Oral Administration of Lactobacillus fermentum I5007 Favors Intestinal

Article pubs.acs.org/JAFC

Oral Administration of Lactobacillus fermentum I5007 Favors Intestinal Development and Alters the Intestinal Microbiota in Formula-Fed Piglets Hong Liu,† Jiang Zhang,† Shihai Zhang,† Fengjuan Yang,† Phil A. Thacker,‡ Guolong Zhang,§ Shiyan Qiao,† and Xi Ma*,† †

State Key Laboratory of Animal Nutrition, China Agricultural University, No. 2, Yuanmingyuan West Road, Beijing 100193, China Department of Animal and Poultry Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, Canada § Department of Animal Science, Oklahoma State University, Stillwater, Oklahoma, United States ‡

ABSTRACT: The present study was conducted to evaluate the effects of early administration of Lactobacillus fermentum I5007 on intestinal development and microbial composition in the gastrointestinal tract using a neonatal piglet model. Full-term 4 day old piglets, fed with milk replacer, were divided into a control group (given placebo of 0.1% peptone water) and a L. fermentum I5007 group (dosed daily with 6 × 109 CFU/mL L. fermentum I5007). The experiment lasted 14 days. On day 14, a significant increase in the jejunum villous height (583 ± 33 vs 526 ± 18) and increases in the concentrations of butyrate (7.55 ± 0.55 vs 5.33 ± 0.39) and branched chain fatty acids in the colonic digesta were observed in piglets in the L. fermentum I5007 treatment (P < 0.05). mRNA expression of IL-1β (1.29 ± 0.29 vs. 0.62 ± 0.07) in the ileum were lower after 14 days of treatment with L. fermentum I5007. Denaturing gradient gel electrophoresis (DGGE) revealed that L. fermentum I5007 affected the colonic microbial communities on day 14 and, in particular, reduced numbers of Clostridium sp. L. fermentum I5007 play a positive role in gut development in neonatal piglets by modulating microbial composition, intestinal development, and immune status. L. fermentum I5007 may be useful as a probiotic for application in neonatal piglets. KEYWORDS: intestinal metabolism, Lactobacillus fermentum I5007, gut microbiota, neonatal piglets

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INTRODUCTION Before birth, the gastrointestinal tract of the fetus is sterile, and during the initiation of the birth process and thereafter, acquisition of microbes is ongoing and time-dependent.1,2 Many factors affect the composition of the microbiota in the intestinal tract before the gut finally gets a dense and stable population as an adult.3 Early colonization with beneficial bacteria could help establish an effective ecosystem and bring about maturity in the physical structure of the gut.4,5 Probiotic and prebiotic administration during the neonatal period can improve gut function by improving the intestinal immune status and maintaining microbial balance during gastrointestinal disturbances.5,6 Although progress has been made in disclosing the mechanisms through which probiotics act on the intestinal immunity and microbial composition of clinical models, knowledge about the interaction between probiotics and intestinal microbial metabolism, as well as microbial composition in healthy animals and humans, is lacking. Lactobacilli are normal inhabitants of the gastrointestinal tract in humans and other mammals, and have been intensively studied during the past decades for their probiotic properties in both clinical and animal models.7−9 Lactobacillus fermentum I5007 was initially isolated in our lab from the colonic mucosa of healthy weaning piglets, identified by the Institute of Microbiology Chinese Academy of Sciences (Beijing, China), and was shown to possess many characteristics of an excellent probiotic, including gastric acid and bile tolerance, high adhesion to CaCo-2 cells, competitive exclusion against © 2014 American Chemical Society

pathogen invasion, and alleviation of weaning stress in piglets.10−12 Our previous study has conferred the optimum dosage (about 1010 CFU/d L. fermentum I5007) for improving growth performance and diarrhea incidence in the neonatal period in piglets (data not published). However, the exact role of these lactobacilli in the intestine during the neonatal period in vivo is still unknown. In contrast to rodent models, the size of the newborn piglet easily allows for clinically relevant nutritional interventions, and the ontogeny and anatomy of the gastrointestinal tract are more similar to those of human infants.13,14 The present study evaluated the effects of an early introduction of an exogenous L. fermentum I5007 strain to gut metabolism and maturation, as well as the indigenous microbiota and their metabolic products, which will determine any implications of L. fermentum I5007 for pediatric nutrition.

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MATERIALS AND METHODS

Chemicals. Rogosa Sharpe medium, Luria−Bertani agar, ampicillin, metaphosphoric acid, and peptone were all purchased from ZSGB-Bio Company (Beijing, China) while Agarose was provided from Gene Company LTD (Hong Kong, China). Skim milk, phosphate buffer saline, ampule, and hematoxylin−eosin were purchased from HuaxingBio Company (Beijing, China). Polyacrylamide gels, Tris-acetateReceived: Revised: Accepted: Published: 860

July 25, 2013 January 2, 2014 January 9, 2014 January 9, 2014 dx.doi.org/10.1021/jf403288r | J. Agric. Food Chem. 2014, 62, 860−866

Journal of Agricultural and Food Chemistry

Article

EDTA buffer, formamide, and urea were obtained from Premedical Lab (Beijing, China). Ethics Statement. The experimental protocol was approved by the China Agriculture University Institutional Animal Care and Use Ethical Committee (Beijing, China). Bacterial Strain, Growth, and Storage Conditions. L. fermentum I5007 was cultured on sterile Man Rogosa Sharpe medium at 37 °C for 24 h in an anaerobic environment, followed by centrifugation at 5000g for 10 min at 4 °C. The cells were resuspended in reconstituted skim milk (20% w/v) and immediately freeze-dried. The freeze-dried powder containing 6 × 109 colony forming units (CFU)/g was stored in sealed packets at a temperature of 4 °C until used. Piglets and Treatment. A total of thirty-six, full-term crossbred (Large White sires × Landrace dams) piglets obtained from four litters were used in this study. The piglets were delivered vaginally at the Langzhong Bei Pig Husbandry Training Farm (Beijing, China) and allowed colostrum for 48 h after birth. After colostrum consumption, the piglets were individually housed in stainless steel pens located over a totally slotted floor in a temperature (33 ± 1 °C) and relative humidity (65−70%) controlled room programmed to deliver a light:dark cycle of 16:8 h. On the third day of life, piglets were trained to suckle from bottles filled with milk replacer (Rosalac Instant, Bonilait Proteins, France, dry matter 89%, lactose 45%, protein 21.5%, fat 18.7%, ash 9.8%, lysine 1.7%; vitamins and minerals meet the nutrient requirements), and the milk replacer was dissolved in warm boiled water (w/v 1:4.5) to provide the same level of nutrients as sowreared piglets. The fresh liquid milk replacer was fed to piglets individually from the feeder six times daily (2, 6, 10, 14, 18, and 22 h). The feeders were cleaned each time before adding the new fresh milk replacer, and the remaining milk was measured. The formula did not contain any antibiotics or other medicine. On day 4 (equivalent to the first day of the experiment), piglets were allocated to one of two treatments (the L. fermentum I5007 group and the control group) balanced for litter of origin and body weight (initial body weight of 2.00 ± 0.31 kg). Each piglet in the L. fermentum I5007 group was orally administered 6 × 109 CFU L. fermentum I5007 dissolved in 3 mL of 0.1% of peptone water once a day for 14 days while the piglets in the control group were given the same volume of 0.1% peptone water. Sample Collection. The health status was recorded for each piglet, and the occurrence of diarrhea was visually assessed each afternoon according to the method of Hart and Dobb.15 Scores were 0 = normal, firm feces; 1 = possible slight diarrhea; 2 = definitely unformed, moderately fluid feces; or 3 = very watery and frothy diarrhea. The occurrence of diarrhea was defined as maintaining score of 3 for one day (monitor time: 10:00 a.m. and 16:00 p.m.). Diarrhea incidence was calculated according to the equation where diarrhea incidence (%) = n (numbers piglets of diarrhea) × days (the diarrhea duration)/n (the total number of experiment piglets) × days (the whole experiment days) × 100%. After being weighed on the morning of the days 7 and 14 of the experiment (equivalent to 11 and 18 days of age), 6 healthy piglets closest to the average body weight balanced for litter of origin from each treatment were euthanized with electricity and their abdominal cavities were opened to remove the gastrointestinal tract. The small intestine was carefully dissected from the mesentery, and 2 cm segments were obtained from the jejunum (the middle of the small intestine). The intestinal segments were placed in neutral formalin for histological analysis. Additional segments 15 cm long, taken from the jejunum, were rinsed with cold 0.01 M sterile PBS (pH 7.4) and blotted dry on filter paper. Jejunal mucosa from the jejunum was obtained by gently scraping the mucosal layer using a glass microscope slide, 0.5 g of mucosal scrapings from each piglet was collected into a sterile Eppendorf tube (Axygen Inc., Union City, California), and 1 mL of 0.01 M PBS containing 2 mg/mL protease inhibitor supplied by the Huaxingbochuang Institute (Beijing, China) was added to each tube. Each sample was centrifuged at 3500g for 10 min at 4 °C after being homogenized (T10, IKA Works, Staufen, Germany). The supernatant was harvested and stored at −20 °C for analysis of

disaccharidase activities. The colonic digesta were used for pH measurements and then gently squeezed into sterile Eppendorf tubes, frozen in liquid nitrogen, and subsequently stored at −80 °C until processing. Two digesta samples per treatment (n = 6) were randomly selected and pooled as three new samples (with equal wet weight) until needed for further genomic DNA isolation. Intestinal Morphology and Enzyme Activities. Samples from jejunal segment were embedded in paraffin and cut into serial sections (5 μm). Five nonsuccessive sections of each sample were selected and stained with hematoxylin−eosin for identification. Five well oriented villi (determined as the distance between the crypt openings and the end of the villi) and their associated crypt (measured from the cryptvillous junction to the base of the crypt) per section were selected and measured under an Olympus Light Microscope (CK-40, Olympus, Tokyo, Japan) at 40× magnification. The means of these measurements were calculated to yield a single value per piglet. These procedures were conducted by an observer unaware of the treatment assignment. Myeloperoxidase (MPO), as an indicator of neutrophil infiltration, was determined in ileal segment on day 14. The activities of the disaccharidase (lactase, maltase, sucrase) and MPO were determined according to assay kit instructions (Nanjingjiancheng, Nanjing, China). One unit of specific MPO activity was defined as that degrading 1 μmol of hydrogen peroxide per min, and data were expressed as units of MPO per gram of tissue. The disaccharidase activities were expressed as units per milligram of protein, and the amount of protein of samples was determined using a commercially available bicinchoninic acid kit (Thermo, Rockford, IL, USA). RNA Isolation and RT-PCR. Total RNA was isolated from the ileal segments using a RNeasy Mini Kit (Qiagen, Hilden, Germany). The purity was determined by the ratio of A260:A280 using a NanoDrop spectrophotometer (P330, Implen, Germany), and then the quality was checked with 1% agarose gel electophoresis. The extracted RNA was converted into first-strand cDNA by reverse transcription of 1 μg of total RNA using a PrimeScript First Strand cDNA Synthesis Kit (Takara, Dalian, China) according to the manufacturer’s protocol and stored at −80 °C. Real-time PCR was performed on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Singapore) using SYBR Green PCR Master Mix (Takara, Dalian, China). The sequences of PCR primer for target genes were as follows: forward 5′-CCTCCTCCCAGGCCTTCTGT-3′, reverse 5′GGGCCAGCCAGCACTAGAGA-3′ for IL-1β;16 forward 5′-AGTTTTCCTGCTTTCTGCAGC T-3′, reverse 5′-TGGCATCGAAGTTCTGCACT-3′ for IL-8;17 forward 5′-ATGGGCGACTTGTTGCTGAC-3′, reverse 5′-CACAGGGCAGAAATTGATGACA-3′ for IL10;18 forward 5′-CCCAAGGACTCAGATCATCG-3′, reverse 5′ATACCCACTCTGCCATTGGA-3′ for TNF-α;16 forward 5′-CATCACCATCGGCAACGA-3′, reverse 5′-GCGTAGAGGTCCTTCCTGATGT-3′ for β-actin.19 Quantification of target mRNA was conducted using a relative standard curve generated by a serial dilution (1:10) covering the appropriate concentration range. The reaction was conducted in a total volume of 10 μL, and the process included predenaturation for 30 s at 95 °C, and then 40 cycles of 5 s at 95 °C, 30 s at 60 °C, and 40 s at 72 °C for the target genes. For the porcine βactin gene, the annealing temperature was set at 58 °C. The relative gene expression was determined for the two treatments on day 14. All reactions were performed in triplicate. The mRNA expression is expressed as the ratio between the target gene and β-actin gene expression. Colonic Short Chain Fatty Acid Concentrations and pH Value. The pH in the colonic digesta was determined for each sample, and the concentrations of short chain fatty acids were determined by a gas chromatographic method following the procedures of Franklin et al.20 with modification. About 1.5 g of thawed digesta was suspended in 1.5 mL of distilled water in a screw-capped tube (Axygen Inc., Union City, CA). The entire sample was centrifuged at 15000g at 4 °C for 10 min. After that, 1 mL of suspernatant was transferred into an ampule and mixed with 200 μL of metaphosphoric acid (HPO3). The ampules were placed in an ice bath for 30 min and then centrifuged again for 10 min. The sample was injected into a HP 6890 series gas chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a HP 861

dx.doi.org/10.1021/jf403288r | J. Agric. Food Chem. 2014, 62, 860−866


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19091N-213 column 30.0 m × 0.32 mm i.d. (Agilent, Palo Alto, CA. The injector and detector temperatures were 185 and 210 °C, respectively. DNA Isolation, PCR-Denaturing Gradient Gel Electrophoresis (DGGE), and Identification of Fragments Extracted from the Gels. The extraction of DNA from colonic digesta for denaturing gradient gel electrophoresis was performed using a QIAamp Stool Mini-Kit (Qiagen, Hilden, Germany). The concentration of extracted DNA was measured using a NanoDrop spectrophotometer (P330, Implen, Germany). The bacterial universal V3 region of the 16S rDNA gene was amplified according to Muyzer et al.21 The PCR products were analyzed by electrophoresis on 1.2% (w/v) agarose gel in SYBR Green I (Sigma, St. Louis, MO) to verify that a single product of the expected size was obtained. DGGE was performed for separation of PCR amplicons in 8% (w/ v) polyacrylamide gels with a vertical gradient of denaturants between 35 and 60% (100% denaturing solution was defined as 40% (v/v) formamide and 7 M urea). For each sample, the same concentration of DNA was loaded to one lane of the gel. Electrophoresis was initiated by prerunning for 5 min at 200 V and then for 16 h at 85 V in 0.5× Tris-acetate-EDTA buffer at a constant temperature of 60 °C in a DCode System Apparatus (Bio-Rad, Hercules, CA). The DGGE gel was stained by SYBR green I (Sigma, St. Louis, MO) for 15 min at room temperature, photographed with a Gel Doc XR+ System (BioRad, Hercules, CA), and analyzed with Quantity One Basic 4.6.6 Software for Windows (Bio-Rad, Hercules, CA). Interesting bands in the DGGE profiles were excised with a sterile needle, reamplified, and purified with a Takara Mini Best DNA Fragment Purification Kit (Takara, Dalian, China) according to the manufacturer’s protocol. The PCR products were cloned into the vector pMD-19T according to the manufacturer’s instructions and transformed into Escherichia coli DH5α cells via chemical transformation. The transformants were grown on Luria−Bertani agar in the presence of 100 μg/mL ampicillin. The positive recombinants were screened and identified by PCR and sent for sequencing. Homology searches were performed using the BLAST program in the NCBI database (http://blast.ncbi.nlm.nih.gov/). Statistical Analysis. Statistical analyses were performed using the Analysis of Variance (ANOVA) procedures of the Statistical Analysis Systems Statistical Software Package Version 8.02 (SAS Institute, Cary, NC). Each piglet served as an experimental unit. Differences at P < 0.05 were considered significant, and P < 0.1 was considered to be indicative of a trend.

(1.19 vs 2.98%). During the second week, two piglets in the control treatment died on days 10 and 13 separately due to serious diarrhea. Maturation of the intestine was assessed by changes in intestinal morphology and the activity ofthe disaccharidases, lactase, sucrase, and maltase in the jejunum (Table 2). In Table 2. Jejunum Structure and Enzyme Activities of L. fermentum I5007 Treated Piglets and the Controla items villus height (μm) crypt depth (μm) lactase (U/mg protein) maltase (U/mg protein) sucrase (U/mg protein) villus height (μm) crypt depth (μm) lactase (U/mg of protein) maltase (U/mg of protein) sucrase (U/mg of protein) MPO (U/g of tissue)

control

L. fermentum I5007 2.01 ± 0.08 2.78 ± 0.08 4.60 ± 0.13

97 ± 5 196 ± 23 b 148 ± 13 b 2.98

110 ± 5 250 ± 10 a 184 ± 6 a 1.19

583 ± 33 a 172 ± 23 185 ± 19 104 ± 11 83 ± 16 0.23 ± 0.01

jejunum, villous height on day 14 was higher in L. fermentum I5007 piglets compared with the control piglets (P < 0.05), while disaccharidase activities on either day 7 or 14 did not differ due to treatment as well as the MPO activity in ileal segment on day 14. Digesta Metabolites. Table 3 shows the concentrations of short chain fatty acids and pH values determined in the colonic digesta. Short chain fatty acid concentrations were not different between the two treatments measured on day 7. However, on day 14, the concentrations of branched chain fatty acids (isobutyric acid and isovaleric acid) and butyric acid were Table 3. pH Values and Short Chain Fatty Acid Concentrations in Colonic Digesta Obtained from the Control and L. fermentum I5007 Treated Pigletsa items

control

pH acetic acid (mmol/kg) propionic acid (mmol/kg) butyric acid (mmol/kg) pentanoic acid (mmol/kg) isobutyric acid (mmol/kg) isovaleric acid (mmol/kg)

Table 1. Effects of L. fermentum I5007 on Piglet Growth Performance and Diarrhea Incidencea 1.99 ± 0.09 2.67 ± 0.09 4.11 ± 0.24

436 ± 43 143 ± 12 211 ± 13 140 ± 22 92 ± 15

All data are means ± SEM. Means within the same row that have no common letters are significantly different (p < 0.05); n = 6.

RESULTS Performance, Jejunum Morphology, and Enzyme Activities. The average daily weight gain during days 8 to 14 and the whole experiment period was 26.9% and 24.3% higher for piglets in the L. fermentum I5007 treatment compared with the control (P < 0.05, Table 1). Diarrhea incidence was lower in piglets fed L. fermentum I5007 compared with the control

items

L. fermentum I5007

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initial body wt (kg) body wt at day 7 (kg) body wt at day 14 (kg) wt gain (g/day) 1−7 days 8−14 days 1−14 days diarrhea incidence (%)

control Day 7 431 ± 31 151 ± 32 172 ± 20 96 ± 17 100 ± 15 Day 14 526 ± 18 b 148 ± 11 190 ± 22 87 ± 4 122 ± 17 0.24 ± 0.02

pH acetic acid (mmol/kg) propionic acid (mmol/kg) butyric acid (mmol/kg) pentanoic acid (mmol/kg) isobutyric acid (mmol/kg) isovaleric acid (mmol/kg)

All data are means ± SEM. Means within the same row that have no common letters are significantly different (p < 0.05); n = 10, the control group; n = 12, L. fermentum I5007 group respectively. a

Day 7 6.46 ± 0.14 27.03 ± 3.68 6.73 ± 0.96 5.62 ± 1.11 2.32 ± 0.39 0.87 ± 0.27 1.10 ± 0.36 Day 14 6.41 ± 0.05 40.00 ± 4.47 15.08 ± 1.54 5.33 ± 0.39 b 2.51 ± 0.38 0.63 ± 0.11 b 1.32 ± 0.13 b

L. fermentum I5007 6.37 ± 0.07 26.19 ± 2.82 7.13 ± 1.23 4.99 ± 0.17 2.16 ± 0.91 0.72 ± 0.21 1.19 ± 0.24 6.27 ± 0.05 45.91 ± 4.90 14.14 ± 1.51 7.55 ± 0.55 a 2.32 ± 0.33 0.95 ± 0.08 a 1.82 ± 0.16 a


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higher in piglets fed L. fermentum I5007 than the control treatment (P < 0.05). Piglets treated with the L. fermentum I5007 tended to exhibited a lower pH (P = 0.06) on day 14 (6.27 vs 6.41). Cytokine Expression. Both inflammatory and antiinflammatory cytokines in the intestinal tissue were measured at the end of the experiment to assess the effects of feeding L. fermentum I5007 on modulations of the immune response (Figure 1). The expression of the inflammatory cytokine IL-1β

sequence analysis. The sequences of excised DGGE bands are available in the EMBL database under accession numbers HF559182−HF559188. These are summarized in Table 5. On Table 5. Clone Relatives to Sequences Retrieved from the DGGE bands of Colonic Digesta band no.

closest relative in GenBank

% similarity

1 2 3 4 5 6, 8 7

Clostridium clostridioforme Clostridium symbiosum Clostridium sp. Clostridium sp. uncultured Clostridiales bacterium Latobacillus fermentum I5007 uncultured bacterium

100 100 100 100 99 100 98

day 7, high individual variation was observed compared with the profile shown on day 14 and the microbial populations became more similar within groups over time. Different bands between two treatments on day 14 were excised from the profile, and a relative decrease of the Clostridium-like phenotype was observed in the L. fermentum I5007 group. The L. fermentum I5007 strain could only be detected in the L. fermentum I5007 group (99% indentity with pure L. fermentum I5007 strain) and was not present in control piglets on either day 7 or 14 (bands 6, 8, Figure 2).

Figure 1. mRNA expression of relevant cytokines in ileal segments. Effects of the L. fermentum I5007 on IL-1β, IL-8, IL-10, and TNF-α mRNA expression in the ileal segment of piglets on day 14. Data are means ± SEM (n = 6). Significant differences between control and L. fermentum I5007 treated piglets: * p < 0.05.

mRNA was observed to be lower in piglets in the L. fermentum I5007 treatment compared with the control treatment (P = 0.05) on day 14. The expression of the anti-inflammatory cytokine IL-10 also tended to be lower in L. fermentum I5007 treated piglets (P = 0.09), while IL-8 and TNF-α mRNA expression did not differ between the two treatments (Figure 1). Colon Microbiota Profiles Generated by PCR-DGGE and Sequence Analysis of DGGE Bands. Analysis of the DGGE fingerprints revealed no significant differences between the treatments in band numbers on day 7 or 14 of the experiment (Table 4). Simpson’s index of diversity and Dice’s Table 4. Effects of L. fermentum I5007 on the Microbial Communities in the Colon of Piglets as Indicated by the Number of Bands, Simpson’s Index of Diversity, and Dice’s Coefficient of Similarity, as Calculated from the DGGEa items number of bands Simpson’s index of diversity Dice’s coeff of similarity number of bands Simpson’s index of diversity Dice’s coeff of similarity

control Day 7 12.33 ± 1.76 1.04 ± 0.06 31.57 ± 6.67 Day 14 16.67 ± 0.67 1.19 ± 0.02 b 43.67 ± 2.84 b

L. fermentum I5007 16.67 ± 2.33 1.47 ± 0.07 38.47 ± 3.24

Figure 2. Bacterial diversity of the colonic microbiota of L. fermentum I5007 treated piglets and the control. DGGE fingerprints of PCR products of the V3 region of 16S rDNA from colonic digesta from piglets. Arrows (1 to 8) indicate excised bands that were different between the treated group and the control, followed by being reamplified (see Table 5); n = 3.

17.33 ± 0.33 1.27 ± 0.01 a 55.13 ± 1.54 a


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DISCUSSION In the present study, neonatal piglets were selected as the model and the effects of L. fermentum I5007 were evaluated during a two-week intervention period. A significant increase in villous height in the jejunum was observed on day 14, indicating a certain rapid maturation of the intestines. The increase in villous height demonstrated improved architecture

coefficient of similarity were not different on day 7. In contrast, Simpson’s index of diversity and Dice’s coefficient of similarity were increased in piglets in the L. fermentum I5007 treatment compared with the control treatment on day 14 (P < 0.05 Table 4). The bands which differed between the two groups were excised from the gels and their species identified by 863



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infection, and the reduction of these opportunistic putrefactive bacteria has been suspected of reducing the probability of pathophysiology disorders.38,42 Thus, it is reasonable to assume that the growth performance and intestinal health were also improved via impact on intestinal metabolism. However, the exact role and its pathway of the branched chain fatty acids are still poorly understood. The microbial ecosystem in the colon is complex and sophisticated.33 L. fermentum I5007 administration increased Simpson’s index of diversity measured using DGGE analysis. This revealed that the population got more complex and diverse by supplementation of the L. fermentum I5007 strain. A more diverse population could allow a more stable ecology when facing environmental disruptions.43,44 This may be the potential reason for the low incidence of diarrhea in the L. fermentum I5007 treatment. In addition, the increase in Dice’s coefficient of similarity within the L. fermentum I5007 treatment in the second week indicated that the microbial profile within this group tended to be more uniform. However, the whole profile revealed that administered L. fermentum I5007 could only make a minor change in the total microbial population. These findings were supported by the work conducted by Fuentes et al.45 and Rauch and Lynch,6 both of whom observed that administration of lactobacilli could shape the gastrointestinal niche and facilitate colonization by a diversity of beneficial bacterial species, but not alter the overall bacteria structure. After L. fermentum I5007 was introduced, no statistical differences were observed between the two groups for any parameters measured during the first week. We speculated that it would take more than one week for the introduction of the exogenous strain to fully interact with the bacterial community for the newborn piglets. The development of the gut microbiota after intervention should be regarded as a gradual and complex process, not only as a succession in the ecological sense but influenced by microbial and host interactions, including strain adhesion, colonization, and proliferation as well as bacterial-host communication.3,46 In addition, due to the immature gut in neonates, the intestinal environment may have undergone turbulence and high dynamics while establishing a new and steady profile. Here the results showed that the profiles were more uniform on day 14 compared with day 7, and the microbiota and concentrations of metabolic products in piglets in the L. fermentum I5007 treatment were significantly different from those in the control on day 14. Furthermore, L. fermentum I5007 was found in the treated group on days 7 and 14 in colonic digesta, which demonstrated that L. fermentum I5007 can tolerate the gastric acid and bile from the small intestine and inhabit the lower gastrointestinal tract. The above-mentioned findings suggest that L. fermentum I5007 exerts its function promptly after initial introduction, but the significant changes in microbial composition and metabolism were time dependent and occur during the following week. In conclusion, our study indicated that after early introduction of L. fermentum I5007, the structure of the neonatal piglets’ microbial composition and their metabolism as well as immune status could be manipulated, while promoting communities more resilient and putatively beneficial to gut homeostasis. The effects are exerted at an early stage following introduction, which suggests that L. fermentum I5007 could be used as a probiotic for application in the neonatal period in piglets. As for the limitations of the study, in our opinion, increased amount of experimental animals will help to reduce

and active sites between the luminal digesta and the epithelium, which might result in an increased digestive and absorptive ability.22 Siggers et al.5 observed that a combination of probiotics supplemented into neonate formulas could increase the villous height. This result was also supported by the work conducted by Wang et al.,12 which revealed that L. fermentum I5007 enhanced the level of mucosal proteins involved in energy metabolism, cell structure, and mobility in the jejunum in weaning piglets. The jejunum is the main organ for nutrient absorption, and therefore it is reasonable that the beneficial effects of L. fermentum I5007 could be mediated via improving the physiological structure of the jejunum. In the present study, lactase, maltase, and sucrase activities were not affected by L. fermentum I5007. Siggers et al.5 also observed that a combination of probiotics supplemented into neonate formulas did not increase disaccharidase activities compared with the control. Our findings show that intervening with desirable microbes at an early age and shifting the microbiota toward a more beneficial composition is a good strategy to maintain a healthy gut and impact later succession of microbiota.23,24 To further illuminate whether the effects of L. fermentum I5007 were mediated through immune regulation, both proinflammatory and anti-inflammatory cytokine mRNA abundance were determined in ileum. IL-1β, mostly considered as a sensitive pro-inflammatory cytokine as the occurrence of host lesions,25 was observed to have lower mRNA expression after 14 days of L. fermentum I5007 administration compared with piglets in the control group. However, myeloperoxidase, which has been considered related to inflammatory response, was not observed significantly different between the two treatments, which indicated that L. fermentum I5007 introduction did not irrigate disturbance in intestine. IL-10 is associated with TH2 cell or Treg cell stimulation and usually is used as a marker for anti-inflammatory effects,26 and it tended to be lower in the L. fermentum I5007 treatment, which was in accordance with the IL-1β expression, while IL-8 and TNF-α expression were not changed. Thus, the beneficial effects of exogenous strain of L. fermentum I5007 could be at least partly mediated via effects on immune modulation. In addition, our results were strengthened by some recent research related to the pathogen invasion in piglets that have been challenged.27,28 Short chain fatty acids are the major end products from the fermentation processes of gut microbiota in the large intestine and play an important role in the maintenance of colonic function such as improving barrier function, inhibiting adherence of pathogens, and contributing to energy utilization.29−32 It is known that changes in the concentration and proportion of individual short chain fatty acids are consistent with changes in bacterial populations.33−37 Our results showed that oral administration of L. fermentum I5007 not only increased the concentration of butyrate and the branched chain fatty acids but also decreased Clostridium strains accompanied by a lowered pH in the colonic digesta, which were consistent with the findings conducted by Hu et al.,35 who found that increase in butyrate-producing bacteria or probiotics as L. fermentum can be related to the reduction of Clostridiales sp. Butyrate has been proved associated with positive effects on pathogen control and gut barrier function,38,39 especially colonic energy utilization, cell proliferation in the gastrointestinal tract, and stabilization of the luminal pH in rodent animals.36,38−41 Recently, via in vivo experiments, it also has been reported that butyrate is capable of reducing the incidence of Clostridium spp. and coliform induced opportunistic 864



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the impact of individual differences among animals; in addition, further research will be performed to reveal the mechanism of L. fermentum I5007 favors on intestinal development.

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AUTHOR INFORMATION

Corresponding Author

*Fax: (8610) 62733688. Phone: (8610) 62733588-1112. Email: [email protected]. Funding

This study was financially supported by the National Natural Science Foundation of China (No. 30930066), the National Basic Research Program of China (973 Program, No. 2012CB124702, 2013CB117302). Notes

The authors declare no competing financial interest.

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REFERENCES

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Oral Administration of Lactobacillus fermentum I5007 Favors Intestinal

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