Analysis of antibiotic resistance genes in pig feces during the weaning transition using whole metagenome shotgun sequencing

Gi Beom Keum1,#, Eun Sol Kim1,#, Jinho Cho2,#, Minho Song3,#, Kwang Kyo Oh4, Jae Hyoung Cho1, Sheena Kim1, Hyeri Kim1, Jinok Kwak1, Hyunok Doo1, Sriniwas Pandey1, Hyeun Bum Kim1,*, Ju-Hoon Lee5,*
Author Information & Copyright
1Department of Animal Resources Science, Dankook University, Cheonan 31116, Korea
2Division of Food and Animal Science, Chungbuk National University, Cheongju 28644, Korea
3Division of Animal and Dairy Science, Chungnam National University, Daejeon 34134, Korea
4Microbial Safety Division, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Korea
5Department of Food Animal Biotechnology, Department of Agricultural Biotechnology, Center for Food and Bioconvergence, Seoul National University, Seoul 08826, Korea

#These authors contributed equally to this work.

*Corresponding author Hyeun Bum Kim, Department of Animal Resources Science, Dankook University, Cheonan 31116, Korea. Tel: +82-41-550-3653, E-mail:
*Corresponding author Ju-Hoon Lee, Department of Food Animal Biotechnology, Department of Agricultural Biotechnology, Center for Food and Bioconvergence, Seoul National University, Seoul 08826, Korea. Tel: +82-2-880-4854, E-mail:

© Copyright 2023 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 ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Oct 12, 2022; Revised: Nov 09, 2022; Accepted: Nov 12, 2022

Published Online: Jan 31, 2023


Antibiotics have been used in livestock production for not only treatment but also for increasing the effectiveness of animal feed, aiding animal growth, and preventing infectious diseases at the time when immunity is lowered due to stress. South Korea and the EU are among the countries that have prohibited the use of antibiotics for growth promotion in order to prevent indiscriminate use of antibiotics, as previous studies have shown that it may lead to increase in cases of antibiotic-resistant bacteria. Therefore, this study evaluated the number of antibiotic resistance genes in piglets staging from pre-weaning to weaning. Fecal samples were collected from 8 piglets just prior to weaning (21 d of age) and again one week after weaning (28 d of age). Total DNA was extracted from the 200 mg of feces collected from the 8 piglets. Whole metagenome shotgun sequencing was carried out using the Illumina Hi-Seq 2000 platform and raw sequence data were imported to Metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) pipeline for microbial functional analysis. The results of this study did not show an increase in antibiotic-resistant bacteria although confirmed an increase in antibiotic-resistant genes as the consequence of changes in diet and environment during the experiment.

Keywords: Antibiotic resistance; Whole metagenome shotgun sequencing; Pig


Antibiotics have been used in livestock production for a longtime [1]. They have been employed in intensive farming to boost productivity. In animals, antibiotics is commonly used for not only treatment but also for increasing the effectiveness of animal feed, promoting animal growth, and preventing diseases at a time when their immunity is low due to stress [2]. However, careless usage of antimicrobials lead to a rise in antibiotic-resistant bacteria and genes [3,4]. Increased antibiotic resistance can raise disease incidence and cause chronic health problems in livestock. So, the use of antibiotics for fostering growth in livestock is no longer permitted in the EU and South Korea [5].

Antibiotic-resistant bacteria can be discharged into the surrounding environment, the soil and water which can act as sources for transmission of antibiotic-resistant bacteria [2]. In addition, there are reports of transmission of antibiotic-resistant genes in a surrounding environment adjacent to the pig farm [6]. During the weaning period, piglets are exposed to a variety of stressors, such as changes in feed composition and environment [7,8]. Prior researches have shown that these stressors alters the piglet gut microbiome during nursing and weaning phases [9]. However, it is uncertain whether the gut microbial shifts are associated with the increased antibiotic-resistant genes in pigs during the weaning transition. In addition, from pre-weaning to weaning pigs are gradually exposed to more environment such as soil and water. Due to such environmental exposure, antibiotic resistance in piglets can increase and remain in animal-derived products for human consumption, so antibiotic-resistant gene is observed as “One Health subject” [2]. Therefore, the whole metagenome shotgun sequencing was used in this study to evaluate changes in microbiome and antibiotic-resistant genes during weaning transition.


Piglet fecal sampling

Fecal samples were collected from 8 piglets in a gap of 1 week, firstly just before weaning (21 d of age) and secondly one week after weaning (28 d of age) and placed in sterile test tubes and stored at −80℃. After weaning, the piglets were fed a conventional nursery feed based on soybean meal and corn, that complied with National Research Council standards [10,11] of nutrient requirements of weaned piglets. The piglets had unrestricted access to feed and water. The piglets received no additional supplements or antibiotics through the whole duration of the experiment.

Fecal DNA extraction

200 mg of feces per sample were used for the total DNA extraction, using QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany), as per the instructions provided by the manufacturer. Cell lysis was achieved by bead-beating the samples twice for 2 min at 300 rpm, followed by a 5-minute incubation period in a water bath at 70℃ between beatings. A Colibri Microvolume Spectrometer (Titertek Berthold, Pforzheim, Germany) was used to measure the concentrations of DNA and only the samples that had OD260/280 ratio between 1.80 and 2.15 underwent further processing.

Whole metagenome shotgun sequencing

The paired-end shotgun sequencing using the Illumina Hi-Seq 2000 platform was used to sequence the extracted DNA representing the fecal microbial communities. The whole metagenome shotgun sequencing was carried out at Macrogen (Seoul, Korea).

Whole metagenome shotgun sequence analysis

Whole metagenome shotgun sequencing was carried out on a subset of eight samples randomly chosen (four samples from the same piglets at 21 and 28 days of age) to examine the microbial diversities and fecal microbial functions present in the piglet fecal samples. For the microbial functional analysis, the obtained raw sequence data in FASTQ format were imported to Metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) pipeline. All the classified subsystem reads were normalized in MG-RAST. To account for differences in sequencing depth of samples, DESeq was utilized within the analysis pipeline [12]. MG-RAST pipeline was used for removing artificial duplicate reads [13]. MG-RAST also removed sequence reads that matched the host’s genome through Bowtie. [14]. The Reference Swine Genome (Sus scrofa, NCBI v10.2) available in MG-RAST was used to filter out the host-derived metagenomic reads [13]. The SEED Subsystems database, which is a collection of functionally related protein families, was used for the functional annotation of the sequence reads [15]. Using an e-value of less than 1 × 10−5, minimum identity of 60%, and a minimum alignment length of 15 amino acids for protein, the similarity search between sequence reads and the SEED databases was performed. For the taxonomic assignment of the sequences, the Greengenes reference database was utilized. Significant variations in functional profiles and taxonomic compositions between the nursing and weaned pigs were determined based on Multiple t-test, using STAMP and GraphPad Prism version 7.00 (GraphPad Software, La Jolla, CA, USA).


Microbial functional characteristics of the piglet gut metagenome associated with “Resistance to antibiotics and toxic compounds”

In general, a total of 50,440,732 sequences was obtained by the whole metagenome shotgun sequencing using HiSeq Illumina platform. After microbial functional analysis using MG-RAST pipeline, 28 level 1 SEED subsystems were identified in both nursing and weaned piglet metagenome, and the functional gene groups associated with level 2 SEED subsystem “Resistance to antibiotics and toxic compounds” within the level 1 SEED subsystem “Virulence, disease, and defense” (Fig. 1A) were studied further. At the level 3 SEED subsystems within the level 2 SEED subsystem “Resistance to antibiotics and toxic compounds”, gene families related to “Resistance to vancomycin”, “Resistance to tetracycline”, “Multidrug resistance_efflux pumps” and “Methicillin resistance in Staphylococcus aureus(MRSA)” tended to increase, although not significantly in the weaned piglets than nursing ones (Fig. 1B). In the “Resistance to vancomycin”, the vancomycin binding blocking protein “B-type resistance protein VanW” was significantly enriched (p < 0.05), and “response regulator VanR” and “Sensor histidine kinase VanS (EC 2.7.3.-) tended to increase (Fig. 2A). In “Resistance to tetracycline”, only ribosomal protection proteins (RPPs) related genes were detected and increased on average (Fig. 2B). In the “Multidrug resistance_efflux pumps”, “Multi antimicrobial extrusion protein MATE family of MDR efflux pumps” and “RND efflux membrane fusion protein” that related in extrusion of toxic substrates into the cellular environment were largely present in the weaned piglets (Fig. 2C). In the “MRSA, “UDP-N-acetylmuramoylalanyl-D-glutamate--2, 6-diaminopimelate ligase (murE)”, “D-alanyl transfer protein DltB”, “Poly D-alanine transfer protein DltD”, “Methicillin resistance and cell wall biosynthesis protein FmtB” and “D-alanine-poly ligase subunit 2 (DltC) (EC” which function as methicillin resistance and cell wall biosynthesis were slightly higher in the weaned piglets than nursing piglets (Fig. 2D).

Fig. 1. Comparison of the functional capacities between nursing and weaned pigs. (a) Level 2 SEED subsystems within the level 1 SEED subsystem “Virulence, disease and defense”. (b) Level 3 SEED subsystems within the level 2 SEED subsystem “Resistance to antibiotics and toxic compounds”.
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Fig. 2. Comparison of normalized sequence abundance at the level 4 SEED subsystem associated with (a) Resistance to vancomycin. (b) Resistance to tetracycline. (c) Multidrug resistance_efflux pumps. (d) Methicillin resistance in Staphylococcus aureus (MRSA). ATP, adenosine triphosphate.
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Vancomycin is a β-lactam glycopeptide antibiotics that binds to acyl-D-ala-D-ala of the peptidoglycan cell wall to inhibit cell wall biosynthesis, but acts only on gram-positive bacteria because it cannot pass through the lipid bilayer of gram-negative bacteria [16]. There are two types of glycopeptide resistance through conversion to D-ala-D-lac [17,18]. As a results of detection, B-type proteins were higher which is resistance to vancomycin and vulnerable to teicoplanin. Tetracycline prevents the binding of aminoacyl-tRNA and ribosome with 30S prokaryotic ribosomal subunit as a target, resulting in translation inhibition [19,20]. Mainly, TetQ is found in anaerobic gram-negative species with a limited host range, TetM is located in specific part of Enterococcus faecalisand TetO is found in Gram-positive species such as Streptococcus pyogenes[2123]. These RPPs restore A-site delivery of aminoacyl-tRNA to give resistance to tetracycline [24]. According to sources of energy utilized (adenosine triphosphate [ATP] or Hydrogen/sodium ions) and amino acid sequences, efflux pumps are classified into five major active super families: Major facilitator superfamily (MFS), Resistance nodulation cell division superfamily (RND), MATE, ATP Binding cassette transporter (ABC) and Small multidrug resistance (SMR) [25]. All bacteria contain several efflux pumps, which extrude not only antibiotic substrates but also non-antibiotic substrates such as heavy metals, dyes, detergents and organic solvents [2628]. There are reports of fluoroquinolone transported by MATE in mammals, and RNDs were distributed in gram-negative bacteria to export β-lactams out of outer-membrane [2933]. The murE and fmtB gene detected in MRSA are associated with cell-wall peptidoglycan biosynthesis, and nothing else is known about the fmtB gene [34,35]. The lipoteichoic acid, which constitutes the cell wall of gram-positive bacteria, is made through D-alanylation of LTA through DltA-D [36]. Due to the mechanism of methicillin, which binds to PBP2a to biosynthesis of cell wall, it is considered that methicillin resistance increased as the number of genes associated with cell wall biosynthesis increased.

Taxonomic classification of the bacteria using whole metagenome shotgun sequences

Using the whole metagenome shotgun sequences, the relative abundance of the gut microbiota was compared at the phylum and genus levels during the weaning transition (Fig. 3). The results from total sequence analysis confirmed significant change in bacterial composition after the weaning transition. At the phylum level, nursing piglet showed presence of Bacteroidetes (43.18%), Firmicutes (40.5%), Spirochaetes (5.21%), Proteobacteria (2.8%), Actinobacteria (1.47%), Tenericutes (1.2%) and other 6 phylum (0.8%) (Fig. 3A). After weaning transition, piglet gut microbiota were consisted of Bacteroidetes (71.5%), Firmicutes (22.46%), Spirochaetes (0.57%), Proteobacteria (0.43%), Actinobacteria (0.71%), Tenericutes (0.28%) and other phylum (0.26%) (Fig. 3A). Compared to before the weaning transition, Bacteroidetes increased significantly from 40.5% to 71.5% on average (p < 0.01). At the genus level, Prevotella showed the largest amount of abundance, and enriched on average 16.57% to 61.09% that significantly increased after weaning (p < 0.001). Streptococcus increased only by a small amount from 0.13% to 0.28%. The relative abundances of other genera were shown in Fig. 3B. The genus Prevotella, which has the ability to break down protein and carbohydrate in feed, increased significantly with the change in diet from milk to grain feed during the weaning transition [37]. Unlike the tendency of antibiotic resistance genes to increase after weaning, there were no significant increase in the relative abundance of major antibiotic resistant bacteria such as Staphylococcus aureus, Enterococcus and Mycobacterium tuberculosis. The data from this study showed that changes in diet and environment can be a potential health risk factor and may affect piglets.

Fig. 3. Comparison of gut microbiome relative abundance at the (a) phylum and (b) genus levels during the weaning transition. (b) Resistance to tetracycline. (c) Multidrug resistance_efflux pumps. (d) Methicillin resistance in Staphylococcus aureus (MRSA). ATP, adenosine triphosphate.
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Competing interests

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

Funding sources

This work was carried out with the support of ‘Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ0162982022)’ Rural Development Administration, Korea.


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: Keum GB, Kim ES, Song M, Kim HB.

Data curation: Kim ES, Kim H.

Methodology: Keum GB, Cho J, Cho JH.

Validation: Oh KK, Kim S, Kim HB.

Investigation: Kwak J, Doo H, Pandey S.

Writing - original draft: Keum GB, Kim ES, Cho J, Song M.

Writing - review & editing: Keum GB, Kim ES, Cho J, Song M, Oh KK, Cho JH, Kim S, Kim H, Kwak J, Doo H, Pandey S, Kim HB, Lee JH.

Ethics approval and consent to participate

Animal care and experimental procedures were conducted according to the guideline of the Institutional Animal Care and Use Committee of Chungnam National University, Daejeon, Korea (Approval number: CNU-00962).



Lekagul A, Tangcharoensathien V, Yeung SM. Patterns of antibiotic use in global pig production: a systematic review. Vet Anim Sci. 2019; 7:100058


Manyi-Loh C, Mamphweli S, Meyer E, Okoh A. Antibiotic use in agriculture and its consequential resistance in environmental sources: potential public health implications. Molecules. 2018; 23:795


Xiao L, Estellé J, Kiilerich P, Ramayo-Caldas Y, Xia Z, Feng Q, et al. A reference gene catalogue of the pig gut microbiome. Nat Microbiol. 2016; 1:16161


Gresse R, Chaucheyras-Durand F, Fleury MA, Van de Wiele T, Forano E, Blanquet-Diot S. Gut microbiota dysbiosis in postweaning piglets: understanding the keys to health. Trends Microbiol. 2017; 25:851-73


Recharla N, Balasubramanian B, Song M, Puligundla P, Kim SK, Jeong JY, et al. Dietary turmeric (Curcuma longa L.) supplementation improves growth performance, short-chain fatty acid production, and modulates bacterial composition of weaned piglets. J Anim Sci Technol. 2021; 63:575-92


Li L, Sun J, Liu B, Zhao D, Ma J, Deng H, et al. Quantification of lincomycin resistance genes associated with lincomycin residues in waters and soils adjacent to representative swine farms in China. Front Microbiol. 2013; 4:364


Namkung H, Li M, Gong J, Yu H, Cottrill M, de Lange CFM. Impact of feeding blends of organic acids and herbal extracts on growth performance, gut microbiota and digestive function in newly weaned pigs. Can J Anim Sci. 2004; 84:697-704


Yun W, Song M, Lee J, Oh H, An J, Kim G, et al. Arginine addition in a diet for weaning pigs can improve the growth performance under heat stress. J Anim Sci Technol. 2020; 62:460-7


Frese SA, Parker K, Calvert CC, Mills DA. Diet shapes the gut microbiome of pigs during nursing and weaning. Microbiome. 2015; 3:28


Godden SM, Wells S, Donahue M, Stabel J, Oakes JM, Sreevatsan S, et al. Effect of feeding heat-treated colostrum on risk for infection with Mycobacterium avium ssp. paratuberculosis, milk production, and longevity in Holstein dairy cows. J Dairy Sci. 2015; 98:5630-41


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


Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol. 2010; 11:R106


Meyer F, Paarmann D, D’Souza M, Olson R, Glass EM, Kubal M, et al. The metagenomics RAST server – a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics. 2008; 9:386


Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10:R25


Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. 2014; 42:D206-14


Nagarajan R. Antibacterial activities and modes of action of vancomycin and related glycopeptides. Antimicrob Agents Chemother. 1991; 35:605-9


Arthur M, Courvalin P. Genetics and mechanisms of glycopeptide resistance in enterococci. Antimicrob Agents Chemother. 1993; 37:1563-71


Bugg TDH, Wright GD, Dutka-Malen S, Arthur M, Courvalin P, Walsh CT. Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA. Biochemistry. 1991; 30:10408-15


Jenner L, Starosta AL, Terry DS, Mikolajka A, Filonava L, Yusupov M, et al. Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis. Proc Natl Acad Sci USA. 2013; 110:3812-6


Nguyen F, Starosta AL, Arenz S, Sohmen D, Dönhöfer A, Wilson DN. Tetracycline antibiotics and resistance mechanisms. Biol Chem. 2014; 395:559-75


Giovanetti E, Brenciani A, Lupidi R, Roberts MC, Varaldo PE. Presence of the tet(O) gene in erythromycin- and tetracycline-resistant strains of Streptococcus pyogenes and linkage with either the mef(A) or the erm(A) gene. Antimicrob Agents Chemother. 2003; 47:2844-9


Leng Z, Riley DE, Berger RE, Krieger JN, Roberts MC. Distribution and mobility of the tetracycline resistance determinant tetQ. J Antimicrob Chemother. 1997; 40:551-9


Su YA, He P, Clewell DB. Characterization of the tet(M) determinant of Tn916: evidence for regulation by transcription attenuation. Antimicrob Agents Chemother. 1992; 36:769-78


Connell SR, Tracz DM, Nierhaus KH, Taylor DE. Ribosomal protection proteins and their mechanism of tetracycline resistance. Antimicrob Agents Chemother. 2003; 47:3675-81


Raj DS, Kesavan DK, Muthusamy N, Umamaheswari S. Efflux pumps potential drug targets to circumvent drug Resistance – Multi drug efflux pumps of Helicobacter pylori. Mater Today Proc. 2021; 45:2976-81


Ramos JL, Duque E, Gallegos MT, Godoy P, Ramos-Gonzalez MI, Rojas A, et al. Mechanisms of solvent tolerance in gram-negative bacteria. Annu Rev Microbiol. 2002; 56:743-68


Zgurskaya HI, Nikaido H. Multidrug resistance mechanisms: drug efflux across two membranes. Mol Microbiol. 2000; 37:219-25


Nies DH. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev. 2003; 27:313-39


Tseng TT, Gratwick KS, Kollman J, Park D, Nies DH, Goffeau A, et al. The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. J Mol Microbiol Biotechnol. 1999; 1:107-25


Terada T, Masuda S, Asaka J, Tsuda M, Katsura T, Inui K. Molecular cloning, functional characterization and tissue distribution of rat H+/organic cation antiporter MATE1. Pharm Res. 2006; 23:1696-701


Tsuda M, Terada T, Asaka J, Ueba M, Katsura T, Inui K. Oppositely directed H+ gradient functions as a driving force of rat H+/organic cation antiporter MATE1. Am J Physiol Renal Physiol. 2007; 292:F593-8


Tanihara Y, Masuda S, Sato T, Katsura T, Ogawa O, Inui K. Substrate specificity of MATE1 and MATE2-K, human multidrug and toxin extrusions/H+-organic cation antiporters. Biochem Pharmacol. 2007; 74:359-71


Morita Y, Tomida J, Kawamura Y. MexXY multidrug efflux system of Pseudomonas aeruginosa. Front Microbiol. 2012; 3:408


Gardete S, Ludovice AM, Sobral RG, Filipe SR, de Lencastre H, Tomasz A. Role of murE in the expression of β-lactam antibiotic resistance in Staphylococcus aureus. J Bacteriol. 2004; 186:1705-13


Komatsuzawa H, Ohta K, Sugai M, Fujiwara T, Glanzmann P, Berger-Bächi B, et al. Tn551-mediated insertional inactivation of the fmtB gene encoding a cell wall-associated protein abolishes methicillin resistance in Staphylococcus aureus. J Antimicrob Chemother. 2000; 45:421-31


Fischer W. Bacterial phosphoglycolipids and lipoteichoic acids.In In: Kates M, editor.editor Glycolipids, phosphoglycolipids, and sulfoglycolipids. New York: Plenum Press. 1990; p. 123-234


Mun D, Kyoung H, Kong M, Ryu S, Jang KB, Baek J, et al. Effects of Bacillus-based probiotics on growth performance, nutrient digestibility, and intestinal health of weaned pigs. J Anim Sci Technol. 2021; 63:1314-27