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Anaerobe 18 (2012) 331e337

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Anaerobe

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Molecular biology, genetics and biotechnology

Gut bacteria profiles of Mus musculus at the phylum and family levels areinfluenced by saturation of dietary fatty acids

Tianyu Liu, Helen Hougen 1, Amy C. Vollmer*, Sara M. HiebertBiology Department, Swarthmore College, 500 College Avenue, Swarthmore, PA 19081-1390, USA

a r t i c l e i n f o

Article history:Received 21 June 2011Received in revised form2 February 2012Accepted 22 February 2012Available online 3 March 2012

We would like to dedicate this report inhonor of the 90th birthday of Dr. Sydney M.Finegold, who has been and continues to bean inspiration for generations ofmicrobiologists.

Keywords:Saturated fatty acidOmega-3 polyunsaturated fatty acidOmega-6 polyunsaturated fatty acid16S rRNA/ssu RNABody mass

* Corresponding author. Tel.: þ1 610 328 8044; faxE-mail addresses: tomliu.swat@gmail.com (T. Liu

(H. Hougen), avollme1@swarthmore.edu (A.swarthmore.edu (S.M. Hiebert).

1 Current address: National Institutes of Health, Natand Human Development, Section on Epigenetics &20892-1103, USA.

1075-9964/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.anaerobe.2012.02.004

a b s t r a c t

Background: Mammalian gut microbiota have been implicated in a variety of functions including thebreakdown of ingested nutrients, the regulation of energy intake and storage, the control of immunesystem development and activity, and the synthesis of novel chemicals. Previous studies have shown thatfeeding mammalian hosts a high-fat diet shifts gut bacteria at the phylum level to reduce the ratio ofBacteroidetes-to-Firmicutes, while feeding hosts a fat-restricted diet increases this ratio. However, fewstudies have investigated the differential effects of fatty acid type on gut bacterial profile.Methods: Over a 14-week period, Mus musculus were fed a diet rich in omega-3 polyunsaturated fattyacids (n-3 PUFAs), omega-6 polyunsaturated fatty acids (n-6 PUFAs), or saturated fatty acids (SFAs). Fecalpellets were collected before and after the treatment period from 12 randomly selected mice (4 pertreatment group). Bacterial DNA was extracted from the pellets and characterized by analysis of thehypervariable V3 region of the 16S rRNA. Nominal logistic regression models were used to assess shifts inmicrobial profile at the phylum and family levels in response to diet.Results: A significant decrease in the proportion of phylumBacteroidetes species was observed formice fedany of the three diets over time. However, the SFA-rich diet group showed a significantly greater decreasein Bacteroidetes proportion (�28%) than did either the n-3 PUFA group (�10%) or the n-6 PUFA group(�12%). At the family level, a significant decrease in proportion of Porphyromonadaceae was observed formice fed the n-6 PUFA-rich diet, and a significant decrease in proportion of Lachnospiraceae was observedfor mice fed the SFA-rich diet. There was no significant effect of diet type on body mass change.Conclusion: Our results indicate that SFAs have stronger effects than PUFAs in shifting gut microbiotaprofiles toward those typical of obese individuals, and that dietary fatty acid saturation influences shiftsin gut microbiota independently of changes in body mass.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Themammalian gut is home to a community of microorganisms(“microbiota”) including bacterial, archeal, and fungal lineages thathave significant influences on host health and function. In humans,bacterial cells outnumber human cells 10:1 [1], and bacterial genesoutnumber human genes 100:1 [2]. Gut microbes help the hostdigest otherwise indigestible components of the host diet,providing humans with as much as 10% of our daily energy [3]. Inaddition, they influence the expression of host genes that promote

: þ1 610 328 8663.), h.yang.hougen@gmail.comC. Vollmer), shieber1@

ional Institute of Child HealthDevelopment, Bethesda, MD

All rights reserved.

storage of energy in adipocytes [4], affect biotransformations thatlead to the synthesis of essential vitamins, and regulate themetabolism of xenobiotics and lipids [5]. As a result, gut microbeshave been implicated in numerous diseases including obesity,diabetes, atopic disorders, gastrointestinal (GI) disorders, andinflammatory bowel disease [6].

In both humans and mice, the majority of gut bacteria fall intoone of two phyla: Firmicutes (60e80%) or Bacteroidetes (20e40%).Other phyla present (�1% each) include Proteobacteria, Actino-bacteria, Cyanobacteria, and TM7 [6]. Factors that have been shownto be associated with changes in the gut microbial profile includediet [7e9], obesity phenotype [10], genetics [11], environmentalexposure [8], and aging [12].

Previous studies have shown that changes in diet can inducesignificant shifts in the gut microbiota at the phylum, class, andfamily levels. Feeding mice a high-fat diet causes a decrease in theBacteroidetes-to-Firmicutes ratio within 24 h, with the new

T. Liu et al. / Anaerobe 18 (2012) 331e337332

proportion stabilizing in as few as seven days [8,9]. Conversely,feeding obese humans fat- or carbohydrate-restricted diets leads toan increase in the Bacteroidetes-to-Firmicutes ratio [7]. However,most studies on high-fat diets have involved diets rich in saturatedfatty acids (SFA); few studies have investigated the differentialeffects of fatty acid type on gut microbial composition.

The traditional Western diet, high in SFAs, has been associatedwith increased risk of cardiovascular disease and colon cancer [13].Epidemiological studies [14e16] have correlated increased SFAconsumption with increased adiposity, obesity, and insulin resis-tance. In addition, human and animal studies [17e19] have shownthat an SFA-rich diet results in increased adiposity and lowermetabolic rate, relative to a polyunsaturated fatty acid (PUFA)-richdiet. In the colon, PUFAs have been shown to exhibit powerfulantibacterial effects, a mild laxative effect [20], and protectiveeffects against colon cancer [13]. Omega-6 (n-6) PUFAs and omega-3 (n-3) PUFAs represent two major subgroups with differentialeffects on health. n-6 PUFAs, dominant in many plant oils, havebeen shown to reduce cholesterol and lower blood pressure [21],but increase production of pro-inflammatory prostaglandins [22]and increase insulin resistance. n-3 PUFAs, found in marine oilsand flaxseed oil, have been shown to reduce inflammation, reduceproduction of reactive oxygen species, and inhibit cytokineproduction, thus alleviating diseases such as rheumatoid arthritis[22] and improving cardiovascular health [23]. Because a diet witha high n-6/n-3 PUFA ratio may be detrimental in terms of adiposityand insulin resistance [24], there is a need to better understand thedifferential effects of these two classes of PUFAs within the body.Furthermore, while PUFAs generally exhibit more potent bacteri-cidal activity than SFAs [25], their effects on the composition of gutbacterial populations have not been well documented.

A previous study [26] showed that BALB/c mice fed diets rich inSFA, n-3 PUFA, or n-6 PUFA did not contain different proportions ofLactobacilli (genus within phylum Firmicutes) or Bacteroides (genuswithin phylum Bacteroidetes), but conclusions were based onculture-derived bacterial samples, whichmay not be representativeof most bacterial species in the gut. New culture-independent,metagenomic methods can detect microbial species that do nothave a cultured representative, which constitute a majority(approximately 78%) of known gut microbes [6]. While the exactcomposition of the gut microbiota differs along the length of the GItract, the feces contain the second-highest density of microbes[after the cecum; 27] and provide a convenient, non-invasivesample source to represent the microbiota of the lower GI tract[28]. Thus, we used 16S rRNA analysis of bacteria present in fecalpellets to investigate the differential effects of SFAs, n-3 PUFAs, andn-6 PUFAs on mouse gut bacteria composition.

2. Materials and methods

2.1. Animals and experiments

All animal care and use procedures were approved by theInstitutional Animal Care and Use Committee at SwarthmoreCollege in accordance with the Guide for the Care and Use of Animals[29]. Forty-five, seven-wk-old outbred male mice [Hsd:ICR (CD-1),Harlan, Madison WI] were maintained in a walk-in environmentalchamber on a 14L:10D photoperiod at 21 �1 �C. Mice were housedindividually in opaque plastic cages (18 cm � 28 cm � 13 cm) linedwith wood shavings and provided with ad libitum access to tapwater and rodent chow (Harlan-Teklad 8664, 6.5% fat by energy).After 14 days, mice were divided into three treatment groups thatwere balanced for bodymass and food consumption (N¼ 15 in eachgroup) for a study on the differential effects of diets rich in SFAs, n-3PUFAs (principally a-linolenic acid, 18:3), or n-6 PUFAs (principally

linoleic acid, 18:2) on physical and cognitive performance at highand low ambient temperatures [H. Hougen, unpubl. data]. Mice inall three treatment groups were fed one of the fat-enriched exper-imental diets (Harlan-Teklad, 32.5% fat by energy) for 10.5 wk, andcontinued on this diet during a 3-wk period duringwhich theyweretested at both 5 and 21 �C for grip strength, skin sensitivity to heat,and memory. Exposures to cold lasted< 3 h at a time, with a periodof 22 h to 4 da at 21 �C between cold exposures. Four spontaneouslyvoided fecal pelletswere collected from the cage of eachmouse 1) atthe beginning of the experiment, when diet was switched frommaintenance rodent chow to the SFA-rich, n-3 PUFA-rich or n-6PUFA-rich diet, and 2) at the conclusion of the performance tests,after the animals had been eating the experimental diets for a totalof 14 weeks. Fecal pellets were stored at �20 �C until DNA extrac-tion. Fecal pellets collected at the beginning and end of diet treat-ment were taken for bacterial analysis from four randomly selectedmice in each treatment group. All mice were weighed before andafter the 14-week experimental period.

2.2. Diet composition and analysis

Isocaloric semisynthetic diets containing a total of 13.4% fat byweight (32.5% of kJ from fat) were prepared by Harlan-Teklad(Madison, WI). The n-3 PUFA-rich (TD.08160), n-6 PUFA-rich(TD.04230) and SFA-rich (TD.04229) diets were created by sup-plementing a nutritionally complete diet with flaxseed oil, soybeanoil or a mixture of soybean oil and fully hydrogenated soybean oil(Archer Daniels Midland, Decatur, IL), respectively. The n-6 PUFA-rich and SFA-rich diets are identical in the distribution of fattyacid chain lengths and thus differ only in the degree of saturation.Flaxseed oil was chosen over fish oil as a source of n-3 PUFAsbecause the distribution of chain lengths of flaxseed oil moreclosely approximated that of the two soybean oil based diets.Flaxseed oil differs substantially from n-3-rich fish oils, however, incontaining only very small amounts of the long chain fatty acidseicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA,22:6). Because trans fatty acids are generated during partialhydrogenation, using a mixture of fully hydrogenated and unhy-drogenated soybean oils allowed production of intermediate levelsof saturation without the inclusion of trans fatty acids. All dietswere kept at 4 �C under nitrogen (to prevent oxidation) until use.

To determine dietary fat composition, fatty acids were extractedusing a method modified from Folch et al. [30]. Lipids froma homogenized diet sample were extracted with a 2:1 mixture ofchloroform:methanol, followed first by two rounds of acidification,centrifugation and removal of the lipid-containing chloroform layerand then by a final filtration. After being dried under nitrogen, fattyacids were methylated by direct transesterification according toa method based on Lepage and Roy [31]. Component fatty acidmethyl esters in toluene were identified on a Varian 3900 gaschromatograph, using helium as a carrier gas in aWCOT fused silicacolumn (100 m � 0.25 mm), a flame ionization detector, and a 5-step temperature program beginning at 150 �C and ending at227 �C for a total program time of 32 min. Peaks were identifiedbased on comparison of retention time with fatty acid standards(Supelco #24056), and composition is expressed as percent ofidentified fatty acid peaks (Table 1).

2.3. DNA extraction and sequencing

In preliminary studies in our laboratory (T. Bice, unpubl. data),bead beating was not shown to be more efficacious in liberatinggenomic DNA prior to PCR amplification. Lysing efficiency wasdetermined bymeasurement ofDNA in the lysate andpellet byA260(structure) and by the measurement of available PCR template

Table 1Fatty acid composition of diets.

Dieta

Standard chow SFA-rich n-3 PUFA-rich n-6 PUFA-rich

16:0 12.9% 12.2% 6.0% 11.6%18:0 3.6% 40.1% 2.3% 3.6%18:1 20.4% 12.0% 20.2% 21.9%18:2 46% 30.0% 19.6% 54.1%18:3 n-3 5.5% 3.5% 50.2% 6.6%Trans fats 0.0% 0.0% 0.0% 0.0%Total n-3 6.7% 3.5% 50.3% 6.6%Total n-6 45.9% 30.2% 19.7% 54.3%n-6:n-3 6.93 8.63 0.39 8.26SFA 18.1% 53.2% 8.8% 16.0%MUFA 21.8% 12.1% 20.5% 22.1%PUFA 52.6% 33.7% 69.9% 60.8%Total UFA 74.5% 45.8% 90.4% 83.0%SFA:UFA 0.24 1.21 0.10 0.19Total % kJb 19.0 34.0 37.0 40.0SFA % kJb 3.4 18.0 3.2 6.4MUFA % kJb 4.1 4.1 7.6 8.9PUFA % kJb 10.0 11.5 25.9 24.3

a Each percentage represents the average of 2e4 samples.b % of total energy.

T. Liu et al. / Anaerobe 18 (2012) 331e337 333

(function). Thus, DNA was extracted from each pellet without beadbeating using the QIAamp DNA Stool Mini Kit (Qiagen). One mlof each DNA sample was used with the GoTaq Green MasterMix (Promega) to perform PCR. Final concentrations of0.72 mM Universal Forward (UF) primer (50-CGGCCCA-GACTCCTACGGGAGGCAGCAG-30) and 0.67 mM Eubacterial Reverse(ER) primer (50-GCGTGGACTACCAGGGTATCTAATCC-30) were usedin each 25 ml PCR sample to amplify the hypervariable V3 region ofthe bacterial 16S rRNA gene. The V3 region was chosen over the V6region because of evidence that the V3 region shows better reso-lution and accuracy in downstream classification [32,33]. Cyclingconditions were 95 �C for 2 min, followed by 30 cycles of 95 �C for0.5 min, 55 �C for 0.5 min, and 72 �C for 0.5 min, with a finalextension period of 3 min at 72 �C. PCR products were purified withthe QIAquick PCR Purification Kit (Qiagen), and then subcloned intopCR4-TOPO TA vector (Invitrogen). Clones were transformed intoEscherichia coli TOP10 or MegaX DH10B cells (Invitrogen), andsequenced unidirectionally using M13 Reverse primer (50-CAG-GAAACAGCTATGAC-30)with anABI 3730 capillary sequencer. UFandER primer sequences were removed using DNA Baser v2 (HeracleBioSoft), and sequences less than 300 bp or more than 900 bp longwere removed as potential truncated sequences or chimeras. Theresulting sequences were classified using the Ribosomal DatabaseProject (RDP, release 10) and assigned a taxonomic classification ifmatched with at least 80% similarity [34]. In light of evidence thatBLAST could achieve an accuracy of essentially 100% while RDP canhave an error rate of up to 20% for certain regions and read lengths[33], we then used the top result of a BLAST search referencing all2358 bacterial, archeal, and fungal genomes (using an E-value ofe�20) to classify all reads that were not classified by RDP.

2.4. Statistical analysis

Because the post-treatment pellets from two mice yieldeda small number of clone sequences, data from these two mice wereexcluded from statistical analysis. Statistical analysis was carriedout on a total of 20 pellets from 10 mice, 4 in the n-6 PUFA groupand 3 each in the n-3 PUFA and SFA groups. Sequence lengths,number of sequences, and percentage of clones belonging tophylum Bacteroidetes or Firmicutes were calculated for each pellet;median values and inter-quartile ranges (IQRs) are given whendistributions were found to be non-normal. Multivariate nominal

logistic models were created in JMP 7.0 (SAS) to assess changes inbacterial profile at the phylum and family levels, with the clone asthe basic unit. Four core variables were initially created: mouse ID(allows for variation among individual mice), treatment (SFA, n-3PUFA, n-6 PUFA), time (pre- or post-diet treatment), and clonephylum. Clone phylum classifies each clone into one of three phyla:B (Bacteroidetes), F (Firmicutes), or U (either could not be classifiedat the phylum level, or belongs to neither Bacteroidetes nor Fir-micutes). “U” sequences were excluded from regression analysisbecause they represented a small minority of all clones (8.3%). Tomeasure shifts in gut bacterial profile at the phylum level over time,a model was created with clone phylum as the output and treat-ment, mouse ID nested within treatment, and time as potentialpredictors. Three interaction variablesdtime � SFA, time � n-3PUFA, and time � n-6 PUFAdwere designed to affect the modelonly if a clone was both in the post time period and belonged to thetreatment of interest. By adding pairs of these interaction variablesto the model, it was possible to discern the effect of each individualtreatment.

To assess shifts at the family level, two additional multivariatenominal regression models were created to look at families withinBacteroidetes and Firmicutes separately. For analysis of Bacter-oidetes clones, Bacteroidetes clones were classified as belonging toeither the Porphyromonadaceae or a non-Porphyromonadaceaefamily, and Firmicutes clones were excluded from analysis. Foranalysis of Firmicutes clones, Firmicutes clones were classified asbelonging to either the Lachnospiraceae or a non-Lachnospiraceaefamily, and Bacteroidetes clones were excluded from analysis. Thesame potential predictors were used as in the phylum level modeldescribed above.

Masses of mice were measured at the beginning and at the endof the 14-week treatment period. Repeated measures ANOVA wasused to assess the effect of time, diet, and interaction between timeand diet on paired before- and after-treatment masses.

3. Results

A total of 1454 sequences were successfully uploaded to RDP,representing 20 pellets from 10 mice. Pellets contained a median of75 sequences (IQR 55e83) each. Sequences were on average 502 bplong (range 301e892). Eighty-eight of the 198 sequences that couldnot be classified at the phylum level with RDP were classified usingBLAST alignment. A median 95.3% (IQR 90.5%e97.6%) of all alignedsequences for a pellet belonged to phylum Bacteroidetes orphylum Firmicutes. The remaining aligned sequences representedthe following phyla: Proteobacteria (1.4%), Actinobacteria (0.5%),TM7 (0.1%), Verrucomicrobia (0.1%), Crenarchaea (0.1%),Deferribacteres (0.1%), and Spirochaeta (0.1%); 6.6% of sequencescould not be classified at the phylum level, and 16.1% of sequencescould not be classified at the family level (Table 2).

Over the course of the 14-week treatment period, all micecollectively experienced a significant decrease in odds ofBacteroidetes-to-Firmicutes by 43.7% (16.1% decrease in proportionof Bacteroidetes). Adding the treatment � time interaction vari-ables showed that the SFA-rich diet significantly reduced theproportion of Bacteroidetes by 28% (P < 0.0001); the n-6 PUFA-richsignificantly reduced the proportion of Bacteroidetes by 12%(P ¼ 0.0015); and the n-3 PUFA-rich diet significantly reduced theproportion of Bacteroidetes by 10% (P ¼ 0.0442; Table 3, Fig. 1).While the effect of the SFA-rich diet differed significantly from thatof either PUFA-rich diet (P ¼ 0.0002), the effects of the two PUFA-rich diets did not differ significantly from each other (P ¼ 0.4317).

Within phylum Bacteroidetes, 74.9% of clones belonged to familyPorphyromonadaceae. Of the three diet treatments, only mice fedthen-6 PUFA-richdiet exhibited significantlydecreasedproportions

Table 2Classification of 16S rRNA sequences from mouse fecal pellets before and after 14-week diet treatment.

Diet Mouse ID Total # sequences Pre-treatmenta,b Post-treatmentb

Pre Post B (%) F (%) O (%) U (%) B (%) F (%) O (%) U (%)

SFA-rich 3 95 28 33 (34.7) 61 (64.2) 0 (0.0) 1 (1.1) 10 (35.7) 17 (60.7) 0 (0.0) 1 (3.6)10 86 84 48 (55.8) 35 (40.7) 1 (1.2) 2 (2.3) 11 (13.1) 51 (60.7) 18 (21.4) 4 (4.8)19 88 78 70 (79.5) 14 (15.9) 0 (0.0) 4 (4.5) 35 (44.9) 35 (44.9) 2 (2.6) 6 (7.7)

n-3 PUFA-rich 5 85 26 37 (43.5) 46 (54.1) 0 (0.0) 2 (2.4) 22 (84.6) 3 (11.5) 1 (3.8) 0 (0.0)13 84 86 74 (88.1) 6 (7.1) 0 (0.0) 4 (4.8) 48 (55.8) 32 (37.2) 1 (1.2) 5 (5.8)15 76 81 59 (77.6) 17 (22.4) 0 (0.0) 0 (0.0) 47 (58.0) 24 (29.6) 2 (2.5) 8 (9.8)

n-6 PUFA-rich 1 75 39 48 (64.0) 19 (25.3) 1 (1.3) 7 (9.3) 31 (79.5) 7 (17.9) 1 (2.6) 0 (0.0)2 76 33 20 (26.3) 25 (32.9) 3 (3.9) 27 (36.8) 14 (42.4) 19 (57.6) 0 (0.0) 0 (0.0)4 95 81 84 (88.4) 4 (4.2) 3 (3.2) 4 (4.2) 68 (84.0) 8 (9.9) 0 (0.0) 5 (6.1)8 87 88 73 (83.9) 8 (9.2) 0 (0.0) 6 (6.9) 49 (55.7) 37 (42.0) 1 (1.1) 1 (1.1)

a Until the beginning of diet treatment, mice were fed standard rodent chow (6.5% fat).b Sequences classified as Bacteroidetes (B), Firmicutes (F), other phyla (O) or unidentified (U).

T. Liu et al. / Anaerobe 18 (2012) 331e337334

of Porphyromonadaceae relative to pre-treatment levels (19%decrease, P ¼ 0.0001; Table 4).

Within phylum Firmicutes, 51.3% of clones belonged to familyLachnospiraceae. Of the three diet treatments, only mice fed theSFA-rich diet exhibited significantly decreased proportions ofLachnospiraceae relative to pre-treatment levels (22% decrease,P ¼ 0.0041; Table 5).

Because two of the 12 mice yielded comparatively fewer clonesin the post-treatment pellet, they were excluded from regressionanalysis. Including them in the analysis, however, did not changethe result that SFA-rich and PUFA-rich diets have distinct effects ongut microbiota shifts, nor does it alter the direction or significanceof the family level analyses.

Although body mass increased significantly from pre-treatmentto post-treatment (rANOVA, P < 0.0001), these changes did notdiffer significantly among the three treatment groups (rANOVA,P ¼ 0.84), and there was no significant interaction between timeand diet (rANOVA, P ¼ 0.12; Table 6).

4. Discussion

The primary novel finding of the present study is that SFAs andPUFAs have distinct effects on gut microbiota: the SFA-rich dietresulted in significantly greater decreases in Bacteroidetes-to-Firmicutes ratio than did either of the PUFA-rich diets. A previousstudy performed in our laboratory [35] examined the effects oftemperature on choice between simultaneously presented SFA-richand n-6 PUFA-rich diets. In contrast to the present results, theprevious study showed that even when CD-1 mice ingestedsignificantly different amounts of the two simultaneously pre-sented diets, proportions of gut Bacteroidetes and Firmicutes didnot differ significantly between diets or shift significantly overa period of 4 weeks. A longer treatment period (14 weeks) and lackof diet choice may have contributed to the significant differencesfound in the present study. Our findings show that in addition tothe percentage of fat in diets [7e9], the type of fat can also

Table 3Regression of log-odds of Bacteroidetes-to-Firmicutes sequences against diet typeover time.

Treatment Beta S.E. P value Proportion of Bacteroidetesb

Pre-treatment

Post-treatment

Changeover time

SFA �1.316 0.237 <0.001a 59% 31% �28%PUFA n-3 �0.458 0.229 0.045a 71% 62% �10%PUFA n-6 �0.711 0.226 0.002a 76% 64% �12%

a Indicates statistical significance.b Proportions were derived by averaging the proportions predicted by the

multivariate regression model.

influence gut microbiota. Two previous studies [8,9] had found thatmice fed a fat-enriched diet (40.6e45% calories from fat) experi-enced significant decreases in Bacteroidetes-to-Firmicutes ratioscompared with mice fed standard chow, concurrent with changesin microbiome gene content and expression. Because the fat-enriched diets (Harlan-Teklad TD.96132; Research Diets D12451)used in those studies were composed of much higher levels of SFAsthan PUFAs (SFA:PUFA ratio of 5.6, compared with 0.1e0.2 in ourPUFA-rich diets), it would be interesting to investigate whether

Fig. 1. Proportions of Bacteroidetes (B), Firmicutes (F), and other/unclassified (U)sequences in fecal pellets of CD-1 mice by treatment group, before (a) and after (b) the14-week diet treatment period.

Table 6Change in body mass of mice in each diet group over 14 weeks.

Treatmentgroup

Before-treatmentbody mass (g)

After-treatmentbody mass (g)

Change inbody mass (g)

SFA 37.3 � 0.6 45.7 � 1.2 8.5 � 1.0n-3 PUFA 36.1 � 0.6 47.8 � 1.5 11.6 � 1.3n-6 PUFA 36.9 � 0.6 47.5 � 1.4 10.8 � 1.0P value 0.39a 0.63b 0.18b

a rANOVA.b Kruskal-Wallis.

Table 4Regression of log-odds of Porphyromonadaceae to non-Porphyromonadaceaesequences within Bacteroidetes clones against diet type over time.

Treatment Beta S.E. P value Proportion of Porphyromonadaceaeb

Pre-treatment

Post-treatment

Changeover time

SFA �0.264 0.366 0.471 77% 72% �5%PUFA n-3 �0.173 0.294 0.556 78% 75% �3%PUFA n-6 �0.923 0.243 <0.001a 77% 58% �19%a

a Indicates statistical significance.b Proportions were derived by averaging the proportions predicted by the

multivariate regression model.

T. Liu et al. / Anaerobe 18 (2012) 331e337 335

those studies would yield less dramatic decreases in Bacteroidetes-to-Firmicutes ratio if repeated with PUFA-rich diets. Furthermore,those studies observed significant shifts in gene content andexpression in response to fat-enriched diets, and it would beinteresting to investigate whether PUFA-rich diets would yielddifferent outcomes.

Conceptually, host diet could alter gut microbial profile byimproving the competitive advantage of certain species, killing orinhibiting the growth of other species, or a combination of both.Since fatty acids have been shown to exhibit antibacterial effectsin vitro, the selective antibacterial effects of different fatty acidsmay be contributing to the differential effects of SFAs and PUFAs ongut bacterial profiles. Several mechanisms have been proposed forthe antibacterial effects of free fatty acids, including the followingprocesses in bacterial cells: incorporation into membranes[13,36,37], interference with enzyme function [38] or nutrientuptake [39], and production of reactive oxygen degradationbyproducts [40]. PUFAsdespecially cis PUFAsegenerally have morepotent antibacterial effects than SFAs [25] and exhibit selectiveantibacterial effects in vitro. Specifically, PUFAs exert strongerantibacterial effects on Gram-positive than on Gram-negativestrains [25,41]. In addition, PUFAs have been shown to inhibit thegrowth of obligate anaerobes but not facultative anaerobes [13,26].The majority of Firmicutes species found in the murine gut belongto the class Clostridia [6], all of which are obligate anaerobes; thus,the tendency for PUFAs to selectively inhibit the Gram-positive,obligately anaerobic Firmicutes species may explain why mice fedeither PUFA-rich diet experienced smaller decreases inBacteroidetes-to-Firmicutes ratio than mice fed the SFA-rich diet.Another mechanism by which dietary fatty acids could modulategut bacteria communities is by increasing production of bile acids,which have been shown to exhibit antimicrobial properties [42].PUFA-rich diets have been associated with increased bile acidproduction compared with SFA-rich diets in rats [43], but themetabolism of conjugated bile acids by bile salt hydrolases isa conserved, adaptive function found across all bacterial divisions[42]. Thus, further research is needed to elucidate whether differ-ential resistance to bile acids contributes to the differential effectsof SFAs and PUFAs on gut bacterial profiles.

Table 5Regression of log-odds of Lachnospiraceae to non-Lachnospiraceae sequenceswithin Firmicutes clones against diet type over time.

Treatment Beta S.E. P value Proportion of Lachnospiraceaeb

Pre-treatment

Post-treatment

Changeover time

SFA �1.001 0.355 0.005a 57% 35% �22%PUFA n-3 0.309 0.529 0.558 57% 64% þ7%PUFA n-6 �0.210 0.427 0.623 58% 53% �5%

a Indicates statistical significance.b Proportions were derived by averaging the proportions predicted by the

multivariate regression model.

In our study, the microbial profiles of outbred mice experiencedsignificant decreases in Bacteroidetes-to-Firmicutes ratio after 14weeks of consuming fat-enriched treatment diets (32.5% energyfrom fat) in comparison with pre-treatment levels, when the micehad been eating standard chow without fat enrichment. Thisfinding is consistent with those of previous studies [8,9] that usedfat-enriched diets (40.6e45% calories from fat). Because we did notinclude a group of mice maintained on standard chow throughoutthe 14-week experimental period, however, we cannot eliminatethe possibility that uncontrolled time-dependent factors contrib-uted to the decline in Bacteroidetes-to-Firmicutes ratio in alltreatment groups over time in our experiment.

We tested the effects of linolenic acid and linoleic acid asrepresentatives of n-3 and n-6 PUFAs, respectively, in order to keepfatty acid chain length consistent among the three treatmentgroups. However, there is evidence that long chain PUFAs, espe-cially EPA (20:5n-3), DHA, (22:6n-3), and arachidonic acid (AA,20:4n-6), have stronger effects on the physiology of the host thanthe shorter-chain n-6 and n-3 PUFAs that dominated our experi-mental diets. For example, a-linolenic acid (18:3n-3) acid is onlyabout one-ninth as potent as an EPA-DHA mixture in decreasinghost cytokine production [22]. Arachidonic acid has been impli-cated in a number of physiological changes [21], but excess dietarylinoleic acid may not substantially alter tissue AA content becausethe conversion process is tightly regulated [44,45]. The effects oflong chain PUFAs (e.g., EPA, DHA and AA) on the gut microbiotain vivo have yet to be studied, and further investigation mayuncover different effects based on chain length as well as on thetwo factors investigated in the present study, level of unsaturationand position of the terminal double bond (n-3 vs n-6).

Interestingly, 16S rRNA analysis of pellets from our mice fedstandard chow revealed Bacteroidetes and Firmicutes proportionsthat differ from proportions found by other laboratories. Gordonet al. [6] determined that the gut microbiota of non-obese adultC57BL/6 mice are composed of 20e40% Bacteroidetes and 60e80%Firmicutes. In contrast, we observed an average of 56% Bacter-oidetes and 31% Firmicutes in each of our pre-treatment CD-1 mice.These differences may be due to a variety of factors, includingmouse strain, age, housing environment, diet composition, ampli-fication methods [46], or other factors. In a previous study con-ducted with CD-1 mice in our laboratory, we observed a similarprominence of Bacteroidetes, suggesting that factors related to ourchoice of an outbred mouse strain may have contributed to theunexpected proportions. It should be noted that the generally lownumbers of Actinobacteria may be due to incomplete lysis [47],although a study of human gut microbiota using bead beatingrevealed similarly low levels of Actinobacteria in the majority ofsamples studied [48]. Given that Actinobacteria levels have beenfound to be elevated in obese individuals [48] and in mice ona high-fat diet [9], further investigations could reveal whetherActinobacteria levels are influenced by type of dietary fat.

While most studies have observed significant shifts at thephylum level in response to diet [8e10], it is also possible thatsignificant shifts are occurring at taxonomic levels below the

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phylum level. Bice et al. [35] showed that mice fed an SFA-rich dietfor four weeks experienced an increase in gut bacteria belonging tofamily Lachnospiraceae, which have been implicated in the bio-hydrogenation of unsaturated fatty acids [49]. Surprisingly, in thepresent study, mice fed either PUFA-rich diet did not experiencesignificant shifts in proportion of Lachnospiraceae, whereas micefed the SFA-rich diet did experience a significant decrease inproportion of Lachnospiraceae. Maia et al. [36] note that bio-hydrogenation is a detoxification process necessary to escape thebacteriostatic effects of PUFAs, and that PUFAs are more toxic tobacteria that cannot biohydrogenate. Thus Lachnospiraceae mayhave a competitive advantage over non-hydrogenating bacteria inhosts fed a PUFA-rich diet, providing a possible explanation for ourfindings. Our study also showed significant decreases in theproportion of Porphyromonadaceae species in mice fed the n-6PUFA-rich diet relative to mice in the other diet groups. Porphyr-omonadaceae is a family of Gram-negative obligate anaerobes [50],which PUFAs have been shown to selectively inhibit [26]. However,this explanation fails to account for the fact that mice fed the n-3PUFA-rich diet did not experience similar decreases. Given thatreduced levels of Porphyromonadaceae in response to metronida-zole treatment of C57BL/6 mice have been associated withincreased levels of pathogenic bacteria [51] and increased intestinalinflammation [52], further studies are needed to understand whyn-3 and n-6 PUFAs might affect Porphyromonadaceae speciesdifferently.

In summary, mice in the three diet treatment groups experi-enced significantly different shifts in gut microbial composition,with mice fed the SFA-rich diet acquiring a gut microbial profilemore typical of obese animals [7,10,53]. Furthermore, after 14weeks of diet treatment in the present study, diet type had affectedshifts in gut microbial populations independently of changes inbody mass. Several reviews [54e56] have explored the role of gutmicrobiota in the development of conditions such as obesity anddiabetes, and further studies may clarify whether the changing gutmicrobiota is a causal factor in subsequent alterations of body massand composition.

Acknowledgments

We would like to thank John Kelly for maintenance of envi-ronmental chambers; Gwen Kannapel for maintenance of labora-tory equipment; Dr. Barbara Mickelson at Harlan-Teklad forassistance in preparing test diets; Archer Daniels Midland fordonating fully saturated soybean oil; Nancy Berner for assistancewith fatty acid analysis; Tami Gura for animal care; Philip Eversonand Steve Wang for assistance with statistical analysis; Frank Chienfor assistance with sequence analysis; Liz Vallen for technicalguidance; Natasha Weiser for collegial support in the laboratory;Michael Mahowald for suggesting ways in which dietary fatty acidmight influence gut microbiota; and Paul Else for encouraging us tobegin this investigation. We gratefully acknowledge support fromthe Howard Hughes Medical Institute to Swarthmore College.

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