Fetal gut colonization: meconium does not have a detectable microbiota before birth Katherine M. Kennedy1,4, Max J. Gerlach2, Thomas Adam3, Markus M. Heimesaat4, Laura Rossi5,6, Michael G. Surette1,5,6, Deborah M. Sloboda1,6,7* and Thorsten Braun2* 1Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada 2Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Obstetrics and Department of 'Experimental Obstetrics', Berlin, Germany 3Labor Berlin, Charité Vivantes GmbH, Berlin, Germany 4Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute for Microbiology, Infectious Diseases and Immunology, Berlin, Germany 5Department of Medicine, McMaster University, Hamilton, Canada. 6Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, Canada 7Department of Pediatrics, McMaster University, Hamilton, Canada. *shared senior authorship Microbial colonization of the human intestine impacts host metabolism and immunity, however when colonization occurs is unclear. Although numerous studies have reported bacterial DNA in first-pass meconium samples, these samples are collected hours to days after birth. We investigated whether bacteria could be detected in meconium prior to birth. Fetal meconium (n = 20) was collected by rectal swab during elective breech Cesarean sections without labour prior to antibiotics and compared to technical and procedural controls (n = 5), first-pass meconium (neonatal meconium; n = 14), and infant stool (n = 25). Unlike first-pass meconium, no microbial signal distinct from negative controls was detected in fetal meconium by 16S rRNA gene sequencing. Additionally, positive aerobic (n = 10 of 20) and anaerobic (n = 12 of 20) clinical cultures of fetal meconium (13 of 20 samples positive in at least one culture) were identified as likely skin contaminants, most frequently Staphylococcus epidermidis, and not detected by sequencing in most samples (same genera detected by culture and sequencing in 2 of 13 samples with positive culture). We conclude that fetal gut colonization does not occur before birth, and that microbial profiles of neonatal meconium reflect populations acquired during and after birth.