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. 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. 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.

中文翻译：

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消除“ kitome”和“ splashome”污染会导致无法检测到独特的胎盘微生物组。

已经描述了胎盘微生物组，其可能在妊娠糖尿病（GDM）中发生改变。然而，出现了对胎盘微生物组的存在与DNA提取试剂盒和试剂（“试剂盒”）中的污染物不同的怀疑的出版物。这项研究的目的是确认存在与污染物不同的胎盘微生物组，并确定其在GDM母亲中是否发生了改变。在一项先导病例对照研究中，我们在足月选择性剖宫产时首先纳入了正常体重，肥胖和GDM母亲（N = 17）。使用标准的Qiagen / MoBio Power Soil试剂盒从胎盘实质，母体和脐带血，母体阴道直肠拭子以及阳性和阴性对照中提取细菌DNA。胎盘具有比阴性对照明显更高的细菌16S rRNA基因拷贝，但胎盘微生物组在所有三个组中均相似，并且无法与空白对照中的污染物区分开。为了确定在初步研究中确定的推定胎盘细菌群落的来源和组成，我们将研究扩大到每组10名受试者（N = 30），并增加了阴性对照的数量和种类（N = 53）。我们修改了方案，使用超净DNA提取试剂盒（带有病原裂解管S的Qiagen QIAamp UCP），该试剂盒减少了“ kitome”污染，但我们仍无法从阴性对照中将胎盘微生物组与污染物区分开。我们注意到来自胎盘和阴性对照样品中高生物量阴道直肠拭子和阳性对照的微生物DNA，并确定这是由于紧密相邻的孔间交叉污染或“斑点”所致。我们通过用至少四个将高生物质与低生物质样品分离的孔重复测序运行，消除了这种污染源。这将胎盘样品中细菌16S rRNA基因的读数减少到无关紧要的数量。在低生物量样品的微生物组研究中，我们确定了井间污染（splashome）的问题是另一个错误来源，并找到了消除它的方法。一旦消除了“ kitome”和“ splashome”污染物，我们将无法鉴定出独特的胎盘微生物组。