Elimination of “kitome” and “splashome” contamination results in lack of detection of a unique placental microbiome

Abstract

Background: A placental microbiome, which may be altered in gestational diabetes mellitus (GDM), has been described. However, publications raising doubts about the existence of a placental microbiome that is different than contaminants in DNA extraction kits and reagents ("kitomes") have emerged. The aims of this study were to confirm the existence of a placental microbiome distinct from contaminants and determine if it is altered in GDM mothers. Results: We first enrolled normal weight, obese and GDM mothers (N = 17) at term elective cesarean section delivery in a pilot case control study. Bacterial DNA was extracted from placental parenchyma, maternal and cord blood, maternal vaginal-rectal swabs, and positive and negative controls with the standard Qiagen/MoBio Power Soil kit. Placentas had significantly higher copies of bacterial 16S rRNA genes than negative controls, but the placental microbiome was similar in all three groups and could not be distinguished from contaminants in blank controls. To determine the source and composition of the putative placental bacterial community identified in the pilot study, we expanded the study to 10 subjects per group (N = 30) and increased the number and variety of negative controls (N = 53). We modified our protocol to use an ultraclean DNA extraction kit (Qiagen QIAamp UCP with Pathogen Lysis Tube S), which reduced the "kitome" contamination, but we were still unable to distinguish a placental microbiome from contaminants in negative controls. We noted microbial DNA from the high biomass vaginal-rectal swabs and positive controls in placental and negative control samples and determined that this resulted from close proximity well-to-well cross contamination or "splashome". We eliminated this source of contamination by repeating the sequencing run with a minimum of four wells separating high biomass from low biomass samples. This reduced the reads of bacterial 16S rRNA genes in placental samples to insignificant numbers. Conclusions: We identified the problem of well-to-well contamination ("splashome") as an additional source of error in microbiome studies of low biomass samples and found a method of eliminating it. Once "kitome" and "splashome" contaminants were eliminated, we were unable to identify a unique placental microbiome.

A philosophical perspective on the prenatal in utero microbiome debate

“…The 16S rRNA gene amplification and sequencing approach used in most studies is so sensitive that it detects dozens if not hundreds of microbial taxa no matter if a sample is added or not (the “kitome”) [ 16 , 17 ]. The approach requires careful sampling, proper controls, bioinformatic tools, and objectivity during the analysis to identify taxa truly overrepresented in samples as compared to controls [ 18 – 25 , 27 , 28 , 38 , 39 ]. Microscopy, qPCR techniques, and culture have also been used to confirm the presence of bacteria [ 27 , 38 ].…”

“…The findings were therefore not sufficient to challenge sterility of the womb, as sterility is defined as the absence of viable life. Over the years, it also became increasingly obvious that contamination [16,17], or the so called "kitome" [18], represented a major problem when next-generation sequencing and PCR-based approaches were applied to low-biomass samples [19]. Consequently, several subsequent sequencing studies that used strict controls for contamination did not support the presence of microbial DNA in utero [18,[20][21][22][23][24][25].…”

In utero human intestine harbors unique metabolome, including bacterial metabolites

“…However, recently Rackaityte and colleagues reported bacterial presence via scanning electron microscopy and limited signatures by 16S rRNA sequencing in a similar sample set (12). The discrepancy between our inability to amplify bacterial DNA and the findings by Rackaityte and colleagues exemplifies the debate over the existence of an in utero microbiome, with some studies identifying a low biomass microbiome (13)(14)(15)(16)(17)(18) and others finding no microbial signatures beyond background levels in animals and humans (7)(8)(9)(10)(11)(46)(47)(48). The controversy about the existence of a placental and fetal microbiome cannot be resolved solely with the data presented in this manuscript.…”

Group therapy on in utero colonization: seeking common truths and a way forward

“…However, they found that the median gene counts for bacterial signal were highest for the maternal side of the placenta regardless of the mode of delivery and indeed detected sporadic bacteria. Olomu et al and Theis et al also used 16S rRNA gene amplicon sequencing of placental parenchyma (villi) with positive and negative controls and did not include maternal decidua in their analyses [ 17 , 18 ]. They found no significant differences in either total biomass or microbial composition when comparing placental samples and negative controls.…”