ORIGINAL RESEARCH published: 21 August 2020 doi: 10.3389/fmicb.2020.02012 Effects of Bifidobacterium longum Subsp. infantis CECT 7210 and Lactobacillus rhamnosus HN001, Combined or Not With Oligofructose-Enriched Inulin, on Weaned Pigs Orally Challenged With Salmonella Typhimurium Agustina Rodríguez-Sorrento 1 , Lorena Castillejos 1* , Paola López-Colom 1 , Gloria Cifuentes-Orjuela 2 , Maria Rodríguez-Palmero 2 , José Antonio Moreno-Muñoz 2 Edited by: and Susana María Martín-Orúe 1 Riadh Hammami, University of Ottawa, Canada 1 Servicio de Nutrición y Bienestar Animal, Departament de Ciència Animal i dels Aliments, Universitat Autònoma Reviewed by: de Barcelona, Bellaterra, Spain, 2 Laboratorios Ordesa S. L., Barcelona, Spain Mikael Lenz Strube, Technical University of Denmark, Salmonella is a common causative agent of enteric disease and is developing Denmark Francesca Valerio, mechanisms of resistance to antimicrobials. Probiotics, such as bifidobacteria and National Research Council (CNR), Italy lactobacilli, and prebiotic fibers are a potential alternative to counteract this pathogen *Correspondence: as they have demonstrated effectiveness in preventing its adhesion, reducing intestinal Lorena Castillejos lorena.castillejos@uab.cat damage, and enhancing the host immune system. Furthermore, the benefits are expected to be potentiated when these compounds are administered together. A trial Specialty section: was performed to evaluate the efficacy of two probiotic strains (Bifidobacterium longum This article was submitted to Food Microbiology, subsp. infantis CECT 7210 (Laboratorios Ordesa S.L.) and Lactobacillus rhamnosus a section of the journal HN001, combined or not with a prebiotic containing oligofructose-enriched inulin, Frontiers in Microbiology against Salmonella Typhimurium. Ninety-six piglets (28 days old) were distributed into Received: 22 January 2020 Accepted: 29 July 2020 32 pens assigned to 5 treatments: one non-challenged (control diet, CTR+) and four Published: 21 August 2020 challenged: control diet (CTR−) or supplemented with probiotics (>3 × 1010 cfu/kg Citation: each strain, PRO), prebiotic (5%, PRE), or their combination (SYN). After 1 week of Rodríguez-Sorrento A, adaptation, animals were orally challenged with Salmonella Typhimurium. Feed intake, Castillejos L, López-Colom P, Cifuentes-Orjuela G, weight, and clinical signs were recorded. On days 4 and 8 post-inoculation (PI), one Rodríguez-Palmero M, animal per pen was euthanized, and samples from blood, digestive content, and Moreno-Muñoz JA and Martín-Orúe SM (2020) Effects ileal tissues were collected to determine Salmonella counts, fermentation products, of Bifidobacterium longum Subsp. ileal histomorphology, and serum TNF-α and Pig-MAP concentrations. The effect of infantis CECT 7210 and Lactobacillus the oral challenge was evidenced by animal performance, fecal consistency, and rhamnosus HN001, Combined or Not With Oligofructose-Enriched Inulin, on intestinal architecture. Regarding the experimental treatments, animals belonging to Weaned Pigs Orally Challenged With the PRO group experienced a faster clearance of the pathogen, with more pigs Salmonella Typhimurium. Front. Microbiol. 11:2012. being negative to its excretion at the end of the study and recovering the impaired doi: 10.3389/fmicb.2020.02012 ileal villi/crypt ratio more rapidly. Animals receiving the PRE diet showed a lower Frontiers in Microbiology | www.frontiersin.org 1 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium intestinal colonization by Salmonella, with no countable levels (<3 cfu/g) in any of the analyzed samples, and an augmented immune response suggested by serum Pig-MAP concentrations. Treatments including the prebiotic (PRE and SYN) showed similar changes in the fermentation pattern, with an increase in the molar percentage of valeric acid concentration in the colon. The SYN group, however, did not show any of the outcomes registered for PRO and PRE in Salmonella colonization or in immunity markers, suggesting the lack of synbiotic action in this animal model. Further research is needed to better understand the complex mechanisms behind these effects. Keywords: synbiotic, Salmonella, piglet, probiotic, prebiotic, inulin, Bifidobacterium, Lactobacillus INTRODUCTION authors have proposed the combined use of probiotics and prebiotics to selectively increase the survival and activity of Salmonellosis is a disease caused by non-typhoidal serotypes the specific probiotic strains, thereby improving their efficacy. of Salmonella; after campylobacteriosis, it is the second most This concept is known as symbiotic therapy (De Vrese and common foodborne in the European Union (91,662 human Schrezenmeir, 2008) and has shown promising results against cases reported in 2017; European Food Safety Authority, acute diarrhea in children (Yang et al., 2019). 2019). It is characterized by symptoms such as an acute The aim of this work was to evaluate a multistrain probiotic onset of fever mainly accompanied by abdominal pain, nausea, composed of Bifidobacterium longum subsp. infantis CECT 7210 and diarrhea. The course of the illness is self-limited, and and Lactobacillus rhamnosus HN001, the prebiotic oligofructose- affected individuals recover without treatment, but it can enriched inulin and their synbiotic combination in a weaned develop to a serious and life-threatening condition in non- piglet model orally challenged with Salmonella Typhimurium. immunocompetent patients, such as children and elderly people (World Health Organization, 2018). Different serovars of Salmonella isolated from humans have MATERIALS AND METHODS been found to be “highly” or “extremely highly” resistant to antimicrobials (European Food Safety Authority, 2019). As A trial was carried out at the Experimental Unit of the a consequence, alternatives to these substances that could Universitat Autònoma de Barcelona (UAB) and received prior potentially fight against these pathogenic bacteria, such approval (Permit No. CEAAH4026) from the Animal and probiotics and prebiotics, are of a great interest. Different Human Experimental Ethical Committee of this institution. The mechanisms could be behind the positive effects against treatment, management, housing, husbandry and slaughtering pathogens reported by different probiotics and prebiotics, conditions conformed to the European Union Guidelines but in general terms, all of them would boost the natural (Directive 2010/63/EU, European Commission, 2010). All efforts mechanisms of colonization resistance (Lawley and Walker, were made to minimize animal suffering. 2013; Fehervari, 2019). Probiotics have demonstrated efficacy against a multitude Animals, Housing, and Experimental of enteropathogens. Some strains of Lactobacillus prevent the Design intestinal damage caused by enterohemorrhagic Escherichia coli This trial was carried out as Level 2 High-Risk Biosecurity (Johnson-Henry et al., 2008), and specifically, the HN001 (DR20) procedure and involved personnel with appropriate training. In strain offers protection against Salmonella Typhimurium by total, 96 male piglets [Landrace × Large White] × Pietrain stimulating the immune response of the host (Gill et al., 2001). of 28 (±3) days of age, weighting 6.81 (±0.13) kg, were used. In addition, the genus Bifidobacterium plays an important role All animals were obtained from high-sanitary-status farms from in the maintenance of gut homeostasis (Tojo et al., 2014), and mothers serologically negative to Salmonella. some strains have also been tested in gastrointestinal pathogen Piglets were transported to the experimental unit located in infections. Specifically, Bifidobacterium longum subsp. infantis the UAB, which consisted of four boxes of eight pens each (32 CECT 7210 has been proven safe and effective against rotavirus in pens, three animals per pen) (see Figure 1). Each 2-m2 pen was a murine model (Muñoz et al., 2011) and against enterotoxigenic equipped with a feeder and water nipple to provide feed and Escherichia coli and Salmonella Typhimurium in a piglet model water ad libitum. All weaning rooms were equipped with an (Barba-Vidal et al., 2017). automatic heater and forced ventilation, and each pen had an Administration of prebiotics also represents a good strategy individual heating light. to fight enteropathogens. Compounds such as inulin and At arrival, animals were distributed according to their initial oligofructose reduce the adhesion of pathogens to intestinal body weight (BW) in order to ensure a homogeneous average epithelium by increasing the bifidobacteria and lactobacilli BW among treatment groups. The trial was conceived as a indigenous population able to use these fibers as growth completely randomized design that included five experimental substrates (Bosscher et al., 2006). In the last years, different groups: non-inoculated control (CTR+) and inoculated control Frontiers in Microbiology | www.frontiersin.org 2 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium TABLE 1 | Ingredients and nutritional composition of the diets. Ingredients (g/kg FM) CTR/PRO PRE/SYN Maize 206.7 196.3 Wheat 179.4 170.4 Barley 2 row 169.4 160.9 Extruded soybean 148.6 141.2 Sweet whey-powder (cattle) 99.6 94.7 Fish meal 59.8 56.8 Soybean meal 44 79.7 75.7 Whey-powder 50% fat 24.9 23.7 Mono-calcium phosphate 6.8 6.4 Calcium carbonate (CaCO3) 3.9 3.7 L-Lysine HCL 5.3 5.1 Vit-Min Premix* 4 3.8 Sodium chloride (marine salt) 2.5 2.4 FIGURE 1 | Distribution of experimental treatments in the facilities. DL-Methionine 99 3.9 3.7 L-Threonine 2.8 2.7 L-Tryptophan 0.8 0.8 L-Valine 2 1.9 (CTR−), probiotic combination (PRO), prebiotic (PRE) and Prebiotic 0 50 synbiotic (SYN). The latter four groups were orally challenged and equally distributed in three of the four rooms, while Analyzed composition (g/kg FM) CTR/PRO PRE/SYN one full room was kept for non-inoculated control pigs. In Dry matter 916.9 917.4 the challenged rooms, probiotic and synbiotic treatments were Ashes 48.2 47.7 distributed within the four pens along one side of the room Crude fat 61.1 58.8 and the control and prebiotic treatments on the other side of Crude protein 203.4 193.1 the room, separated by a corridor. Each experimental group had Neutral detergent fiber 80.5 82 six replicates, except for the non-challenged groups, which had Acid detergent fiber 25.7 27.1 eight replicates. *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 B6, 0.025 mg vitamin B12, 20 mg calcium pantothenate, 60 mg nicotinic Probiotic Strains, Prebiotic Mixture, and acid, 0.1 mg biotin, 0.5 mg folic acid, 150 mg Fe, 156 mg Cu, 0.5 mg Co, 120 mg Diets Zn, 49.8 mg Mn, 2 mg I, 0.3 mg Se. The tested probiotics were Bifidobacterium longum subsp. infantis CECT 7210, supplied by Ordesa S.L., and Lactobacillus rhamnosus HN001 (Danisco USA Inc.) strains. Both were stored Salmonella Strain in lyophilized form containing 5 × 1010 and 3 × 1010 colony- The bacterial strain used for the oral challenge was Salmonella forming units [cfu] per gram of product, respectively, in a enterica serovar Typhimurium var. Monophasic (formula: maltodextrin base. The lyophilized probiotics were daily mixed 4,5,12:i:-, resistance profile: ACSSuT-Ge, Fagotype: U302), isolated into the feed for a final dosage of 5.5 × 107 and 3.3 × 107 cfu/g, from a salmonellosis outbreak of fattening pigs in Spain, respectively, totally replacing the feed each day. The viability of and provided by the Infectious Diseases Laboratory (Ref. the probiotic throughout the day in the dry feed was confirmed 301/99) of the UAB. The preparation of the oral inoculum prior to the trial. With this aim, one hundred grams of feed consisted of 24-h incubation at 37◦ C in buffered peptone water belonging to each treatment was analyzed at 0, 6, 12, 24 and 48 h (Oxoid; Hampshire, United Kingdom) and diluted (1:10) with after feed replacement. sterile phosphate buffered saline (PBS) (Sigma-Aldrich; Madrid, The oligofructose-enriched inulin (OF) was in powder form Spain). The final concentration of the inoculum was 1 × 109 and was manually mixed into the feed up to a final concentration cfu/mL. Inoculum concentrations were determined prior to of 5% (w/w); OF was mixed previously to the probiotic inoculation by McFarland standards and were double-plated in in the SYN diet. Tryptic Soy Agar (TSA) (Liofilche; Italy) on the same day for Pre-starter diets were formulated in concordance with the manual plate counting. nutrient requirement standards for pigs (NRC, 2012) and given in a mashed form. Synthetic amino acids were added to PRE and Experimental Procedure SYN diets in order to compensate for the possible amino acid The experimental schedule is depicted in Figure 2. The dilution produced by prebiotic incorporation: 0.5 g L-valine, 0.9 g experimental duration was 15 days. After an adaptation period L-lysine HCL, 1.2 g DL-methionine, 0.5 g L-threonine, and 0.2 g of 7 days, animals were orally challenged with the pathogen. L-tryptophan per kg of feed. Details regarding the ingredient and One animal of each pen was euthanized on days 4 and 8 post- chemical composition are given in Table 1. inoculation (PI). Frontiers in Microbiology | www.frontiersin.org 3 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium FIGURE 2 | Experimental schedule. After 1 week of adaptation, the inoculum containing the (0 PI) and on days 1, 3, and 7 PI. Fecal samples were always pathogenic bacterial culture was orally given to each animal obtained from the same animal, corresponding to the largest one of the challenged groups. Each animal received a total volume of each pen at the beginning of the trial (N = 32). of 2 mL, corresponding to a 2 × 109 cfu total dose of On days 4 and 8 PI, one pig per pen was euthanized. On Salmonella Typhimurium. The same amount of sterile broth day 4 PI, the selected animal was the one with the medium was administered to non-challenged piglets. To ensure that the initial BW, while on day 8 PI, the chosen piglet was the heaviest animals had a full stomach at the moment of the oral challenge, one of each pen. Animals were euthanized and sequentially pigs were starved for a period of 12 hs, and feed was reintroduced sampled during the morning of each sampling day (between 30 min prior to inoculation. 8:00 and 13:00 h). Prior to injection of the euthanasic drug, From the challenge onward, animals were checked daily to 10 mL of blood were taken from each animal via venepuncture determine clinical signs and to evaluate their post-inoculation of the cranial cava vein, using 10-mL blood collection tubes status (i.e., dehydration, anorexia, apathy, general behavior, and without anticoagulant (Aquisel; Madrid, Spain). Immediately fecal score), always by the same person. Fecal score was measured after blood sampling, pigs were administered a lethal dose using the following scale: 1 = solid and cloddy, 2 = soft with shape, injection of sodium pentobarbital intravenously (140 mg/kg BW; 3 = very soft or viscous liquid, and 4 = watery or with blood. Euthasol, Le Vet B.V.; Oudewater, Netherlands). Once dead, Rectal temperature was assessed with a digital thermometer animals were bled, the abdomen was immediately opened, and (Accuvet, Sanchung City, Taiwan) on days 1 and 3 PI. the gastrointestinal tract was extracted. To monitor animal performance, individual body weight was A sample from caecal content was obtained for microbial registered at arrival and on days 0, 4, and 8 PI. Feed intake was analysis and kept on ice until analysis within the first 4 h. monitored daily. Average daily gain (ADG), average daily feed Subsequently, digesta of ileum and proximal colon were collected intake (ADFI), and gain: feed ratio (G:F) were calculated per pen. and homogenized prior to pH determination with a pH Mortality rate was also registered, and no antibiotic treatment meter (Crison 52–32 electrode, Net Interlab; Barcelona, Spain). was given to the animals. Subsamples of ileal and colonic digesta were preserved for For microbiological analysis, fecal samples were collected different analyses. A set of samples was stored for ammonia aseptically after spontaneous defecation or by digital stimulation. determination (NH3 ) (frozen at −20◦ C in H2 SO4 solution (3 mL Fecal samples were taken at arrival, the day of the inoculation of content plus 3 mL of 0.2 N H2 SO4 )), and an additional set Frontiers in Microbiology | www.frontiersin.org 4 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium (∼10 g) for determination of short-chain fatty acids (SCFA) and of each one were performed with a light microscope (BHS, lactic acid (stored at −20◦ C). Olympus; Barcelona Spain), using as guideline the procedure For the histological study, 1-cm sections from the ileum were described in Nofrarías et al. (2006). removed, opened longitudinally, and washed thoroughly The concentration of B. infantis CECT 7210 was determined and carefully with 4% formaldehyde solution (Panreac; by quantitative real-time PCR. To do so, DNA was extracted Castellar del Vallès, Spain) before being fixed by immersion from the colonic content samples from day 8 PI and the in the same solution. probiotic strain quantified by PCR using the forward (50 - Blood samples were centrifuged after blood clotting (3,000 × g CACAGCGGGCAGATCGGTAT-30 ) and reverse primers for 15 min at 4◦ C), and the serum obtained stored at −20◦ C. (50 -CGCCGGTGCCAGTCA-30 ) and a TaqMan probe (50 - [6FAM]CCGGTTAGTCCTCTACCGTACGCAAGC[TAM]-30 ). Analytical Procedures The master mix used was “HOT FIREPol Probe qPCR Mix Plus” Chemical analyses of the diets, including dry matter (DM), (Solis BioDyne; Tartu, Estonia). ash, crude protein, and diethyl ether extract, were performed according to the Association of Official Agricultural Chemists Statistical Analysis standard procedures (AOAC International, 1995). Neutral The effect of the experimental treatments on the performance detergent fiber and acid-detergent fiber were determined and slaughter measurements was determined using free software according to the method of Van Soest et al. (1991). R v.3.4.4 (R Development Core Team; Franklin Lakes, NJ, For microbiological analysis of Salmonella, samples were United States) and the stats package (R Core Team, 2013) transferred to buffered peptone water (BPW) solution in a lm function for a one-way ANOVA, with diet as a fixed concentration of 1:10. Quantitative analysis was performed by effect. Salmonella spp. count levels and Pig-MAP concentration seeding serial dilutions of 10−2 , 10−4 , and 10−6 in xylose- levels were previously categorized and subsequently analyzed lactose-tergitol-4 plates (XLT-4) (Merck; Madrid, Spain). For with the fisher.test function for frequency analysis. Pig-MAP the qualitative analysis, samples were incubated in BPW concentration levels were categorized considering the normal (37◦ C, 24 h) and 100 µL of the culture were transferred to range (0.3–1 mg/mL), borderline (1–2 mg/mL) and high levels 10 mL of Rappaport-Vassiliadis for a second incubation (42◦ C, (>2 mg/mL) (Piñeiro et al., 2009). Fisher test was applied to 48 h) to finally seed them in XLT4 plaques to observe H2 S- all groups together and also by pairwise. Regarding values for positive colonies. daily fecal consistency data were also assessed using the lme4 Short-chain fatty acid and lactic acid analyses were performed package (Douglas et al., 2015) lmer function for an adjusted linear using gas chromatography after the samples had undergone acid- mixed model, with a treatment-by-time interaction term. When base treatment prior to ether extraction and derivatization with treatment effects were established, means were compared by the N-(tertbutyldimethylsilyl)-N-methyl-trifluoroacetamide plus 1% Tukey–Kramer test (TukeyHSD function). Considering the large tert-butyldimethylchlorosilane agent, using the method of number of omnibus test included in this study and with the aim Richardson et al. (1989), modified by Jensen et al. (1995). of controlling possible type I errors, P-values were also adjusted Ammonia concentration was assed using a gas-sensitive using the Benjamini–Hochberg procedure. electrode (Hatch Co., Colorado Springs, CO, United States) For all analyzed data, the pen was the experimental unit. combined with a digital voltmeter (Crison GLP 22, Crison The alpha level for the determination of significance for all the Instruments, S.A.; Barcelona, Spain). We followed the procedure analyses was P = 0.05. The statistical trends were also considered described by Hermes et al. (2013), which was adapted from for P values >0.05 and <0.10. Data are presented as means and Diebold et al. (2004). Samples were diluted (1:2) in 0.16 M their residual standard error (RSE) unless otherwise stated. NaOH and, after homogenization, centrifuged at 1500 × g for 10 minutes. Once the ammonia was released, it was measured in the supernatants as a change in voltage in mV. RESULTS Serum concentrations of Tumor Necrosis Factor-α (TNF- α) were determined by Quantikine Porcine TNF-α kits (R&D The course of the experiment went as expected, without Systems; Minneapolis, MN, United States), and pig major acute- any remarkable incidences. Feed consumption was within the phase protein (Pig-MAP) concentration was determined by a expected values, with piglets receiving the calculated daily sandwich-type ELISA (Pig MAP Kit ELISA, Pig CHAMP Pro amounts of probiotics and prebiotics. Europe S.A.; Segovia, Spain) according to the manufacturer’s After oral challenge with the pathogenic bacteria, animals instructions. Antibodies against Salmonella were also assed presented mild to moderate clinical signs of diarrhea. We using an ELISA Salmonella Herdcheck (Idexx; Hoofddorp, registered only one casualty at day 2 PI (CTR− group), and no Netherlands); the established cut-off for positivity in optic euthanasia was required. density was ≥40%. For histological study, tissue samples were dehydrated and Probiotic Viability in Feed and embedded in paraffin wax: 4-µm thick sections were stained Gastrointestinal Survival with hematoxylin and eosin. Measurements of 10 different villus- As described in the previous section, the viability of both crypt complexes per sample and counting of intraepithelial probiotic strains in animal’s feed was tested. In fact, both of them lymphocytes (IEL), Goblet cells (GC), and number of mitosis maintained their viability in the feed for 24 h (L. rhamnosus Frontiers in Microbiology | www.frontiersin.org 5 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium TABLE 2 | Effects of experimental treatments on body weight (BW), average daily feed intake (ADFI), and average daily weight gain (ADG). Treatment RSD P-value Adjusted P-value CTR+ PRO PRE SYN CTR− BW (kg) Initial 6.80 6.76 6.78 6.88 6.81 0.140 0.687 0.777 Final 10.45a 9.34ab 8.86ab 8.21b 9.44ab 1.067 0.009* 0.045* ADFI (g) Adaptation 291.2a 242.2ab 236.4ab 225.2b 249.0ab 40.16 0.040* 0.131 0–4 PI 395.9a 316.1ab 287.4b 255.2b 315.8ab 68.55 0.009* 0.045* 4–8 PI 481.5a 314.9b 299.9b 219.1b 316.1b 103.10 0.001* 0.013* ADG (g) Adaptation 206.2 172.3 143.0 150.9 146.5 64.96 0.334 0.477 0–4 PI 274.7a 223.7ab 153.9ab 85.5b 222.9ab 110.60 0.042* 0.131 4–8 PI 270.0a 156.3ab 136.4ab 38.7b 150.3ab 97.46 0.003* 0.021* Adaptation: pre-inoculation week (days 1–8); 0–4 PI: acute post-inoculation period (days 8-11); 4–8 PI: sub-acute post-inoculation period (days 11–15). CTR+: Non- Inoculated animals receiving placebo; PRO: Inoculated animals receiving the probiotic; PRE: Inoculated animals receiving the prebiotic; SYN: Inoculated animals receiving the synbiotic; CTR−: Inoculated animals receiving placebo. N = 6 for all groups except for non-challenged animals, N = 8. P- values were obtained by an ANOVA, using the linear model procedure in the R software and adjusted by the Benjamini–Hochberg procedure. Means in the same row with different superscripts are different from each other (Tukey’s < P 0.05). HN001: 7.59 vs 7.57 log cfu/g, B. infantis CECT 7210: 7.45 vs days 5 (P = 0.116) and 7 PI (P = 0.140), all inoculated groups 7.33 log cfu/g at 0 and 24 h, respectively), suggesting that animals recovered, with no significant differences between treatments. belonging to PRO and SYN groups were receiving the desired Febrile response (> 40.5◦ C) could not be registered in the daily amount of probiotics. animals after the challenge (Figure 4). However, on day 3 Regarding the survival in the gastrointestinal tract of the PI, a trend for an increased rectal temperature was found B. infantis CECT 7210 strain, animals belonging to PRO group in the challenged animals except for those receiving the SYN showed a presence of 5.75 log cfu/g of colonic content, whereas treatment (P = 0.056). the concentration in those included in SYN group was of 3.96 log cfu/g. No significant difference was found between Microbiological Analysis treatments (P = 0.212). Serologic analysis confirmed that none of the animals was exposed to the pathogen prior to transfer, with all of them being Performance Parameters seronegative at the end of the experimental trial (data not shown). Results for BW, average daily feed intake (ADFI), and average To analyze data from Salmonella fecal plate counts, animals daily gain (ADG) are presented in Table 2. were classified into five different levels: high (between 107 and 108 The first effects on average daily feed intake were observed cfu per gram), medium (between 106 and 105 cfu per gram), low immediately after the oral inoculation. During the acute phase (between 104 and 103 cfu per gram), non-quantifiable positive of the infection (days 0–4 PI), PRE and SYN groups also (between 102 and 10 cfu per gram), or negative. This way, reached significant decreases compared to CTR+ (P = 0.049 and the effect of diets was analyzed by Fisher’s exact test, and no P = 0.006, respectively), and during the sub-acute phase (days 4–8 significant differences could be found related to experimental PI), all challenged groups showed a reduced feed consumption. treatments (Figure 5). Regarding average daily gain, no significant differences related However, it should be mentioned that when the same test was to diets were found after adaptation nor during the first phase sectorally applied to compare treatments, it was seen on day 7 of the infection. However, between days 4 and 8 PI, although PI that a greater percentage of animals receiving the PRO diet all challenged groups showed numerical reductions compared to became negative to Salmonella compared to CTR−animals (65 CTR+, only for the SYN group, this reduction was statistically vs 0%), with significant P-values (P = 0.028). In addition, when significant (P = 0.001), leading to a lower BW at the end of the the test was applied to compare treatments in terms of percentage experiment (P = 0.004). of animals with non-countable Salmonella throughout the entire experimental period (negative + very low levels), the only Clinical Signs treatment with no animal with countable levels was the PRE The evolution of fecal consistency along the post-inoculation treatment, with significant differences when compared to the period is represented in Figure 3. Clinical signs of diarrhea were CTR−group (P = 0.013). mild after the challenge, but fecal scores showed a clear increase in all inoculated groups the day after (P < 0.001, at day 1 PI). On Intestinal Fermentation day 2 PI, the difference to the CTR+ group was only significant The values of intestinal pH, ammonia, lactic acid, and short-chain for the SYN group (P = 0.006), whereas on day 3 PI, all challenged fatty acid concentrations in ileal and colonic contents for the groups except PRE were different from CTR+ (P < 0.001). On different experimental treatments are presented in Table 3. Frontiers in Microbiology | www.frontiersin.org 6 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium FIGURE 3 | Evolution of average fecal scores for the different experimental groups in the post-inoculation period. CTR+: Non-inoculated animals receiving placebo; PRO: Inoculated animals receiving the probiotic; PRE: Inoculated animals receiving the prebiotic; SYN: Inoculated animals receiving the synbiotic; CTR−: Inoculated animals receiving placebo. N = 6 for all groups except for non-challenged animals, N = 8. Bars correspond to standard error. FIGURE 4 | Effects of experimental treatments on rectal temperature on days 1 and 3 post-inoculation. CTR+: Non-inoculated animals receiving placebo; PRO: Inoculated animals receiving the probiotic; PRE: Inoculated animals receiving the prebiotic; SYN: Inoculated animals receiving the synbiotic; CTR−: Inoculated animals receiving placebo. N = 6 for all groups except for non-challenged animals, N = 8. Bars correspond to standard error. At the ileal level, no significant differences were seen for pH or to decrease with the challenge (P = 0.025 & adjusted P = 0.104). ammonia concentrations related to the experimental treatments. No statistical differences were found in the fermentation profile The concentration of total short-chain fatty acids showed a trend in terms of molar ratio. Frontiers in Microbiology | www.frontiersin.org 7 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium FIGURE 5 | Percentage of animals included in the different fecal and caecal excretion levels of Salmonella. All samples were obtained from the same animal of each pen (with the highest weight) except for the caecal sample on day 4 PI, obtained from the medium-weight animal. PRO: Inoculated animals receiving the probiotic; PRE: Inoculated animals receiving the prebiotic; SYN: Inoculated animals receiving the synbiotic; CTR−: Inoculated animals receiving placebo. N = 6 for all groups except for non-challenged animals, N = 8. P-values were obtained using Fisher’s Exact Test in the R software. Regarding the colon, no differences related to the experimental was numerically higher in those animals receiving de prebiotic treatments were found in ammonia, lactic acid, or total SCFA (PRE and SYN) at day 4 and 8 PI when compared to the rest concentrations. However, on day 4 PI, pH values tended to of the groups, although statistical significances were only found be lower for the challenged groups, especially for PRE diet (P < 0.05) when compared to CTR+ (PRE at day 4 and 8 PI) and compared to CTR+ (P = 0.018 & adjusted P = 0.082). Although to CTR− (PRE and SYN at day 8 PI). the concentration of total SCFA was not modified by the experimental treatments, there was an impact of the molar Immune Response ratio for the different fermentation products. On day 4 PI, The TNF-α did not respond to the challenge and only tended the PRE group showed a lower percentage of acetic acid than to be higher with the PRE diet at day 4 PI (106.5, 122.6, the control groups (P = 0.004 and P = 0.012 for CTR+ and 147.8, 140.8, and 111.2 pg/mL for CTR+, PRO, PRE, SYN, and CTR−, respectively). Moreover, the percentage of valeric acid CTR−, respectively; P = 0.094). Pig-MAP concentrations did Frontiers in Microbiology | www.frontiersin.org 8 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium TABLE 3 | Effects of experimental treatments on ileal and colonic fermentation. PI Day Treatment RSD P-value Adjusted P-value CTR+ PRO PRE SYN CTR− ILEUM pH 4 6.66 6.50 6.55 6.62 6.61 0.253 0.812 0.842 8 6.53 6.63 6.86 6.85 6.88 0.299 0.130 0.296 NH3 (mmol/L) 4 0.59 0.48 0.44 0.54 0.53 0.206 0.715 0.777 8 0.98 0.99 0.79 1.06 0.70 0.397 0.468 0.616 Lactic acid (mmol/kg) 4 9.97 10.48 5.60 3.93 12.26 10.27 0.589 0.718 8 22.1 22.42 5.38 16.47 5.77 25.10 0.586 0.718 SCFA (mmol/kg) 4 6.40 7.12 3.11 3.39 6.27 3.934 0.266 0.474 8 8.22a 6.76ab 4.26ab 3.09b 4.81ab 2.954 0.025* 0.104 SCFA molar ratio (%) Acetic 4 97.3 96.3 94.1 94.7 96.2 4.06 0.623 0.741 8 96.5 97.3 94.4 85.5 94.9 8.75 0.158 0.330 Propionic 4 3.22 3.36 10.19 6.95 7.20 3.709 0.253 0.474 8 1.93 1.92 4.55 6.72 4.21 3.544 0.105 0.262 Butyric 4 1.43 2.51 0.83 3.50 2.74 2.085 0.275 0.474 8 1.52 1.03 1.23 9.26 1.07 7.032 0.286 0.474 COLON pH 4 5.82a 5.65ab 5.17b 5.42ab 5.67ab 0.340 0.018* 0.082 8 5.74 5.76 5.85 5.94 5.92 0.390 0.826 0.843 NH3 (mmol/L) 4 4.52 8.43 5.31 8.97 9.84 5.517 0.325 0.477 8 4.66 8.92 10.22 9.07 11.64 6.651 0.369 0.512 Lactic acid (mmol/kg) 4 1.16 0.37 1.36 1.41 1.35 1.173 0.695 0.777 8 0.41 0.38 0.00 0.81 2.15 1.993 0.514 0.660 SCFA (mmol/kg) 4 158 152 138 155 147 29.7 0.756 0.804 8 177 171 165 157 146 41.4 0.706 0.777 SCFA molar ratio (%) Acetic 4 59.7a 57.8ab 51.6b 53.9ab 59.4a 3.83 0.002* 0.020* 8 55.7 58.8 51.6 58.4 61.6 6.03 0.077 0.214 Propionic 4 25.7 24.1 28.3 26.3 24.4 3.51 0.268 0.474 8 26.9 23.7 29.7 23.8 23.9 4.82 0.145 0.315 Butyric 4 10.8 13.8 14.4 14.6 11.8 3.87 0.297 0.477 8 12.1 12.9 11.9 11.5 10.3 2.86 0.398 0.538 Valeric 4 2.25a 2.59a 4.85b 4.11ab 2.79ab 1.246 0.003* 0.021* 8 2.68ab 2.77ab 4.81c 4.34bc 2.23a 1.127 0.001* 0.013* BCFA 4 1.55 1.70 0.86 1.11 1.59 0.593 0.087 0.223 8 1.56 1.77 1.95 2.04 1.97 0.932 0.863 0.863 The table includes pH values, ammonia concentration (NH3 ) (mmol/L), lactic acid (mmol/kg of FM), total short-chain fatty acids (SCFA) (mmol/kg of FM), and molar ratio of these SFCA. BCFA = branched-chain fatty acids. CTR+: Non-Inoculated animals receiving placebo; PRO: Inoculated animals receiving the probiotic; PRE: Inoculated animals receiving the prebiotic; SYN: Inoculated animals receiving the synbiotic; CTR−: Inoculated animals receiving placebo. N = 6 for all groups except for non-challenged animals, N = 8. P-values were obtained by an ANOVA, using the linear model procedure in the R software and adjusted by the Benjamini–Hochberg procedure. Means in the same row with different superscripts are different from each other (Tukey’s < P 0.05). not adjust to a normal distribution, and therefore, data were value (P = 0.033 & adjusted P = 0.118). Regarding crypt analyzed by frequencies using Fisher’s exact test (Figure 6). In depth measurements, they were numerically increased with this way, statistical differences were found when PRE treatment the challenge, albeit only with significant differences for SYN was compared to both control groups (CTR+ and CTR−) on (P < 0.001). Consequently, a significantly reduced villous height: day 4 PI, showing a higher percentage of animals with levels crypt depth ratio was found in all challenged groups (P < 0.001, above those considered normal (>2 mg/mL) (Piñeiro et al., 2009) adjusted P = 0.003) at day 4 PI, with a faster recovery at (P = 0.002 and P = 0.006, respectively). day 8 PI with the PRO diet, which was the only one not different from CTR+. Intestinal Histological Structure Regarding the number of mucosal intraepithelial lymphocytes, As depicted in Table 4, 4 days after the challenge, the on day 8 PI, animals receiving the PRE diet tended to have height of the ileal villi showed a numerical decrease in higher values when compared to CTR+ and CTR−, showing all inoculated groups, almost reaching the statistical trend PRO and SYN intermediate levels (P = 0.028, adjusted P = 0.108). Frontiers in Microbiology | www.frontiersin.org 9 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium FIGURE 6 | Effect of experimental treatments in serum levels of acute-phase protein Pig-MAP in piglets on day 4 after oral Salmonella challenge. Figure represents frequencies for values between a normal (0.3–1 mg/mL) or abnormal (>2 mg/mL) range. CTR+: Non-Inoculated animals receiving placebo; PRO: Inoculated animals receiving the probiotic; PRE: Inoculated animals receiving the prebiotic; SYN: Inoculated animals receiving the synbiotic; CTR−: Inoculated animals receiving placebo. N = 6 for all groups except for non-challenged animals, N = 8. P-values were obtained using Fisher’s Exact Test in the R software [Piñeiro et al., 2009: normal (≤1 mg/mL), borderline (1–2 mg/mL) high levels (>2 mg/mL)]. TABLE 4 | Effects of treatments on histomorphological parameters on days 4 and 8 post-inoculation. PI Day Treatment RSD P-value Adjusted P-value CTR+ PRO PRE SYN CTR− Villous height (µm) 4 282a 229ab 213ab 184b 246ab 52.5 0.033* 0.118 8 259 256 227 227 242 36.1 0.320 0.477 Crypt depth (µm) 4 228a 263abc 249ab 299c 282cb 26.2 <0.001* 0.003* 8 245 266 275 267 282 31.3 0.241 0.474 Villous height: crypt depth ratio 4 1.24a 0.88b 0.85b 0.64b 0.87b 0.201 <0.001* 0.003* 8 1.06a 0.96ab 0.84b 0.84b 0.85b 0.123 0.008* 0.045* IEL (Cell no./100 µm) 4 0.46 0.68 1.02 0.54 0.57 0.776 0.320 0.477 8 0.50a 0.67ab 0.98b 0.65ab 0.45a 0.286 0.028* 0.108 CTR+ – Non-Inoculated animals receiving placebo; PRO- Inoculated animals receiving the probiotic; PRE- Inoculated animals receiving the prebiotic; SYN- Inoculated animals receiving the synbiotic; CTR− - Inoculated animals receiving placebo. N = 6 for all groups except for non-challenged animals, N = 8. P-values were obtained by an ANOVA, using the linear model procedure in the R software and adjusted by the Benjamini–Hochberg procedure. Means in the same row with different superscripts are different from each other (Tukey’s < P 0.05). A similar pattern was seen on day 4 PI, although without bacteria. Among them, Lactobacillus rhamnosus HN001 is able statistical significance. to produce bacteriocin-like substances (Aguilar-Uscanga et al., 2013), fact that is not demonstrated for Bifidobacterium longum subsp. infantis CECT 7210 yet, which has just been proven DISCUSSION to synthetize a peptide against rotavirus (Muñoz et al., 2011). This experimental trial shows that the probiotic compound One of the main characteristics of probiotics is their ability to formed by Bifidobacterium longum subsp. infantis CECT7210 antagonize pathogenic bacteria and, thus, to improve host health. and Lactobacillus rhamnosus HN001 (DR20) exerted a negative As summarized by Markowiak and Śliżewska (2017), the capacity impact on Salmonella Typhimurium infection, as it reduced its to reach this goal is achieved by four different mechanisms, fecal shedding 7 days after the challenge. Moreover, the animals such as the production of antimicrobial substances (bacteriocins, recovered faster from the intestinal damage produced by the SCFA, etc.), competition for the adhesion sites in the intestinal challenge as suggest the improved villi/crypt ratio registered at epithelium and for nutrients, modulation of the immune system day 8 PI with this diet. It is not clear, however, which one of the of the host, and blockage of the toxin production by pathogenic two strains was the main actor of this action or if the effect was, Frontiers in Microbiology | www.frontiersin.org 10 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium precisely, due to the administration of both strains. Regarding activation (Vogt et al., 2013), which can provoke an up- the strain Lactobacillus rhamnosus HN001, previous works have regulation of the IL-6 gene expression (Libermann and Baltimore, demonstrated its effectiveness against Salmonella Typhimurium 1990), although the literature data are sometimes contradictory. in a murine model by reducing pathogen loads in visceral organs In this regard, Shukla et al. (2016) reported an increase in and enhancing the immune system (Gill et al., 2001). This interleukin in Giardia-infected mice treated with inulin, while strain has also been considered to be responsible for increasing Zhang et al. (2018) and Marciano et al. (2015) observed a the blood leucocyte phagocytic activity of mice challenged with decrease in the IL-6 concentration in rats administered inulin Escherichia coli O157:H7 (Shu and Gill, 2002). Other strains, (3%) and inulin/oligofructose (10%), respectively. This apparent such as Lactobacillus rhamnosus (GG), can also reduce the inconsistency between studies could be due to a differential effect levels of Salmonella Infantis in the jejunum of inoculated pigs, of these substances, depending on the dose. In this sense, Song thereby mitigating intestinal inflammation response caused by et al. (2018) reported that in chickens, while the use of a low this bacterium (Yang et al., 2017). These outcomes are, therefore, dose of inulin (0.25%) was translated into a reduction of IL-6 consistent with the improved Salmonella clearance found in our gene expression, a high dose (2%) was associated with an increase. study in the PRO group. However, as we did not determine the levels of this interleukin in Regarding Bifidobacterium longum subsp. infantis, benefits our animals, we cannot confirm if the increase observed in Pig- against infectious agents have been described. Mice pre- MAP with a 5% inclusion level of inulin + OF was or was not treated with the 35624 strain, in a scenario of a Salmonella related to a down-regulation of IL-6 expression. infection, showed a diminished enterocyte damage and a reduced Regarding IEL, these cells are part of the gut-associated expression of interleukins IL-8 and IL-10 (Symonds et al., 2012). lymphoid tissue (GALT), and it is believed that they can have This down-regulation of pro-inflammatory cytokine is consistent a suppressant activity in the development of oral tolerance with results reported by O’Mahony et al. (2008), where mice (Trejdosiewicz, 1992). There are numerous works supporting the consuming this probiotic showed a decrease in the release of effects of prebiotics (including inulin and OF) on the GALT. IFN-γ, TNF-α, and IL-10 following CD3/CD28 stimulation by Some of these studies have been reviewed by Schley and Field a challenge with Salmonella Typhimurium or LPS. In addition, (2002), suggesting different hypotheses about the underlying other authors have reported more CD4+CD25+ cells in the mechanisms of these effects; however, in all cases, they are spleen, associated with pro-inflammatory cytokine inhibition mediated by an increase in the lactic acid bacterial population, (Maloy et al., 2003; Scully et al., 2013). More specifically, promoted by prebiotics. The proposed mechanisms include the tested CECT 7210 strain manifested effectiveness against direct contact of lactic acid bacteria (or bacterial products) with rotavirus infection in mice (Muñoz et al., 2011), with a good immune cells in the intestine, synthesis of short-chain fatty outcome against digestive pathogens (Salmonella and ETEC) acids by the microbiota, and modulation of mucin production. in piglets (Barba-Vidal et al., 2017). In this case, the probiotic However, from our results, it is difficult to confirm any of these reduced pathogen intestinal colonization and modulated the hypotheses, although in the treatments with the prebiotic, the immune response with an increase in the intraepithelial molar proportion of SCFA was modified to a greater extent. lymphocytes at ileal level. In the present trial, however, we A complementary analysis of the microbiota composition would did not find such an effect on IEL numbers with the PRO have helped to clarify the possible role of microbial shifts in the treatment, neither in TNF-α nor in Pig-MAP. This suggests that local immune response. the combined use of these two strains does not result in a clear Together with changes in immunity markers, the PRE immunomodulatory activity. treatment was also able to limit the colonization of the gut Nonetheless, the inclusion of the prebiotic mixture of inulin by Salmonella, considering that this was the only treatment and oligofructose in the PRE treatment showed changes in in which Salmonella numbers in the digesta were always immunity parameters, suggesting a possible immunomodulatory below the countable levels. Other authors have also described effect for this additive. The PRE diet promoted an increase in the the potential of non-digestible oligosaccharides to control the concentration of serum Major Acute-phase Protein (Pig-MAP) presence of this pathogen in the gut by different mechanisms. and also tended to increment the number of ileal IEL. Pig-MAP For example, oligofructose has been shown to reduce Salmonella is mainly synthetized in the liver when an acute phase response is enteriditis counts in the cecum of laying hens (Adhikari et al., occurring, representing an unspecific reaction to tissue damage. 2018) and to diminish Salmonella adhesion to HT-29 cells by We assume that the registered increase in Pig-MAP with the 50% (Wang et al., 2015), which was directly related to its PRE diet was a response to a higher damage induced by the concentration. Moreover, Kanjan and Hongpattarakere (2017), Salmonella infection; however, with this treatment, we did neither testing the efficacy of inulin in a proximal colon model, find changes in histomorphological parameters nor in Salmonella also observed a competitive exclusion of Salmonella due to loads, which were actually lower compared to CTR−. In this nutrient limitation and antibacterial metabolites produced by regard, it should be remembered that the expression of Pig- stimulated bifidobacteria. In our case, we hypothesize that any MAP is, in a certain way, conditioned by cytokine IL-6, as of these mechanisms could be behind the lower colonization its presence induces a higher production (González-Ramón levels found for the PRE treatment, although we cannot et al., 2000). Simultaneously, IL-6 is affected by the ingestion provide evidences. of some prebiotic fibers, such as inulin and oligofructose. In Despite the beneficial effects of the two-strain-probiotic a previous study, β2→1-fructans could induce NF-κB/AP-1 or the inulin+OF mixture, we could not find any synbiotic Frontiers in Microbiology | www.frontiersin.org 11 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium behavior when both strategies were combined. No benefits provide benefits in a Salmonella Typhimurium infection were found in Salmonella prevalence in the colon or in the scenario. While the two-strain probiotic appears to speed- immunity response compared to CTR−. The negative impacts up the clearance of the pathogen probably by competitive- of the challenge on fecal consistency or on the villi/crypt exclusion mechanisms, the tested prebiotic mixture reduced ratio were similar for SYN and CTR−. Despite this, the lower colonic colonization possibly by modulation of the local weight gains reported for this treatment during the post- and systemic immune response. However, these desirable challenge period are remarkable. While in formula-fed infants, effects are not synergistic when the two compounds are a moderate weight gain might be desirable to prevent risk administered in a synbiotic combination. Results of this of obesity (Appleton et al., 2018), in challenged animals, it work give insights regarding the safety of the use of these is an unmistakable sign of unwellness. This can be partially probiotics to fight enteropathogens like Salmonella in infants explained by the drop in feed intake registered in the post- and, moreover, open the possibility of its use in livestock challenged period, particularly in the acute phase (0–4), which as feed additives to be included in the pre-starter diets. was only significant for the SYN treatment. Nevertheless, More studies are therefore required to better understand the decrease in ADG was higher than expected, suggesting the mechanisms involved and why synergic effects were not that other factors were involved. A decreased digestibility or seen in the synbiotic combination. A better understanding profitability of the diet could be associated with an increased of the changes induced in the intestinal microbiota, and transit time of digesta with this treatment. In this regard, in its cross-talk with the host, are probably the key for a the inclusion of inulin-type prebiotics has been reported to mechanistic approach. be responsible for stool softening and increased defecation frequency (Euler et al., 2005; Kapiki et al., 2007), and in our study, numerical increases were seen in fecal scores few DATA AVAILABILITY STATEMENT days after challenge with the SYN treatment. However, these differences were not significant when compared to the rest of the The datasets generated for this study are available on request to challenged groups. An impaired digestibility with the SYN diet the corresponding author. could also be due to the decreased ileal villous height registered after the challenge, which was more profound with the SYN treatment in the 0–4 PI period. Nevertheless, similar to fecal ETHICS STATEMENT consistency, the differences to the remaining challenged groups were not significant. Another explanation for the retarded growth The animal study was reviewed and approved by the would be a potential overgrowth of the probiotic along the Animal and Human Experimental Ethical Committee of small intestine, boosted by the administration of the prebiotic, Universitat Autònoma de Barcelona (UAB) and its competent resulting in nutrient competition with the host. However, these authorities (Permit No. CEEAH: 4026 DMAH: 10118). The phenomena would have also taken place during the adaptation treatment, management, housing, husbandry and slaughtering week before the challenge, and the results obtained do not conditions conformed to European Union Guidelines (Directive support this assumption. Finally, it should not be discarded that 2010/63/EU, European Commission, 2010). Euthanasia was the lower gains registered for this treatment would have been performed by intravenous injection of sodium pentobarbital. All a random effect associated with a limited number of replicates efforts were made to minimise animal suffering. for assessing the effects on performance (N = 6 pens), even more critical in challenging scenarios when a higher residual variability is expected. AUTHOR CONTRIBUTIONS The probiotic and prebiotic combination tested in the present study did not exhibit any synbiotic effect against AR-S participated in the experimental design and was responsible oral Salmonella Typhimurium challenge. Previous studies of for the animal trial, laboratory analysis, data analysis, and other authors evaluating the effect of synbiotic combinations writing. PL-C participated in the experimental design, animal have, contrarily, demonstrated positive results. For example, trials, data analysis, and writing. GC-O, JM-M, and MR-P Baffoni et al. (2011) observed a reduction in campylobacter participated in the experimental design and contributed to data shedding in chickens challenged with this pathogen and analysis and writing. SM-O participated in the experimental treated with Bifidobacterium longum subsp. longum and design, animal trials, laboratory analysis, data analysis, and fructo-oligosaccharides. Similarly, Naqid et al. (2015) reduced writing. All authors contributed to the article and approved the Salmonella Typhimurium excretion in infected pigs by using submitted version. a combination of Lactobacillus plantarum and lactulose. Different outcomes could be due to the distinct synbiotic combinations tested, but also to many other factors, such as diet FUNDING and prebiotic dose. The probiotic strains Bifidobacterium longum subsp. infantis This study was supported by the Spanish Center for the CECT 7210 and Lactobacillus rhamnosus HN001 and the Development of Industrial Technology (CDTI) and the Project prebiotic mixture of inulin, enriched with oligofructose, can SMARTFOODS (IDI-20141216). Frontiers in Microbiology | www.frontiersin.org 12 August 2020 | Volume 11 | Article 2012 Rodríguez-Sorrento et al. Synbiotic Combination Against Salmonella Typhimurium REFERENCES Johnson-Henry, K. C., Donato, K. A., Shen-Tu, G., Gordanpour, M., and Sherman, P. M. (2008). Lactobacillus rhamnosus strain GG prevents enterohemorrhagic AOAC International, (1995). Official Methods of Analysis of AOAC International. Escherichia coli O157:H7-induced changes in epithelial barrier function. Infect. AOAC: Washington, DC. Immun. 76, 1340–1348. doi: 10.1128/IAI.00778-07 Adhikari, P., Cosby, D. E., Cox, N. A., Franca, M. S., Williams, S. M., Gogal, R. M. 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Vogt, L., Ramasamy, U., Meyer, D., Pullens, G., Venema, K., Faas, M. M., et al. Copyright © 2020 Rodríguez-Sorrento, Castillejos, López-Colom, Cifuentes-Orjuela, (2013). Immune modulation by different types of β2→1-fructans is toll-like Rodríguez-Palmero, Moreno-Muñoz and Martín-Orúe. This is an open-access article receptor dependent. PLoS One 8:e68367. doi: 10.1371/journal.pone.0068367 distributed under the terms of the Creative Commons Attribution License (CC BY). Wang, S., Wang, J., Mou, H., Luo, B., and Jiang, X. (2015). Inhibition of adhesion of The use, distribution or reproduction in other forums is permitted, provided the intestinal pathogens (Escherichia coli, Vibrio cholerae, Campylobacter jejuni, and original author(s) and the copyright owner(s) are credited and that the original Salmonella Typhimurium) by common oligosaccharides. Foodborne Pathog. publication in this journal is cited, in accordance with accepted academic practice. 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