Quantification of the absolute abundance of the jejunal mucosa-associated microbiota of pigs using spike-in control

Abstract

The objective was to quantify the absolute abundance of the jejunal mucosa-associated microbiota in pigs and develop a standardized spike-in control protocol. The spike-in control, containing bacteria that are Gram-negative (Imtechella halotolerans) and Gram-positive (Allobacillus halotolerans), was diluted 100 times and 10 μL (100,000 bacterial cells of each species) was added to 100 mg of porcine jejunal mucosa samples. The relative abundance of the jejunal mucosa-associated microbiota differed between the diet groups, whereas no difference was observed in their absolute abundance. In conclusion, the spike-in control containing 100,000 cells of Imtechella halotolerans and Allobacillus halotolerans can be used to quantify the absolute abundance of microbiota in 100 mg of pig jejunal mucosa.

Keywords

Absolute abundance / mucosa-associated microbiota / pig / spike-in control

References

[1] Snoeck V,Verfaillie T,Verdonck F,Goddeeris BM,Cox E. The jejunal Peyer’s patches are the major inductive sites of the F4-specific immune response following intestinal immunisation of pigs with F4 (K88) fimbriae Vaccine. 2006 24 3812 20

[2] Cera KR,Mahan DC,Cross RF,Reinhart GA,Whitmoyer RE. Effect of age, weaning and postweaning diet on small intestinal growth and jejunal morphology in young swine J Anim Sci. 1988 66 574 84

[3] Mowat AM,Agace WW. Regional specialization within the intestinal immune system Nat Rev Immunol. 2014 14 667 85

[4] Duarte ME,Kim SW. Significance of mucosa-associated microbiota and its impacts on intestinal health of pigs challenged with F18⁺ E. coli Pathogens. 2022 1 589

[5] Adhikari B,Kim SW,Kwon YM. Characterization of microbiota associated with digesta and mucosa in different regions of gastrointestinal tract of nursery pigs Int J Mol Sci. 2019 20 1630 PMC6480377

[6] Davenport M,Poles J,Leung JM.et al. Metabolic alterations to the mucosal microbiota in inflammatory bowel disease Inflamm Bowel Dis. 2014 20 723 31

[7] Gormley AR,Duarte ME,Deng Z,Kim SW. Saccharomyces yeast postbiotics mitigate mucosal damages from F18⁺ Escherichia coli challenges by positively balancing the mucosal microbiota in the jejunum of young pigs Anim Microbiome. 2024 6 73

[8] De Rodas B,Youmans BP,Danzeisen JL,Tran H,Johnson TJ. Microbiome profiling of commercial pigs from farrow to finish J Anim Sci. 2018 96 1778 94 PMC6140882

[9] McCormack UM,Curião T,Buzoianu SG.et al. Exploring a possible link between the intestinal microbiota and feed efficiency in pigs Appl Environ Microbiol. 2017 83 e00380 17

[10] Zaramela LS,Tjuanta M,Moyne O,Neal M,Zengler K. synDNA-a synthetic DNA spike-in method for absolute quantification of shotgun metagenomic sequencing mSystems. 2022 7 e0044722

[11] Bruijning M,Ayroles JF,Henry LP,Koskella B,Meyer KM,Metcalf CJE. Relative abundance data can misrepresent heritability of the microbiome Microbiome. 2023 11 222 PMC10561453

[12] Aitchison J. The statistical analysis of compositional data J R Stat Soc Ser B (Stat Methodol). 1982 44 139 60

[13] Maghini DG,Dvorak M,Dahlen A.et al. Quantifying bias introduced by sample collection in relative and absolute microbiome measurements Nat Biotechnol. 2024 42 328 38

[14] Wang S,Healy D,Patangia D.et al. Assessment of absolute abundance in mother-infant gut microbiome using marine-sourced bacterial DNA spike-in and comparison with conventional quantification methods Microbiome Res Rep. 2025 4 23 PMC12367464

[15] Seiler CL,Kiflen M,Stefanolo JP.et al. Probiotics for celiac disease: a systematic review and meta-analysis of randomized controlled trials Am J Gastroenterol. 2020 115 1584 95

[16] Stämmler F,Gläsner J,Hiergeist A.et al. Adjusting microbiome profiles for differences in microbial load by spike-in bacteria Microbiome. 2016 4 28 PMC4915049

[17] Odom AR,Faits T,Castro-Nallar E,Crandall KA,Johnson WE. Metagenomic profiling pipelines improve taxonomic classification for 16S amplicon sequencing data Sci Rep. 2023 13 13957 PMC10460424

[18] Oshiro M,Nakamura K,Tashiro Y. Challenge of validation in whole-cell spike-in amplicon sequencing to comprehensively quantify food lactic acid bacteriota Biosci. Biotechnol. Biochem. 2025 89 294 303

[19] Tourlousse DM,Yoshiike S,Ohashi A,Matsukura S,Noda N,Sekiguchi Y. Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing Nucleic Acids Res. 2017 45 e23 PMC5389483

[20] Rao C,Coyte KZ,Bainter W,Geha RS,Martin CR,Rakoff-Nahoum S. Multi-kingdom ecological drivers of microbiota assembly in preterm infants Nature. 2021 591 633 8 PMC7990694

[21] Tkacz A,Hortala M,Poole PS. Absolute quantitation of microbiota abundance in environmental samples Microbiome. 2018 6 110 PMC6009823

[22] Kallastu A,Malv E,Aro V.et al. Absolute quantification of viable bacteria abundances in food by next-generation sequencing: quantitative NGS of viable microbes Curr Res Food Sci. 2023 6 100443 PMC9860258

[23] Chen K,Hu Z,Xia Z,Zhao D,Li W,Tyler JK. The overlooked fact: fundamental need for spike-in control for virtually all genome-wide analyses Mol Cell Biol. 2015 36 662 7

[24] Lyra A,Forssten S,Rolny P.et al. Comparison of bacterial quantities in left and right colon biopsies and faeces World J Gastroenterol. 2012 18 4404 11 PMC3436058

[25] NRC. Nutrient requirements of swine. 11th rev. ed. Washington. DC: Natl. Acad. Press; 2012

[26] Zymo Research. Instruction Manual: ZymoBIOMICS™ Spike-in Control I (High Microbial Load). Ver. 1.1.5. Available from https://files.zymoresearch.com/protocols/_d6320_zymobiomics_spike-in_control_i.pdf. [accessed 16 March 2026]

[27] Pan P,Gu Y,Sun DL,Wu QL,Zhou NY. Microbial diversity biased estimation caused by intragenomic heterogeneity and interspecific conservation of 16S rRNA genes Appl Environ Microbiol. 2023 89 e0210822 PMC10231250

[28] Barlow JT,Bogatyrev SR,Ismagilov RF. A quantitative sequencing framework for absolute abundance measurements of mucosal and lumenal microbial communities Nat Commun. 2020 11 2590 PMC7244552

[29] Yao H,Lu S,Williams BA,Flanagan BM,Gidley MJ,Mikkelsen D. Absolute abundance values reveal microbial shifts and co-occurrence patterns during gut microbiota fermentation of dietary fibres in vitro Food Hydrocolloids. 2022 127 107422

[30] Bonk F,Popp D,Harms H,Centler F. PCR-based quantification of taxa-specific abundances in microbial communities: quantifying and avoiding common pitfalls J Microbiol Methods. 2018 153 139 47

[31] Fierer N,Leung PM,Lappan R.et al. Guidelines for preventing and reporting contamination in low-biomass microbiome studies Nat Microbiol. 2025 10 1570 80