International Journal of Molecular Sciences Article Effects of Rifaximin on Luminal and Wall-Adhered Gut Commensal Microbiota in Mice Marina Ferrer 1,2 , Mònica Aguilera 1,2 and Vicente Martinez 1,2,3, * 1 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; marinaferrerclotas@gmail.com (M.F.); monica.aguilerap@gmail.com (M.A.) 2 Neuroscience Institute, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 3 Centro de Investigación Biomédicaen Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, 28029 Madrid, Spain * Correspondence: vicente.martinez@uab.es; Tel.: +34-093-581-3834 Abstract: Rifaximin is a broad-spectrum antibiotic that ameliorates symptomatology in inflamma- tory/functional gastrointestinal disorders. We assessed changes in gut commensal microbiota (GCM) and Toll-like receptors (TLRs) associated to rifaximin treatment in mice. Adult C57BL/6NCrl mice were treated (7/14 days) with rifaximin (50/150 mg/mouse/day, PO). Luminal and wall-adhered ceco-colonic GCM were characterized by fluorescent in situ hybridization (FISH) and microbial pro- files determined by terminal restriction fragment length polymorphism (T-RFLP). Colonic expression of TLR2/3/4/5/7 and immune-related markers was assessed (RT-qPCR). Regardless the period of treatment or the dose, rifaximin did not alter total bacterial counts or bacterial biodiversity. Only a modest increase in Bacteroides spp. (150 mg/1-week treatment) was detected. In control conditions, only Clostridium spp. and Bifidobacterium spp. were found attached to the colonic epithelium. Rifax- imin showed a tendency to favour their adherence after a 1-week, but not 2-week, treatment period. Minor up-regulation in TLRs expression was observed. Only the 50 mg dose for 1-week led to a  significant increase (by 3-fold) in TLR-4 expression. No changes in the expression of immune-related  markers were observed. Rifaximin, although its antibacterial properties, induces minor changes in Citation: Ferrer, M.; Aguilera, M.; luminal and wall-adhered GCM in healthy mice. Moreover, no modulation of TLRs or local immune Martinez, V. Effects of Rifaximin on systems was observed. These findings, in normal conditions, do not rule out a modulatory role of Luminal and Wall-Adhered Gut rifaximin in inflammatory and or dysbiotic states of the gut. Commensal Microbiota in Mice. Int. J. Mol. Sci. 2021, 22, 500. Keywords: dysbiosis; gut commensal microbiota; host-bacterial interaction systems; immune mark- https://doi.org/10.3390/ ers; rifaximin; toll-like receptors ijms22020500 Received: 20 November 2020 Accepted: 1 January 2021 Published: 6 January 2021 1. Introduction Rifaximin is a semi-synthetic non-absorbable antibiotic derived from rifamycin and Publisher’s Note: MDPI stays neu- with a broad-spectrum activity against Gram-positive and Gram-negative microorganisms tral with regard to jurisdictional clai- proposed to act on the gut microenvironment [1,2]. The main advantage of rifaximin over ms in published maps and institutio- similar antibiotics is that it is virtually unabsorbable, which minimizes systemic exposure nal affiliations. and adverse events in all patient populations [3]. Rifaximin showed to be effective for a variety of clinical uses and was initially approved for the treatment of traveler’s diarrhea caused by noninvasive strains of E. coli [1] and hepatic encephalopathy (due to its inhibition of ammonia-producing enteric bacteria and consequent reduction of circulating ammonia Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. in patients with cirrhosis) [4]. Further clinical evidence led to the approval of the use of This article is an open access article rifaximin for the treatment of diarrhea-predominant irritable bowel syndrome (IBS) [5]. distributed under the terms and con- Moreover, in the clinical practice, rifaximin is often prescribed for other gastrointestinal ditions of the Creative Commons At- disorders, such as inflammatory bowel disease (IBD), small intestinal bacterial overgrowth tribution (CC BY) license (https:// (SIBO), and diverticular disease, because of its theoretical capability to modulate the creativecommons.org/licenses/by/ intestinal microbiota [6]. 4.0/). Int. J. Mol. Sci. 2021, 22, 500. https://doi.org/10.3390/ijms22020500 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 500 2 of 19 IBS is a chronic, functional gastrointestinal disorder characterized by abdominal pain/discomfort, associated with altered bowel habits. The etiology of IBS is unknown and the pathophysiology is complex, heterogeneous, and not well understood. There is evi- dence for a number of underlying mechanisms, including altered intestinal barrier function, altered motility, visceral hypersensitivity and, possibly, a chronic, low-grade inflammatory or immunological response [7–9]. Moreover, interactions between environmental factors, such psychosocial stress and anxiety, led to the inclusion of brain-gut interactions in the etiology of the disease [7,8]. During the last years, gut microbiota has also been implicated in the pathogenesis of IBS. In this sense, some IBS patients have reduced gut microbial biodiversity [8] and acute enteric infections have been associated to the development of IBS, the so called post-infectious IBS [10]. Overall, the presence of dysbiosis, temporal or permanent, has been seen in more than 70% of patients with IBS [8], although great variability has been observed [11]. Nevertheless, the connection between dysbiosis and IBS is not completely understood, and a causal relationship has not been demonstrated [11,12]. It is known that gut commensal microbiota (GCM) contributes to maintenance of gastroin- testinal homeostasis [13]. Therefore, modifying the GCM is a therapeutic approach of growing interest for IBS. In this context, rifaximin is used to treat SIBO and IBD [14], and, as mentioned above, was approved for the treatment of diarrhea-predominant IBS [15]. There is some clinical overlap between IBS and IBD, and some authors consider IBS and IBD at the two extremes of the same spectrum, being IBS a low-grade IBD without structural alterations [16]. In any case, dysbiosis seems to be a common finding in IBS and IBD. Besides its antibacterial effects, pre-clinical evidence suggests that rifaximin might have anti-inflammatory activity, reducing mucosal inflammation and visceral hypersen- sitivity and restoring epithelial barrier function [17–20]. Whether or not these effects are secondary to its microbial actions or are direct, non-microbial-related, is still a matter of debate. Moreover, direct effects of rifaximin on intestinal epithelial cell physiology, associ- ated with reductions in bacterial attachment and internalization and epithelial responses to inflammatory mediators, have also been suggested [19,21,22]. In this study, we assessed the effects of rifaximin on GCM in healthy mice. For this, we used fluorescence in situ hybridization (FISH) and a terminal restriction fragment length polymorphism (T-RFLP) analysis to determine rifaximin-induced changes in colonic luminal and wall adhered bacteria. To determine if changes in GCM might be associated to the local modulation of host immune-related responses, we also assessed (real-time qPCR) changes in the expression of toll-like receptors (TLR)-dependent host-bacterial interaction systems and immune-related markers. 2. Results 2.1. Effects of Rifaximin on Body Weight, Weight of Body Organs and Colonic Histology Body weight was stable over the treatment period, without treatment-related sig- nificant changes (Table 1). At necropsy, the relative weight of the liver was slightly, but significantly reduced in rifaximin-treated animals, in similar proportion regardless the dose and the duration of treatment (p < 0.05 vs. respective vehicle-treated group). Relative weight of the spleen, thymus, and adrenal glands were similar across groups, (data not shown). At necropsy, no macroscopic alterations were observed in the colon or cecum, irre- spective of the experimental group considered. Similarly, cecal and colonic content had a normal consistency in all experimental groups. Colonic and cecal relative weight was simi- lar across groups (Table 1). Consistent with these observations, no histological alterations were observed in the colon in rifaximin-treated animals, regardless the treatment time or dose. In all cases, total histological scores ranged between 0 and 2 (data not shown). Int. J. Mol. Sci. 2021, 22, 500 3 of 19 Table 1. Body weight and relative weight of colon and cecum in the different experimental groups 1 . Cecum Body Body Colon Relative Treatment Weight at Weight (% Relative Treatment n Weight Duration Necropsy Change Weight (mg/g Body (g) from Day 0) (mg/cm) Weight) Vehicle 4 17.6 ± 0.6 −4.6 ± 2.7 23.2 ± 1.7 21.5 ± 0.9 Rifaximin 7-day 6 17.8 ± 0.2 2.7 ± 2.4 21.8 ± 0.8 25.1 ± 2.3 (50 mg/kg) Rifaximin 5 18.3 ± 0.2 −1.6 ± 1.5 21.3 ± 1.6 25.9 ± 0.9 (150 mg/kg) Vehicle 4 17.4 ± 0.7 1.9 ± 2.2 21.7 ± 0.2 22.2 ± 0.8 Rifaximin 6 18.3 ± 0.3 −2.5 ± 2.1 22.9 ± 1.3 23.7 ± 1.9 14-day (50 mg/kg) Rifaximin 6 18.2 ± 0.2 2.1 ± 1.4 24.5 ± 0.7 21.2 ± 0.9 (150 mg/kg) 1 Data represent mean ± SEM of the number of animals indicated (n). 2.2. Effects of Rifaximin on Luminal GCM In vehicle-treated animals, regardless the duration of treatment, total bacterial counts within the luminal content (EUB338-probe) oscillated between 2 × 1010 cells/mL and 1.5 × 1011 cells/mL (Figure 1); consistent with previous observations [23–25]. There was a good coincidence between total bacterial counts assessed by FISH (EUB338-probe) and DAPI staining (Figure 1). The most abundant bacterial group was Clostridium spp. (EREC482-probe), being within the order of 1010 cells/mL; followed by Bacteroides spp. (BAC303-probe) at 109 cells/mL and Verrucobacteria (VER620-probe) at 108 cells/mL. On the other hand, Lactobacillus/Enterococcus spp. and Bifidobacterium spp. were ranging between detection levels (106 cells/mLL) and 108 –109 cells/mL. Enterobacteria appeared below or at the threshold of detection levels (Figures 1 and 2). Total bacterial counts remained stable after the treatment with rifaximin, regard- less the dose (50 or 150 mg/kg/day) or the duration of treatment considered (7-day or 14-day). Total counts oscillated between 2 × 1010 cells/mL and 7 × 1010 cells/mL and 1.5 × 1010 cells/mL and 1.5 × 1011 cells/mL for the 7-day and 14-day treatment period, respectively (Figure 1). Assessment of specific bacterial groups showed an increase in Bacteroides spp. (BAC303-probe) counts after a 7-day treatment period, but not after a 14-day period (Figures 1 and 2). Although not significant, during the 14-day treatment, a dose-dependent reduction in the proportion of Clostridium spp. (EREC482-probe) was observed, at the expense of an increase in the relative abundance the other bacterial groups assessed, particularly Bacteroides spp. (BAC303-probe) (Figure 2). The ecological characterization of the luminal microbiota was performed with a T- RFLP analysis. The dendrogram representation of the similarity indexes of the T-RFLP profiles of the ceco-colonic microbiota did not show a clustering of the different experimen- tal groups (Figure 3). The number of t-RFs and their size distribution, taken as a measure of biodiversity, was similar across groups, regardless the dose (50 or 150 mg/kg/day) or the duration of treatment considered (7-day or 14-day) (p = 0.166; Figure 4). Int. Int. J.J. Int. Mol. J. Mol. Sci. Mol. Sci. 2021, Sci.2021, 22, 22, xx500 2021,22, FOR PEER FOR PEER REVIEW REVIEW 444 of of 19 of 19 19 Figure 1. Figure 1. Colonic Colonic microbiota, microbiota, as as quantified quantified by by FISH FISH after after aa 7-day 7-day or or aa 14-day 14-day treatment treatment period period with with rifaximin. EUB: Total rifaximin. EUB: Total bacteria; BAC: bacteria; BAC: Bacteroides Bacteroides spp.; spp.; ENT-D: ENT-D: Enterobacteria Enterobacteria group; group; VER: VER: Verrucobacteria VER:Verrucobacteria group; BIF: Verrucobacteria group; BIF: Bifidobacterium Bifidobacterium spp.; spp.; LAB: LAB: LAB: Lactobacillus/Enterococcus spp.; Lactobacillus/Enterococcus Lactobacillus/Enterococcus spp.; EREC: spp.;EREC: Clostridium Clostridium EREC: Clostridiumspp. spp. cluster cluster spp. XIVa XIVa cluster group. group. XIVa DataData Data group. are media are media (interquartile (interquartile are media range) range) (interquartile ±± SD, SD,± range) nn =4– =4– SD, 66n per per group. *: p < 0.05 vs. corresponding vehicle. #: p < 0.05 vs. rifaximin at 50 mg/kg. V: Vehicle; R50: rifaximin = 4–6 per group. *: p < 0.05 vs. corresponding vehicle. #: p < 0.05 vs. rifaximin at 50 mg/kg. V: Vehicle; R50: rifaximin50 group. *: p < 0.05 vs. corresponding vehicle. #: p < 0.05 vs. rifaximin at 50 mg/kg. V: Vehicle; R50: rifaximin at at 50 at mg/kg; R150: mg/kg; R150: rifaximin rifaximin atat 150 150 mg/kg. mg/kg. 50 mg/kg; R150: rifaximin at 150 mg/kg. Figure 2. Figure Relative distribution 2. Relative distribution ofof the the colonic colonic microbiota, microbiota, asas quantified quantified byby FISH, FISH, in in the the different different experimental experimental groups. Data groups. Data Data Figure 2. Relative distribution of the colonic microbiota, as quantified by FISH, in the different experimental groups. represent the represent the represent relative the relative abundance relative abundance (percent) abundance (percent) of (percent) ofthe bacterial of the groups the bacterial bacterial groups characterized groups characterized [Bacteroides characterized [Bacteroides spp. (BAC), [Bacteroides spp. Bifidobacterium spp. (BAC), spp. (BAC), Bifidobacterium Bifidobacterium (BIF), spp. Clostridium spp. (BIF), (BIF), spp. (EREC), Clostridium Clostridium spp. Enterobacteria spp. (EREC), (EREC), Enterobacteria Enterobacteria Lactobacillus (ENT),(ENT), (ENT), spp. (LAB), Lactobacillus Lactobacillus and Verrucobacteria spp. (LAB), spp. (LAB), (VER)].(VER)]. and Verrucobacteria and Verrucobacteria RelativeRelative (VER)]. percent Relative percent composition composition percent of the the microbiota of the microbiota composition of microbiota was calculated was calculated was calculated taking astaking 100%as taking as 100% the100% the totalthe totalof counts total counts of the the bacterial the bacterial counts of bacterial groups assessed. groups assessed. groups assessed. nn == n = 4–6 per 4–6 4–6 perV: per group. group. group. V: Vehicle; V: Vehicle; Vehicle; R50: rifaximin R50: R50: rifaximin rifaximin at 50 at at 50 mg/kg;50 mg/kg; mg/kg; R150: rifaximin R150: R150: rifaximinrifaximin at 150 at 150 mg/kg. at 150 mg/kg. mg/kg. Int. Int.J.J.Mol. Mol.Sci. Sci.2021, 2021,22, 22,xxFOR FORPEER PEERREVIEW REVIEW 55of of19 19 Int. J. Mol. Sci. 2021, 22, 500 5 of 19 100 50 55 60 65 70 75 80 85 90 95 100 50 55 60 65 70 75 80 85 90 95 R50 R507-day 7-day R50 R507-day 7-day VV7-day 7-day R150 R15014-day 14-day R50 R507-day 7-day R50 R5014-day 14-day R50 R5014-day 14-day R50 R5014-day 14-day R150 R15014-day 14-day R150 R15014-day 14-day R50 R507-day 7-day R150 R1507-day 7-day VV7-day 7-day R150 R15014-day 14-day R150 R1507-day 7-day R50 R5014-day 14-day R50 R507-day 7-day VV14-day 14-day R50 R507-day 7-day R50 R5014-day 14-day R150 R1507-day 7-day R50 R5014-day 14-day R150 R15014-day 14-day VV7-day 7-day VV14-day 14-day R150 R1507-day 7-day R150 R15014-day 14-day VV14-day 14-day VV14-day 14-day VV7-day 7-day R150 R1507-day 7-day Figure 3. Ecological characterization of the luminal microbiota by T-RFLP analysis. Dendrogram Figure Figure 3. Ecological 3.the Ecological characterization characterization of of the luminal luminal microbiota theexperimental microbiota by T-RFLP T-RFLP analysis. Dendrogram showing distribution of the different groups by according analysis. to the T-RFLPDendrogram banding showing showing the the distribution distribution ofof the the different different experimental experimental groups groups according according to to the the T-RFLP T-RFLP banding banding patterns patterns obtained from the analysis of the ceco-colonic samples. Each line represents an animal patterns obtained obtained from from the the analysis analysisof of the the ceco-colonic ceco-colonic samples. samples.Each Each line linerepresents represents an an animal animal identified identified by either R50 (rifaximin at 50 50 mg/kg),R150 R150 (rifaximinatat150150 mg/kg) or V (vehicle), fol- identified by byeither either R50 R50 (rifaximin (rifaximin at at 50 mg/kg), mg/kg), R150 (rifaximin (rifaximin at 150 mg/kg) mg/kg) or or VV (vehicle), (vehicle), fol- fol- lowed lowed by by the the experimental experimental period period (7-day (7-day oror14-day). The 14-day). The dendrogram dendrogram lowed by the experimental period (7-day or 14-day). The dendrogram distances representdistances distances represent representpercentage percent- percent- of agesimilarity. age of The The of similarity. similarity. different experimental The different different experimental experimental groups did did groups groups not cluster did not together not cluster cluster togetherindicating together indicatingthethe indicating absence the absence absenceof of treatment-related treatment-related changes changes in in the the microbiota microbiota composition. composition. of treatment-related changes in the microbiota composition. Figure Figure 4. Figure 4. Ecological characterization Ecological 4.of the luminal Ecological characterization microbiota byof characterization the the luminal T-RFLP of microbiota analysis: luminal by Effects of microbiota T-RFLP T-RFLP analysis: byrifaximin Effects Effectsof on biodiversity analysis: of rifax- rifax- imin the ceco-colonic microbiota. imin on (A) Numberbiodiversity of of tRFs of on biodiversity the ceco-colonic detected. Each symbol the ceco-colonic microbiota. (A) represents microbiota. Number of an individual (A) Number tRFs of tRFs detected. animal; Each symbol the horizontal detected. Each symbol lines with errors correspondrepresents represents to the mean an an individual ± SEM. Foranimal; individual animal; the sake theofhorizontal the horizontal clarity, andlines lines since with noerrors with errors correspond correspond differences to to the were observedthe mean mean ±± SEM. among SEM. For them, vehicle-treated animalsFor the have sake thebeen of clarity, sakemerged of clarity, and in aand since since single no differences no differences group. were V: Vehicle;were observed R50:observed among rifaximinamong them, vehicle-treated R150: rifaximinani- them, vehicle-treated at 50 mg/kg; ani- mals mals have been merged in in aa single singletogroup. V: V: Vehicle; R50: rifaximin at 50 50 mg/kg; mg/kg; R150: R150: rifaximin at 150 mg/kg. (B) Distribution of have the tRFsbeen mergedaccording detected group. their size. Vehicle; Each line R50: rifaximinan represents atindividual animal rifaximin and at at 150 150 mg/kg. mg/kg. (B)(B) Distribution Distribution of of the the tRFs tRFs detected detected according according to to their their size. size. Each Each line line represents represents an an each column a tRF size. tRF distribution indicates a similar microbial biodiversity in all experimental groups, regardless the individual individual animal animal and and each each column column aa tRFtRF size. size. tRF tRF distribution distribution indicates indicates aa similar similar microbial microbial biodi- biodi- treatment applied. See also Table versity 2 forall details regardinggroups,taxonomical classification of the tRFs detected. versity inin all experimental experimental groups, regardless regardless the the treatment treatment applied. applied. See See also also Table Table 22 for for details details regarding taxonomical classification of the tRFs regarding taxonomical classification of the tRFs detected. detected. Int. J. Mol. Sci. 2021, 22, 500 6 of 19 Table 2. Theoretical restriction 50 -fragment (tRF) size predicted for the major mouse gut bacteria and prevalence in the different experimental groups. Frequency 1 Compatible Bacterial Group tRFSize V 7-day V 14-day R50 7-day R50 14-day R150 7-day R150 14-day (n = 4) (n = 4) (n = 6) (n = 6) (n = 5) (n = 6) Unidentified 54–55 3 (75) 4 (100) 4 (67) 3 (50) 3 (60) 5 (83) Bacillus spp./Lactococcus lactis spp. 61–62 1 (25) 2 (50) 1 (17) 0 (0) 1 (20) 1 (17) Salinicoccusroseus 63 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Burkholderia spp./Bordetella spp./Thiomonas spp./ 67 0 (0) 0 (0) 0 (0) 0 (0) 1 (20) 1 (17) uncultured bacterium Mycobacterium spp./ 68 0 (0) 2 (50) 1 (17) 2 (33) 1 (20) 2 (33) uncultured rumen bacterium Uncultured rumen bacterium 69 4 (100) 4 (100) 5 (83) 5 (83) 4 (80) 6 (100) Uncultured rumen bacterium/ 71 2 (50) 1 (25) 4 (67) 1 (17) 2 (40) 0 (0) Leptotrichia spp. Uncultured rumen bacterium 72 1 (25) 1 (25) 1 (17) 3 (50) 2 (40) 1 (17) Photorhabdus sp. 74–75 1 (25) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Uncultured bacterium 77 2 (50) 1 (25) 3 (50) 3 (50) 1 (20) 5 (83) Uncultured rumen bacterium/ 78 1 (25) 3 (75) 1 (17) 1 (17) 1 (20) 3 (50) naphthalene-utilizing bacterium Uncultured bacterium 80 1 (25) 0 (0) 2 (33) 0 (0) 1 (20) 0 (0) Sphingomonas spp./ 81 0 (0) 1 (25) 0 (0) 0 (0) 0 (0) 0 (0) uncultured bacterium Uncultured bacterium 84 3 (75) 2 (50) 2 (33) 1 (17) 0 (0) 1 (17) Desulfovibriodefluvii/ 86–87 2 (50) 1 (25) 1 (17) 2 (33) 1 (20) 5 (83) Roseiflexus spp. Flavobacterium psychrophilum 88–89 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (17) Flavobacterium johnsoniae 90 1 (25) 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) Anaeromyxobacter dehalogenans/ 91 0 (0) 1 (25) 0 (0) 1 (17) 2 (40) 0 (0) uncultured bacterium Geobacter spp./uncultured Bacteroidetes/ 92 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Cytophaga spp./ Algoriphagus spp. Flavobacteriaceae bacterium/uncultured rumen 93–94 2 (50) 0 (0) 3 (50) 4 (67) 2 (40) 5 (83) bacterium/ Desulfovibrio spp. Desulfovibrio profundus/ 95 2 (50) 0 (0) 3 (50) 1 (17) 1 (20) 0 (0) uncultured bacterium Uncultured bacterium 96 1 (25) 1 (25) 3 (50) 4 (67) 2 (40) 4 (67) Desulfococcus oleovorans/ Desulfomonile limimaris/ 97–98 1 (25) 0 (0) 4 (67) 4 (67) 2 (40) 4 (67) Helicobacter pylori Helicobacter pylori/ 99 2 (50) 1 (17) 2 (33) 1 (20) 5 (83) uncultured rumen bacterium Bacteroides spp./ 100 3 (75) 3 (75) 2 (33) 1 (17) 2 (40) 0 (0) uncultured rumen bacterium Bacteroides fragilis/ uncultured rumen bacterium/ 101–102 1 (25) 2 (50) 0 (0) 0 (0) 0 (0) 0 (0) Prevotella ruminicola Uncultured rumen bacterium 103–104 1 (25) 1 (25) 1 (17) 0 (0) 0 (0) 0 (0) Uncultured bacterium 105 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Desulfitobacterium hafniense 107–108 1 (25) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Thiobacillus spp. 110–111 1 (25) 2 (50) 0 (0) 0 (0) 1 (20) 0 (0) Uncultured bacterium 112 0 (0) 1 (25) 0 (0) 1 (17) 0 (0) 0 (0) Unidentified 113 0 (0) 0 (0) 0 (0) 1 (17) 0 (0) 1 (17) Uncultured bacterium 116 2 (50) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Unidentified 117 1 (25) 2 (50) 4 (67) 3 (50) 2 (40) 5 (83) Desulfitobacterium hafniense 118 1 (25) 0 (0) 2 (33) 1 (17) 1 (20) 0 (0) Unidentified 123–124 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Unidentified 127–129 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Unidentified 136 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Uncultured rumen bacterium 137 1 (25) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Microbacterium spp. 144–145 0 (0) 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) Leucobacter spp./ 147 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Janibacter spp. Int. J. Mol. Sci. 2021, 22, 500 7 of 19 Table 2. Cont. Frequency 1 Compatible Bacterial Group tRFSize V 7-day V 14-day R50 7-day R50 14-day R150 7-day R150 14-day (n = 4) (n = 4) (n = 6) (n = 6) (n = 5) (n = 6) Unidentified 148–149 0 (0) 0 (0) 2 (33) 0 (0) 1 (20) 0 (0) Pseudomonas aeruginosa 155 0 (0) 0 (0) 0 (0) 1 (17) 1 (20) 1 (17) Unidentified 156 1 (25) 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) Unidentified 163 0 (0) 0 (0) 0 (0) 1 (17) 1 (20) 1 (17) Synechococcus spp. 164 1 (25) 0 (0) 0 (0) 3 (50) 0 (0) 0 (0) Unidentified 165–167 0 (0) 0 (0) 3 (50) 0 (0) 1 (20) 0 (0) Uncultured bacterium 178 1 (25) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Uncultured rumen bacterium 179 0 (0) 0 (0) 0 (0) 1 (17) 0 (0) 1 (17) Uncultured rumen bacterium 180 0 (0) 0 (0) 0 (0) 2 (33) 0 (0) 0 (0) Uncultured rumen bacterium 181–182 1 (25) 0 (0) 2 (33) 2 (33) 0 (0) 0 (0) Uncultured rumen bacterium 183 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Uncultured rumen bacterium 184–185 1 (25) 0 (0) 2 (33) 0 (0) 1 (20) 0 (0) Listeria monocytogenes 186 1 (25) 0 (0) 0 (0) 0 (0) 1 (20) 0 (0) Uncultured rumen bacterium 187 1 (25) 0 (0) 1 (17) 0 (0) 1 (20) 1 (17) Uncultured rumen bacterium 193 0 (0) 1 (25) 0 (0) 0 (0) 0 (0) 0 (0) Psychrobacter spp./ uncultured bacterium/ 194–195 1 (25) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Francisella spp. Unidentified 199 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Bacillus spp. 200 0 (0) 0 (0) 1 (17) 1 (17) 0 (0) 0 (0) Clostridium rectum/ uncultured bacterium/ 201 1 (25) 0 (0) 3 (50) 0 (0) 1 (20) 1 (17) Mycobacterium spp. Fervidobacterium spp./ Dehalococcoides spp./ 202 0 (0) 0 (0) 0 (0) 0 (0) 1 (20) 0 (0) uncultured bacterium Uncultured rumen bacterium 203–204 2 (50) 1 (25) 3 (50) 1 (17) 1 (20) 5 (83) Clostridium spp. 231–232 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Clostridium perfringens 234–235 0 (0) 0 (0) 1 (17) 0 (0) 1 (20) 1 (17) Clostridium botulinum 237 0 (0) 0 (0) 0 (0) 1 (17) 0 (0) 1 (17) Bacillus subtilis subsp. Subtilis 238 1 (25) 0 (0) 1 (17) 0 (0) 0 (0) 0 (0) Bacillus subtilis subsp. subtilis/Bacillus 241–242 1 (25) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) licheniformis/Bacillus spp. Geobacillus 244 1 (25) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) stearothermophilus/Paenibacillus spp. Microbispora spp./ Pseudonocardia compacta/Nonomuraea 356 0 (0) 0 (0) 0 (0) 1 (17) 0 (0) 0 (0) bangladeshensis/Kineosporia aurantiaca Microbispora spp./ 357 0 (0) 0 (0) 0 (0) 0 (0) 1 (20) 0 (0) Herbidospora spp. Kribbella spp./ Actinomadura spp./ 358 0 (0) 0 (0) 2 (33) 2 (33) 0 (0) 1 (17) Pseudonocardia spp./ Anaplasma marginale Arthrobacter spp. 359 0 (0) 0 (0) 0 (0) 1 (17) 0 (0) 1 (17) Uncultured bacterium 500 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (17) 1 Data represent the number of animals within each group presenting the bacterial group predicted by the corresponding tRF size and the incidence, in percentage (between brackets). Table 2 summarizes the main bacterial groups, as detected by the T-RFLP analysis, with differential presence in the six experimental groups (see also Figure 4B for distribution of the different tRF detected in function of their size). Overall, the T-RFLP analysis reveals high similarities in bacterial composition among the different experimental groups, without evident dose- or period of treatment-related changes in the diversity of the microbiota. Although similar bacterial groups were detected (according to the theoretical restriction 50 -fragment size), in many cases these groups could not be identified phylogenetically and were classified as “unidentified” or “uncultured bacterium”. According to the T-RFLP, tRFs with a size between 356 and 359 appeared in some rifaximin-treated animals, regardless Int. J. Mol. Sci. 2021, 22, 500 8 of 19 the dose or the duration of treatment (Figure 4B). Although this might indicate some treatment-related effect, the low incidence observed (17–33%; Table 2) complicates its interpretation. 2.3. Effects of Rifaximin on Bacterial Adherence to the Colonic Wall In vehicle-treated mice, Bifidobacterium spp. and Clostridium spp. were the only bacte- rial group attached to the colonic wall (epithelium). The overall incidence of attachment ranged from 12.5 to 37.5% (Figures 5 and 6). Figure 5. Bacterial wall adherence. Representative images (×100) showing bacterial adherence to the colonic epithelium for Bifidobacterium spp. and Clostridium spp. (A,A’) non-adhered bacteria within the intestinal lumen. (B,B’) adhered bacteria in a vehicle-treated mice (7-day), (C,C’) adhered bacteria in a rifaximin-treated mice (50 mg/kg, 7-day); (D,D’) adhered bacteria in a rifaximin-treated mice (150 mg/kg, 7-day). Figure 5. Bacterial wall adherence. Representative images (×100) showing bacterial adherence to the colonic epithelium for Bifidobacterium spp. and Clostridium spp. (A) non-adhered bacteria within the intestinal lumen. (B) adhered bacteria in a vehicle-treated mice (7-day), (C) adhered Int. J. Mol. Sci. 2021, 22, 500 9 of 19 bacteria in a rifaximin-treated mice (50 mg/kg, 7-day); (D) adhered bacteria in a rifaximin-treated mice (150 mg/kg, 7-day). Incidenceofofbacterial Figure6.6.Incidence Figure bacterialwall walladherence. adherence.DataData represent represent thethe percentage percentage ofof animals animals showing showing bacterialwall bacterial walladherence. adherence. Numbers Numbers onontoptop of of columns columns represent represent thethe number number of animals of animals showing showing bacterialwall bacterial walladherence adherence over over the total number numberof ofanimals. animals.For Forthe thesake sakeofofclarity, and clarity, since and differences since differ- ences werewere not observed not observed amongamong them,them, vehicle-treated vehicle-treated animals animals have merged have been been merged in a single in a single groupgroup (n = 8). (nV:=Vehicle; 8). V: Vehicle; R50: rifaximin R50: rifaximin at 50 mg/kg; at 50 mg/kg; R150: rifaximin R150: rifaximin at 150 mg/kg. at 150 mg/kg. Because Because of technical of technical problems, problems, one of theone of thefrom samples samples groupfrom group R150 7-dayR150 was7-day was lost lost during itsduring its processing. processing. During antibiotic treatment for a 7-day period, there was a tendency to increase the incidence of bacterial wall adherence for Clostridium spp. (from an incidence of 37.5% in con- trol conditions to 50 and 75% at 50 mg/kg and 150 mg/kg, respectively) and Bifidobacterium spp. (from an incidence of 12.5% in control conditions to 50% for both antibiotic-treated groups). However, this tendency disappeared in animals treated for a 14-day period (Figures 5 and 6). 2.4. Effects of Rifaximin on Colonic Expression of TLRs and Immune-Related Markers Expression of TLR2, 3, 4, 5 and 7 was detected in all colonic samples. Overall, rifax- imin induced minor changes in TLRs expression with only a moderate (2- to 3-fold), but significant, up-regulation observed for TLR3 and 4 for the 50 mg/kg dose during a 7-day period (Figure 7). Expression of all immune-related markers assessed was detected in colonic tissues, although in some cases with relatively high variability. Regardless the dose and time of treatment, no changes were observed for pro-inflammatory cytokines (IL-6, IL-18, IFNγ and IL-12p40), anti-inflammatory cytokines (IL-10) or antimicrobial peptides (Defα24, RELMβ and RegIIIγ) (Figure 8). 2.4. Effects of Rifaximin on Colonic Expression of TLRs and Immune-Related Markers Expression of TLR2, 3, 4, 5 and 7 was detected in all colonic samples. Overall, rifaxi- min induced minor changes in TLRs expression with only a moderate (2- to 3-fold), but Int. J. Mol. Sci. 2021, 22, 500 significant, up-regulation observed for TLR3 and 4 for the 50 mg/kg dose during a 10 7-day of 19 period (Figure 7). Effectsof Figure7.7.Effects Figure ofrifaximin rifaximinon onthe the colonic colonic expression expression ofof TLRs. TLRs. Each Each point point represents represents an an individual individ- animal; the horizontal lines with errors correspond to the mean ± SEM. For ual animal; the horizontal lines with errors correspond to the mean ± SEM. For the sake of the sake of clarity, and clarity, and since no differences were observed among them, vehicle-treated animals have been mergedinina since no differences were observed among them, vehicle-treated animals have been merged asingle singlegroup group(n (n == 8). *:*:pp<<0.05 0.05vs.vs. V;V;&:&: p