Differentiating between contamination and the genuine presence of 16S rRNA genes in gestational tissue samples is the gold standard for supporting the in utero colonization hypothesis. During gestation, the fetus undergoes significant physiological changes that may be directly affected by maternal colonization of key bacterial genera. In this study, lab benches, necropsy tables, and air ducts were swabbed at the same time as clinical sampling. The relative and absolute abundance of bacteria present in sheep samples was determined by culture-independent and culture-dependent means. Of 14 healthy pregnant ewes, there was no evidence of any bacteria in the fetal liver, spleen, or brain cortex using culture-independent techniques despite evidence of the presence of bacteria in various locations of the necropsy room used for 11 of these 14 sheep. Of the 336 bacterial genera found in the room swabs, only 12 (5%) were also found in the saliva and vaginal swabs among the three ewes for which bacteria were detected. These 12 taxa represent 1.32% of the relative abundance and approximately 393 16S rRNA copies/swab in these three ewes. Using careful necropsy protocols, bacterial contamination of sheep tissues was avoided. Contamination of saliva and vaginal samples was limited to less than 2% of the bacterial population.IMPORTANCE Recent evidence for a gestational microbiome suggests that active transfer between mother and fetus in utero is possible, and, therefore, actions must be taken to clarify the presence versus absence of these organisms in their respected sources. The value of this study is the differentiation between bacterial DNA identified in the necropsy rooms of animals and bacterial DNA whose origin is purely clinical in nature. We do not know the extent to which microorganisms traverse maternal tissues and infiltrate fetal circulation, so measures taken to control for contamination during sample processing are vital for addressing these concerns.
Keywords: community-level analysis; contamination; environment; gestation; microbiome.
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