes and polymorphonuclear cells, dilation of capillaries and lymphatic vessels, and thickening with edema formation in the submucosa and external muscle layers. Each histological variable was scored from 0 (average) to 3 (maximum damage), and based on the findings, the points were added for each animal. For morphometric evaluation of the intestinal epithelium structure, 20 villi and 20 crypts were measured per animal. The measurements were performed with the help of ImageJ software on images from random regions of the histological section.

**2.6.2. Goblet cells counting**

Additionally, the quantification of goblet cells per crypt was established using histology images. Five images were captured from random ileum sections for each animal. Goblet cell counting was conducted for the entire image and presented as the goblet cell count per field.

**2.7. Myeloperoxidase (MPO) enzyme activity**

Myeloperoxidase enzyme activity was measured to determine the rate of neutrophil recruitment into the small intestine. Fragments of the distal ileum of the animals were collected, weighed, and stored at -80°C. After thawing, the tissue was homogenized with ice-cold buffer (Buffer 1) (NaCl 0.1 M; Na₂EDTA 0.015 M, pH 4.7) (ratio: 1.9 mL/100 mg tissue) and centrifuged at 4°C for 15 min at 10,000 rpm. Immediately afterward, the supernatant was discarded, and the precipitate was submitted to hypotonic lysis by adding ice-cold 0.2% NaCl and 1.6% NaCl supplemented with 5% glucose (ratio: 1.5 mL/100 mg of tissue) to break up red blood cells. The samples were homogenized and centrifuged again at 4°C for 15 min at 10,000 rpm. The supernatant was discarded, and the precipitate resuspended with buffer (Buffer 2) (NaH₂PO₄ 0.05 M; Hexadecyltrimethylammonium bromide (HTAB) 0.5%, pH 5.4) (ratio: 1.9 mL/100 mg tissue) and homogenized again at room temperature. Successively, 1 mL aliquots of the
suspension were transferred to 1.5 mL microtubes, frozen in liquid nitrogen, and thawed in water at room
temperature alternately three times. Subsequently, the samples were centrifuged at 4°C for 15 minutes
at 10,000 rpm, and the supernatant was collected for the enzymatic assay. In a 96-well plate (Nunc-
Immuno, MaxiSorp), 25 µL of the collected supernatant was added along with 25 µL of the 1.6 mM TMB
substrate (3,3',5,5'-tetramethylbenzidine) (Sigma-Aldrich®), previously diluted in dimethyl sulfoxide
(DMSO). Subsequently, the samples were incubated at 37°C for 5 min, and then 100 µL of 0.002%
hydrogen peroxide was added to each well and again incubated at 37°C for 5 min. Soon after, 100 µL of 1
M H₂SO₄, whose function is to stop the reaction, was added. After this step, the absorbance was
measured by spectrophotometry at a wavelength of 450 nm using a microplate reader (Bio-Rad model
450, Bio-Rad Laboratories, Hercules, CA, USA), and the results were plotted as enzyme units per
milligram of tissue (U/mg).

## 2.8. Gene expression analysis of inflammatory and barrier markers

Quantitative PCR was carried out with the PowerUp™ SYBR® Green Master Mix (Thermo Fisher) on the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems™) under the following steps: 95°C for 10 min, and 40 cycles of 95°C for 15 s, and 60°C for 1 min. The modulation of gene expression was performed for genes coding for the nuclear factor-kappa B (*NFKB1*), interleukin 1 beta (*IL1B*) and 17 alfa (*IL17A*), mucin 2 (*MUC2*), occludin (*OCLN*), tight junction protein 1 (*TJP1*), and claudin 1 (*CLDN1*). Gene expression results were analyzed following the 2^−ΔΔCT^ method using glyceraldehyde 3-phosphate dehydrogenase (*GAPDH*) and eukaryotic elongation factor 2 (*EEF2*) genes as endogenous references (Table 1).
<table><caption>Table 1
Sequence of primers used in this study</caption><thead><tr><th>Gene</th><th>Primer sequence</th><th>Reference</th></tr></thead><tbody><tr><td>Gapdh*</td><td>F: TCACCACCATGGAGAAGGC<br>R: GCTAAGCAGTTGGTGGTGCA</td><td>[32]</td></tr><tr><td>Eef2*</td><td>F: TGTCAGTCATCGCCCATGTG<br>R: CATCCTTGCGAGTGTCAGTGA</td><td>[33]</td></tr><tr><td>Nfkb1</td><td>F: GTGGAGGCATGTTCGGTAGTG<br>R: TCTTGGCACAATCTTTAGGGC</td><td>[34]</td></tr><tr><td>Il1b</td><td>F: CAACCAACAAGTGATATTCTCCATG<br>R: GATCCACACTCTCCAGCTGCA</td><td>[35]</td></tr><tr><td>Il17a</td><td>F: GCTCCAGAAGGCCCTCAGA<br>R: AGCTTTCCCTCCGCATTGA</td><td>[36]</td></tr><tr><td>Muc2</td><td>F: GATGGCACCTACCTCGTTGT<br>R: GTCCTGGCACTTGTTGGAAT</td><td>[37]</td></tr><tr><td>Ocln</td><td>F: ACTCCTCCAATGGCAAAGTG<br>R: CCCCACCTGTCGTGTAGTCT</td><td>[38]</td></tr><tr><td>Tjp1</td><td>F: CCACCTCTGTCCAGCTCTTC<br>R: CACCGGAGTGATGGTTTTCT</td><td>[39]</td></tr><tr><td>Cldn1</td><td>F: TCCTTGCTGAATCTGAACA<br>R: AGCCATCCACATCTTCTG</td><td>[40]</td></tr><tr><td colspan="3">*Reference genes</td></tr></tbody></table>

## 2.9. Statistical Analysis

The test for the normality of the data was performed using the Shapiro-Wilk test. Data were evaluated using one-way ANOVA followed by Tukey's post-hoc test (for parametric data) or Kruskal-Wallis test and further tested by Dunn's test (for non-parametric data). The Mann-Whitney test was used to analyze food and liquid intake before and after induction of inflammation by 5-FU. Spearman's test was used for correlation analysis. All data were performed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, United States), p-value < 0.05. Results are presented as mean ± standard deviation.

## 3. RESULTS
**3.1. *Escherichia coli* CEC15, as well as its inactivated form limited weight loss during mucositis**

During the 10 days of treatment administration, there was no change in food and fluid intake, but a notable weight gain in the Ct- (negative control – healthy animals) (1.2 ± 0.5 g), MUC (mucositis control – healthy until day 10) (0.8 ± 0.6 g), CEC (Live CEC15 treatment) (1.1 ± 0.2 g), CECi (Heat inactivated CEC15 treatment) (1.2 ± 0.7 g), and CECs (CEC15 cell-free supernatant treatment) (1.9 ± 0.5 g) treated groups was observed (Fig. 2A). No increase in weight gain was observed in the groups treated with EcN (Live EcN treatment) (0 ± 1.5 g) and its derivatives EcNi (Heat inactivated EcN treatment) (0.5 ± 1 g) and EcNs (EcN cell-free supernatant treatment) (0.4 ± 1.6 g) (Fig. 2B). The treatment with 5-FU drastically decreased feed and water consumption (data not shown), which consequently led to a significant weight loss (-2.7 g ± 0.5, p < 0.0001), while healthy animals (Ct- group) gained weight (1.0 g ± 0.09) (Fig. 2a and 2b). Treatment with EcN (-2.1 g ± 0.71, p = 0.4208), EcNi (-2.0 g ± 0.64, p = 0.2238), and CECs (-2.1 g ± 0.44, p = 0.2692) could not prevent animal weight loss. In contrast, animals treated with CEC (-1.6 g ± 0.49, p = 0.0035), CECi (-1.5 g ± 0.34, p = 0.0004), and EcNs (-1.9 g ± 0.54, p = 0.0462) showed limited weight loss, when compared to the MUC (Intestinal mucositis control – no treatment) group (Fig. 2c). However, there was no significant difference when the different treatments were compared.

**3.2. *Escherichia coli* CEC15, as well as its postbiotic fractions, limited intestinal architecture damages**

The effects of CEC15, EcN, and their derivatives on the intestinal structure were also evaluated at the macroscopic and microscopic levels. The protective effect of the treatments on the intestinal mucosa can also be observed in the histological images (Fig. 3a), where we can see great destruction of the epithelial architecture of the ileum in the MUC group and its preservation, more pronounced, in the groups treated with CEC15 and its derivatives. The induction of mucositis led to a relevant shortening on small intestine length (MUC: 29.8 cm ± 2.03, p < 0.0001) compared with the control group (36.7 cm ± 2.03) with an average loss of 6.9 cm. The treatments with CEC (34.5 cm ± 1.75, p = 0.0012), CECi (36.4 cm ± 2.54, p < 0.0001), and EcNs (36.1 cm ± 0.88, p < 0.0001) maintained intestinal length (Fig. 3b), preventing thus the shortening of the small intestine promoted by 5-FU administration. In contrast, EcN (30.3 cm ± 3.04, p = 0.9999), EcNi (30.6 cm ± 2.5, p = 0.9943), and CECs (32.9 cm ± 2.18, p = 0.0973) did not help prevent small intestine shortening.

All treatments reduced the damage caused by 5-FU in the intestinal mucosa, as can be observed by the histological score, which was preserved in all treatments (Fig. 3c). Healthy animals presented a very low score (Ct-, 0.8 ± 0.46), which was increased by 5- FU administration (MUC, 13.1 ± 1.89, p < 0.0001). However, only the group treated with live CEC15 (CEC, 3.3 ± 2.38, p < 0.0001 to MUC) did not show significant differences compared with the healthy group. Furthermore, apart from the EcN group (6.9 ± 2.1, p < 0.0001 to MUC), the other treatments did not show difference between them (EcNi, 5.4 ± 1.99; EcNs, 5.3 ± 1.83; CECi, 4.7 ± 2.06; CECs, 4.5 ± 0.76, p < 0.0001 for all to MUC group). 5-FU treatment induced a considerable reduction in villus height (Fig. 3d) (MUC, 254.3 µm ± 16.56, p < 0.0001) and crypt
depth (Fig. 3E) (MUC, 128.9 µm ± 25.33, p < 0.0001) when compared to healthy animals (Ct-, 501.8 µm ± 47.5 and 197.1 µm ± 32.74, respectively). Although no treatment prevented villus shortening, all treatments (CEC, 389.0 µm ± 22.15; CECi, 394.4 µm ± 22.54; CECs, 425.1 µm ± 27.11; EcNi, 363.6 µm ± 45.81; EcNs, 383.4 µm ± 22.15, p < 0.0001 for all), except for the EcN group (285.2 µm ± 34.45, p = 0.6164), showed a reduction in shortening, with this protection being more pronounced in groups containing CEC15. Regarding the depth of the crypts, no group could prevent or reduce the reduction of the crypts (Fig. 3e) (CEC, 150.2 µm ± 20.04, p = 0.5176; CECi, 145.8 µm ± 16.06, p = 0.7764; CECs, 144.2 µm ± 12.38, p = 0.8509; EcN, 148.3 µm ± 20.19, p = 0.6304; EcNi, 154.8 µm ± 25.80, p = 0.2726; EcN, 154.9 µm ± 14.16, p = 0.2681).

### 3.3. *Escherichia coli* CEC15, as well as its postbiotic fractions, restored goblet cells counts

The variation in the number of goblet cells was evaluated in PAS staining slides (Fig. 4a). Significant reduction on goblet cells' number was also observed in the 5-FU-treated (MUC: 22.6 ± 3.96, p < 0.0001) ileum-inflamed mice compared to the Ct- group (109.1 ± 12.38). Goblet cell loss was minimized in mice treated with CEC15 (CEC, 68.4 ± 16.39, p < 0.0001) and its derivatives (CECi, 66.5 ± 5.32, p < 0.0001; CECs, 63.9 ± 14.62, p < 0.0001). Similar results were observed in groups treated with the postbiotic forms of EcN (EcNi, 58.3 ± 8.83, p < 0.0001; EcNs, 59.8 ± 6.76, p < 0.0001), while the treatment with live EcN (31.3 ± 16.47, p = 0.8103) was unable to alleviate this loss (Fig. 4b).

### 3.4. *Escherichia coli* CEC15, as well as its postbiotic fractions, limited gut permeability increase

The 5-FU administration triggered an increased intestinal permeability (Ct-, 0.014% ± 0.003; MUC, 0.698% ± 0.289, p < 0.0001). Although all tested treatments reduced the permeability caused by 5-FU (CEC, 0.067% ± 0.043, p < 0.0001; CECi, 0.064% ± 0.043, p < 0.0001; CECs, 0.103% ± 0.048, p < 0.0001; EcN, 0.316% ± 0.159, p = 0.0001; EcNi, 0.106% ± 0.062, p < 0.0001; EcNs, 0.233% ± 0.185, p < 0.0001), the EcN group was the only one that showed a significant difference with groups Ct- (p = 0.0038), CEC (p = 0.0297), and CECi (p = 0.0266) (Fig. 5). Consumption of CEC and of its postbiotic fractions led to permeability values close to that of the control healthy group. Consumption of EcN only partially limited (5-FU-induced permeability increase.

### 3.5. *Escherichia coli* CEC15, as well as its postbiotic fractions, limited induction of gut inflammation

Neutrophil infiltrate, which is an important marker of the inflammatory process, was evaluated by monitoring myeloperoxidase enzyme activity (Fig. 6a). Myeloperoxidase activity was highly increased by 5-FU administration (Ct-, 0.36 ± 0.17; MUC, 1.14 ± 0.28, p < 0.0001) and was only reduced in the groups treated with CEC (0.43 ± 0.12, p < 0.0001), CECs (0.52 ± 0.24, p = 0.0003), and EcNi (0.70 ± 0.29, p = 0.0250), while the other groups did not differ from the MUC group (EcN, 1.25 ± 0.41, p = 0.9928; EcNs,
0.82 ± 0.16, p = 0.2373; CECi, 0.76 ± 0.28, p = 0.0748) (Fig. 6a). 5-FU administration also induced the expression of the pro-inflammatory related genes *Il1b* (p < 0.0001), *Il17a* (p = 0.0021), and *Nfkb1* (p = 0.0006) compared to the control group (Ct-). Modulation of these markers was also observed after treatment with both strains and their postbiotic derivatives. We noticed a reduction of *Il1b* mRNA transcription only in group CECi (p = 0.0246) and EcNi (p = 0.0239) (Fig. 6b), while *Nfkb1* transcription was reduced in groups CEC (p = 0.0471), EcN (p = 0.0009) and EcNs (p = 0.0006) (Fig. 6d). Finally, a decrease of *Il17a* expression (p = 0.0477) was only detected in the CEC group when compared to to MUC group (Fig. 6c). Among the tested fractions, inactivated CECi and EcNi were the most efficient at reducing *Il1b* induction, while live CEC was the most efficient regarding *Il17a* expression.

## 3.6. Escherichia coli CEC15, as well as its postbiotic fractions, modulated expression of proteins involved in gut barrier function

The expression of intestinal barrier genes (*Muc2*, *Tjp1*, *Cldn1*, and *Ocln*), which are closely related to intestinal mucosa integrity, was also evaluated. No significant variation in the expression of these genes was observed between the control group (Ct-) and the MUC group (Fig. 7). Despite of this, among the treatments, there was a higher transcription of *Muc2* gene in the CEC group compared to the groups MUC (p < 0.0001), EcN (p < 0.0001), EcNi (p < 0.0001) and EcNs (p < 0.0001) (Fig. 7a). The *Tjp1* gene was also overexpressed in the CEC group compared with the groups MUC (p = 0.0294), EcN (p < 0.0001), EcNi (p = 0.0002) and EcNs (p = 0.0004). Although there was no difference between CECi and MUC groups, the CECi treatment enhanced the *Tjp1* expression when compared to EcN (p = 0.0095) and EcNi (p = 0.0433) groups (Fig. 7b). The *Cldn1* gene transcript was enhanced in the CECi group when compared to the MUC (p = 0.0023), EcN (p < 0.0001), EcNi (p = 0.0002) and EcNs (p = 0.0176) groups (Fig. 7c). Finally, while all treatments with EcN and its derivatives downregulated *Ocln* expression, no difference in its transcription was observed between the Ct-, MUC, CEC, CECi, and CECs groups (Fig. 7d).

## 4. DISCUSSION

Probiotics are considered essential tools in modulating inflammatory and infectious intestinal diseases, as well as a promising therapeutic alternative to alleviate symptoms of 5-FU induced mucositis [24]. Nonetheless, questions have been raised related to the safety of administering live bacteria in immunocompromised individuals and there is a constant search for solutions to this issue. The number of studies regarding the use of postbiotics, inactivated microorganisms and/or their metabolites, as an alternative to probiotics in these cases have risen with promising results [24, 41–43].

In this study, we have evaluated the postbiotic activity of formulations, heat-inactivated bacteria, and cell-free supernatant, derived from the *E. coli* strains Nissle 1917 and CEC15, in a murine model of 5-FU-induced intestinal mucositis. Beneficial activity of the two probiotic strains has been already established *in vivo* in various mouse models of inflammation [11, 14, 19, 44–46]. In particular, we previously
compared the beneficial activity of both strains on 5-FU-induced intestinal mucositis in mice and showed
the better protective effect of the CEC15 strain [20]. Here we confirmed these results, notably in showing
the specific effect of CEC15, compared to EcN, in the prevention of animal weight loss, villous height
reduction, goblet cell depletion, neutrophil infiltrate, intestinal permeability increases, and inflammatory
related genetic markers.

Mucositis is a global inflammation in the GIT, giving rise to various symptoms such as bleeding, diarrhea, abdominal pain, fatigue, malnutrition, electrolyte imbalance, and infections. These symptoms can cause life-threatening complications of cancer chemotherapy [47-49]. The cytotoxic effects of 5-FU on epithelial cells of the GIT represent a major challenge for cancer treatment, as they impair the patient's ability to tolerate therapy, consequently reducing the quality of life and influencing treatment success [50]. The inflammation caused by 5-FU, which extends throughout the whole GIT, is highly associated with weight loss and reduced fluid and food intake. This weight loss can occur either by reduced consumption or by decreased ability to absorb nutrients [48]. As expected, the induction of intestinal mucositis with 5-FU reduced significantly the ingesta of food and liquid and, therefore, induced a considerable weight loss on mice. Here, none of the treatments was able to maintain food and liquid intake in face of 5-FU administration. This inability to attenuate the decrease of food and liquid intake has been reported for other probiotic species in this animal model, like *Lactobacillus delbrueckii* [51, 52], *Lactiplantibacillus plantarum* (formerly *Lactobacillus plantarum*) [21], and *Lacticaseibacillus rhamnosus* (formerly *Lactobacillus rhamnosus*) [42]. Among the four postbiotic formulations tested, only two, heat-inactivated CEC15 (CECi) and EcN cell-free supernatant (EcNs), were able to attenuate weight loss. Similar results were found after administration of heat-inactivated *L. delbrueckii* CIDCA133 were the weight loss reduced from 10-6.5% from the initial weight [41], and with administration of heat-inactivated *L. rhamnosus* CGMCC1.3724, reducing weight loss from 2.4 g to 1.7 g [42].

Looking further on the effects of 5-FU administration, changes in the architecture of the intestinal
epithelium are one of the commonly seen in the inflammatory process promoted in the intestinal
mucositis [48]. We observed that all postbiotic treatments could reduce the damage associated with the
intestinal mucosa by maintaining the integrity of the brush border of enterocytes and reducing the
reactivity of the submucosa. The structural damage caused by the administration of 5-FU led to changes
in the height of intestinal villi, the deepening of the crypts, and the reduction of goblet cells, whose main
function is the production of mucin, a key component of the protective intestinal mucus layer. These are
parameters for assessing the severity of the damage associated with mucositis and are also associated
with the signs mentioned above [31, 53]. As expected, we could see a reduction on villus height and
crypts depth, and a depletion on the number of goblet cells after 5-FU administration. All postbiotic
formulations derived from both strains were able to mitigate the reduction of villus height and depletion
of goblet cells. As reported before, the effectiveness of live CEC15 was confirmed as well as the absence
of protection from live EcN facing 5-FU administration, the results here presented consolidate the CEC15
effect, as well as the effect of its postbiotic preparations in the protection of intestinal epithelium
structure, in special the inactivated CEC15. As for EcN preparations, cell-free supernatant presented
promising results, as it has already been demonstrated before [53] with the heat-inactivated form
showing some level of protection in a lower extent.

The damage caused to the intestinal mucosa by 5-FU is associated with increased intestinal
permeability. This last is associated with an increased risk of infection and transit of complex molecules,
including toxins, through the epithelium and promotes intestinal bleeding and fluid and nutrient loss. All
treatments were found to prevent increased intestinal permeability. Nevertheless, the permeability result
is partially in accordance with the results on gene expression modulation of barrier related genes The
groups with the lowest protection against increased permeability (EcN and EcNs, also EcNi and CECs to
a lesser extent) are also the ones with reduced expression of *Tjp1* and *Ocln* genes, highly associated
with the integrity of intestinal barrier. CEC15 groups, in special the CEC and CECi ones, present the best
results in both permeability and gene expression of barrier related genes with an over expression of
*Muc2* and *Tjp1* on the CEC group and of *Cldn1* on the CECi group. These results express the important
role of CEC15, live or inactivated, in the maintenance of intestinal barrier integrity, by elevating the
expression of tight junction proteins and the production of mucus, maintaining barrier integrity, and
reducing the transit of toxic compounds and microorganisms. Previous research has highlighted the
enhancement of mucins and tight junction proteins following treatment with heat-inactivated probiotics
and/or their respective supernatants. An illustrative example is the supernatant of mulberry leaf extract
fermented by *L. acidophilus*, whose consumption induced upregulation of *Muc2* and *Muc5ac* gene
expression in 5-FU-inflamed mice [54]. Additionally, the EcN-derived supernatant was found to mitigate
the epithelial barrier disruption induced by enteropathogenic *E. coli*, in Caco-2 cells, by elevating the gene
expression of tight junction proteins such as *Tjp1*, *Cldn14*, and *Cldn2* [55].

All preparations of both strains promoted a protective effect on the intestinal mucosa, reducing the
damage associated with mucositis. The infiltration of neutrophils in tissue is associated with
inflammation as these cells are recruited in response to tissue damage [56]. The enzyme
myeloperoxidase is produced exclusively by neutrophils and serves as an indirect measure of the
number of cells present in the tissue [52]. MPO enzyme activity in injured tissue was attenuated by all
treatments with the CEC15-derived preparations, in contrast to what is observed with those derived from
the EcN strain. Similar results were seen after administration of postbiotics preparations of *L. delbrueckii*
CIDCA133 [41]. The lower levels of MPO indicates that the CEC15 preparations are more efficient than
the EcN in limiting neutrophil recruitment into the intestinal epithelium.

Mucositis is associated with altered expression of pro- and anti-inflammatory cytokine genes. These
changes generate greater activation of inflammatory pathways such as NFkB and TNF, loosening of the
intestinal barrier, and weakening the protection of the epithelium through the modulation of genes such
as *Il1b*, *Il17a*, *Nfkb1*, among others [24]. Here we have seen an elevation of the expression of these three
genes after administration of 5-FU, we have also observed a protection against *Il1b* by treatment with
CECi and EcNi, and of *Nfkb* after treatment with EcNs in addition to live CEC15 and EcN. Expression of
*Il17a* was only reduced by administration of live CEC15. Other groups, like EcNi and EcNs, have shown a
tendency for protection (no difference to the Ct- group), however they did not present statistical
difference from the MUC group. The increase on //1b expression has been correlated to increase on permeability by degradation of the occludin mRNA *in vitro* and in a murine model [57] and it seems to be the case on treatments with EcN and its derivates. The //17a gene is known to be overexpressed in intestinal inflamed tissue [58], its overexpression, however, is not the sole responsible for the inflammatory process and there have been studies showing //17 as an anti-inflammatory cytokine, depending on the co-expression of other cytokines such as //10 and Inf-γ [59–62].

It's important to emphasize that not all probiotics demonstrated anti-inflammatory effects in the context of 5-FU-induced epithelial damage [53, 63]. Despite sharing similar probiotic characteristics [64], the mechanisms involved in mitigating 5-FU-induced inflammation may vary among strains or species. This variability could be attributed to factors such as the probiotic dosage, the type of antineoplastic agents, the specific experimental protocols used to induce mucositis, and the form in which beneficial microorganisms are administered, extending to their postbiotic forms (i.e., live, inactivated, or their secreted products) as we have demonstrated in this study [24, 65–67].

# 5. CONCLUSION

The results so far demonstrate that the *E. coli* CEC15 strain can protect the intestinal epithelium from the deleterious effects of 5-FU, maintaining its integrity besides modulating the inflammatory response. The study shed light on the protective role of CEC15 in preserving intestinal barrier integrity, promoting the upregulation of tight junction proteins and of mucin. Additionally, the observed reduction in neutrophil infiltration and modulation of pro- and anti-inflammatory cytokine genes confirms the anti-inflammatory potential of this probiotic. This protective effect of CEC15 may be mediated by bacterial structures and/or secreted metabolites considering that postbiotic preparations of the strain promoted beneficial effects like those promoted by live bacteria. Such postbiotic fractions may be used with less risk to patients. By contrast, despite being a recognized probiotic bacterium and having proven efficacy in several models of inflammatory bowel disease, EcN demonstrated here no protective effects in the model of intestinal mucositis used here, confirming that probiotic effects may be highly strain dependent. Overall, this study contributes to the growing understanding of probiotics and postbiotics as potential therapeutic strategies in mitigating mucositis-associated intestinal damage. Future research should further explore and refine these approaches to optimize their therapeutic benefits.

# Declarations

**Ethics approval and consent to participate:** The experiment was conducted in agreement with the Brazilian College of Animal Experimentation (COBEA) and approved by the "Use of Animals Ethics Committee" from UFMG (CEUA – UFMG) under the protocol 210/2022.

**Availability of data and materials:** Raw data can become available upon reasonable request to the corresponding author.

**Competing interests:** The authors declare no competing interests.
**Funding:** This work was supported by the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and National Council for Scientific and Technological Development (CNPq) from Brazil.

**Authors' contributions:** Conceptualization, RG, TdS and VA; methodology, RG, TdS, and VA; formal analysis, RG, TdS, TG, KV, EF; resources, SF, VC and VA; writing-original draft preparation, RG and TdS; writing-review and editing, RG, TdS, GJ, YIL, CC, PL, JC, EG and VA; supervision, GJ, EG and VA; funding acquisition, GJ, EG and VA. All authors have read and agreed to the published version of the manuscript.

**Acknowledgements:** This work was conducted in the frame of BactInflam International Associated Laboratory between INRAE (France) and UFMG (Brazil).

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## Figures

<figure><img src="image_2.png" /><figcaption>**Figure 1**</figcaption></figure>

Experimental design. The experimental groups received doses of live, heat-inactivated *E. coli* Nissle 1917 or CEC15 or cell-free culture supernatant for 12 days. On the 10th day of experimentation, the animals received 300 mg/kg of 5-FU (or PBS) intraperitoneally and were euthanized after 72 hours.
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>

Treatment and 5-FU-induced weight modulation. (a) CEC15 groups weight variation during 10 days of treatment and 3 days after 5-FU-induction of mucositis. (b) EcN groups weight variation during 10 days of treatment and 3 days after 5-FU-induction of mucositis. The dotted red line indicated the date of mucositis induction. (c) Weight change after mucositis induction by administering 5-FU. Statistical analysis was performed by 2-way ANOVA followed by a post-test of Tukey. Different letters indicated significant differences.
<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>

Modulation of macro and microscopic aspects of 5-FU-induced intestinal mucositis. (a) Histology of the iliac portion of the intestine stained in HE. (b) Modulation of intestinal length. (c) Histological score of the iliac portion. (d) Height of intestinal villus. (e) Depth of intestinal crypts. Villus height and crypt depth measurements were performed using ImageJ 1.51j.8 software (NIH, Bethesda, MD, United States). Statistical analysis was performed by 2-way ANOVA followed by a post-test of Tukey. Different letters indicated significant differences.
<figure><img src="image_7.png" /><figcaption>Figure 4</figcaption></figure>

Goblet cell counting. (a) Histology of the iliac portion of the intestine stained in PA. (b) Goblet cell count per field. Statistical analysis was performed by 2-way ANOVA followed by a post-test of Tukey. Different letters indicated significant differences.
Permeability

<figure><img src="image_8.png" /><figcaption>Figure 5</figcaption></figure>

Intestinal permeability. Serum ⁹⁹mTc-DTPA dosage. Statistical analysis was performed by 2-way ANOVA followed by a post-test of Tukey. Different letters indicated significant differences.
<figure><img src="image_9.png" /></figure>

MPO

b

<figure><img src="image_10.png" /></figure>

IL1B

c

<figure><img src="image_11.png" /></figure>

IL17A

d

<figure><img src="image_12.png" /></figure>

NFKB1

<caption>Figure 6</caption>

Modulation of inflammation by regulation of MPO activity and pro-inflammatory gene expression by CEC15, EcN, and their derivatives. MPO activity (a) and relative expression level of (b) //1b, (c) //17a, and (d) Nfkb1 genes in all treated groups versus the Ct- control group. Statistical analysis was performed by 2-way ANOVA followed by a post-test of Tukey. Different letters indicated significant differences.
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>Figure 7</figcaption></figure>

Modulation of barrier gene expression by CEC15, EcN and their derivatives. Relative expression level of (a) *Muc2*, (b) *Tjp1*, (c) *Cldn1*, and (d) *Ocln* genes in all treated *groups* versus the Ct- (control group). Statistical analysis was performed by 2-way ANOVA followed by a post-test of Tukey. Different letters indicated significant differences.