During early ontogeny, microbiome affects development of the gastrointestinal tract, immunity, and survival in vertebrates. Bird eggs are thought to be (1) initially sterile (sterile egg hypothesis) and (2) colonized after oviposition through horizontal trans-shell migration, or (3) initially seeded with bacteria by vertical transfer from mother oviduct. To date, however, little empirical data illuminate the contribution of these mechanisms to gut microbiota formation in avian embryos. We investigated microbiome of the egg content (day 0; E0-egg), embryonic gut at day 13 (E13) and female faeces in a free-living passerine, the great tit (Parus major), using a methodologically advanced procedure combining 16S rRNA gene sequencing and microbe-specific qPCR assays. Our metabarcoding revealed that the avian egg is (nearly) sterile, but acquires a slightly richer microbiome during the embryonic development. Of the three potentially pathogenic bacteria targeted by qPCR, only Dietzia was found in E0-egg (yet also in negative controls), E13 gut and female samples, which might indicate possible vertical transfer. Unlike in poultry, we have shown that major bacterial colonization of the gut in passerines does not occur before hatching. We emphasize that protocols that carefully check for environmental contamination are critical in studies with low-bacterial biomass samples.

Division of Air Quality Czech Hydrometeorological Institute Tušimice Observatory Tušimice 6 432 01 Kadaň Czech Republic

Faculty of Science Department of Parasitology Campus Universitario de Fuentenueva University of Granada Profesor Adolfo Rancano 18071 Granada Spain

Faculty of Science Department of Zoology Charles University Viničná 7 128 43 Prague Czech Republic

Institute of Paleonatomy Domestification Research and History of Veterinary Medicine Ludwig Maxmilian University of Munich Kaulbachstr 37 3 80539 Munich Germany

Institute of Vertebrate Biology v v i The Czech Academy of Sciences Květná 8 Brno 603 65 Czech Republic

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Aagaard K, Ma J, Antony KMet al. . The placenta harbors a unique microbiome. Sci Transl Med May. 2014;21:237–65. PubMed PMC

Akinyemi FT, Ding J, Zhou Het al. . Dynamic distribution of gut microbiota during embryonic development in chicken. Poult Sci. 2020;99:5079–90. PubMed PMC

Bäckhed F, Ley RE, Sonnenburg JLet al. . Host-bacterial mutualism in the human intestine. Science. 2005;307:1915–20. PubMed

Barbosa A, Palacios MJ. Health of Antarctic birds: a review of their parasites, pathogens and diseases. Pol Biol. 2009;32:1095–115. PubMed PMC

Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc. 1995;57:289–300.

Benskin CMWH, Wilson K, Jones Ket al. . Bacterial pathogens in wild birds: a review of the frequency and effects of infection. Biol Rev. 2009;84:349–73. PubMed

Bi Y, Tu Y, Zhang Net al. . Multiomics analysis reveals the presence of a microbiome in the gut of fetal lambs. Gut. 2021;70:853–64. PubMed PMC

Bruce J, Drysdale EM. Trans-shell transmission BT—microbiology of the avian egg. In: Board RG, Fuller R (eds.), Microbiology of the Avian Egg. Boston: Springer, 1994, 63–91.

Byrd AL, Belkaid Y, Segre JA. The human skin microbiome. Nat Rev Micro. 2018;16:143–55. PubMed

Callahan BJ, McMurdie PJ, Rosen MJet al. . DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581–3. PubMed PMC

Campos-Cerda F, Bohannan BJM. The nidobiome: a framework for understanding microbiome assembly in neonates. Trends Ecol Evol. 2020;35:573–82. PubMed

Canty A, Ripley BD. boot: bootstrap R (S-Plus) functions. CRAN, 2021.

Cuperus T, Coorens M, van Dijk Aet al. . Avian host defense peptides. Dev Compar Immunol. 2013;41:352–69. PubMed

D'Alba L, Shawkey MD. Mechanisms of antimicrobial defense in avian eggs. J Ornithol. 2015;156:399–408.

Davis NM, DiM P, Holmes SPet al. . Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome. 2018;6:1–14. PubMed PMC

de Goffau MC, Lager S, Sovio Uet al. . Human placenta has no microbiome but can contain potential pathogens. Nature. 2019;572:329–34. PubMed PMC

Ding J, Dai R, Yang Let al. . Inheritance and establishment of gut microbiota in chickens. Front Microbiol. 2017;8:1–11. PubMed PMC

Ding P, Liu H, Tong Yet al. . Developmental change of yolk microbiota and its role on early colonization of intestinal microbiota in chicken embryo. Animals. 2022;12:1–15. PubMed PMC

Dorn-In S, Bassitta R, Schwaiger Ket al. . Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA. J Microbiol Methods. 2015;113:50–6. PubMed

Edgar RC, Haas BJ, Clemente JCet al. . UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011;27:2194–200. PubMed PMC

Eisenhofer R, Minich JJ, Marotz Cet al. . Contamination in low microbial biomass microbiome studies: issues and recommendations. Trends Microbiol. 2019;27:105–17. PubMed

Funkhouser LJ, Bordenstein SR. Mom knows best: the universality of maternal microbial transmission. PLoS Biol. 2013;11:1–9. PubMed PMC

Gantois I, Ducatelle R, Pasmans Fet al. . Mechanisms of egg contamination by Salmonella enteritidis: review article. FEMS Microbiol Rev. 2009;33:718–38. PubMed

Grond K, Lanctot RB, Jumpponen Aet al. . Recruitment and establishment of the gut microbiome in arctic shorebirds. FEMS Microbiol Ecol. 2017;93:1–9. PubMed

Hansen CM, Meixell BW, Van Hemert Cet al. . Microbial infections are associated with embryo mortality in Arctic-nesting geese. Appl Environ Microb. 2015;81:5583–92. PubMed PMC

Honda K, Littman DR. The microbiome in infectious disease and inflammation. Annu Rev Immunol. 2012;30:759–95. PubMed PMC

Chen CY, Chen CK, Chen YYet al. . Maternal gut microbes shape the early-life assembly of gut microbiota in passerine chicks via nests. Microbiome. 2020;8:1–13. PubMed PMC

Jiang H, Lei R, Ding SWet al. . Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinf. 2014;15:1–12. PubMed PMC

Keller LH, Benson CE, Krotec Ket al. . Salmonella enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens. Infect Immun. 1995;63:2443–9. PubMed PMC

Kennedy KM, de Goffau MC, Perez-Muñoz MEet al. . Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies. Nature. 2023;613:639–49. PubMed PMC

Kerr KG, Snelling AM. Pseudomonas aeruginosa: a formidable and ever-present adversary. J Hosp Infect. 2009;73:338–44. PubMed

Kizerwetter-Świda M, Binek M. Bacterial microflora of the chicken embryos and newly hatched chicken. J Anim Feed Sci. 2008;17:224–32.

Klindworth A, Pruesse E, Schweer Tet al. . Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 2013;41:e1–. PubMed PMC

Koerner RJ, Goodfellow M, Jones AL. The genus Dietzia: a new home for some known and emerging opportunist pathogens. FEMS Immunol Med Microbiol. 2009;55:296–305. PubMed

Kreisinger J, Kropáčková L, Petrželková Aet al. . Temporal stability and the effect of transgenerational transfer on fecal microbiota structure in a long distance migratory bird. Front Microbiol. 2017;8:1–19. PubMed PMC

Kropáčková L, Pechmanová H, Vinkler Met al. . Variation between the oral and faecal microbiota in a free-living passerine bird, the great tit (Parus major). PLoS ONE. 2017a;12:e0179945. 10.1371/journal.pone.0179945. PubMed DOI PMC

Kropáčková L, Těšický M, Albrecht Tet al. . Codiversification of gastrointestinal microbiota and phylogeny in passerines is not explained by ecological divergence. Mol Ecol. 2017b;26:5292–304. PubMed

Lagier JC, Dubourg G, Million Met al. . Culturing the human microbiota and culturomics. Nat Rev Micro. 2018;16:540–50. PubMed

Lee SW, La TM, Lee HJet al. . Characterization of microbial communities in the chicken oviduct and the origin of chicken embryo gut microbiota. Sci Rep. 2019;9:1–11. PubMed PMC

Liong JWW, Frank JF, Bailey S. Visualization of eggshell membranes and their interaction with Salmonella enteritidis using confocal scanning laser microscopy. J Food Prot. 1997;60:1022–8. PubMed

Liu C, Cui Y, Li Xet al. . Microeco: an R package for data mining in microbial community ecology. FEMS Microbiol Ecol. 2021;97:1–9. PubMed

Lüdecke D. ggeffects: create tidy data frames of marginal effects for “ggplot” from model outputs. GitHub, 2018.

Lunam CA, Ruiz J. Ultrastructural analysis of the eggshell: contribution of the individual calcified layers and the cuticle to hatchability and egg viability in broiler breeders. Br Poult Sci. 2000;41:584–92. PubMed

Mann K. The chicken egg white proteome. Proteomics. 2007;7:3558–68. PubMed

Mishra A, Lai GC, Yao LJet al. . Microbial exposure during early human development primes fetal immune cells. Cell. 2021;184:3394–409.e20. PubMed PMC

Olowo-okere A, Ibrahim YKE, Lo CIet al. . Bhargavaea massiliensis sp. nov. and Dietzia massiliensis sp. nov., novel bacteria species isolated from human urine samples in Nigeria. Curr Microbiol. 2022;79:1–8. PubMed

Ost KS, Round JL. Communication between the microbiota and mammalian immunity. Annu Rev Microbiol. 2018;72:399–422. PubMed PMC

Pan X, Yang Y, Zhang JR.. Molecular basis of host specificity in human pathogenic bacteria. Emerg Microbes Infect. 2014;3:1. PubMed PMC

Pedroso AA. Which came first: the egg or its microbiota?. Poult Inf Prof. 2009;48:1–5.

Perez-Muñoz ME, Arrieta MC, Ramer-Tait AEet al. . A critical assessment of the “sterile womb” and “in utero colonization” hypotheses: implications for research on the pioneer infant microbiome. Microbiome. 2017;5:1–19. PubMed PMC

Primm TP, Lucero CA, Falkinham JO. Health impacts of environmental Mycobacteria. Clin Microbiol Rev. 2004;17:98–106. PubMed PMC

Quast C, Pruesse E, Yilmaz Pet al. . The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41:D590–6. PubMed PMC

R Core Team . R: a language and environment for statistical computing. RFoundation for Statistical Computing, Vienna, Austria, 2017. https://www.R-project.org/.

Rackaityte E, Halkias J, Fukui EMet al. . Viable bacterial colonization is highly limited in the human intestine in utero. Nat Med. 2020;26:599–607. PubMed PMC

Risely A, Waite DW, Ujvari Bet al. . Active migration is associated with specific and consistent changes to gut microbiota in Calidris shorebirds. J Anim Ecol. 2018;87:428–37. PubMed

Roto SM, Kwon YM, Ricke SC. Applications of in ovo technique for the optimal development of the gastrointestinal tract and the potential influence on the establishment of its microbiome in poultry. Front Vet Sci. 2016;3:1–13. PubMed PMC

Ryan MP, Adley CC. Ralstonia spp.: emerging global opportunistic pathogens. Eur J Clin Microbiol Infect Dis. 2014;33:291–304. PubMed

Salter SJ, Cox MJ, Turek EMet al. . Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 2014;12:1–12. PubMed PMC

Stinson LF, Keelan JA, Payne MS. Identification and removal of contaminating microbial DNA from PCR reagents: impact on low-biomass microbiome analyses. Lett Appl Microbiol. 2019;68:2–8. PubMed

Strandwitz P. Neurotransmitter modulation by the gut microbiota. Brain Res. 2018;1693:128–33. PubMed PMC

Sundaram S, Auriemma M, Howard Get al. . Application of membrane filtration for removal of diminutive bioburden organisms in pharmaceutical products and processes. PDA J Pharm Sci Technol. 1999;53:186–201. PubMed

Svensson L, Baker K. Identification Guide to European Passerines. 4th edn.Stockholm: British Trust for Ornithology, 1992.

Těšický M, Krajzingrová T, Eliáš Jet al. . Inter-annual repeatability and age-dependent changes in plasma testosterone levels in a longitudinally monitored free-living passerine bird. Oecologia. 2022;198:53–66. PubMed

Těšický M, Krajzingrová T, Świderská Zet al. . Longitudinal evidence for immunosenescence and inflammaging in free-living great tits. Exp Gerontol. 2021;154:111527. PubMed

Teyssier A, Lens L, Matthysen Eet al. . Dynamics of gut microbiota diversity during the early development of an avian host: evidence from a cross-foster experiment. Front Microbiol. 2018;9. 10.3389/fmicb.2018.01524. PubMed DOI PMC

Trevelline BK, MacLeod KJ, Knutie SAet al. . In ovo microbial communities: a potential mechanism for the initial acquisition of gut microbiota among oviparous birds and lizards. Biol Lett. 2018;14:3–6. PubMed PMC

Tsiodras S, Kelesidis T, Kelesidis Iet al. . Human infections associated with wild birds. J Infect. 2008;56:83–98. PubMed PMC

Van Veelen HPJ, Salles JF, Tieleman BI. Microbiome assembly of avian eggshells and their potential as transgenerational carriers of maternal microbiota. ISME J. 2018;12:1375–88. PubMed PMC

Walker RW, Clemente JC, Peter Iet al. . The prenatal gut microbiome: are we colonized with bacteria in utero?. Pediatr Obesity. 2017;12:3–17. PubMed PMC

Wang Q, Garrity GM, Tiedje JMet al. . Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microb. 2007;73:5261–7. PubMed PMC

Wickham H. Ggplot2: Elegant Graphics for Data Analysis. New York: Springer-Verlag, 2016.

Wright ES. DECIPHER: harnessing local sequence context to improve protein multiple sequence alignment. BMC Bioinf. 2015;16:1–14. PubMed PMC

The strength of gut microbiota transfer along social networks and genealogical lineages in the house mouse