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

Olomu et al. BMC Microbiology (2020) 20:157 https://doi.org/10.1186/s12866-020-01839-y RESEARCH ARTICLE Open Access Elimination of “kitome” and “splashome” contamination results in lack of detection of a unique placental microbiome Isoken Nicholas Olomu1* , Luis Carlos Pena-Cortes2, Robert A. Long3,4, Arpita Vyas5, Olha Krichevskiy3, Ryan Luellwitz6, Pallavi Singh7 and Martha H. Mulks8 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. Keywords: Placenta, Microbiome, Kits, Reagents, ‘Splashome’, Contaminants * Correspondence: 1 Department of Pediatrics & Human Development, Division of Neonatology, Michigan State University, East Lansing, MI, USA Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Olomu et al. BMC Microbiology (2020) 20:157 Background The Human Microbiome Project (HMP) was initiated to characterize and compare the complex microbial communities that inhabit different niches of the healthy adult human body, including the skin, nasal passages, oral cavity, gastrointestinal tract, and urogenital tract in an attempt determine whether a core healthy human microbiome exists in each of these sites [1, 2]. This project has generated an extensive database using sequencing of bacterial 16S rRNA genes. By comparing extracted 16S rRNA gene results to historic HMP data, Aagaard et al. [3], published the existence of a unique placental microbiome that was described as most comparable to the oral microbiome. The placental samples used in that study were from diverse sources, including term and preterm pregnancies and vaginal and cesarean section deliveries, and included mothers with remote history of infection during the pregnancy [3]. Subsequently, there have been reports of the possible involvement of the placenta in fetal macrosomia in gestational diabetes mellitus (GDM) [4], an altered placental microbiome in pregnancies complicated by GDM [5, 6] and of correlations between the placental microbiome and fetal macrosomia in mothers with GDM [7]. These studies are of particular interest given extensive data from humans and laboratory animals indicating obesity and insulin resistance are associated with alterations in the “normal” gastrointestinal microbiome [8, 9], and suggestions there is an “obesity-associated” gut microbiome [10, 11]. We embarked on this study to investigate the possible existence of a “macrosomia-associated” placental microbiome in mothers with GDM. In designing our investigation, we acknowledged and took heed of the growing volume of published data refuting the existence of a unique placental microbiome, calling into question the methods and controls of the above studies, all of which lack positive and negative controls [12– 15]. We were mindful of likely sources of error and attempted to control for potential contamination wherever possible. We recruited patients equally into three groups: normal weight (control), obese (control), and GDM. To limit contamination by rectovaginal microorganisms or concurrent infection, our enrolled study and control mothers were all delivered by scheduled cesarean section at term gestation, without labor and with intact fetal membranes. We also took cognizance of criticisms leveled at the initial description of the placental microbiome by including maternal and fetal blood specimens to control for organisms that may be present in the bloodstream (so called ‘dormant blood microbiome’ [16]) that may seed the placenta [17]. We further included a vaginalrectal (VR) swab (high biomass sample) obtained from each mother to serve as a positive control. More importantly, several studies had drawn atte (...truncated)