Prebiotic Properties of Galursan HF 7K on Mouse Gut Microbiota

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PrebioticPropertiesofGalursanHF7KonMouseGutMicrobiota

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Cell Physiol Biochem 2013;32(suppl 1):96-110DOI: 10.1159/000356631Published online: December 18, 2013

© 2013 S. Karger AG, Baselwww.karger.com/cpb 96

Engevik et al.: Galursan HF 7K on Mouse Gut Microbiota

Cellular Physiology and Biochemistry


1421-9778/13/0327-0096$38.00/0

Original Paper

Copyright © 2013 S. Karger AG, Basel

Accepted: November 27, 2013

This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.

Prebiotic Properties of Galursan HF 7K on Mouse Gut Microbiota Melinda A. Engevika,b Carla J. Falettic Markus Paulmichlc Roger T. Worrella,b

aDepartment of Molecular and Cellular Physiology, University of Cincinnati College of MedicineCincinnati, bDigestive Health Center of Cincinnati Children’s Hospital, Cincinnati, OH, USA; cInstitute of Pharmacology and Toxicology, Center for Pharmacogenetics and Pharmacogenomics, Paracelsus Medical University, Salzburg, Austria

Key WordsIntestine • Antibiotics • Sugars • Diarrhea

Abstract Background: With the rise of antibiotic resistance, new alternatives are being sought to effectively modulate the characteristics of gut microbiota to obtain pathogen resistance without the use of antibiotics. In the past, an oligosaccharide derivative of carrots, galursan HF 7K (GHF7K), has been used clinically in Austria and recently in the fowl-industry to promote health. This study examined the potential role of GHF7K as a prebiotic to alter the gut microbiota in mice. Methods: Mice were fed either a control diet (CT) or a diet containing 2% GHF7K in the water and chow for 2 weeks, and weight, food and water consumption, gut microbiota and ion composition of the intestinal fluid were examined. Results: Dietary supplement of GHF7K did not alter mouse weight or daily food consumption. Additionally, no changes were observed in the total number of luminal or mucosa-associated bacteria populations in GHF7K-fed mice. GHF7K supplementation significantly altered the composition of luminal, and to a less extent, mucosa-associated bacterial populations at the level of the phyla, with region-specific differences. Similar to antibiotic use, Proteobacteria number was increased in the ileum and colon of GHF7K–fed mice, with no changes in the number of beneficial Lactobacillus and Bifidobacterium genera of phylum Firmicutes. Corresponding with the altered gut microbiota, changes in the ion composition of the intestinal fluid were observed. An increased Cl- concentration was observed in the duodenum and jejunum, while the Na+ concentration was increased in the cecum of GHF7K-fed mice. Decreases were observed in the K+ concentration in the cecum and distal colon. Conclusions: Dietary supplement of GHF7K is capable of altering the gut microbiota, which correlates to changes in the intestinal environment. These data suggest that GHF7K dietary supplement can purposefully be used to alter the gut microbiota, and thus could potentially represent an alternative approach to prophylactic antibiotic use.

Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267 (USA)Tel. +1513-558-6489, E-Mail [email protected]

Roger T. Worrell, Ph.D.

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Introduction

The intestinal microbiota contains over 1014 bacteria and plays a role in health and disease [1-3]. The microbiota-host interaction plays a key role in maintaining intestinal homeostasis. Alterations in either the gut microbiota or the host genetics can result in alterations in the normal gut flora, termed microbial dysbiosis. In an attempt to control the gut microbiota, antibiotic use has become a routine procedure for treating both humans [4] and livestock [5]. Studies have shown that antibiotic treatment of chickens, cattle and swine reduces Salmonella and E. coli infection [6-10]. However, antibiotics have been shown to increase Proteobacteria, a phylum which contains a large number of pathogenic bacteria [11-16]. In addition, long term use of antibiotics has also been shown to contribute to the increased prevalence of antibiotic-resistant bacteria [5]. Additional risk lies in the potential transfer of antibiotic resistance genes to resident microflora, resulting in propagation of antibiotic-resistant bacteria. Livestock in particular serve as a reservoir of bacteria that are resistant to antibiotics. These resistant bacteria tend to spread from manure, commonly used as fertilizer, to the water supply and plants which are consumed by other animals or humans [17].

As a result of the adverse effects of continued antibiotic use, studies have begun to explore alternative methods of controlling and maintaining the normal gut microbiota while preventing infection. Dietary supplements have gained attention in recent years. Soluble dietary oligosaccharides extracted from various sources have been shown to promote the proliferation of specific beneficial bacterial groups and thus are termed prebiotics [18-23]. Examples of prebiotics include fructooligosaccharides, galactooligosaccharides, arabinose, galactose, inulin, raffinose, mannose, lactulose, stachyose, mannanoligosaccharides, xylooligosaccharides, palatinose, lactosucrose, glycooligosaccharides, isomaltooligosaccharides, and soybean oligosaccharides [20, 24-26]. A dietary supplement approach has been used throughout history for the prevention and treatment of diarrhea. Ernst Moro, an Austrian physician and pediatrician, demonstrated in 1908 that a carrot soup dramatically decreased the infant death rate by diarrhea in Germany [27, 28]. Preliminary studies in fowl farming suggest an oligosaccharide derivative of carrot, oligo(2-7)-galacturonic acid can be substituted for antibiotics to prevent gastrointestinal tract (GI) infections (data not shown). These polysaccharides are hypothesized to be responsible for the anti-diarrheal effect of the carrot soup. The aim of this study was to examine the ability of a related carrot compound, galursan HF 7K (GHF7K), to alter the intestinal microbiota, by determining both the luminal and mucosa-associated microbiota profiles from the duodenum to the distal colon in mice.

Materials and Methods

Mice All experimental protocols were approved by the University of Cincinnati Animal Care and Use

Committee and complied with National Institutes of Health guidelines. Friend virus B-type susceptibility / NIH (FVB/N) WT mice [29] were used for all experiments. At 6-8 weeks post-weaning, mice were maintained on either a control mouse diet (CT, 7922 NIH-07 Mouse diet, Harlan Laboratories, Indianapolis, IN) or a GFH7K diet (Richter Pharma AG; Wels, Austria), which consists of 2% GFH7K added to the control diet chow and drinking water. The CT diet consisted of 22.5% crude protein, 5.2% fat, 3.7% crude fiber, 3.1 kcal/g energy density, 29% calories from protein, 15% calories from fat and 56% calories from carbohydrates. The diet was fed for 14 days and water was replaced daily. This experiment was repeated separately two times (1st: CT n=8, GHF7K n=7, 2nd: CT n=6, GHF7K n=4). On day 15, ~5cm of duodenum, jejunum, terminal ileum (hereafter referred to as ileum), cecum, and colon (proximal and distal) segments were collected from CT and GFH7K diet fed mice. Individual intestinal segments were flushed with phosphate buffered saline (PBS, pH 7.4) and mucosal scrapings were collected as previously described [30-33]. Briefly, intestinal segments were flushed with 500 µl PBS. The segments were then opened lengthwise, washed thoroughly with PBS

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and glass slides were used to scrape the epithelia and mucus layer. Luminal flushes and mucosal scrapings were processed for total DNA which was stored at -20 °C until the samples were evaluated by quantitative real time PCR (qPCR).

Bacterial strains and culture conditions Pure cultures of bacterial strains were used to generate standard curves to correlate bacterial

cell number with qPCR cycle threshold (CT) values. Micrococcus luteus, Peptostreptococcus anaerobius, Staphylococcus aureus, Escherichia coli, Faecalibacterium prausnitzii and Burkoholdena cepacia were a gift from Dr. Daniel J. Hassett. Lactobacillus acidophilus and Rhibozium legaminosarum were purchased from Carolina Biological Supply Company (Carolina Biological Supply Company, Burlington, NC), Bacteroidetes thetaiotaomicron ATCC 29741 and Prevotella melaninogenica ATCC 25845 were purchased from Fisher Scientific (Thermo Fisher Scientific, Waltham, MA) and Ruminococcus productus ATCC 27340D-5 was purchased from ATCC (American Type Culture Collection, Manassas, VA). E. coli, S. aureus, M. luteus, B. cepacia, L. acidophilus and R. legaminosarum were grown in Luria–Burtani (LB; Thermo Fisher Scientific) broth at 37 °C in a shaking incubator. R. productus and F. prausnitzii were grown in tryptone-yeast extract-glucose (TYG; Thermo Fisher Scientific) broth, P. melaninogenica and B. thetaiotaomicron were grown in tryptone-soy broth (TSB; Thermo Fisher Scientific), and Peptostreptococcus anaerobius was grown in brain-heart-infusion (BHI; Thermo Fisher Scientific) broth at 37 °C in a dual-port anaerobic chamber (Coy Systems, Coy Lab Products, Grass Lake, MI). Bacterial cell counts were determined via a Petroff Hauser chamber (Hausser Scientific; Horsham, PA) and by colony forming units (CFU) [30, 34-36].

qPCR amplification of bacterial 16S genomic DNA sequences QIAamp DNA Stool kit (Qiagen, Valencia, CA, USA) was used to extract total DNA according to the

manufacturer’s instructions and as previously described with the addition of lysozyme (10 mg/ml, 37 °C for 30 min) and 95 °C lysis temperature [30, 33, 37, 38]. qPCR was used to analyze the amount of total bacteria and bacterial phyla, class, genus and species using a Step One Real Time PCR machine (Applied Biosystems, Carlsbad, California USA) with SYBR Green PCR master mix (Applied Biosystems) and bacteria-specific primers to 16S genomic DNA (Table 1) in a 20 µl final volume. Standard curves were generated from pure bacterial cultures and used to calculate bacteria number from cycle of threshold values (CT) [38, 39]. Bacterial phyla composition was determined using calculated CFU values for each bacterial phyla as a percentage of the total bacteria. Total bacteria were calculated using the universal bacterial primer and represent the total number of bacteria per intestinal segment examined.

Ion concentration measurements Intestinal segments flushed with 100 µl of double deionized water were used to analyze the ion

composition of the intestinal content of CT and GHF7K diet- fed mice as previously described [30]. Flushes were performed on the same length intestinal segments as used to collect bacterial content. Collected flush samples were weighed and the intestinal volume calculated from the total weight minus the 100 µl of flush water assuming a density of 1. GHF7K diet did not alter intestinal fluid volume (data not shown). Flushes were then centrifuged at 1,400 g for 10 min at 4 °C to pellet intestinal solids. Flame photometry was used to determine supernatant Na+ and K+ concentrations (digital Flame photometer, Single-Channel Digital Flame Photometer Model 02655-10; Cole-Parmer Instrument Company Vernon Hills, IL). Chloridometry was used to determine the Cl- ion concentration (digital Chloridometer, Model 4425100, Labconco Kansas City, MO). Calculated ion concentration was normalized to intestinal volume and presented as mM. An electronic pH meter (Orion Model 720A; Thermo Fisher Scientific Waltham, MA) was used to measure pH electrochemically.

Statistics The data presented herein as the mean ± SEM. Differences between groups determined by two factors

(diet and gut region) were determined using the two-way analysis of variance (ANOVA). The Holme-Sidak post-hoc test was applied to determine significance between pairwise comparisons, using SigmaPlot (Systat Software Inc, San Jose, CA). P < 0.05 was considered significant and n is the number of experiments.

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Results

WT FVB/N mice were fed either a control diet (CT) or a diet supplemented with 2% GHF7K in the water and chow for a period of 2 weeks. Mouse weight was examined at the beginning and end of the study. No significant changes in weight were observed during the course of the study (Table 2). In addition, mice supplemented with GHF7K consumed the same food and water quantity as CT diet fed mice, indicating that the sugar itself did not stimulate increased food or water consumption or weight gain. In order to examine if the total number of bacteria was changed in mice supplemented with

Table 1. Primer sequences for qPCR of total bacteria and specific bacterial phyla and groups

Table 2. Weight, food and water consumption for mice on control and GHF7K diet. Weight was recorded at the end of the study for mice on the control diet and GHF7K diet. Dry food was weighed and water quantity was recorded daily to determine the total volume of food and water consumed by mice on the control diet and GHF7K diet. n=14 for control and n=11 for GHF7K diet. No significant differences were observed between the control and GHF7K diet. Analyzed by two-way ANOVA with Holme-Sidak post-hoc test

GHF7K, DNA was extracted from luminal flushes and mucosal scrapings and analyzed by qPCR using a universal 16S RNA primer. No changes were observed in the total number of luminal bacteria (total bacterial load) in any intestinal segment (Fig. 1A). As with the luminal bacterial population, no changes were observed in the mucosa-associated total bacterial load (Fig. 1B). This indicates that no general overgrowth or depletion of bacteria occurred with the GHF7K supplemented diet. This is advantageous since increased total bacteria have been associated with increased bacterial translocation, sepsis and inflammation [40-46]. As a result, minimal changes in the total luminal and mucosa-associated bacterial populations is likely beneficial because it minimizes potentially detrimental epithelia- immune response interactions.

In order to determine if GHF7K was able to alter the gut microbiota in a similar manner to antibiotics, the bacterial composition was analyzed by qPCR using bacterial phyla specific primers. Studies have shown that the mouse and human GI tract is dominated by Firmicutes and Bacteroidetes, while the Actinobacteria, Proteobacteria, Fusobacteria and Verruomicrobia phyla are present in lower abundance [2, 47-53]. The major mouse intestinal bacterial phyla were compared as a percentage of total bacteria as shown in Fig. 2. In the duodenum or cecum (Fig. 2A) there were no significant changes in the luminal bacterial population due to the GHF7K-diet. However, there was a decrease in Firmicutes (Δ23.1 ± 3.7% between CT and

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GHF7K) and an increase in Bacteroidetes (22.3 ± 4.1%) in the jejunum from GHF7K-fed mice. Similarly, the ileum of GHF7K-fed mice showed a decrease in Firmicutes (19.8 ± 2.4%) and an increase in Bacteroidetes (11.2 ± 2.3%), while in the proximal colon there was a decrease in Bacteroidetes (15.8 ± 1.2%) but no significant change in Firmicutes (0.9 ± 0.2 %). In the distal colon there was a decrease in both Firmicutes (10.7 ± 2.2%) and Bacteroidetes (14.0 ± 3.0%) in the experimental group. Actinobacteria did not change in any of the GHF7K-fed mouse intestinal segments. Interestingly, α,β,γ-Proteobacteria was increased in ileum (8.6 ± 1.8%), proximal (15.0 ± 2.8%) and distal colon (24.2 ± 4.7%). This increase in Proteobacteria is similar to that observed with antibiotic use [11-16]. In the GHF7K fed mice, the increase in Proteobacteria was due to an increase in γ-Proteobacteria (ileum: 8.46 ± 1.9%, proximal colon: 14.0 ± 2.5%, distal colon: 2.4 ± 0.8%) and α-Proteobacteria (distal colon: 21.1 ± 3.8%). Proteobacteria, particularly γ-Proteobacteria, harbor a number of pathogenic bacteria [54], such as Escherichia coli, Salmonella, Yersinia, Vibrio, and Pseudomonas. The increased γ-Proteobacteria in the GHF7K-fed mice may minimize growth of non-resident pathogenic species, and thus infection, via competition since members of the same phyla occupy the same niche.

In contrast with the luminal bacterial population, no significant changes were observed in the mucosa-associated bacterial population for the lower intestinal segments (Fig. 2B). However, changes were observed in the upper intestinal segments, duodenum and jejunum. In the GHF7K-fed mice, there was a decrease in α,β,γ-Proteobacteria (3.0 ± 1.5%) and a modest increase in Firmicutes (2.1 ± 0.7%) in the duodenum. In jejunum, there was an increase in Actinobacteria (4.2 ± 1.1%) with a decrease in other unspecified bacteria species (5.9 ± 1.1%). This indicates that GHF7K primarily affects the luminal bacterial population, and that those effects are region-specific.

Next, subgroups of the major phyla from the luminal bacterial population were examined by qPCR using subgroup-specific primers and bacterial cell number calculated from comparison to standard curves generated from each individual species. Antibiotics have been shown to decrease the majority of the Clostridium groups including C. coccoides

Fig. 1. GHF7K-fed mice have no changes in total bacteria compared to control diet fed mice. Total bacteria were quantified by qPCR using universal bacterial 16S genomic DNA sequence specific primers. The bacterial cell number was calculated from a standard curve and normalized to intestinal flush volume. (A) Bacteria contained within the luminal flushes of control (n=14, black bars) and GHF7K diet (n=11, white bars) fed mice. (B) Mucosa-associated (adherent) bacterial levels between control (n=14, black bars) and GHF7K diet (n=11, white bars) mice. As expected, the number of total bacteria in the colon is significantly greater than the total number in the small intestine (P < 0.001) GHF7K diet did not significantly alter the total bacteria number for any given segment (P = 0.665), indicating that GHF7K diet does not change the total bacteria load in the mouse intestine overall. Analyzed by two-way ANOVA with Holme-Sidak post-hoc test.

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cluster XIVa and C. leptum cluster IV [55, 56]. In addition, antibiotics have been shown to decrease Bacteroides [55], Prevotella [57], Lactobacillus [12, 55], Bifidobacteria [58-60] and Prevotella, and Mouse Intestinal Bacteroidetes (MIB, see [38, 61] references for classification) [55]. These groups were examined within the bacterial luminal population of GHF7K fed mice to determine if GHF7K decreases these groups, similarly to antibiotics. No changes were observed in the Firmicutes subgroup Lactobacillus or the Actinobacteria subgroup Bifidobacterium in any of the GHF7K-fed mice intestinal segments (Fig. 3). Lactobacillus and Bifidobacterium are commonly used as probiotics because of their therapeutic and prophylactic properties [62-70]. Studies have shown that probiotic bacteria can be beneficial for treatment against diarrhea [64, 71-74] and pathogen infection [37, 75]. Therefore, it is beneficial that the GHF7K diet did not decrease either of these valuable bacteria.

Fig. 2. GHF7K-fed mice exhibit region-specific alterations in the luminal and mucosa-associated gut microbiota. Relative abundance was calculated as the percentage of bacterial phyla in comparison to total bacteria for luminal (A) and mucosa-associated bacteria (B). Differences at the level of segment were observed for all bacterial populations (P < 0.001), indicating different colonization patterns depending on region. More dramatic changes were found in the luminal bacterial population compared to the mucosa-associated bacterial population. In the luminal population, Firmicutes had significant changes in jejunum (P = 0.029), ileum (P = 0.030), and distal colon (P = 0.048). Bacteroidetes had significant changes in the jejunum (P = 0.042), ileum (P = 0.029), proximal (P < 0.001) and distal colon (P < 0.001). No significant changes were observed between diet and segment for Actinobacteria (P = 0.263). α, β, γ-Proteobacteria had significant increases in the ileum (P = 0.031), proximal (P = 0.022) and distal colon (P < 0.001). In the mucosa-associated bacterial population, changes were observed in Firmicutes (P = 0.033) and α, β, γ-Proteobacteria in the duodenum (P = 0.025), and Actinobacteria (P = 0.014) and unspecific bacteria (P = 0.02) in the jejunum. n=14 for control and n=11 for GHF7K diet fed mice. Analyzed by two-way ANOVA with Holme-Sidak post-hoc test.

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When the Firmicutes subgroup C. coccoides cluster XIVa and C. leptum cluster IV were examined, changes were observed with the GHF7K diet (Fig. 4). Decreased C. coccoides was observed in GHF7K-fed mice jejunum while increased C. coccoides occurred in the cecum, proximal and distal colon. C. leptum was found to be significantly decreased in both duodenum and jejunum of the GHF7K-fed mice. The Bacteroidetes subgroups Bacteroides, Prevotella, and MIB were also examined (Fig. 5). MIB was decreased in the proximal and distal colon and Prevotella was significantly decreased in the duodenum of GHF7K fed mice. No changes were observed in Bacteroides in any intestinal segment. With the exception of decreased MIB, the changes in the Firmicutes and Bacteroidetes subgroups do not directly mirror changes observed with antibiotics. Together, this data suggests that the GHF7K diet can alter the gut microbiota at the level of the phyla and subgroups.

Certain bacterial groups have the capacity to alter ion transport [76-78]. To determine if GHF7K affected luminal ion concentration either directly or indirectly, Na+ and K+ concentrations of the intestinal content were determined by flame photometry and Cl- concentration was determined by chloridometry as depicted in Fig. 6. In comparison to CT diet fed mice, GHF7K fed mice showed increased luminal Na+ concentration in the cecum with no changes in any other segment (Fig. 6A). The K+ concentration was also affected: K+ was decreased in the cecum and distal colon of the experimental group (Fig. 6B). The

Fig. 3. GHF7K diet does not alter beneficial bacterial groups Lactobacillus and Bifidobacterium. Bacterial number was examined from luminal flushes from CT and GHF7K-fed mice by qPCR. No changes were observed in the levels of the beneficial bacterial groups Lactobacillus (A) and Bifidobacterium (B) in the luminal population of control (n=14, black bars) or GHF7K (n=11, white bars) diet fed mice. Analyzed by two-way ANOVA with Holme-Sidak post-hoc test.

Fig. 4. GHF7K diet alters Clostridial groups in the colon. Bacterial number was examined from luminal flushes from CT and GHF7K-fed mice by qPCR. Regional changes were observed in the levels of Firmicutes members (A) C. coccoides and (B) C. leptum. C. coccoides was decreased in jejunum, but increased in the cecum and colon of GHF7K diet fed mice (n=11, white bars). C. leptum was decreased only in the duodenum and jejunum of GHF7K diet fed mice (n=11, white bars) compared to control mice (n=14, black bars). * P < 0.05 by two-way ANOVA with Holme-Sidak post-hoc test.

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Cl- concentration was increased only in the duodenum and jejunum of GHF7K fed mice (Fig. 6C). The differences in the Na+ and K+ concentrations in the cecum, and Cl- concentrations in the upper portion of the small intestine, observed between CT and GH7FK fed mice, could represent changes in intestinal reabsorption. However, these changes were likely resolved by the colon, since no differences in Na+ or Cl- concentration were observed in this segment. Decreases were observed in the K+ concentration in the distal colon, which may indicate decreased K+ secretion. It is not clear whether this effect is host or bacterial mediated, but does represent another level of bacterial-host interaction. Changes in ion composition can affect the gut microbiota [30, 34, 35, 79-81], and an altered ion composition may represent a mechanism by which newly proliferating bacteria are able to maintain a niche. These data indicate that the GHF7K dietary supplement can alter luminal ion composition along with altered bacterial composition. No changes in intestinal pH were observed in any intestinal segment of the GHF7K-fed mice (Fig. 6D), similar to results found in dogs fed galacturonic acid [82]. Taken together, these studies demonstrate that a GHF7K supplemented diet can induce regional specific changes in the gut microbiota on the phyla and subgroup level and ion composition of the intestinal content, which correspond to changes in the intestine environment.

Discussion

It has been well documented that oligosaccharides are decomposed in the intestinal tract of mammals [82], and can act as a prebiotic by modifying the host microbiota in a beneficial manner [83]. The concept of modulating gut health through diet has been used throughout history, but recently, scientific advances have begun to provide mechanistic insight on how diet supplementation is capable of benefiting the host. The aim of the present work was to assess the prebiotic properties of the acidic oligosaccharide GHF7K. Our data demonstrate that GHF7K diet supplementation increases the phylum Proteobacteria by increasing α- and γ-Proteobacteria. Dietary supplement of GHF7K also changes Firmicutes and Bacteroidetes

Fig. 5. GHF7K diet alters MIB and Prevotella, but does not alter Bacteroides. Bacterial number was examined from luminal flushes from CT and GHF7K-fed mice by qPCR. No changes were observed in Bacteroidetes member Bacteroides (B), but regional changes were observed in the levels of (C) Prevotella and (A) MIB. GHF7K (n=11, white bars), control (n=14, black bars). * P < 0.05 by two-way ANOVA with Holme-Sidak post-hoc test.

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subgroup members in a region-specific manner. This substance is of particular interest for the livestock industry, which currently relies on antibiotics for animal health purposes. If GHF7K can reproduce the beneficial effects observed with antibiotics while maintaining constant levels of beneficial bacteria, then it could serve as an antibiotic alternative. This would be of great importance to reduce the propagation of antibiotic resistance within the food chain. In this paper, we demonstrate that GHF7K mimics antibiotic use in that it increases Proteobacteria in a mouse model and could potentially be used as an antibiotic alternative.

Bacteroidetes and Firmicutes are the two dominant phyla in all vertebrates [50, 84, 85]. Studies have demonstrated that mice and humans share similar composition at the high-taxonomic levels (phyla, class, order), but differ greatly at the lower-taxonomic levels (genera, species, subspecies) [50, 85, 86]. Despite lower-taxonomic differences, broad trends exist at the phylum level [85]. Additionally, the gut microbiota of higher vertebrates (including humans, mice, and livestock) have been shown to be similar in core functions [86], adding the value of using mouse models to extrapolate large trends in the gut microbiota. Interestingly, no changes were observed in the cecum from mice fed GHF7K. The cecum represents a “bioreactor” and has been shown to be a relatively stable environment [87, 88]. It may be possible that conditions within the cecum prevent dramatic changes from occurring, thereby preserving the gut microbiota. The cecum has been shown to harbor a complex microbial community [87-89] and although changes were not observed in the phyla

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Fig. 6. GHF7K diet alters the intestinal environment with regional changes in Na+, K+ and Cl- concentrations, but does not change pH. Ion concentrations in luminal fluid from control (black bars) and GHF7K (white bars) diet fed mice intestinal segments. (A) The Na+ concentration, as determined by flame photometry, was increased only in the GHF7K-fed mice cecum. (B) The K+ concentration, as determined by flame photometry, was significantly decreased only in GHF7K-fed mice cecum and distal colon. C) The Cl- concentration, as determined by chloridometry, was significantly increased in the GHF7K-fed mice duodenum and jejunum. D) No changes in pH along the length of the intestinal tract between GHF7K and control diet fed mice were observed. GHF7K (n=11, white bars), control (n=14, black bars). * P < 0.05 by two-way ANOVA with Holme-Sidak post-hoc test.

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or major subgroups in this area, it is possible that changes do occur at the level of the species. However, sequencing would be required to fully address these changes.

Prebiotics have been shown to selectively stimulate the growth and/or activity of specific bacteria [19, 21, 90, 91]. Pre-clinical studies indicate that prebiotics have the potential to be used in disease treatment [92] and for the prevention of intestinal infections [91, 93]. Data presented in this paper and data in the literature suggest that prebiotics predominantly affect the luminal bacterial population, whereas the mucosa-associated bacteria appear to be more affected by modifications at the level of the host epithelia [30, 31, 33, 38]. In addition to prebiotics and probiotics, antibiotics have also been used to modify the intestinal gut microbiota [12-15]. Multiple antibiotics have been shown to decrease the beneficial bacteria Lactobacillus [12, 55] and Bifidobacteria [58-60]. It is advantageous that GHF7K does not decrease these two groups because Lactobacillus and Bifidobacteria have been shown to reduce luminal pH, produce short-chain fatty acids, secrete antimicrobial compounds (bacteriocins), induce production of antimicrobial compounds (defensins) by the host epithelium, prevent pathogenic bacterial adhesion to epithelial cells, and actively compete for nutrients that might be used by pathogenic bacteria [83, 94-98]. Previous studies have shown that supplementation of acidic oligosaccharides, such as galacturonic acids, in formula does not affect levels of Bifidobacteria or Lactobacillus in infants [99, 100]. This is consistent with the current study, which demonstrates no change in Bifidobacteria or Lactobacillus in GHF7K-fed mice.

GHF7K administration also resulted in region-specific changes in the phyla Bacteroidetes and Firmicutes, and in the subgroups C. coccoides, C. leptum, Prevotella and MIB. Whether or not these changes are beneficial, detrimental or neutral is not clear. Dietary changes have been shown to cause rapid changes in intestinal metagenomics [86], which represents all the host and gut microbial genes as well as corresponding functions [1]. However, as long as the core metagenomic functions are conserved within a host, the changes in specific species are counterbalanced and have no detrimental effect [86]. At this time, we can speculate that GHF7K does not have a negative impact on the gut microbiota as it did not alter the gross morphology of the intestine or result in large scale changes, such as weight gain, in the mice. However, further experiments are warranted to fully address this question.

Vancomycin, metronidazole, amoxicillin-clavulanic acid, clindamycin, ancomycin, amipenem, and neomycin have all been shown to increase the phylum Proteobacteria [11-13, 16, 55-60]. Although Proteobacteria represent a minor taxon in the gut microbiota, this group has been shown to play a significant, active role in overall gut metabolism and host interaction [101]. This study has demonstrated that GHF7K alters the gut microbiota by increasing the levels of α and γ-Proteobacteria, providing evidence of its application as a potential antibiotic alternative.

A potential downside to antibiotic use is that antibiotics can result in increased susceptibility to further pathogen infection [12]. Whether GHF7K increases pathogen susceptibility or protects against pathogen colonization is currently unkown. In the future, it would be interesting to challenge GHF7K fed mice with a pathogen such as Salmonella typhymurium, Escherichia coli, or Clostridium difficile, in order to determine if it actually provides benefit. Although GHF7K does increase Proteobacteria, it does not mirror antibiotic use completely, indicating that there might be some potential for benefit. Additionally, preliminary studies with oligo(2-7)-galacturonic acids in fowl farming (data not shown) and traditional application of Moro’s carrot soup (citations 27 & 28?), suggest that GHF7K may provide benefit to the host and may prevent and/or resolve pathogen infection. It is therefore plausible that dietary supplement of GHF7K may prevent infection by either (1) causing the proliferation of resident Proteobacteria, thereby limiting further colonization by other pathogenic Proteobacteria or (2) that the addition of GHF7K is a preferred binding substrate of Proteobacteria members and luminal GHF7K may prevent pathogenic γ-Proteobacteria from binding to oligosaccharides on the host mucosa.

In addition to the potential for GHF7K to be used as an antibiotic alternative, GHF7K may be beneficial for other disease states associated with an altered intestinal environment,

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or microbial dysbiosis. One example of this is seen in human gastric bypass surgery as a treatment for obese patients. Gastric bypass surgery has been shown to promote the proliferation of γ -Proteobacteria with a corresponding decrease in Firmicutes and methanogens that are responsible for increased energy consumption in obese individuals [51, 102, 103]. Herein we demonstrate that GHF7K acts in a similar manner as gastric bypass surgery: decreasing Firmicutes in the small intestine and increasing γ –Proteobacteria. This suggests that GHF7K could also be used as a food additive for obese patients and may produce similarly “beneficial” effects as those observed following gastric bypass. Although many oligosaccharides have been shown to alter the gut microbiota, it is important to note that each oligosaccharide has its own properties and not all act on the same bacterial groups or species. The data in this study suggest that the galursan HF 7K sugar could potentially be used to alter the gut microbiota in a beneficial manner with defined changes in the luminal bacterial populations.

Conflict of Interests

None.

Acknowledgements

This work was supported by the FWF (P18608) and the FP-7 (PIRSES-GA-2008-230661) grants to M. Paulmichl.

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