Journal of Animal Science and Technology
Korean Society of Animal Sciences and Technology
RESEARCH ARTICLE

Effects of supplemental bacteriophage on the gut microbiota and nutrient digestibility of ileal-cannulated pigs

Hyunwoong Jo1https://orcid.org/0000-0002-3837-6041, Geongoo Han2https://orcid.org/0000-0003-4866-1185, Eun Bae Kim3https://orcid.org/0000-0002-6475-2012, Changsu Kong4https://orcid.org/0000-0002-3876-6488, Beob Gyun Kim1,5,*https://orcid.org/0000-0003-2097-717X
1Monogastric Animal Feed Research Institute, Konkuk University, Seoul 05029, Korea
2Molecular Microbiology and Immunology, Brown University, Providence 02912, Rhode Island, USA
3Department of Applied Animal Science, Kangwon National University, Chuncheon 24341, Korea
4Department of Animal Science, Kyungpook National University, Sangju 37224, Korea
5Department of Animal Science and Technology, Konkuk University, Seoul 05029, Korea
*Corresponding author: Beob Gyun Kim, Monogastric Animal Feed Research Institute, Konkuk University, Seoul 05029, Korea. Tel: +82-2-2049-6255, E-mail: bgkim@konkuk.ac.kr

© Copyright 2024 Korean Society of Animal Science and Technology. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Aug 07, 2023; Revised: Sep 02, 2023; Accepted: Sep 10, 2023

Published Online: Mar 31, 2024

Abstract

This study measured the potential changes of the microbiota in the gastrointestinal tract and energy and nutrient digestibility by supplemental bacteriophages in pigs. Twelve castrated male pigs (initial mean body weight = 29.5 ± 2.3 kg) were surgically cannulated using T-cannula. The animals were housed individually in pens equipped with a feeder and a nipple waterer. The pigs were allotted to 1 of 3 experimental diets in a quadruplicated 3 × 2 Latin square design with 3 experimental diets, 2 periods, and 12 pigs resulting in 8 replicates per diet. The 3 diets were a control mainly based on corn and soybean meal with no antibiotics or bacteriophages, a diet containing 0.1% antibiotics, and a diet containing 0.2% bacteriophages. On day 5 of the experimental period, feces were collected and on days 6 and 7, ileal digesta were collected. Genomic DNA for bacteria were extracted from the ileal digesta and feces and the V4 region of the 16S rRNA gene was amplified. The ileal and fecal digestibility of energy, dry matter, organic matter, crude protein, and fiber was unaffected by dietary antibiotics or bacteriophages. At the phylum level, the supplemental antibiotic or bacteriophage tended to result in a higher proportion of Firmicutes (p = 0.059) and a lower proportion of Bacteroidetes (p = 0.099) in the ileal digesta samples compared with the control group with no difference between the antibiotic and bacteriophage groups. At the genus level, the supplemental antibiotic or bacteriophage tended to result in a higher proportion of Lactobacillus (p = 0.062) and a lower proportion of Bacteroides (p = 0.074) and Streptococcus (p = 0.088) in the ileal digesta compared with the control group with no difference between the antibiotic and bacteriophage groups. In the feces, supplemental antibiotics or bacteriophages reduced the proportion of Bifidobacterium compared with the control group (p = 0.029) with no difference between the antibiotic and bacteriophage groups. Overall, supplemental antibiotics and bacteriophages showed positive effect on the microbiota of in the ileal digesta without largely affecting energy or nutrient digestibility, with no differences between the antibiotic and bacteriophage groups in growing pigs.

Keywords: Additive; Antibiotic; Digestibility; Probiotic; Pig; Lactobacillus

INTRODUCTION

For several decades, antibiotics (AB) have been used in pig diets to promote growth, improve feed efficiency, and prevent diseases [1]. However, the use of antibiotics is a concern because of the potential occurrence of antibiotic-resistant bacteria and residual antibiotics in pork [2,3]. Hence, the swine feed industry in many countries has prohibited the use of AB in swine diets, and identifying alternatives has become a research focus [4-7]. Bacteriophages (BP) have received attention as appropriate AB alternatives and many studies on BP have been conducted in pigs [8-10].

As the BP are non-hazardous and self-replicating agents that are parasitic on bacteria as host, they have been suggested to infect and grow on bacteria [11]. Therefore, supplemental BP in pig diets may mimic the antimicrobial effects of AB, promoting the digestion and absorption of nutrients by eliminating detrimental bacteria in the intestinal tract. Previous studies reported that supplemental BP in pig diets increases the apparent total tract digestibility (ATTD) of energy and nutrients [12] and decreases the concentration of Escherichia coli and Salmonella in feces [13]. In addition, a recent study reported that supplemental BP improved intestinal barrier function and intestinal microbiota in nursery pigs [10]. However, information on the influence of BP on changes of microbial concentrations in the small intestine and ileal nutrient digestibility is lacking. Thus, to bridge this gap, the present work aimed to determine the effects of BP on the relative proportions of microbiota in the ileal digesta and feces and on nutrient digestibility in pigs.

MATERIALS AND METHODS

Ethical declaration

The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Konkuk University (KU15016).

Animals, diets, and experimental design

Twelve crossbred castrated male pigs with an initial mean body weight of 29.5 kg (standard deviation = 2.3) were employed to assess the effect of BP on microbial concentrations in the small and large intestines, as well as energy and nutrient digestibility. Pigs were fitted with a T-cannula at the distal ileum using the surgical procedure described by Stein et al. [14]. The animals were individually housed in pens (2.0 × 2.2 m) equipped with a feeder and a nipper waterer. The 12 animals were randomly allotted to one of 3 dietary treatment groups in a quadruplicated 3 × 2 Latin square design, with 3 experimental diets and 2 periods per square. Potential residual effects were balanced using a spreadsheet program developed by Kim and Kim [15].

The 3 experimental diets were (Table 1): a control diet mainly based on corn and soybean meal, with 0.2% ground corn cob and wheat shorts as BP carriers; an AB diet, supplemented with 0.1% AB (avilamycin; Avilamix, CTCBIO, Seoul, Korea); and a BP diet supplemented with 0.2% BP at the expense of the BP carriers in the control diet. The energy and nutrient concentrations in all diets met or exceeded the requirement estimates suggested by the NRC [16]. All experimental diets contained chromic oxide at 0.5% as an indigestible index. The BP product was obtained from CTCBIO Inc. (Seoul, Korea). The product contained a mixture of BP consisted of Clostridium perfringens types A and C, Escherichia coli (f41, k88, and k99), Salmonella spp. (Salmonella choleraesuis, Salmonella derby, Salmonella enteritidis, and Salmonella typhimurium), and Staphylococcus aureus. The BP product contained 109 plaque-forming units per gram.

Table 1. Ingredient and analyzed nutrient composition of experimental diets (as-fed basis, %)
Item Control Treatment
Antibiotic (AB)1) Bacteriophage (BP)2)
Ingredient composition
 Corn (yellow dent) 64.35 64.35 64.35
 Soybean meal (48% crude protein) 30.00 30.00 30.00
 Soybean oil 2.00 2.00 2.00
 AB - 0.10 -
 BP - - 0.20
 BP carrier3) 0.20 0.10 -
 L-Lys·HCl, 78.8% 0.10 0.10 0.10
 Dicalcium phosphate 1.10 1.10 1.10
 Limestone 0.85 0.85 0.85
 Vitamin-mineral premix4) 0.50 0.50 0.50
 Salt 0.40 0.40 0.40
 Chromic oxide 0.50 0.50 0.50
Analyzed nutrient composition
 Dry matter 92.2 92. 6 92.6
 Organic matter 86.5 87.0 87.1
 Crude protein 20.2 20.2 20.2
 Neutral detergent fiber 11.9 11.8 11.8
 Acid detergent fiber 3.6 3.7 3.6
 Gross energy (kcal/kg) 4,175 4,198 4,198

Avilamix (CTCBIO, Seoul, Korea) contained 20 g of avilamycin/kg.

BacterPhage (CTCBIO) contained contained a mixture of BP consisted of Clostridium perfringens types A and C, Escherichia coli (f41, k88, and k99), Salmonella spp. (Salmonella choleraesuis, Salmonella derby, Salmonella enteritidis, and Salmonella typhimurium), and Staphylococcus aureus. The BP product contained 109 plaque-forming units per gram.

Consisted of ground corn cob and wheat shorts.

Provided the following quantities per kg of complete diet: vitamin A, 25,000 IU; vitamin D3, 4,000 IU; vitamin E, 50 IU; vitamin K, 5.0 mg; thiamin, 4.9 mg; riboflavin, 10.0 mg; pyridoxine, 4.9 mg; vitamin B12, 0.06 mg; pantothenic acid, 37.5 mg; folic acid, 1.10 mg; niacin, 62 mg; biotin, 0.06 mg; Cu, 25 mg as copper sulfate; Fe, 268 mg as iron sulfate; I, 5.0 mg as potassium iodate; Mn, 125 mg as manganese sulfate; Se, 0.38 mg as sodium selenite; Zn, 313 mg as zinc oxide; butylatedhydroxytoluene, 50 mg.

Download Excel Table
Feeding and sample collection

The animals were fed the experimental diets at 3 times the daily maintenance requirement for energy (i.e., 197 kcal metabolizable energy/kg body weight (BW)0.60; [16]). The amount of feed allowance per pig was divided into 2 equal meals and provided to pigs at 0800 and 1700 h. Water was freely available. An experimental period was consisted of 4 days of adaptation to the experimental diet, 1 day of fecal collection, and 2 days of ileal digesta collection. Fecal samples were collected from 0900 to 1800 on day 5 by grab sampling, and rectal massage was carried out to acquire fresh fecal samples for microbial community analysis. Ileal digesta samples were collected from 0900 to 1630 on days 6 and 7 using a wired plastic bag fixed to a T-cannula. The plastic bags were replaced whenever filled with ileal digesta, or at least every 30 min. Collected feces and ileal digesta were immediately stored at −20°C for further analyses. After the termination of the first period, 4 days of buffering period was used to prevent potential carryover effects.

Microbial community and chemical analyses

Bacterial genomic DNA were extracted from fresh ileal and fecal samples and the V4 region of the 16S rRNA gene was amplified. Amplicons were sequenced on an Illumina MiSeq, and microbial communities and alpha diversity were analyzed using Quantitative Insights into Microbial Ecology as described by Han et al. [17].

Fecal samples were dried in a forced-air drying oven and ileal digesta samples were lyophilized in a freeze dryer and finely ground (< 1 mm) before chemical analysis. Diets, feces, and ileal digesta samples were analyzed for dry matter (AOAC [18]; method 930.15), crude protein (AOAC [18]; method 990.03), and chromium (AOAC [18]; method 990.08) contents. The fibers were analyzed (D200; Ankom Technology, Macedon, NY, USA) for neutral detergent fiber and acid detergent fiber contents. All samples and diets were analyzed for gross energy using bomb calorimetry (Model C2000, IKA, Staufen, Germany).

Calculations and statistical analyses

The apparent ileal digestibility (AID) and ATTD of energy and nutrients were calculated using the index method [19]. Experimental data were analyzed using the MIXED procedure of SAS 9.4 (SAS Institute, Cary, NC, USA). The statistical model included dietary treatment as the fixed variable and replication, period within replication, and animal within replication as random variables. The proportion of microbial communities in each treatment was expressed as a percentage of the total 16S rRNA gene sequences. Least squares means for the response variables were calculated for each treatment. Orthogonal contrasts were used to compare the control versus the AB and BP groups and the AB group versus the BP group. An individual pig was the experimental unit. Statistical significance and trend were determined at p ≤ 0.05 and 0.05 < p ≤ 0.10, respectively.

RESULTS

One pig that was provided with the control diet declined to consume the diet during the collection period and was subsequently excluded from the data for statistical analysis.

Microbial communities at different gut locations

A total of 528,719 (mean = 11,494 ± 575) 16S rRNA reads were generated, with an average of 12,361 (±1,465) and 9,970 (±1,627) reads in ileal digesta and feces from the pigs fed the control diet, 12,787 (±699) and 10,633 (±1,627) reads in ileal digesta and feces from the pigs fed the AB diet and 12,932 (±1,761) and 10,198 (±1,059) reads in ileal digesta and feces from the pigs fed the BP diet, respectively.

The alpha diversity of the microbial communities in the ileum and feces was not affected by AB or BP supplementation (Figs. 1 and 2). At the phylum level, Firmicutes accounted for the largest proportion of the microbiota in the ileal and fecal samples of all groups, followed by Bacteroidetes (Table 2). The proportion of Bacteroidetes in the ileal digesta tended to be less (p = 0.099) in the pigs fed the AB- or BP-supplemented diets compared with the control group, but there was no difference between AB and BP groups. The proportion of Firmicutes in the ileal digesta tended to be greater (p = 0.059) in the pigs fed the AB or BP diets than in the control group but there was no difference between the AB and BP groups. In the feces, there were no differences in the relative proportions of Bacteroidetes and Firmicutes among all treatments.

jast-66-2-340-g1
Fig. 1. The alpha diversity of Shannon index for ileum microbial community in pigs fed control, antibiotic (AB), or bacteriophage (BP) diet. The X symbols represent the mean values.
Download Original Figure
jast-66-2-340-g2
Fig. 2. The alpha diversity of Shannon index for fecal microbial community in pigs fed control, antibiotic (AB), or bacteriophage (BP) diet. The X symbols represent the mean values.
Download Original Figure
Table 2. Effects of supplemental antibiotic (AB) and bacteriophage (BP) on relative proportion of phylum and Firmicutes-to-Bacteroidetes ratio (F:B) in the ileal and fecal microbiota of growing pigs1)
Item2) Treatment SEM p-values
Control AB3) BP4) Control vs. AB and BP AB vs. BP
Ileum (%)
Actinobacteria 4.5 4.9 10.3 3.1 0.395 0.179
Bacteroidetes 18.4 14.2 11.9 3.0 0.099 0.492
Firmicutes 60.5 68.7 68.8 3.6 0.059 0.987
Proteobacteria 5.9 6.6 3.8 1.8 0.686 0.172
 Others5) 10.5 6.1 5.1 2.4 0.102 0.741
Feces (%)
Actinobacteria 5.2 2.3 2.6 1.3 0.056 0.867
Bacteroidetes 27.2 26.9 26.1 4.1 0.892 0.895
Firmicutes 56.9 60.0 59.4 3.5 0.525 0.900
Proteobacteria 2.5 2.9 4.2 0.9 0.275 0.226
 Others5) 7.6 8.4 7.5 0.9 0.796 0.530

Each least squares mean represents 8 observations except the control diet (n = 7).

The taxa at the phylum level with more than 3% average proportion in all samples were selected.

Avilamix (avilamycin 20 g/kg, CTCBIO, Seoul, Korea) was supplemented at 0.1%.

BacterPhage (CTCBIO) was supplemented at 0.2%. The product contained a mixture of BP consisted of Clostridium perfringens types A and C, Escherichia coli (f41, k88, and k99), Salmonella spp. (Salmonella choleraesuis, Salmonella derby, Salmonella enteritidis, and Salmonella typhimurium), and Staphylococcus aureus. The BP product contained 109 plaque-forming units per gram.

Phylum less than 0.1% of the average in all groups.

Download Excel Table

At the genus level, the proportion of Lactobacillus in the ileal digesta was the most dominant and tended to be greater (p = 0.062) in the pigs fed the AB- or BP-supplemented diets than in the control group, with no difference between the AB and BP groups (Table 3). The proportions of Bacteroides (p = 0.074) and Streptococcus (p = 0.088) in the ileal digesta tended to be less in the AB and BP groups compared to the control group with no difference between the AB and BP groups. In the fecal samples, the relative proportion of Lactobacillus was the most dominant. The relative proportion of Bifidobacterium was less (p = 0.029) in the AB and BP groups compared with the control group with no difference between the AB and BP groups. In contrast, the relative proportions of Parabacteroides and Succinivibrio were less (p < 0.05) in the AB group compared with the BP group.

Table 3. Effects of supplemental antibiotic (AB) and bacteriophage (BP) on relative proportion of genus in the ileal and fecal microbiota of growing pigs1)
Item2) Treatment SEM p-values
Control AB3) BP4) Control vs. AB and BP AB vs. BP
Ileum (%)
Clostridium 1.7 1.9 1.5 0.5 0.915 0.496
Lactobacillus 28.0 36.6 38.4 4.1 0.062 0.729
Bacillus 2.7 1.2 1.5 0.6 0.105 0.702
Bacteroides 4.6 3.0 2.4 0.8 0.074 0.560
Bifidobacterium 4.0 4.5 9.7 2.9 0.345 0.142
Megasphaera 2.8 4.1 3.7 0.8 0.295 0.751
Mitsuokella 1.9 1.4 1.7 0.6 0.697 0.762
Prevotella 7.0 6.0 4.9 1.3 0.246 0.407
Streptococcus 2.2 1.4 1.6 0.3 0.088 0.702
Veillonella 0.4 1.2 1.5 0.7 0.277 0.782
 Others5) 44.4 38.7 33.1 4.0 0.110 0.320
Feces (%)
Clostridium 0.6 0.6 0.6 0.1 0.954 0.734
Lactobacillus 20.2 20.5 20.6 3.7 0.933 0.988
Bacteroides 3.9 4.9 4.0 1.1 0.688 0.484
Bifidobacterium 3.5 1.6 2.0 0.8 0.029 0.589
Faecalibacterium 1.5 1.3 1.2 0.2 0.262 0.737
Megasphaera 3.3 3.4 7.6 2.2 0.386 0.142
Oscillospira 1.4 1.5 1.2 0.2 0.723 0.107
Parabacteroides 1.0 1.4 5.5 1.6 0.245 0.080
Prevotella 7.0 6.5 6.5 1.4 0.801 0.997
Streptococcus 3.4 0.7 0.6 1.4 0.152 0.985
Ruminococcus 1.4 2.4 1.6 0.5 0.344 0.293
Succinivibrio 0.8 0.7 2.3 0.8 0.417 0.090
Treponema 1.4 0.8 1.6 0.5 0.830 0.215
 Others5) 50.1 54.0 44.4 3.1 0.784 0.032

Each least squares mean represents 8 observations except the control diet (n = 7).

The taxa at the genus level with more than 1% average proportion in all samples were selected.

Avilamix (avilamycin 20 g/kg, CTCBIO, Seoul, Korea) was supplemented at 0.1%.

BacterPhage (CTCBIO) was supplemented at 0.2%. The product contained a mixture of BP consisted of Clostridium perfringens types A and C, Escherichia coli (f41, k88, and k99), Salmonella spp. (Salmonella choleraesuis, Salmonella derby, Salmonella enteritidis, and Salmonella typhimurium), and Staphylococcus aureus. The BP product contained 109 plaque-forming units per gram.

Genus less than 0.1% of the average in all groups and the unclassified genera.

Download Excel Table
Energy and nutrient digestibility at different gut locations

The AID and ATTD of energy and nutrients were not affected by the supplemental AB or BP (Table 4).

Table 4. Effects of supplemental antibiotic (AB) and bacteriophage (BP) on apparent ileal and total tract digestibility of energy and nutrients in growing pigs1)
Item Diet SEM p-values
Control AB2) BP3) Control vs. AB and BP AB vs. BP
Ileal digestbility (%)
 Energy 80.7 79.1 79.5 1.4 0.347 0.818
 Dry matter 79.6 78.2 78.5 1.5 0.413 0.845
 Organic matter 81.5 79.9 80.3 1.6 0.426 0.809
 Crude protein 83.8 82.4 81.0 1.4 0.109 0.276
 Neutral detergent fiber 45.8 42.1 41.7 3.7 0.411 0.929
 Acid detergent fiber 26.9 28.4 30.2 3.9 0.570 0.708
Total tract digestibility (%)
 Energy 81.4 82.5 82.4 0.9 0.206 0.917
 Dry matter 82.0 82.9 83.0 0.7 0.199 0.966
 Organic matter 84.1 84.7 85.0 0.7 0.376 0.752
 Crude protein 80.7 82.5 81.9 1.2 0.242 0.626
 Neutral detergent fiber 49.8 49.6 52.8 2.5 0.403 0.100
 Acid detergent fiber 39.1 37.7 39.9 3.9 0.886 0.380

Each least squares mean represents 8 observations except the control diet (n = 7).

Avilamix (avilamycin 20 g/kg, CTCBIO, Seoul, Korea) was supplemented at 0.1%.

BacterPhage (CTCBIO) was supplemented at 0.2%. The product contained a mixture of BP consisted of Clostridium perfringens types A and C, Escherichia coli (f41, k88, and k99), Salmonella spp. (Salmonella choleraesuis, Salmonella derby, Salmonella enteritidis, and Salmonella typhimurium), and Staphylococcus aureus. The BP product contained 109 plaque-forming units per gram.

Download Excel Table

DISCUSSION

The BP bind to specific receptors on the surface of bacteria prior to introducing their genetic materials [20,21]. The relationship between BP and bacteria is either lytic or lysogenic. During lytic infection, the phages adhere to the surface of bacteria and they inject their chromosomes into the bacterial cells. After that, the phages reproduce and release virulent phages. In the lysogenic cycle, the genetic materials of phages incorporate into bacterial chromosomes. This incorporation permits bacteria to reproduce generally along with phage genetic material known as prophages. Consequently, temperate phages are released, and these have the potential to convert into virulent phages at any time [22]. Thus, the antimicrobial potential of virulent phages is greater than that of temperate phages. Each type of BP can efficiently infect specific bacteria more efficiently than others [23]. As the BP product used in this study contained a cocktail of BP for Clostridium perfringens types A and C, Escherichia coliSalmonella spp., and Staphylococcus aureus, the phage product may be specifically effective against these bacteria. Kim et al. [24] reported that supplemental BP at 0.1% or 0.15% decreased ileal Escherichia coli and Clostridium proportion in pigs. Furthermore, a previous study found that supplemental BP against Salmonellatyphimurium increased the body weight gain and feed efficiency in Salmonella-challenged growing pigs [25].

Avilamix containing 20 g/kg avilamycin was used in the present study. Avilamycin is an AB that inhibits protein synthesis by selectively targeting bacterial ribosomes. This action disrupts protein production, effectively suppressing bacterial growth and demonstrating antibacterial activity [26]. A previous study observed a significant reduction in Salmonella count in chicks treated with 100 ppm avilamycin compared to the control group [27].

The overall microbial diversity comprised prevalent intestinal microbial groups, with Firmicutes (35%), Bacteroidetes (21%), Proteobacteria (3%), and Spirochaetes (2%) dominating the total 16S rRNA gene sequences, as previously observed in pigs [28,29]. However, according to a meta-analysis to define the core microbiota in the gastrointestinal tract of pigs, the ileal digesta primarily consisted of Firmicutes and Proteobacteria, whereas the phylum composition in the cecum and colon exhibited a high level of consistency [30]. In the ileal digesta in the present study, Bacteroidetes were 10 percentage unit greater than that reported in the literature. This inconsistency might be explained by different ileal digesta sampling methods. Most studies in the meta-analysis obtained ileal samples by necropsy at the end of the experiment [31-33]. In contrast, the collection of ileal digesta in this study used a T-cannula inserted into the terminal ileum, approximately 15 cm from the ileocecal valve [14]. Because the flow of ileal digesta in the small intestine is not consistent in one direction, the ileal digesta collected using a T-cannula may contain some cecal digesta. The analytical procedure, such as the targeted region of the 16S rRNA gene and DNA purification methods, also affects the composition of the microbial community [34].

Intestinal microbiota plays a critical role in maintaining immune function, nutritional status, and physiology in pigs. [35,36]. Accordingly, major or frequent changes in intestinal microbiota are often associated with ill health [37-39]. Firmicutes and Bacteroidetes are the 2 dominant phyla in the intestine of pigs [40]. The proportions of Firmicutes and Bacteroidetes in the intestine are associated with the maintenance of homeostasis and changes in their proportions can lead to various pathologies. It has been reported that a decrease in the proportion of Bacteroidetes together with an increase in the proportion of Firmicutes can create an intestinal environment conducive to energy production and absorption in the intestine [41,42]. To our knowledge, we first report the effects of BP on microbiota at the phylum level in the ileal digesta of pigs. The effects of BP on the alteration of microbiota at the phylum level in the ileal digesta were similar to those of AB. However, the effects of AB or BP on fecal microbiota at the phylum level have shown inconsistent results compared to the ileal digesta, which may be explained by the experimental period. Fecal samples were collected after 4 days of the adaptation period, which could be insufficient to detect noticeable changes. However, ileal digesta samples were collected after 5 days of adaptation and the fecal collection period, which might be sufficient to detect the effects of supplemental AB or BP.

Bifidobacterium and Lactobacillus exert beneficial effects on the intestinal environment of animals [43-45]. The growth of these beneficial bacteria was enhanced by the addition of AB. Because the antimicrobial action of AB reduces the colonization of harmful bacteria, it is likely to result in an increase in probiotics in a less competitive intestinal environment [46]. The avilamycin used in this study is recognized for its primary targeting of Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes [26]. However, our study did not detect these specific harmful bacteria and previous research indicated no significant impact on the normal gut flora [47]. This aligns with our findings, suggesting that microbial diversity remains unaffected by avilamycin administration. In this study, we hypothesized that microbes affected by avilamycin could be found among the low-abundance microbes.

The increased proportion of Lactobacillus in the ileum with supplemental AB or BP was likely due to the inhibitory action of AB or BP on pathogenic bacteria. To our knowledge, only one study determined the effect of BP on the microbiota of this genus in the ileal digesta of pigs. Kim et al. [24] reported that supplemental BP at 0.10% or 0.15% increased the ileal Lactobacillus proportion but had no effect on the fecal Lactobacillus proportion in pigs, which agrees with the present results. However, previous studies have reported that supplemental BP increased the fecal Lactobacillus proportion in pigs [8,12,13]. The reason for the inconsistency between the present and previous studies is unclear.

The less proportion of Bifidobacterium in the feces observed in the AB or BP group compared with the control group was inconsistent with previous studies [8,12,24], the reason for which remains unclear. However, another study reported that a supplemental AB mixture consisting of ampicillin, gentamycin, and metronidazole resulted in decreased Bifidobacterium abundance in the feces of pigs [48], which agrees with the present study. They observed that the fecal Bifidobacterium proportion was positively correlated with branched-chain fatty acid concentration in the feces of pigs fed AB. Branched-chain fatty acids are formed from branched-chain amino acids that originate exclusively from the breakdown of proteins and thus serve as indicators of microbial branched-chain amino acid deamination. Thus, they suggested that the reduction in branched-chain fatty acids due to the effect of AB on microbial nitrogen results in a decreased Bifidobacterium proportion in the feces [48].

At the genus level in the gut, the target bacteria of BP containing Clostridium perfringens, Escherichia coli, Salmonella spp., and Staphylococcus aureus were not detected in this study. However, the proportion of Streptococcus in the ileal digesta decreased with the addition of AB or BP. Streptococcus is a genus of bacteria and certain species within this genus are known to cause various diseases. However, the results of this observation are unclear because of the known host specificity of BP.

The ATTD of energy and nutrients in the control diet was within the range of previous data [49,50]. The index method used to calculate digestibility in this study relies greatly on the accurate chemical analysis of index compounds in the feed and digesta, which requires sufficient adaptation to achieve a constant fecal index concentration. Choi and Kim. [51] suggested that a minimum adaptation time of 3.5 days is needed to achieve a constant fecal index concentration if a high-fiber diet containing 30% neutral detergent fiber is fed, whereas 5.5 days are needed for a low-fiber diet containing 5% neutral detergent fiber. Because the neutral detergent fiber concentration of the experimental diets in this study was greater than 5%, an adaptation period of 4 days was sufficient.

The intestinal microbiota helps the host utilize nutrients and defend against pathogens [52]. However, in the present study, BP supplementation did not affect nutrient digestibility despite having positive effects on the intestinal microbial community. There are two potential reasons for these results. First, the species of microbes are critical for significant changes in nutrient digestibility. Niu et al. [53] suggested that only specific microbial species improve nutrient digestibility. In their study, the proportions of 3 phyla of Proteobacteria, Tenericutes, and TM7, and 11 genera including Anaeroplasma, Campylobacter, and Clostridium were positively correlated with apparent crude fiber digestibility. However, in the present study, there were no differences in the abundance of these phyla or genera among the treatments. Second, the experimental conditions, such as the use of a variety of AB, experimental diet composition, and experimental period, differed among the studies. There are discrepancies among studies on the relationships between microbial communities and nutrient digestibility in pigs. A strong relationship between the intestinal microbiota and nutrient digestibility in pigs has been reported in multiple studies [12,13,48] whereas no relationship was observed in other studies [54,55]. These discrepancies were attributed to the fact that the magnitude of microbial changes was not large and perhaps would not improve nutrient digestibility because of the short experimental period compared to previous studies. Previous studies fed diets containing 0.05% [13] or 0.1% [12] BP for more than 30 days, whereas this experiment was conducted for only 7 days. In addition, Kim et al. [24] reported that supplementation with BP at 0.15% resulted in increased apparent total tract dry matter digestibility for 35 days, but had no effect when fed for 7 days.

In conclusion, although BP supplementation had no effect on energy and nutrient digestibility and seemed to require a relatively long adaptation time to improve digestibility, the alteration of the intestinal microbiota in the ileal digesta and feces in the BP group was similar to that in the AB group. This might be the basis for supplemental BP to show a function similar to that of AB. However, the effects of supplemental BP on the intestinal microbiota were inconsistent with those reported in previous studies. In addition, studies on the effect of BP on ileal microbiota at the phylum level and ileal digestibility in pigs are scarce. Therefore, further studies to determine the effects of BP on the ileal microbiota should be conducted to bridge this gap.

Competing interests

No potential conflict of interest relevant to this article was reported.

Funding sources

Not applicable.

Acknowledgements

Not applicable.

Availability of data and material

Upon reasonable request, the datasets of this study can be available from the corresponding author.

Authors’ contributions

Conceptualization: Kim EB, Kong C, Kim BG.

Data curation: Jo H, Han G.

Formal analysis: Jo H, Han G.

Methodology: Kong C, Kim BG.

Validation: Kim EB.

Supervision: Kim BG.

Writing - original draft: Jo H, Han G.

Writing - review & editing: Jo H, Han G, Kim EB, Kong C, Kim BG.

Ethics approval and consent to participate

The experimental protocol was approved by the Institutional Animal Care and Use Committee at Konkuk University (KU15016).

REFERENCES

1.

Cromwell GL. Why and how antibiotics are used in swine production. Anim Biotechnol. 2002; 13:7-27

2.

Monger XC, Gilbert AA, Saucier L, Vincent AT. Antibiotic resistance: from pig to meat. Antibiotics. 2021; 10:1209

3.

Mitchaothai J, Srikijkasemwat K. Antimicrobial resistance in fecal Escherichia coli from different pig production systems. Anim Biosci. 2022; 35:138-46

4.

Costa LB, Luciano FB, Miyada VS, Gois FD. Review article: herbal extracts and organic acids as natural feed additives in pig diets. S Afr J Anim Sci. 2013; 43:181-93

5.

Liu Y, Espinosa CD, Abelilla JJ, Casas GA, Lagos LV, Lee SA, et al. Non-antibiotic feed additives in diets for pigs: a review. Anim Nutr. 2018; 4:113-25

6.

Wang H, Long W, Chadwick D, Zhang X, Zhang S, Piao X, et al. Dietary acidifiers as an alternative to antibiotics for promoting pig growth performance: a systematic review and meta-analysis. Anim Feed Sci Technol. 2022; 289:115320

7.

Kim YJ, Cho SB, Song MH, Lee SI, Hong SM, Yun W, et al. Effects of different Bacillus licheniformis and Bacillus subtilis ratios on nutrient digestibility, fecal microflora, and gas emissions of growing pigs. J Anim Sci Technol. 2022; 64:291-301

8.

Lee S, Hosseindoust A, Goel A, Choi Y, Kwon IK, Chae B. Effects of dietary supplementation of bacteriophage with or without zinc oxide on the performance and gut development of weanling pigs. Ital J Anim Sci. 2016; 15:412-8

9.

Desiree K, Mosimann S, Ebner P. Efficacy of phage therapy in pigs: systematic review and meta-analysis. J Anim Sci. 2021; 99skab157

10.

Zeng Y, Wang Z, Zou T, Chen J, Li G, Zheng L, et al. Bacteriophage as an alternative to antibiotics promotes growth performance by regulating intestinal inflammation, intestinal barrier function and gut microbiota in weaned piglets. Front Vet Sci. 2021; 8:623899

11.

Huff WE, Huff GR, Rath NC, Balog JM, Donoghue AM. Alternatives to antibiotics: utilization of bacteriophage to treat colibacillosis and prevent foodborne pathogens. Poult Sci. 2005; 84:655-9

12.

Kim KH, Ingale SL, Kim JS, Lee SH, Lee JH, Kwon IK, et al. Bacteriophage and probiotics both enhance the performance of growing pigs but bacteriophage are more effective. Anim Feed Sci Technol. 2014; 196:88-95

13.

Yan L, Hong SM, Kim IH. Effect of bacteriophage supplementation on the growth performance, nutrient digestibility, blood characteristics, and fecal microbial shedding in growing pigs. Asian-Australas J Anim Sci. 2012; 25:1451-6

14.

Stein HH, Shipley CF, Easter RA. Technical note: a technique for inserting a T-cannula into the distal ileum of pregnant sows. J Anim Sci. 1998; 76:1433-6

15.

Kim BG, Kim T. A program for making completely balanced Latin Square designs employing a systemic method. Rev Colomb Cienc Pecu. 2010; 23:277-82

16.

NRC [National Research Council]. Nutrient requirements of swine. 11th ed Washington, DC: National Academies Press. 2012

17.

Han GG, Kim EB, Lee J, Lee JY, Jin G, Park J, et al. Relationship between the microbiota in different sections of the gastrointestinal tract, and the body weight of broiler chickens. SpringerPlus. 2016; 5:911

18.

AOAC [Association of Official Analytical Chemists] International. Official methods of analysis of AOAC International. 18th edArlington, VAAOAC International 2005

19.

Kong C, Adeola O. Evaluation of amino acid and energy utilization in feedstuff for swine and poultry diets. Asian-Australas J Anim Sci. 2014; 27:917-25

20.

Weinbauer MG. Ecology of prokaryotic viruses. FEMS Microbiol Rev. 2004; 28:127-81

21.

Sime-Ngando T. Environmental bacteriophages: viruses of microbes in aquatic ecosystems. Front Microbiol. 2014; 5:355

22.

Bhargava K, Nath G, Bhargava A, Aseri GK, Jain N. Phage therapeutics: from promises to practices and prospectives. Appl Microbiol Biotechnol. 2021; 105:9047-67

23.

Sulakvelidze A, Alavidze Z, Morris JG. Bacteriophage therapy. Antimicrob Agents Chemother. 2001; 45:649-59

24.

Kim JS, Hosseindoust A, Lee SH, Choi YH, Kim MJ, Lee JH, et al. Bacteriophage cocktail and multi-strain probiotics in the feed for weanling pigs: effects on intestine morphology and targeted intestinal coliforms and Clostridium. Animal. 2017; 11:45-53

25.

Gebru E, Lee JS, Son JC, Yang SY, Shin SA, Kim B, et al. Effect of probiotic-, bacteriophage-, or organic acid-supplemented feeds or fermented soybean meal on the growth performance, acute-phase response, and bacterial shedding of grower pigs challenged with Salmonella enterica serotype Typhimurium. J Anim Sci. 2010; 88:3880-6

26.

Wolf H. Avilamycin, an inhibitor of the 30 S ribosomal subunits function. FEBS Lett. 1973; 36:181-6

27.

Sadeghi A, Izadi W, Shawrang P, Chamani M, Amin Afshar M. A comparison of the effects of dietary ginger powder and avilamycin on growth performance and intestinal salmonella count of challenged broiler chickens. Iran J Appl Anim Sci. 2013; 3:769-75

28.

Lamendella R, Santo Domingo JW, Ghosh S, Martinson J, Oerther DB. Comparative fecal metagenomics unveils unique functional capacity of the swine gut. BMC Microbiol. 2011; 11:103

29.

Looft T, Allen HK, Cantarel BL, Levine UY, Bayles DO, Alt DP, et al. Bacteria, phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations. ISME J. 2014; 8:1566-76

30.

Holman DB, Brunelle BW, Trachsel J, Allen HK. Meta-analysis to define a core microbiota in the swine gut. mSystems. 2017; 2e00004-17

31.

Mann E, Schmitz-Esser S, Zebeli Q, Wagner M, Ritzmann M, Metzler-Zebeli BU. Mucosa-associated bacterial microbiome of the gastrointestinal tract of weaned pigs and dynamics linked to dietary calcium-phosphorus. PLOS ONE. 2014; 9e86950

32.

Luo Y, Yang C, Wright ADG, He J, Chen D. Responses in ileal and cecal bacteria to low and high amylose/amylopectin ratio diets in growing pigs. Appl Microbiol Biotechnol. 2015; 99:10627-38

33.

Hedegaard CJ, Strube ML, Hansen MB, Lindved BK, Lihme A, Boye M, et al. Natural pig plasma immunoglobulins have anti-bacterial effects: potential for use as feed supplement for treatment of intestinal infections in pigs. PLOS ONE. 2016; 11e0147373

34.

Darwish N, Shao J, Schreier LL, Proszkowiec-Weglarz M. Choice of 16S ribosomal RNA primers affects the microbiome analysis in chicken ceca. Sci Rep. 2021; 11:11848

35.

Lee YK, Mazmanian SK. Has the microbiota played a critical role in the evolution of the adaptive immune system?. Science. 2010; 330:1768-73

36.

Brestoff JR, Artis D. Commensal bacteria at the interface of host metabolism and the immune system. Nat Immunol. 2013; 14:676-84

37.

Mazmanian SK, Round JL, Kasper DL. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature. 2008; 453:620-5

38.

Handelsman J. Microbial symbiosis: in sickness and in health. DNA Cell Biol. 2009; 28:359-60

39.

Chow J, Lee SM, Shen Y, Khosravi A, Mazmanian SK. Host–bacterial symbiosis in health and disease. Adv Immunol. 2010; 107:243-74

40.

Rinninella E, Raoul P, Cintoni M, Franceschi F, Miggiano GAD, Gasbarrini A, et al. What is the healthy gut microbiota composition? A changing ecosystem across age, environment, diet, and diseases. Microorganisms. 2019; 7:14

41.

Abenavoli L, Scarpellini E, Colica C, Boccuto L, Salehi B, Sharifi-Rad J, et al. Gut microbiota and obesity: a role for probiotics. Nutrients. 2019; 11:2690

42.

Shen ZH, Zhu CX, Quan YS, Yang ZY, Wu S, Luo WW, et al. Relationship between intestinal microbiota and ulcerative colitis: mechanisms and clinical application of probiotics and fecal microbiota transplantation. World J Gastroenterol. 2018; 24:5-14

43.

Aizawa E, Tsuji H, Asahara T, Takahashi T, Teraishi T, Yoshida S, et al. Bifidobacterium and Lactobacillus counts in the gut microbiota of patients with bipolar disorder and healthy controls. Front Psychiatry. 2019; 9:730

44.

Gomes AMP, Malcata FX. Bifidobacterium spp. and Lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics. Trends Food Sci Technol. 1999; 10:139-57

45.

Kang J, Lee JJ, Cho JH, Choe J, Kyoung H, Kim SH, et al. Effects of dietary inactivated probiotics on growth performance and immune responses of weaned pigs. J Anim Sci Technol. 2021; 63:520-30

46.

Li D, Zang S, Li T, Qiao Q, Thacker PA, Kim JH. Effect of feed antibiotics on the performance and intestinal microflora of weanling pigs in China. Asian-Australas J Anim Sci. 2000; 13:1554-60

47.

Castillo M, Martín-Orúe SM, Roca M, Manzanilla EG, Badiola I, Perez JF, et al. The response of gastrointestinal microbiota to avilamycin, butyrate, and plant extracts in early-weaned pigs. J Anim Sci. 2006; 84:2725-34

48.

Pi Y, Gao K, Peng Y, Mu CL, Zhu WY. Antibiotic-induced alterations of the gut microbiota and microbial fermentation in protein parallel the changes in host nitrogen metabolism of growing pigs. Animal. 2019; 13:262-72

49.

Ji F, Casper DP, Brown PK, Spangler DA, Haydon KD, Pettigrew JE. Effects of dietary supplementation of an enzyme blend on the ileal and fecal digestibility of nutrients in growing pigs. J Anim Sci. 2008; 86:1533-43

50.

Liu JB, Liu ZQ, Chen L, Zhang HF. Effects of feed intake and dietary nutrient density on apparent ileal and total tract digestibility of nutrients and gross energy for growing pigs. J Anim Sci. 2016; 94:4251-8

51.

Choi H, Kim BG. A low-fiber diet requires a longer adaptation period before collecting feces of pigs compared with a high-fiber diet in digestibility experiments using the inert marker method. Anim Feed Sci Technol. 2019; 256:114254

52.

Zoetendal EG, Cheng B, Koike S, Mackie RI. Molecular microbial ecology of the gastrointestinal tract: from phylogeny to function. Curr Issues Intest Microbiol. 2004; 5:31-48

53.

Niu Q, Li P, Hao S, Zhang Y, Kim SW, Li H, et al. Dynamic distribution of the gut microbiota and the relationship with apparent crude fiber digestibility and growth stages in pigs. Sci Rep. 2015; 5:9938

54.

Jin Z, Shinde PL, Yang YX, Choi JY, Yoon SY, Hahn TW, et al. Use of refined potato (Solanum tuberosum L. cv. Gogu valley) protein as an alternative to antibiotics in weanling pigs. Livest Sci. 2009; 124:26-32

55.

Choi Y, Hosseindoust A, Goel A, Lee S, Jha PK, Kwon IK, et al. Effects of Ecklonia cava as fucoidan-rich algae on growth performance, nutrient digestibility, intestinal morphology and caecal microflora in weanling pigs. Asian-Australas J Anim Sci. 2017; 30:64-70