ORIGINAL RESEARCH published: 08 March 2022 doi: 10.3389/fmicb.2022.831737 Assessment of the Effects of the Synbiotic Combination of Bifidobacterium longum subsp. infantis CECT 7210 and Oligofructose-Enriched Inulin Against Digestive Bacterial Infections in a Piglet Model Agustina Rodríguez-Sorrento 1, Lorena Castillejos 1*, Paola López-Colom 1,2, Gloria Cifuentes-Orjuela 3, José A. Moreno-Muñoz 3 and Susana M. Martín-Orúe 1 1 Servicio de Nutrición y Bienestar Animal, Departament de Ciència Animal i Dels Aliments, Universitat Autònoma de Edited by: Barcelona, Bellaterra, Spain, 2 Departamento de Producción Animal, Facultad de Medicina Veterinaria y Zootecnia, Diana Luise, Universidad Agraria del Ecuador (UAE), Guayaquil, Ecuador, 3 Laboratorios Ordesa SL, Parc Científic de Barcelona, University of Bologna, Italy Barcelona, Spain Reviewed by: Ester Arévalo Sureda, University of Liège, Belgium The use of bifidobacteria as probiotics has proven to be beneficial in gastroenteric Corina-Diana Ceapa, infections. Furthermore, prebiotics such as inulin can enhance the survival and growth of Universidad Nacional Autónoma de México, Mexico these bacteria. Two trials were performed to evaluate the effects of the administration of *Correspondence: Bifidobacterium longum subsp. infantis CECT 7210 and oligofructose-enriched inulin Lorena Castillejos against Salmonella enterica serovar Typhimurium or enterotoxigenic Escherichia coli (ETEC) lorena.castillejos@uab.cat F4. A total of 72 (Salmonella trial) and 96 (ETEC F4 trial) weaned piglets were used in a Specialty section: 2 × 2 design (with or without synbiotic, inoculated or not with the pathogen). After This article was submitted to adaptation, animals were orally inoculated. Performance and clinical signs were evaluated. Microbiotechnology, a section of the journal On days 4 and 8 (Salmonella trial) and 3 and 7 (ETEC F4 trial) post-inoculation (PI), one Frontiers in Microbiology animal per pen was euthanized. Blood, digestive content and tissue samples were Received: 08 December 2021 collected and microbiological counts, fermentation products, serum inflammatory markers Accepted: 11 February 2022 and ileum histomorphometry analysis were performed. Both challenges had an impact Published: 08 March 2022 on faecal consistency (p < 0.001), including the faecal shedding of Salmonella and Citation: Rodríguez-Sorrento A, Castillejos L, increased numbers of enterobacteria and coliforms. The synbiotic administration did not López-Colom P, Cifuentes-Orjuela G, have any effect on pathogen loads but induced changes in the fermentation profile, such Moreno-Muñoz JA and Martín-Orúe SM (2022) Assessment as increased valeric acid in both trials as well as decreased acetic acid, except for of the Effects of the Synbiotic Salmonella-challenged animals. The effect on propionate varied among trials, increasing Combination of Bifidobacterium in challenged synbiotic-treated pigs and decreasing in non-challenged ones in the longum subsp. infantis CECT 7210 and Oligofructose-Enriched Inulin Salmonella trial (P interaction = 0.013), while the opposed occurred in the ETEC F4 trial Against Digestive Bacterial Infections (P interaction = 0.013). The administration of the synbiotic increased intraepithelial in a Piglet Model. Front. Microbiol. 13:831737. lymphocytes (IEL; p = 0.039) on day 8 PI in the Salmonella trial and a similar trend occurred doi: 10.3389/fmicb.2022.831737 in non-challenged pigs in the ETEC F4 trial (P interaction = 0.086). The results did not Frontiers in Microbiology | www.frontiersin.org 1 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens provide evidence of reduced pathogen load with the synbiotic, although a modulation in fermentative activity could be identified depending on the challenge. Consistent increases were found in IEL, suggesting that this synbiotic combination has some immunomodulatory properties. Keywords: probiotic, piglet, ETEC, Salmonella, fructo oligosaccharides, prebiotic, inulin, bifidobacteria INTRODUCTION illness. The aim of this work is to determine the efficacy of a synbiotic combination of B. longum subsp. infantis CECT Acute enteritis is a pathology that consists of a loss of faecal 7210 and inulin enriched with oligofructose against Salmonella consistency and/or increased stool frequency, with a duration Typhimurium and enterotoxigenic E. coli F4, using weaned of less than 7 days. In the vast majority of cases, it is piglets as an animal model. precipitated by an infectious agent, this being a frequent cause of global childhood mortality and morbidity, especially in the developing world (Thapar and Sanderson, 2004). The MATERIALS AND METHODS list of organisms that can provoke acute infectious enteritis includes viruses, bacteria and parasites. As many as 70% of Two different experiments were executed to evaluate the efficacy cases are generated by a virus (principally rotavirus), while of the synbiotic combination against an oral challenge with a non-negligible 20% are caused by a bacterial infection either Salmonella enterica serovar Typhimurium (Salmonella (Koletzko and Osterrieder, 2009). Among these bacteria, trial) or enterotoxigenic E. coli F4 (ETEC F4 trial). Both Salmonella and Escherichia coli can often be isolated, with experiments were performed at the Experimental Unit of the a total of 25,708 (20, 385 Salmonella; 5, 323 E. coli) cases Universitat Autònoma de Barcelona (UAB) and received prior reported to the National Outbreak Reporting System in the approval (Permit No. CEEAH: 4026 DMAH: 10118) from the United States between 2015 and 2018 (CDC, 2021). These Animal and Human Experimental Ethical Committee of this infections commonly have their origin in food contamination, institution and its competent authorities. The treatment, although the infection can also be produced via contact management, housing, husbandry and slaughtering conditions with infected animals (Conrad et al., 2017). New-born and conformed to European Union Guidelines (Directive 2010/63/ young children are particularly susceptible to infections EU, European Commission, 2010). All efforts were made to because their immune systems are not mature and they are minimise animal suffering. not fully immunocompetent. An important form of protection is provided by the mother through passive IgG transplacental Animals, Housing and Experimental transfer, as well as in the milk (Simon et al., 2015) as it Design has been demonstrated that breastfeeding diminishes the These trials were carried out as biosafety Level 2 procedures incidence and severity of infectious diarrhoea (Farthing et al., and all personnel involved received appropriate training. A 2013; Hartman et al., 2019). total of 168 male piglets were distributed between the two Probiotics are live beneficial microorganisms that, when trials as follows: 72 (Landrace × Large White) × Pietrain of 24 administered in infant formulas, can help reduce the number (± 4) days of age weighing 7.70 (± 0.15) kg for the Salmonella of episodes and duration of diarrhoea associated with acute trial and 96 (Landrace × Large White) × Pietrain piglets of 21 infections (Szajewska and Mrukowicz, 2001). Nonetheless, this (± 4) days of age weighing 4.98 (± 0.07) kg for the ETEC effect is strain-dependent and different outcomes have been F4 trial. All animals came from high-sanitary-status farms and reported in the literature (Skórka et al., 2017). Bifidobacterium mothers that were serologically negative to Salmonella in the longum subsp. infantis CECT 7210 is a bifidobacteria isolated Salmonella trial or were not vaccinated against E. coli in the from infant faeces that when given as a supplement to healthy ETEC F4 trial. children, has been associated with a reduction in diarrhoea Piglets were transported to the experimental unit located events (Escribano et al., 2018). Moreover, by the use of different at the Autonomous University of Barcelona, comprising three animal models, previous results have evidenced the ability of boxes (Salmonella trial) and four boxes (ETEC F4 trial) of this strain to provide protection against rotavirus infection eight pens each (24 and 32 pens, respectively, with three animals using murine models (Moreno-Muñoz et al., 2011) and reduce per pen). Each 2m2 pen was separated by a solid fence that Salmonella shedding in oral challenged piglets (Barba-Vidal prevented any contact between animals of different pens. Each et al., 2017). Prebiotics such as inulin or its derivatives can pen had a feeder and water nipple to provide feed and water potentially benefit the survival and multiplication of ad libitum. All weaning rooms were equipped with an automatic bifidobacteria (Vandeputte et al., 2017) and combat enteric heater and forced ventilation and each pen had an individual pathogens (Tran et al., 2018). heating light. Therefore, it is hypothesised that the synbiotic administration At arrival, the animals were distributed according to their of an advantageous Bifidobacterium strain with these prebiotics initial body weight (BW) in order to ensure a homogeneous will result in an improved outcome regarding digestive bacterial average body weight between treatment groups. Trials consisted Frontiers in Microbiology | www.frontiersin.org 2 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens of a factorial design 2 × 2 that included two treatments (control TABLE 1 | Ingredient and nutritional composition of the diets. vs. synbiotic) and challenged or not with the pathogen (yes Salmonella Trial ETEC F4 Trial vs. no), resulting in a total of four experimental groups: Ingredients (g/kg FM) control non-inoculated animals, NC; non-inoculated animals Control Synbiotic Control Synbiotic receiving synbiotic treatment, NS; control inoculated animals, IC; and inoculated animals receiving synbiotic treatment, Maize 280.8 266.8 207.4 196.3 Wheat 170.0 161.5 180.0 170.0 IS. In the case of the first trial, the design was unbalanced, Barley 2 row 150.0 142.5 170.0 160.9 as piglets in two of the three rooms were challenged while Extruded soybean 122.4 116.3 149.1 141.1 the third room remained non-challenged. In contrast, for Sweet whey powder (cattle) 100.0 95.0 100.0 94.6 the second trial, we utilised four rooms and therefore had Fish meal 50.0 47.5 60.0 56.8 a balanced design. In each room, synbiotic treatment was Soybean meal 44 50.0 47.5 80.0 75.7 Whey powder 50% fat 30.3 28.8 25.0 23.6 distributed within four pens on one side of the room, with Mono-calcium phosphate 21.3 20.2 6.8 6.4 the four control pens on the other side of the room separated Calcium carbonate (CaCO3) 8.2 7.8 3.9 3.6 by a corridor to prevent contact between animals. Each L-Lysine HCL 4.5 4.3 4.5 5.0 experimental group had eight replicates, except for the Vit-Min Premix* 4.0 3.8 4.0 3.7 non-challenged groups in the Salmonella trial, which had Sodium chloride (marine 3.0 2.9 2.5 2.3 salt) four replicates instead. DL-Methionine 99 2.4 2.3 2.6 3.6 L-Threonine 2.3 2.2 2.3 2.6 Probiotic Strains, Prebiotic and Diets L-Tryptophan 0.9 0.9 0.6 0.7 In both trials, the tested probiotic was B. longum subsp. infantis L-Valine 1.0 1.0 1.5 1.9 CECT 7210 strain, supplied by Ordesa SL in a lyophilized Prebiotic 0 50 0 50 Analysed composition form and containing 5 × 1010 colony-forming units (cfu) per Control Synbiotic Control Synbiotic (g/kg FM) gram of product in a maltodextrin carrier. The estimated dosage Dry matter 903.4 902.9 909.6 912.7 during the procedures was the same for both trials (1 × 109 cfu Ashes 68.5 61.6 52.9 50.4 per piglet and day). In the Salmonella trial, the animals received Crude fat 57.7 53.8 60.2 56.1 the probiotic orally each morning using disposable syringes Crude protein 174.0 161.4 204.7 180.7 Neutral detergent fibre 89.8 119.2 92.2 83.0 without a needle. To this end, the lyophilized bacteria were Acid-detergent fibre 29.4 27.3 30.3 29.4 re-suspended in 2 ml of phosphate-buffered saline (PBS) no more than 1 h prior to administration. The control groups * Provided per kilogram of complete diet: 10, 200 IU vitamin A, 2,100 IU vitamin D3, 39.9 mg vitamin E, 3 mg vitamin K3, 2 mg vitamin B1, 2.3 mg vitamin B2, 3 mg vitamin were administered the same amount of sterile PBS as a placebo. B6, 0.025 mg vitamin B12, 20 mg calcium pantothenate, 60 mg nicotinic acid, 0.1 mg In the ETEC F4 trial, the piglets received the probiotic mixed biotin, 0.5 mg folic acid, 150 mg Fe, 156 mg Cu, 0.5 mg Co, 120 mg Zn, 49.8 mg Mn, in their feed with the aim of simplifying daily work and 2 mg I, and 0.3 mg Se. minimise the manipulation and the stress of the animals. Each day, the lyophilized probiotic was thoroughly mixed manually with fresh feed, with the dose adjusted considering the average Salmonella and ETEC Strains feed intake (1 g of lyophilized probiotic per 1,000 g of feed). In the first trial, the bacterial strain used for the oral challenge The stability of the probiotic into the feed and feeders was was a Salmonella enterica serovar Typhimurium var. monophasic previously assessed in a viability test to ensure an accurate (formula: 4,5,12:i:-, resistance profile: ACSSuT-Ge, Fagotype: dosage of the product per day. U302) that had been isolated from a salmonellosis outbreak The prebiotic consisted of a mixture of oligofructose (FOS) of fattening pigs in Spain, provided by the Infectious Diseases and inulin (Orafti® Synergy1, Beneo; Mannheim, Germany) Laboratory (Ref. 301/99) of the UAB. Preparation of the oral that was administered through the feed (5%) in both trials. inoculum consisted of 24-h incubation at 37°C in buffered Pre-starter diets were formulated in concordance with the peptone water (BPW; Oxoid; Hampshire, United Kingdom) nutrient requirement standards for pigs (NRC, 2012) and given and diluted (1:10) with sterile PBS (Sigma-Aldrich; Madrid, in a mash form. In the Salmonella trial, both experimental Spain). The final concentration of the inoculum was 1 × 109 cfu/ diets were manufactured from the same single basal batch ml. Inoculum concentrations were determined prior to the (with the same formula) being the prebiotic subsequently mixed inoculation by McFarland standards and were doubly plated with half of the batch. In the ETEC F4 trial, potential amino in Tryptic Soy Agar (Liofilche; Italy) on the same day in order acid dilution in the synbiotic diet due to the incorporation that they could be checked by manual plate counting. of the prebiotic was compensated by the addition of synthetic In the second trial, the bacterial strain of enterotoxigenic E. amino acids with different adjusted formulae for each coli F4 used was isolated from the faeces of 14-week-old pigs experimental diet (synbiotic diet included as: 0.5 g L-valine, and provided by the Infectious Diseases Laboratory (Ref. 30/14) 0.9 g L-lysine HCL, 1.2 g DL-methionine, 0.5 g L-threonine and of the UAB. This strain presented the following virulence factors: 0.2 g L-tryptophan per kg of feed); in this way, we could F4ab, F4ac, LT, STb and EAST1 and was negative for K99, F6, guarantee the same level of the most limiting amino acids in F18, F41, STa, VT1 and VT2 y EAE. The oral inoculum was both experimental diets. Details of the ingredient and chemical prepared via 12-h overnight incubation at 37°C in Brain Heart composition are given in Table 1. Infusion broth (Oxoid; Hampshire, England) with slow agitation Frontiers in Microbiology | www.frontiersin.org 3 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens (250 rpm) in an orbital incubator. The culture was directly given injecting the euthanasia drug, 10 ml sample blood was taken to the animals with a final concentration of 1·109 cfu/ml. Inoculum from each animal via venepuncture of the cranial cava vein concentrations were also determined before the inoculation by using 10 ml blood collection tubes without anticoagulant (Aquisel; McFarland standards and were plated in Luria Agar (LA; made Madrid, Spain). Immediately after blood sampling, pigs were in-house: tryptase, yeast extract, NaCl, agar and Oxoid; Hampshire, intravenously administered a lethal dose injection of sodium UK) the same day for manual plate counting. pentobarbital (140 mg/kg BW; Euthasol, Le Vet B.V.; Oudewater, Netherlands). Once dead, the animals were bled, the abdomen Experimental Procedure opened and the gastrointestinal tract extracted. Both experiments lasted 15 days. One animal from each pen A faecal sample from the rectum was used for traditional was euthanized on days 4 and 8 post-inoculation (PI) in the microbiology in the ETEC F4 trial, whereas a caecal sample Salmonella trial and on days 3 and 7 PI in the ETEC F4 trial. was obtained for microbiology in the Salmonella trial. They After an adaptation period of 7 days in the Salmonella trial were kept on ice and analysed within 4 hours. and 8 days in the ETEC F4 trial, an inoculum containing the In both experiments, the digesta of the ileum and the proximal pathogenic bacteria culture was given to the challenged groups colon were collected and homogenised prior to pH determination orally: in the first trial, this was one 2 ml dose (2 × 109 cfu) of with a pH meter calibrated on each day of use (Crison 52–32 Salmonella Typhimurium, whereas in the second trial, it was electrode, Net Interlab; Barcelona, Spain) and the digesta score one 6 ml dose (6 × 109 cfu) of ETEC F4. The same amount of was registered on a scale as follows: 1 = liquid; 2 = liquid with sterile broth was administered to the non-challenged piglets. some formed material; 3 = thick; and 4 = semi-solid. Subsamples In order to ensure that the animals’ stomachs were full at the of the ileal and colonic digesta were preserved for different time of the oral challenge, pigs were starved for a period of analyses. One aliquot of colonic content was kept at −80°C for 12 h and feed was reintroduced 30 min before inoculation. ETEC F4 (ETEC F4 trial) and probiotic quantification by qPCR. From the challenge onwards, the animals’ clinical signs were A set of ileal and colonic digesta samples were conserved frozen checked daily to evaluate their post-inoculation status (i.e. at −20°C in H2SO4 solution (3 ml of content plus 3 ml of 0.2 N dehydration, anorexia, apathy, general behaviour and faecal H2SO4) for ammonia (NH3) determination and an additional score), always by the same person. Faecal score was measured set (~10 g) was also frozen (−20°C) for future analysis of short- using a scale whereby 1 = solid and cloddy, 2 = soft with shape, chain fatty acids (SCFA) and lactic acid. 3 = very soft or viscous liquid and 4 = watery or with blood. In the ETEC F4 trial, to determine the number of Rectal temperature was assessed using a digital thermometer enterobacteria, coliforms and ETEC F4 attached to the intestinal (Accuvet, Sanchung City, Taiwan) on days 1, 2 and 3 PI in mucosa, 5 cm sections of distal ileum were collected from each the Salmonella trial and days 1 and 2 PI in the ETEC F4 trial. animal, washed thoroughly with sterile PBS, opened longitudinally The animals’ performance was also monitored as: individual and scraped with a microscopy glass slide to obtain the body weight (BW) was registered on arrival and on days 0, mucosa scraping. 4 and 8 PI (0, 3 and 7 PI in the ETEC F4 trial) and feed For the histological study, 1 cm sections from the ileum intake was determined on days 0, 4 and 8 PI in the Salmonella were removed, opened longitudinally, thoroughly and carefully trial, whereas in the ETEC F4 trial, feed intake was registered washed with 4% formaldehyde solution (Panreac; Castellar del daily, concurring with the regular feed replacement aimed at Vallès, Spain) and fixed by immersion in the same solution. maintaining probiotic viability. The average daily gain (ADG), Blood samples were centrifuged (3,000 × g for 15 min at 4°C) average daily feed intake (ADFI) and the gain:feed ratio (G:F) after clotting and the serum obtained was stored at −20°C. were calculated by pen. The mortality rate was also registered and no antibiotic treatment was given to the animals in any of the experiments. Analytical Procedures For microbiological analysis, faecal samples were collected Feed Analysis aseptically after spontaneous defecation or by digital stimulation Chemical analyses of the diets, including dry matter (DM), at arrival on the day of the inoculation (0 PI): in the Salmonella ash, crude protein and diethyl ether extract, were performed trial, this was from the animal with the highest initial BW according to Association of Official Agricultural Chemists in each pen (N = 24), whereas in the ETEC F4 trial, faecal standard procedures (AOAC, 1995). Neutral detergent fibre samples were obtained from the animal with the medium BW and acid-detergent fibre were determined according to the in each pen (N = 32). Furthermore (and just for the Salmonella method of Van Soest et al. (1991). trial), additional faecal samples were taken on days 1, 3 and 7 PI from the same animal. Microbiological Analysis On days 4 and 8 PI (3 and 7 PI in the ETEC F4 trial), For the microbiological analysis of Salmonella, samples were one pig per pen was euthanized. Slight differences in sampling transferred to buffered peptone water solution in a concentration days between trials were due to logistic reasons. On day 4 of 1:10. The quantitative analysis was performed by seeding PI, the animal selected was the one with the medium initial serial dilutions of the samples 10−2, 10−4 and 10−6 in Xylose- BW, while on day 8 PI, it was the heaviest piglet in each pen. Lactose-Tergitol-4 plates (XLT-4; Merck; Madrid, Spain). For The animals were euthanized and sequentially sampled during the qualitative analysis, samples were incubated in BPW (37°C, the morning of each day (between 8:00 and 13:00 h). Before 24 h), transferring 100 μl of the culture to 10 ml of Frontiers in Microbiology | www.frontiersin.org 4 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens Rappaport-Vassiliadis for a second incubation (42°C, 48 h) to Histological Analysis finally seed them in XLT4 plates to observe H2S positive colonies. For histological study, tissue samples were dehydrated and For the enterobacteria and coliform counts, samples were embedded in paraffin wax, sectioned at a thickness of 4 μm serially diluted in Lactate Ringer Solution (Sigma-Aldrich; and stained with haematoxylin and eosin. The measurements Madrid, Spain) and proper dilutions seeded in MacConkey of 10 different villus-crypt complexes per sample and the agar (Oxoid; Madrid, Spain) and eosin methylene blue agar counting of intraepithelial lymphocytes (IEL), goblet cells (GC) (Scharlab; Barcelona, Spain). Plaques were incubated for 24 h and the number of mitosis of each were performed with a at 37°C and colonies were manually counted. light microscope (BHS, Olympus; Barcelona Spain), using as The presence of ETEC F4 in the colonic digesta and ileal a guideline the procedure described in Nofrarías et al. (2006). scrapings was determined by real-time PCR. To extract the Briefly, the villus height and crypt depth were measured using DNA from these samples, the commercial QIAmp DNA stool a linear ocular micrometre (Olympus, Microplanet). The same minikit (Qiagen; West Sussex, United Kingdom) was utilised. villus and crypt columns were used to determine the number Afterwards, several aliquots of DNA eluted in Qiagen buffer of IEL, GC and mitosis. On the basis of the cellular morphology, AE (total volume; 200 μl) were stored frozen at −80°C. A qPCR differences between the nuclei of enterocytes, mitotic figures, targeting the gene coding the F4 fimbria of ETEC F4 using goblet cells and lymphocytes were clearly distinguishable at the SYBR green dye was performed according to the procedure 400× magnification. All morphometric analysis was done by described by Hermes et al. (2013). To express the results, the the same person, who was blinded to the treatments. animals were distributed across five levels according to the number of gene copies per gram of fresh matter that they Statistical Analysis showed when qPCR was performed. Ranges were defined as The results are expressed as means with their standard errors follows: negative = under 4 logarithmic units of gene copies per unless otherwise stated. Microbiological counts were log gram of fresh matter; low = 4–5.5 logarithmic units of gene copies transformed for analysis. A two-way analysis of variance per gram of fresh matter; medium = 5.5–7 logarithmic units of (ANOVA) was used to examine the effect of the two experimental gene copies per gram of fresh matter; high = 7–8.5 logarithmic treatments and the inoculation with the following model: units of gene copies per gram of fresh matter; and very high = more than 8.5 logarithmic units of gene copies per gram of fresh matter.    Y ijk  m  treat i  challenge j  treat challengeij  eij where Yijk relates to each observation of the outcome variable, Short-Chain Fatty Acids, Lactic Acid and m is the global mean, treati is the main effect of treatment, Ammonia Analyses challengej is the main effect of the oral pathogen challenge Short-chain fatty acids and lactic acid analyses were performed and treat*challengeij corresponds to the interaction between using gas chromatography, after the samples had undergone treatment and the challenge. Finally, eij is the experimental acid–base treatment followed by ether extraction and error term. Regarding the interaction term, it was removed derivatization with N-(tertbutyldimethylsilyl)-N-methyl- from the model when found not to be significant. trifluoroacetamide plus 1% tert-butyldimethylchlorosilane agent, The effects on the post-slaughter measurements were examined using the method of Richardson et al. (1989) that was using the R v3.4 (R Core Team, 2013) lm function for two-way subsequently modified by Jensen et al. (1995). ANOVA, with the treatment and the challenge effects as main Ammonia concentrations were assessed using a gas-sensitive effects. For Salmonella and E. coli F4 prevalence values and electrode (Hatch Co.; Colorado, United States) combined with a Pig-MAP serum concentrations, data were subjected to frequency digital voltmeter (Crison GLP 22, Crison Instruments, SA; Barcelona, analysis using the fisher. Test function in the same package. Spain), following a procedure described by Hermes et al. (2009) ADFI and daily faecal scores were also analysed using the that was adapted from Diebold et al. (2004). Samples were diluted lme4 package (Bates et al., 2015) lmer function for a generalised (1:2) in 0.16 M NaOH and, after homogenisation, were centrifuged linear mixed model with a treatment-by-time interaction term at 1500 × g for 10 min. Once the ammonia was released, it was and considering the animal as the random effect. measured in the supernatants as a change in voltage in mV. The experimental unit was the pen. For all analyses, significance was set at p < 0.05 and values of p ≥0.05 but ≤0.10 were considered to indicate a statistical trend. When treatment Serum Analysis effects were established, the mean comparison was adjusted Serum concentrations of Tumour Necrosis Factor-α (TNF-α) with the Tukey–Kramer test. Data are presented as means and were determined by Quantikine Porcine TNF-α kits (R&D residual standard deviation. Systems; Minneapolis, United States) and pig major acute-phase protein (Pig-MAP) concentration was determined by a sandwich- type enzyme-linked immunosorbent assay (ELISA; Pig MAP RESULTS Kit ELISA, Pig CHAMP Pro Europe SA; Segovia, Spain) according to the manufacturer’s instructions. In the Salmonella Both experiments proceeded as expected, without any trial, antibodies against Salmonella were also assessed using remarkable incidence. an ELISA Salmonella Herdcheck (Idexx; Hoofddorp, Netherlands), The oral challenge with the pathogenic bacteria induced establishing the cut-off for positivity in optic density ≥ 40%. moderate clinical signs in the animals that were slightly more Frontiers in Microbiology | www.frontiersin.org 5 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens severe after the Salmonella challenge (Salmonella trial). In these pathogen in faeces and intestinal digesta, Figure 2 shows trials, humane euthanasia of two pigs was indicated (1 IS; 1 the evolution of Salmonella plate counts along sampling days NIS). Furthermore, two spontaneous casualties were registered in the challenged animals. In general terms, the non-challenged in the Salmonella trial (1 IC; 1 IS) and the ETEC F4 trial (1 piglets remained negative during the study, with the exception NIS; 1 IC). of three piglets that recorded a positive result in at least one sample, albeit always at low-to-uncountable levels (< 102 cfu/g). All challenged animals were positive in at least one faecal Performance Parameters sample and all presented Salmonella in the caecal content. Changes in ADG, ADFI and gain:feed ratio (G:F) with the Regarding synbiotic administration, only a trend on day 1 experimental treatments are shown in Table 2. PI was found, with the faeces revealing that the animals that The challenge with the pathogen caused a decrease in ADFI received the synbiotic presented lower shedding compared to and ADG in the Salmonella trial (p = 0.010 and p = 0.024, the control group (p = 0.145). Moreover, regarding the caecal respectively). The effects of the ETEC F4 challenge were milder, digesta, 25% of piglets treated with the synbiotic turned negative with only a numerical trend seen for a lower ADFI in the to Salmonella excretion (vs. 0% control) on day 8 PI (P positive/ post-inoculation period (333.65 vs. 367.95 g for challenged and negative excretion = 0.076). non-challenged groups, respectively, p = 0.152). Regarding the ETEC F4 trial, no differences between groups No significant changes in ADFI nor ADG were registered were registered in the plate counts of faecal enterobacteria or that might be associated with the synbiotic treatment regardless coliforms on the day of the piglets’ arrival. Furthermore, no of the trial, aside from a numerical difference (p = 0.130) in changes caused by the synbiotic treatment were observed before the ETEC F4 trial for reduced ADG during the post-inoculation the oral challenge, although differences could be noted related phase. Moreover, in the Salmonella trial, a reduction in the to the box (9.79, 10.69, 8.12 and 8.44 log cfu/g FM for IC, G:F ratio was seen after the adaptation period (p = 0.020). IS, NIC and NIS, respectively; p < 0.001). Table 3 shows the impact of the experimental treatments on the enterobacteria Clinical Signs and coliform plate counts post-inoculation. The oral challenge On day 1 PI, an increment of 1°C of rectal temperature was promoted an increase in enterobacteria and coliforms either caused by the Salmonella challenge (38.9 ± 0.13°C vs. in the faeces (p < 0.013) or the ileal scrapings (p < 0.013) on 39.8 ± 0.18°C, p < 0.001), whereas the ETEC F4 challenge did day 3 PI. The effects produced by the challenge were no longer not alter the piglets’ temperatures (39.1 ± 0.07°C vs. 39.1 ± 0.17°C, detected at day 7 PI. Moreover, no significant effects were p = 0.974). No significant differences were found related to the found related to the synbiotic supplementation in any of the synbiotic administration. parameters analysed. However, a trend towards interaction in Figure 1 shows the evolution in faecal consistency after enterobacteria (p = 0.057) and coliform (p = 0.104) counts in the oral challenge for each trial. In both trials, the challenge the ileal scrapings could be identified at day 7 PI. On this was able to significantly impair faecal consistency (p < 0.001) day, non-challenged animals that received the synbiotic presented with an increase in the incidence of diarrhoea. However, the lower counts than the control, whereas challenged piglets progression of the faecal score over time differed between the exhibited the opposite effect. trials: whereas with the Salmonella challenge, the impairment The results corresponding to qPCR targeting the coding of faecal consistency was registered up to the end of the trial; gen of the F4 fimbria of E. coli F4 are summarised in Figure 3. in the ETEC F4 trial, the faecal consistency returned to normal Given that the pathogen could not be quantified in all animals, within 3–4 days. No significant differences were found related the data were analysed as frequencies. The figure shows the to the synbiotic treatment. distribution of the percentage of animals within each of the Regarding the consistency of the ileal and colonic digesta, five defined ranges based on the number of copies/g fresh the oral challenge did not instigate significant changes in any matter found in the analysis. The effect of the oral challenge of these parameters, although some effects were observed was clearly evidenced on day 3 PI through a significant increase regarding the colon digesta’s consistency following the in the percentage of animals showing large or very large administration of the synbiotic. In the Salmonella trial, the numbers of copies in colonic content (p < 0.001) or ileal synbiotic treatment was associated with improved consistency scrapings (p = 0.045). No significant differences were found at day 8 PI (3.44 vs. 2.62; p = 0.010); moreover, in the ETEC related to the synbiotic administration. F4 trial, it caused a trend towards interaction (p = 0.061) with an improvement with supplementation in challenged animals Intestinal Fermentation (3.62 vs. 3.00), but a looser consistency in the non-challenged Tables 4 and 5 show all the changes induced by the different group (3.87 vs. 3.62). experimental treatments in the ileal and colonic fermentation products. Microbiological Analysis As displayed, the challenge with Salmonella stimulated a In the Salmonella trial, the serological analysis revealed that significant increase in ammonia levels in the colon (34.77 vs. all the animals remained seronegative throughout the study, 14.48 mmol/l; p = 0.001) on day 8 PI. It did not influence the confirming that they had not been exposed to the pathogen total amount of SCFA or lactic acid, but challenged animals prior to the oral challenge. Regarding the presence of the presented a higher molar percentage of valeric acid in the Frontiers in Microbiology | www.frontiersin.org 6 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens TABLE 2 | Effects of experimental treatments on feed intake and weight gain. Treatment RSD p-value IC IS NIC NIS Challenge Treatment Salmonella trial BW (kg) Initial 7.70 7.74 7.70 7.66 0.156 0.540 0.878 Final 9.23 9.71 10.52 10.26 1.311 0.120 0.662 ADFI (g) pre 181.1 189.1 192.1 214.5 43.66 0.347 0.480 post 288.2 287.4 398.7 403.2 93.08 0.010* 0.979 ADG (g) pre 121.6 89.4 126.1 111.1 42.85 0.146 0.488 post 85.2 109.2 249.9 165.9 104.90 0.024* 0.781 G:F pre 0.67 0.44 0.66 0.52 0.197 0.704 0.020* post 0.26 0.33 0.60 0.36 0.268 0.125 0.737 ETEC F4 trial BW (kg) Initial 4.99 5.02 4.97 4.96 0.790 0.107 0.723 Final 7.46 6.94 7.20 7.14 0.775 0.915 0.290 ADFI (g) pre 82.8 77.5 98.2 79.2 17.03 0.506 0.215 post 359.3 308.0 370.5 365.4 65.95 0.152 0.237 ADG (g) pre 55.5 57.3 69.1 48.7 32.30 0.830 0.421 post 283.2 219.2 250.5 235.2 72.01 0.745 0.130 G:F pre 0.61 0.73 0.76 0.53 0.349 0.853 0.674 post 0.79 0.70 0.68 0.63 0.152 0.102 0.207 Body weight (BW; kg), average daily feed intake (ADFI; g/day), average daily gain (ADG; g/day) and feed efficiency (gain:feed ratio, G:F) for the pre-inoculation period (pre: days 1–8 Salmonella trial and days 1–9 ETEC trial) and post-inoculation period (post: days 8–15 Salmonella trial and days 9–15 ETEC trial). IC, Inoculated animals receiving placebo; IS, Inoculated animals receiving the synbiotic; NIC, Non-inoculated animals receiving placebo; and NIS, Non-inoculated animals receiving the synbiotic. N = 8 for all groups except for non-challenged animals in Salmonella trial, N = 4. No interaction effects between challenge and treatment were found. RSD, residual standard deviation. *indicates statistical difference. FIGURE 1 | Evolution of average faecal scores for the different experimental groups during the post-inoculation (PI) period. IC, Inoculated animals receiving placebo; IS, Inoculated animals receiving the synbiotic; NIC, Non-inoculated animals receiving placebo; and NIS, Non-inoculated animals receiving the synbiotic. N = 8 for all groups except for non-challenged animals in Salmonella trial, N = 4. Bars correspond to standard error. *indicates statistical difference. colon on day 4 PI (4.61 vs. 3.02%; p = 0.044) and also tended it promoted a higher molar percentage of valeric acid in the to have more branched-chain fatty acids on day 8 PI (1.09 colon on day 8 PI (6.28 vs. 2.49%; p = 0.014) and provoked vs. 0.53%; p = 0.088). Although the administration of the synbiotic two interactions in the molar percentages of acetic (p = 0.012) did not modify the ammonia, lactic or SCFA concentrations, and propionic acids (p = 0.002). Whereas in non-challenged Frontiers in Microbiology | www.frontiersin.org 7 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens FIGURE 2 | Percentage of animals included in the different faecal and caecal excretion levels of Salmonella. All samples were obtained from the heaviest animal in each pen, except for the caecal sample on day 4 PI, which was obtained from the anima of medium weight. IC, inoculated animals receiving placebo; IS, Inoculated animals receiving the synbiotic; and N = 8 for IC and IS. Values of p were obtained using Fisher’s Exact Test on R software. TABLE 3 | Effects of experimental treatments on enterobacteria and coliform counts in faecal samples and ileal scrapings. Treatment RSD p-value IC IS NIC NIS Challenge Treatment Interaction Enterobacteria (log cfu/g FM) Faeces Day 3 PI 10.44 10.51 9.15 8.83 1.564 0.012* 0.828 0.725 Day 7 PI 8.52 8.07 8.15 8.62 2.581 0.925 0.993 0.615 Ileal scrapings Day 3 PI 8.56 8.37 6.57 7.06 1.564 0.012* 0.828 0.726 Day 7 PI 7.08y 8.53xy 9.25x 7.98xy 1.942 0.247 0.892 0.057 Total Coliforms (log cfu/g FM) Faeces Day 3 PI 9.60 10.36 8.66 8.26 1.630 0.013* 0.753 0.323 Day 8 PI 7.90 7.51 8.10 8.55 2.446 0.480 0.974 0.632 Ileal scrapings Day 3 PI 8.30 8.07 6.25 6.95 1.630 0.013* 0.753 0.323 Day 7 PI 6.70 8.55 8.21 7.86 1.850 0.532 0.263 0.104 IC, Inoculated animals receiving placebo; IS, Inoculated animals receiving the synbiotic; NIC, Non-inoculated animals receiving placebo; and NIS, Non-inoculated animals receiving the synbiotic. N = 8 for all experimental groups. Values of p were obtained by ANOVA using the generalised linear procedure in R software. Letters x and y express differences considered for p < 0.07. RSD, residual standard deviation. *indicates statistical difference. piglets administration of the synbiotic mixture reduced the was responsible for a drop in pH on day 3 PI (6.11 vs. 6.38, molar percentage of acetic acid and increased that of propionic p = 0.035) and an increase in the total amount of SCFA on day acid, in the challenged animals, the effect was the opposite. 7 PI (129.2 vs. 111.3 mmol/kg; p = 0.026). The molar proportion In the ETEC F4 trial, the effects of the challenge in terms of of propionic was also increased on day 3 PI (24.10 vs. 21.10%; fermentative activity were more apparent. In the colon, the challenge p = 0.016), especially in the synbiotic group (P interaction = 0.022). Frontiers in Microbiology | www.frontiersin.org 8 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens FIGURE 3 | Percentage of animals in each presence level of ETEC F4 on days 3 and 7 post-inoculation. Different animals were sampled on day 3 PI (medium weight) and 7 PI (greatest weight). IC, inoculated animals receiving placebo; IS, Inoculated animals receiving the synbiotic; NIC, Non-inoculated animals receiving placebo; and NIS, Non-inoculated animals receiving the synbiotic. N = 8 for all experimental groups. Values of p were obtained using Fisher’s Exact Test on R software. Related to the effects of the synbiotic mixture on the Immune Response fermentation parameters, an interaction was found in the ileum No significant differences related to the synbiotic treatment on day 4 PI regarding the concentration of acetic acid and were found in the serum levels of TNF-α or Pig-MAP. However, lactic acid as the main products of fermentation. Acetic acid changes were noted associated with the pathogen inoculation. showed the greatest concentration in the non-challenged animals Regarding TNF-α, animals challenged with ETEC F4 presented not receiving the synbiotic (P interaction = 0.033), whereas lactic higher concentrations than non-challenged pigs on day 7 PI acid presented the highest values in the non-challenged and (58.9, 57.2, 48.1 and 43.1 pg./ml for IC, IS, NIC and NIS, non-supplemented animals (P interaction = 0.004). At the colonic respectively; p = 0.010) and a similar pattern was found after level, no effects were found for the pH, total concentration the Salmonella challenge, although in this case, the differences of SCFA or lactic acid, but changes did occur in the profile did not reach statistical significance (125, 135, 124 and 101 pg./ of fermentation. The percentage of acetic acid fell on both ml for IC, IS, NIC and NIS, respectively, day 8 PI; p = 0.132). days (56.05 vs. 60.65% for day 3 PI, p = 0.012 and 55.9 vs. When analysing the Pig-MAP, the values did not adjust to 59.55% for day 7 PI, p = 0.029), in line with an increase in a normal distribution and therefore the results were analysed valeric acid (4.34 vs. 2.43% for day 3 PI, p = 0.010 and 4.07 as frequencies using Fisher’s exact test. Three range levels were vs. 2.20% for day 7 PI, p < 0.001) and a trend towards a higher defined as: high (> 2 mg/ml); borderline (1–2 mg/ml) and normal butyric acid molar percentage (14.52 vs. 11.88% for day 3 PI, (< 1 mg/ml) according to Piñeiro et al. (2009). The results analysed p = 0.093 and 14.22 vs. 12.78% for day 7 PI, p = 0.140). in this way are shown in Figure 4. Only the Salmonella challenge Frontiers in Microbiology | www.frontiersin.org 9 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens TABLE 4 | Effects of experimental treatments on ileal and colonic fermentation in the Salmonella trial. Day PI Treatment RSD p-value IC IS NIC NIS Challenge Treatment Interaction Salmonella trial ILEUM Lactic acid 4 16.56 17.09 7.07 9.52 20.010 0.341 0.911 0.913 (mmol/kg) 8 22.38 6.01 7.37 3.10 14.260 0.173 0.064 0.351 Acetic acid 4 4.43 2.40 3.34 2.06 2.301 0.479 0.077 0.711 (mmol/kg) 8 2.95 2.97 2.37 2.40 2.441 0.599 0.984 0.999 COLON 4 6.00 5.89 6.20 6.03 0.547 0.475 0.559 0.900 pH 8 6.03 6.21 6.02 5.67 0.335 0.070 0.986 0.819 4 7.99 6.66 11.56 7.44 3.877 0.210 0.169 0.414 NH3 (mmol/L) 8 30.75 38.80 15.39 13.57 12.620 0.001* 0.366 0.377 Lactic acid 4 6.20 1.80 0.24 1.11 6.984 0.571 0.437 0.628 (mmol/kg) 8 5.76 5.72 23.46 0.24 14.340 0.376 0.272 0.155 SCFA (mmol/ 4 93.1 89.6 107.9 97.1 40.90 0.537 0.725 0.839 kg) 8 129.3 97.4 96.9 105.6 45.16 0.543 0.332 0.313 SCFA molar ratio (%) 4 53.8 54.3 57.8 54.8 9.51 0.589 0.873 0.673 Acetic 8 55.5ab 61.3ab 64.4a 45.3b 10.50 0.437 0.571 0.012* 4 25.2 24.5 26.2 25.5 6.03 0.695 0.777 0.998 Propionic 8 28.5b 22.1c 24.5c 36.4a 6.22 0.069 0.891 0.002* 4 13.1 13.1 11.9 14.0 4.79 0.944 0.706 0.619 Butyric 8 11.7 10.8 8.8 9.6 4.22 0.268 0.865 0.650 4 4.19 5.04 2.16 3.89 1.714 0.044* 0.117 0.557 Valeric 8 3.33b 4.36b 1.66b 8.21a 2.607 0.345 0.014* 0.023* 4 1.74 1.81 1.43 1.28 1.388 0.491 0.997 0.856 BCFA 8 0.82 1.36 0.65 0.42 0.335 0.088 0.343 0.223 This table includes values corresponding to pH, ammonia concentration (NH3; mmol/L of FM), lactic acid (mmol/L of FM) and total short-chain fatty acids (SCFA; mmol/L of FM and molar ratio) analysed in samples of ileal and colonic digesta. IC, Inoculated animals receiving placebo; IS, Inoculated animals receiving the synbiotic; NIC, Non-inoculated animals receiving placebo; and NIS, Non-inoculated animals receiving the synbiotic. N = 8 for all groups except for non-challenged animals, N = 4. Values of p were obtained by ANOVA using the generalised linear procedure in R software. Letters a,b,c indicate statical differences between groups. FM, Fresh matter; NH3, ammonia concentration; SFCA, short-chain fatty acids; BCFA, branched-chain fatty acids; and RSD, residual standard deviation. *indicates statistical difference. was able to promote an increase in the number of animals with on day 3 PI in the ETEC F4 trial, although only in the borderline-high levels of Pig-MAP at day 8 PI (p = 0.017), with non-challenged animals (P interaction = 0.086). Mitosis were no significant change induced by the ETEC F4 inoculation. reduced by the synbiotic at day 8 PI in the Salmonella trial (p = 0.008), especially in the challenged animals (P interaction = 0.094). In contrast, in the ETEC F4 trial, mitosis Intestinal Histological Structure increased thanks to the synbiotic at day 3 PI (p = 0.006). The effects of the experimental treatments on ileal histomorphology are summarised in Table 6. The Salmonella challenge promoted shorter villi on day 4 PI (p < 0.001) and deeper crypts on days DISCUSSION 4 and 8 PI (p = 0.013 and p = 0.042, respectively). After the ETEC F4 challenge, no significant differences were The aim of this work was to assess the potential of the found apart from a greater number of mitosis on day 7 PI combination of B. longum subsp. infantis CECT 7210 and a (p = 0.014). mixture of inulin and FOS as a synbiotic strategy to fight two Regarding impact of synbiotic administration on ileal common gastrointestinal pathogens: Salmonella and ETEC F4. histomorphology, different outcomes were found depending on In our previous research (Barba-Vidal et al., 2017), the strain the trial. In the ETEC F4 trial, villous height was greater on B. longum subsp. infantis CECT 7210 was found to have an day 3 PI in the animals receiving the synbiotic, but only in effect against Salmonella Typhimurium and ETEC F4 colonisation the non-challenge group (P interaction = 0.032). However, the in pigs with a stimulation of local immune response by increasing opposite numerical effect was observed on the second sampling the number of intraepithelial lymphocytes. Furthermore, it is day in both trials (P interaction <0.16). Crypt depth showed widely acknowledged that fructo-oligosaccharides (FOS) and an increasing trend due to the synbiotic administration on inulin are selectively fermented by most strains of bifidobacteria day 4 PI in the Salmonella trial (p = 0.078), but, contrarily, a (Wang and Gibson, 1993; Kaplan and Hutkins, 2000) due to decreasing one on day 3 in the ETEC F4 trial (p = 0.081). IEL the production of β-fructofuranosidases (Imamura et al., 1994). presence was enhanced by the synbiotic at day 8 PI in the In fact, FOS and inulin are two of the most studied prebiotics Salmonella trial (p = 0.039) and the same trend was observed with bifidogenic properties (Meyer and Stasse-Wolthuis, 2009). Frontiers in Microbiology | www.frontiersin.org 10 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens TABLE 5 | Effects of experimental treatments on ileal and colonic fermentation in ETEC F4 trial. Day PI Treatment RSD p-value IC IS NIC NIS Challenge Treatment Interaction ETEC F4 trial ILEUM Lactic acid 3 7.55 10.58 54.33 10.12 15.880 0.013* 0.116 0.004* (mmol/kg) 7 14.42 18.06 15.96 14.52 15.330 0.854 0.840 0.643 Acetic acid 3 2.45 2.31 2.13 5.57 1.727 0.034* 0.092 0.033* (mmol/kg) 7 3.93 3.85 3.86 6.20 3.704 0.390 0.395 0.362 COLON 3 6.14 6.09 6.26 6.38 0.383 0.035* 0.548 0.096 pH 7 6.05 6.08 6.21 6.15 0.332 0.363 0.894 0.699 3 9.56 10.14 5.8 4.82 7.008 0.097 0.906 0.767 NH3 (mmol/L) 7 2.17 2.04 2.44 1.22 1.288 0.532 0.150 0.253 Lactic acid 3 0.65 3.10 1.65 2.64 2.858 0.940 0.140 0.575 (mmol/kg) 7 0.44 0.72 0.25 0.69 0.397 0.667 0.106 0.704 SCFA 3 99.8 99.3 81.8 103.4 40.60 0.716 0.517 0.489 (mmol/kg) 7 132.6 126.1 115.2 107.2 21.94 0.026* 0.359 0.921 SCFA molar ratio (%) 3 60.4 54.4 60.9 57.7 4.53 0.261 0.012* 0.444 Acetic 7 61.0 55.5 58.1 56.3 4.45 0.515 0.029* 0.241 3 22.6ab 25.6a 22.8ab 19.4b 3.37 0.016* 0.982 0.022* Propionic 7 22.6 23.7 25.0 24.4 3.44 0.217 0.846 0.485 3 11.9 13.5 11.8 15.5 3.78 0.483 0.093 0.464 Butyric 7 13.1 15.2 12.4 13.1 2.67 0.154 0.140 0.459 3 2.82 4.07 2.05 4.62 1.741 0.959 0.010* 0.336 Valeric 7 1.88 3.74 2.52 4.40 1.328 0.174 <0.001* 0.981 3 1.74 1.32 1.37 2.08 0.893 0.469 0.729 0.117 BCFA 7 1.31 1.69 1.92 1.64 0.779 0.325 0.862 0.243 This table includes values corresponding to pH, ammonia concentration (NH3; mmol/L of FM), lactic acid (mmol/kg of FM) and total short-chain fatty acids (SCFA; mmol/kg of FM and molar ratio) analysed in samples of ileal and colonic digesta. IC, Inoculated animals receiving placebo; IS, Inoculated animals receiving the synbiotic; NIC, Non-inoculated animals receiving placebo; and NIS, Non-inoculated animals receiving the synbiotic. N = 8 for all groups except. Values of p were obtained by ANOVA using the generalised linear procedure in R software. Letters a and b indicate statical differences between groups. FM, Fresh Matter; NH3, ammonia concentration; SFCA, short-chain fatty acids; BCFA, branched-chain fatty acids; and RSD, residual standard deviation. *indicates statistical difference. Considering these facts, we hypothesised that combining B. of a dilution of the energy or limiting of amino acids in the infantis CECT 7210 with FOS and inulin could therefore SYN diet following the inclusion of 5% of the prebiotic. It may enhance its beneficial effects against pathogens, contributing also have owed to changes in the transit time and digestibility to improved gut health. of nutrients related to the inclusion of 5% of FOS/inulin (Kelly, With this objective, two different trials were performed to 2009; Chen et al., 2017), being the changes that we observed challenge animals with either Salmonella or ETEC F4. As is in the consistency of caecal digesta at day 8 PI supported by well known, these pathogens exhibit differences in terms of this hypothesis. Furthermore, the reduction of gain:feed may pathogenicity, mediated by distinct virulence factors and have been due to modifications in the gut microbiota promoted mechanisms (Boyen et al., 2008; Clements et al., 2012). Thus, by the synbiotic with an impact on host energy homeostasis the animals’ responses to the challenge and the clinical course (Rosenbaum et al., 2015; Ley et al., 2016). However, this is mere differed depending on the pathogen. Whereas after the Salmonella speculation, as we lack the evidence to support these ideas. challenge growth and feed intake were markedly reduced, with Clinical signs such as diarrhoea incidence were not improved a clear effect on faecal score and an increase in rectal temperature, by the administration of the synbiotic compound to the animals, the challenge with ETEC F4 exhibited a much milder course but it is also fair to highlight that no deterioration was observed of diarrhoea, the differences not being statistically significant in any of the trials, verifying the safety of the probiotic strain, for several parameters. Similar effects were observed by Barba- as proved by other authors (Moreno-Muñoz et al., 2011; Barba- Vidal et al. (2017), viewing the challenge by ETEC F4 as Vidal et al., 2017; Rodríguez-Sorrento et al., 2020) even when milder than that of Salmonella Typhimurium. it is combined with inulin and fructo-oligosaccharides. The performance of the animals before the oral challenge Numerous previous studies have demonstrated that was not modified by the administration of the synbiotic in terms bifidobacteria, inulin and FOS can have a beneficial effect for of feed intake or weight gain, but in terms of gain:feed, a significant the host, helping it to maintain a healthy gut environment. reduction in feed efficiency was seen in the Salmonella trial Indeed, bifidobacteria may increase the colonic intraluminal (0.48 vs. 0.66 for synbiotic vs. control diet, p = 0.02). We might concentration of short-chain fatty acids (SCFA; Servin, 2004), hypothesise that this decrease in feed efficiency was the result which are responsible for a wide range of effects in the Frontiers in Microbiology | www.frontiersin.org 11 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens Higher amounts of acetate and butyrate have been shown to have favourable effects on the colonic structure; for example, acetate promotes colonic epithelial proliferation and butyrate is responsible for the maintenance of mucosal integrity, reparation and colonocyte proliferation, given that it is the preferred energetic source for these cells (Topping, 1996). Inulin and FOS have been reported as exerting a bifidogenic effect in infants (Meyer and Stasse-Wolthuis, 2009; Paineau et al., 2013) as well as in modulating fermentation products in the gut. Furthermore, various in vitro studies have shown how inulin and oligofructose can increase butyrate, propionate (van de Wiele et al., 2007) and acetate (van der Beek et al., 2018) production. Differential effects may be related to the contrasting chemical structure of these compounds as well as to the specific microbial ecosystems in which they are introduced. In this regard, Rossi et al. (2005) has reported differences between inulin and FOS: whereas for inulin, the main fermentation product was butyric acid with lower amounts of acetic, lactic and propionic acids, for FOS, the main fermentation products were lactic acid and acetic acid, alongside lower amounts of butyric acid and no propionic acid. In vivo studies have also shown a variable impact on intestinal fermentation. Scholtens et al. (2006) have demonstrated increases in acetate and decreases in butyrate in the faeces of humans receiving 25–30 g/d of FOS for a period of 2 weeks, while Boets et al. (2015) have used stable isotope technology to demonstrate how in humans inulin is mainly fermented into acetate as well FIGURE 4 | Effect of experimental treatments in serum levels of acute-phase protein Pig-MAP in piglets on day 8 (Salmonella trial) and 7 (ETEC F4 trial) as to a lesser extent into butyrate and propionate. following a pathogen oral challenge. Figure represents value frequencies Considering the varied findings of previous works, it was between a normal (0.3–1 mg/ml) and abnormal (>2 mg/ml) range. IC, difficult to anticipate what to expect when combining Inoculated animals receiving placebo; IS, Inoculated animals receiving the bifidobacteria, inulin and FOS; moreover, the impact of the synbiotic; NIC, Non-inoculated animals receiving placebo; and NIS, Non- synbiotic would seem to be contingent on the trial in question. inoculated animals receiving the synbiotic. N = 8 for all experimental groups except for non-challenged animals in Salmonella trial, N = 4. Values of p were In the Salmonella trial, the synbiotic reduced the molar proportion obtained using Fisher’s Exact Test on R software. of acetate in non-challenged animals but increased it in challenged ones. Differently, in the ETEC F4 trial, acetate was consistently reduced. These lower levels of acetate might be explained by gastrointestinal system. Topping (1996) has described how the a cross-feeding phenomena, supported in the ETEC F4 trial reduction in pH associated with an increase in SCFA might by the observable increasing trend of butyrate. In the same help to control the proliferation of pathogenic microorganisms. vein, the increase observed for acetate in the Salmonella- In addition, inulin and the fructo-oligosaccharides derived can challenged animals with the synbiotic, could correspond to a produce the same effect on SCFA concentrations in vivo and more acute dysbiosis that might disturb the normal cross- in vitro (Pompei et al., 2008; Van der Beek et al., 2018; Baxter feeding phenomena within bifidobacteria and colonic bacteria. et al., 2019). In our trials, however, the concentration of SCFA Similar kinds of interactions have been described in the in the colonic content was not enhanced by the synbiotic literature. For instance, regarding FOS supplements to dogs, treatment. According to Nyman (2002), this outcome should Pinna et al. (2018) found increases in the acetate:propionate be expected because the concentration of measured SCFA is ratio in low-protein diets but a decrease in high-protein diets. contingent on the balance between production and absorption, Another SCFA that is rarely considered in the literature and and commonly SCFA produced by fermentation are rapidly whose concentration was augmented by the synbiotic treatment absorbed or utilised by the colonic mucosa. in both trials is valeric acid. This fatty acid, which is capable Regarding changes in the fermentation profile, different effects of inhibiting the growth of pathogenic bacteria like Clostridium can be attributed to bifidobacteria and inulin or FOS. Several difficile (McDonald et al., 2018), originates in 5-aminovalerate, authors have described increases in the molar percentage of butyrate which is a product of the anaerobic degradation of previously with probiotic bifidobacteria (Rossi et al., 2005; Belenguer et al., hydrolyzed protein by gut bacteria (Barker et al., 1987). Recent 2006), although the main fermentation product of bifidobacteria investigations have proved that a strain of Megasphaera elsdenii is acetate. Butyrogenic effects owe to a stimulation of acetate- (a major inhabitant of the pig intestine) can utilise lactic acid depending, butyrate-producing colon bacteria by cross-feeding as a fermentation substrate and convert it into valerate (Yoshikawa interactions that, in parallel, are required by some other bacteria et al., 2018). It is feasible that a similar effect occurred in our that can convert lactate into butyrate, albeit only when acetate experiments, as colonic lactic acid was augmented by the synbiotic, is present (De Vuyst and Leroy, 2011; Moens et al., 2016, 2017). albeit only in the ETEC F4 trial. Frontiers in Microbiology | www.frontiersin.org 12 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens TABLE 6 | Effects of treatments on histomorphological parameters on days 4 and 8 (Salmonella trial) and 3 and 7 (ETEC F4 trial) post-inoculation. PI Day Treatment RSD p-value IC IS NIC NIS Challenge Treatment Interaction Salmonella trial Villi height (μm) 4 128 149 230 228 50.4 <0.001* 0.532 0.604 8 215 202 227 270 44.7 0.061 0.716 0.150 4 290 314 240 277 24.4 0.013* 0.076 0.665 Crypt depth (μm) 8 327 327 282 296 40.1 0.042* 0.769 0.705 4 1.22 1.28 1.19 0.69 0.636 0.276 0.626 0.320 IEL (Cell no./100 μm) 8 1.01 1.41 1.14 1.53 0.443 0.510 0.039* 0.994 4 0.90 0.93 0.60 0.85 0.136 0.171 0.598 0.310 Mitosis (Cell no./100 μm) 8 0.84 0.59 0.63 0.60 0.408 0.142 0.008* 0.094 ETEC F4 trial 3 256ab 267ab 287b 216a 50.8 0.584 0.106 0.032* Villi height (μm) 7 285 261 261 294 55.1 0.830 0.825 0.156 3 244 231 249 201 47.8 0.475 0.081 0.308 Crypt depth (μm) 7 223 219 217 245 24.8 0.264 0.178 0.068 3 0.57 0.45 0.43 0.62 0.252 0.871 0.705 0.086 IEL (Cell no./100 μm) 7 0.49 0.40 0.45 0.43 0.131 0.885 0.243 0.452 3 0.29 0.33 0.27 0.40 0.084 0.546 0.006* 0.173 Mitosis (Cell no./100 μm) 7 0.43 0.38 0.33 0.32 0.091 0.014* 0.332 0.483 IC, Inoculated animals receiving placebo; IS, Inoculated animals receiving the synbiotic; NIC, Non-inoculated animals receiving placebo; and NIS, Non-inoculated animals receiving the synbiotic. N = 8 for all groups except for non-challenged animals in Salmonella trial, N = 4. Values of p were obtained by ANOVA using the generalised linear procedure in R software. Letters a and b express differences considered for p < 0.05. IEL, villous intraepithelial lymphocytes; RSD, residual standard deviation. *indicates statistical difference. Together with the inhibitory effects promoted by probiotics use these fermentable sources of carbohydrates as growth on enteropathogens throughout changes in the fermentation substrates, increasing its concentration when it is seeded in products, probiotics have also been shown to fight pathogens faecal cultures supplemented with inulin or FOS. However, via other mechanisms. In particular, several species of despite these effects on coliforms, it is also important to Bifidobacterium are deemed capable of enhancing and modulating remember that in both the present study and that of Barba- the immune response (Wagner et al., 2009; Bermudez-Brito Vidal et al. (2017), no significant effects (nor increases or et al., 2013; Presti et al., 2015), of releasing bacteriocins and decreases) were detected in the numbers of ETEC F4 either bacteriocin-like substances (Poltavska and Kovalenko, 2012; in the digesta or in the ileal scrapings. Martinez et al., 2013) and of completing/displacing pathogens Although this investigation’s results do not provide evidence from their adhesion sites on the intestinal epithelium (Collado of the ability of the synbiotic to reduce the number of pathogens et al., 2005; Candela et al., 2008). The probiotic strain tested—B. in the intestine, some insights regarding the potential positive longum subsp. infantis CECT 7210—has proven to be effective effects can be provided. In the Salmonella trial, the reduction in reducing Salmonella loads in piglets in previous works of villi height associated with the pathogen challenge (Olivares- (Barba-Vidal et al., 2017). Interestingly, the former strain Villagómez and Van Kaer, 2018) was not attenuated by the administered together with L. rhamnosus HN001 was also synbiotic mixture, although a decline in the number of mitosis capable of diminishing Salmonella and ETEC F4 presence as was seen, suggesting that the amount of damaged tissue that shown by our research group in the past (Rodríguez-Sorrento needed to be replaced was reduced. Furthermore, the synbiotic et al., 2020, 2021). Nevertheless, in the present trial, combining may have also modulated the immune response at the gut level, this probiotic with inulin and FOS was unable to reduce as an increase in the number of IEL with the synbiotic was Salmonella excretion, suggesting that the combination of this found in the Salmonella trial (p = 0.039) at day 8 PI as well as Bifidobacterium strain with these prebiotics does not improve in the ETEC F4 trial, albeit only in those animals challenged its power to fight the pathogen. Regarding the potential of B. with the pathogen (P interaction = 0.086). Similarly with this longum subsp. infantis CECT 7210 to exclude ETEC F4, previous probiotic strain, Barba-Vidal et al. (2017) have also reported studies have demonstrated this strain’s ability to reduce the an increase in IEL in the ileum of piglets (whether challenged number of coliforms adhered to ileal mucosa in ETEC or not) with Salmonella of ETEC F4. A higher presence of IEL F4-challenged animals (Barba-Vidal et al., 2017), but in the might be regarded as beneficial considering that these cells are present study, we were only able to find such an effect in the responsible for the healing and protection of the integrity of non-challenged animals. When challenged with the ETEC F4, the intestinal epithelium as well as acting as early response the synbiotic treatment was associated with a numerical increase effectors against mucosal pathogens (Olivares-Villagómez and in attached coliforms, although not significantly. This interaction Van Kaer, 2018). Supporting the immunomodulatory properties (day 7 PI; p = 0.057) may be explained by inoculated ETEC of this strain, previous studies with a murine model of rotavirus F4 potentially profiting from the supplemented inulin and FOS, infection (Moreno-Muñoz et al., 2011) have reported increases considering that as stated by Rossi et al. (2005) E. coli can in the levels of secretory Immunoglobulin A (IgA) in the faeces. Frontiers in Microbiology | www.frontiersin.org 13 March 2022 | Volume 13 | Article 831737 Rodríguez-Sorrento et al. Synbiotic Efficacy Against Digestive Pathogens In the light of these results, increases in ileal IEL in the present ETHICS STATEMENT study with the synbiotic could be attributed to the probiotic strain. However, a possible additional impact of the prebiotic The animal study was reviewed and approved by the Animal fibres should not be discounted, considering that in the literature and Human Experimental Ethical Committee of the Autonomous several works have reported the ability of inulin and FOS to University of Barcelona (Permit No. CEEAH: 4026; enhance local immune responses (Shukla et al., 2016; Le Bourgot DMAH: 10118). et al., 2017; Myhill et al., 2018). To summarise, combining B. longum subsp. infantis CECT 7210 with inulin and FOS was not able to reduce Salmonella or coliforms loads in the gut, as has been previously reported AUTHOR CONTRIBUTIONS for the single probiotic strain. Nonetheless, this synbiotic AR-S participated in the experimental design and was responsible combination was able to modify the fermentative activity of the for the animal trial, laboratory analysis, data analysis, and intestine with differential effects depending on the pathogen writing. LC participated in the experimental design, animal challenge, most likely disturbing the expected cross-feeding trials, data analysis, and writing. PL-C participated in animal processes between the bifidobacteria and the indigenous trials and data analysis. GC-O and JM-M participated in the butyrogenic colonic bacteria. The combination of this probiotic experimental design and contributed to data analysis and writing. strain with inulin and FOS was also able to increase the numbers SM-O participated in the experimental design, animal trials, of IEL at the ileal level, suggesting certain immunomodulatory laboratory analysis, data analysis, and writing. All authors properties. A more in-depth study of the changes produced in contributed to the article and approved the submitted the gut ecosystem is necessary in order to develop a greater version. understanding of the role of this synbiotic combination in a scenario of well-balanced or dysbiotic microbiota. FUNDING DATA AVAILABILITY STATEMENT This project has been supported by CDTI (Ministerio de Ciencia The original contributions presented in the study are included e Innovación de España) and Fondo Europeo de Desarrollo in the article/Supplementary Material, further inquiries can Regional (FEDER; Acronym: SMARTFOODS, project number be directed to the corresponding author. IDI-20141206). REFERENCES Boyen, F., Haesebrouck, F., Maes, D., Van Immerseel, F., Ducatelle, R., and Pasmans, F. (2008). Non-typhoidal Salmonella infections in pigs: a closer AOAC (1995). International Official Methods of Analysis of AOAC International. look at epidemiology, pathogenesis and control. Vet. Microbiol. 130, 1–19. Washington, DC: AOAC. doi: 10.1016/j.vetmic.2007.12.017 Barba-Vidal, E., Castillejos, L., López-Colom, P., Rivero-Urgell, M., Candela, M., Perna, F., Carnevali, P., Vitali, B., Ciati, R., Gionchetti, P., et al. Moreno-Muñoz, J. A., and Martín-Orúe, S. M. (2017). Evaluation of the (2008). 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The prebiotic inulin improves substrate metabolism and promotes short-chain fatty acid production Copyright © 2022 Rodríguez-Sorrento, Castillejos, López-Colom, Cifuentes-Orjuela, in overweight to obese men. Metabolism 87, 25–35. doi: 10.1016/j. Moreno-Muñoz and Martín-Orúe. This is an open-access article distributed under metabol.2018.06.009 the terms of the Creative Commons Attribution License (CC BY). The use, distribution Van Soest, P. J., Robertson, J. B., and Lewis, B. A. (1991). Methods for dietary or reproduction in other forums is permitted, provided the original author(s) and fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal the copyright owner(s) are credited and that the original publication in this journal nutrition. J. Dairy Sci. 74, 3583–3597. doi: 10.3168/jds.S0022-0302(91) is cited, in accordance with accepted academic practice. No use, distribution or 78551-2 reproduction is permitted which does not comply with these terms. 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