Sampling strategy

Six women were selected for study with uncomplicated singleton pregnancies at term in spontaneous labor (regular contractions, cervical dilation) or following spontaneous rupture of membranes. Demographic and clinical characteristics of participants are listed in Additional file 1: Table S1. Sample types collected are listed in Table 1. Placental samples were isolated as 0.5 × 0.5 × 0.5 cm cuboidal sections from internal structures within the placenta (maternal and fetal sides) to eliminate surface contamination. Maternal saliva samples and vaginal swab samples were also collected by study personnel from participants at admission to the hospital preceding delivery. Analysis of paired samples from the same patient is a notable difference from [3], where non-placental samples (vaginal/oral) were from historical controls.

Table 1 Sample types studied Full size table

Three types of negative controls were collected. Following each delivery, a swab was waved in the air in the clinical laboratory where the placental biopsies were carried out (adjacent to the labor and delivery rooms), then sealed in a closed container (designated “air swab”). Unused sterile swabs were also collected (“sterile swab”). Thirdly, samples containing the purification reagents only were purified by each method to document bacterial sequences introduced during downstream sample processing (“extraction blank”).

DNA was then purified from each specimen. Two different kits were compared, the STRATEC PSP Spin Stool DNA Plus Kit (henceforth “PSP”) and the MO BIO PowerSoil DNA Isolation Kit (abbreviated “MO BIO”), both of which have been used extensively in microbiome research, and the second of which was used by Aagaard et al. [3], which proposed the existence of a commensal microbiota. An additional reason for comparing these two methods is to identify reagent contamination, because contaminating 16S rRNA gene sequences introduced by DNA purification kits tend to differ by kit [9, 10].

Analysis of total 16S rRNA gene copies using quantitative PCR

Quantification of 16S rRNA gene copies by quantitative PCR is shown in Fig. 1a, b. For qPCR, equal volumes of the purified DNA of all samples were used in the assay. In this and subsequent figures, results for DNA samples prepared using the two purification kits are shown side by side. For the PSP kit, the mean 16S rRNA gene copies in the saliva samples were 2.3 × 107 ± 1.42 × 107 SEM (standard error of the mean). Mean copy number in the vaginal samples was 5.97 × 106 ± 2.92 × 106 SEM. Results for placental samples were much lower, with mean copy number for maternal side (MS) at 5.72 × 102 ± 3.48 × 102 SEM and fetal side (FS) at 1.24 × 102 ± 2.1 × 101 SEM. For controls, the mean copy number was between 9.7 × 101 ± 4.1 × 101 and 1.93 × 102 ± 3.07 × 101 SEM. For the MO BIO kit, mean copy number for the saliva samples was high, 3.69 × 108 ± 2.98 × 108 SEM, while mean copy numbers for the placental samples were again much lower and indistinguishable from contamination controls; maternal side placenta was 2.56 × 102 ± 1.05 × 102 SEM, fetal side was 2.61 × 102 ± 6.3 × 101 SEM, and for controls mean copy numbers were between 8.77 × 101 ± 1.1 × 101 SEM and 1.29 × 102 ± 2.87 × 101 SEM.

Fig. 1 Quantitative PCR analysis of 16S rRNA gene copy numbers. 16S rRNA gene copies per reaction were quantified in the six study subjects using (a) PSP extraction method and (b) MO BIO extraction method. Fetal side (FS), maternal side (MS). c, d Comparison of mean cycle threshold values for 16S rRNA gene qPCR. Limit of detection is 38.29 (PSP) and 34.04 (MO BIO). All data sets were analyzed by Kruskal-Wallis with Dunn’s post test. Comparison of any pair of placental samples to controls yielded p > 0.05 Full size image

For statistical analysis, we compared the cycle of threshold values for all sample sets, because this represents the rawest form of the data (Fig. 1c, d; amplification curves in Additional file 2: Figure S1). The cycle of threshold indicates the value at which exponential increase in the fluorescence signal initiated, so larger values indicate lower numbers of starting templates. For tests of samples purified using either method, there was no significant difference between median values for any pair of placental and control samples (p > 0.05; Kruskal-Wallis test with Dunn’s post test). In comparisons without correction for multiple comparisons, one pair showed a difference in the direction of the negative control air swab amplifying at lower cycle of threshold (more template copies) than the fetal side placental sample (Fig. 1c).

Using a Bayesian approach, it is possible to interrogate these data further (Additional file 3: Figure S2). A posterior probability distribution was calculated for the difference in mean cycle numbers between air swabs and all other sample types. Based on the posterior probability distribution, we calculated that for both placental samples extracted by either of the methods, comparison to air swabs yielded p > 0.05 for the probability of greater abundance of 16S rRNA gene DNA in the placental samples, as was seen with conventional hypothesis testing. Further, we calculate the probability that the pooled placental samples contain 10-fold or more 16S rRNA gene copies than air swabs as p = 0.00004 for PSP and p = 0.004 for MO BIO. In addition, the analysis shows that there is a 95 % probability that the placental samples have <3.5× more DNA than air swabs in MO BIO and <0.8× DNA for PSP. For comparison, the vaginal or oral swabs have ~30,000-fold more DNA than controls when compared by this method.

In summary, all placental and negative control samples were at the extreme low end of the range detectable by qPCR (Additional file 2: Figure S1A–C). For both purification methods, we conclude that placental and control samples showed low and indistinguishable numbers of 16S rRNA gene copies.

Analysis of bacterial community structure using deep sequencing

Samples were next compared based on the proportions of bacterial lineages in each specimen using deep sequencing, investigating the hypothesis that the types of bacteria present in placental and control samples differed. Samples were pooled for sequencing by adding equal volumes of the purified DNA from placental and control samples and equal masses of DNA for the higher biomass oral and vaginal samples.

DNA samples were PCR amplified using bar-coded primers flanking the V1V2 region of the 16S rRNA gene, and samples were sequenced using the Illumina method (Fig. 2a, b). The V1V2 region is useful with low biomass samples because the shorter length allows more efficient amplification of rare template sequences, as shown, for example, in studies of bronchoalveolar lavage samples [11–15], and V1V2 has been used extensively in previous work [16–18]. An alternative approach is to use shotgun metagenomic sequencing to characterize all DNA present in a sample [3]—however, this has the disadvantage for placental samples that the great majority of the sequencing effort is expended resequencing human DNA and so provides little additional information at a much higher cost when only a characterization of bacterial taxa present is desired. Each sample returned an average of 59,198 (PSP extraction) and 25,044 (MO BIO extraction) 16S rRNA gene reads. After forming operational taxonomic units at 97 % identity, a total of 12,813 OTUs were recovered for the PSP samples and 12,032 OTUs for the MO BIO samples.

Fig. 2 Heat maps illustrating the major bacterial lineages detected in each sample. Heat maps of major bacterial lineages for (a) PSP- and (b) MO BIO-extracted samples. Columns indicate the patient ID and sample types, and rows indicate the OTU detected in sequencing. Fetal side (FS), maternal side (MS), family (f), genus (g), order (o) Full size image

For the high biomass saliva samples, the major bacterial lineages were consistent between the two DNA purification methods. The saliva samples were high in Streptococcus, Veillonella, and Prevotella, paralleling many previous studies [11, 14, 19]. The lineages detected for samples purified with each of the two methods were identical at the OTU level.

Vaginal samples were collected as single swabs and thus could be assayed with only one DNA purification method. We chose PSP because it yielded particularly good DNA recovery from other types of microbiome samples [20]. Vaginal samples were high in Lactobacillus, as has been seen for vaginal samples in many studies [19, 21–23].

For the placental and background contamination samples, results diverged radically and were associated with the kit used to purify the DNA. For samples purified using the PSP kit, all placental samples and background controls contained Sediminibacterium, Bradyrhizobiaceae, Methylobacterium, and Propionibacterium. In most samples, the first three contributed the majority of the sequence reads. Sediminibacterium, from the family Sphingobacteriaceae, is a known soil bacterium. Bradyrhizobiaceae contains both soil- and plant-associated bacteria as well as animal-associated bacteria. Methylobacterium is a normal inhabitant of soil and water. Propionibacterium is a ubiquitous human skin organism that is common in house dust. All four lineages have all been reported to be common contaminants of DNA extraction kits [9, 10].

Additional lineages were prominent in a few samples. One of the maternal side biopsy samples was high in Enterobacteraceae and a second in Prevotella. Several contamination controls were high in Phyllobacteriaceae, another bacteria associated with soil and plants that has been identified as a contaminant of DNA extraction kits [9, 10].

For DNA purified from the MO BIO kit, the dominant lineage in the controls was an OTU aligning 100 % to Clostridium difficile. This OTU was predominant in 22 out of 23 samples, including fetal side and maternal side placenta, extraction blanks, unopened sterile swabs, and swabs exposed to air in the clinical laboratory. This OTU was found in a previous negative control sample from our laboratory in another study extracted with this kit, but not in negative control samples extracted with other kits, suggestive of contamination in commercial reagents. We have previously amplified C. difficile 16S rRNA gene sequences in our laboratory, so we cannot rule out that the low-level contamination originated from this source. However, as C. difficile only appeared in negative controls worked up with the MO BIO kit, we tentatively associate this contamination with the kit. Additionally, one sample was high in a lineage of the order Streptophyta, possibly a sequence derived from chloroplasts in pollen.

We attempted to investigate the origin of the sample-specific high proportion OTUs in the maternal side placental data set but were unable to specify the source. Of particular interest were connections with oral microbiota, since oral sites were proposed to donate lineages to placenta in the previous report on a commensal microbiome by Aagaard and colleagues [3]. For the PSP-extracted samples, although Prevotella lineages could be detected in subject 67’s oral samples, the OTU enriched in the maternal side placental biopsy was not among those present in the subject’s saliva sample, nor in her vaginal or fetal side placental samples. Thus, the data did not support the idea that the Prevotella detected in placenta originated at an oral site. For the Enterobacteraceae OTU enriched in the maternal side sample from subject 61, no reads were detected from this OTU in saliva or vaginal samples, and three reads were detected for fetal side placental samples (out of 7633). Thus, we were unable to trace the origin of these outlier lineages to specific body sites in our paired samples.

Analysis of clustering by sample type

We next compared community structure by calculating distances between all pairs of samples and interrogating these data for clustering associated with sample type. In our first approach, we calculated Bray-Curtis distances for data pooled at the phylum level, paralleling the approach used by Aagaard et al. [3]. For each purification method, when all sample types were analyzed together, the difference among groups was highly significant (PERMANOVA p < 0.001 for comparison of the centroids of each group), driven by the formation of discrete clusters for the vaginal and oral samples. The analysis was then repeated, excluding the vaginal and saliva samples. In this case, PERMANOVA p values were >0.05 for PSP-extracted samples and 0.033 for MO BIO. The p value lower than 0.05 in the MO BIO analysis could be attributed to outliers in the unopened sterile swab controls—comparison of placental samples to swabs exposed to air in the clinical laboratory and extraction blanks only (dropping out the unopened swabs) showed a p value of 0.11. Thus, we conclude that we could not distinguish placental samples from contamination controls by PERMANOVA analysis of Bray-Curtis distances.

We repeated the PERMANOVA tests using weighted and unweighted UniFrac distances generated from operational taxonomic units (OTUs) clustered at 97 % sequence similarity, a more conventional approach (Additional file 4: Figure S3A-H). Once again, there was significant clustering by group when all groups were analyzed together (p = 0.001 for both weighted and unweighted UniFrac). When the vaginal and salivary samples were removed, p values were >0.05 for PSP-extracted samples and 0.024 and 0.049 for MO BIO (unweighted and weighted, respectively). Paralleling the results of the Bray-Curtis analysis, removal of the sterile swab data resulted in p values >0.05 for use of both weighted and unweighted UniFrac (comparing placental samples to air swabs and extraction blanks). Thus, we conclude that we could not distinguish placental samples from contamination controls using UniFrac and PERMANOVA, though we could readily distinguish oral and vaginal samples.

A heat map summarizing OTU representation in the sample set is shown in Fig. 3. Clustering by resemblance among samples pulled the oral samples together, with pairs of samples from each of the six individuals clustering together despite purification using different DNA purification kits. The placental samples and contamination controls were interspersed and mostly clustered by the kit used for purification (27/28 for MO BIO and 28/29 for PSP). The vaginal samples mostly clustered together (5/6) within the PSP group.

Fig. 3 Analysis of microbial communities by co-occurrence. The heat map compares taxa observed within each sample, where each row represents a sample and each column an OTU. OTU sample combinations are colored to indicate abundance relative to the maximum proportion observed in any sample for that OTU. Thus, the red cell in each column indicates the sample with the highest proportional abundance for that OTU. Sample characteristics are given to the right of the plot and are color coded for convenience; numbers refer to the research subject ID. Row and columns are clustered by co-occurrence with trees indicating complete-linkage clustering of the Euclidean distance between the abundances of samples (rows) or OTUs (columns) Full size image

Analysis of enriched lineages

We next asked whether any single lineages could be extracted from the data that might represent authentic placental microbiota. We reasoned that any lineages that were seen in placental samples extracted by both methods, but less abundant or absent in controls, would be candidates (though we note that there is evidence for occasional recovery of different lineages dependent on the DNA purification kit used [24]).

An overview of OTU sharing between pairs of samples extracted by the two methods is presented in Fig. 4, summarizing the community recovered over a wide range of abundance filters. Placental and salivary OTUs were first filtered by requiring recovery in both the PSP and MOBIO procedures, then the reproducible OTUs were compared to contamination. In this method, a reproducible OTU seen in placental or oral samples at a given proportion was counted if absent in contamination controls at or above that proportion. By this means, we scored the placental or oral OTUs enriched over contamination in comparisons spanning 5 logs of abundance values (Additional file 5: Table S3).

Fig. 4 Enrichment of bacterial lineages detected reproducibly in placental or oral samples. The number of OTUs present in at least one saliva (red) or maternal (green) or fetal (blue) side fetal sample in both DNA extractions and not in negative controls at that abundance is shown over a range of abundance values (x-axis) Full size image

There were no placental OTUs that achieved at least 1 % abundance in both PSP and MOBIO in at least one sample and not in controls, In contrast, the same filtering procedure recovered 28 OTUs from salivary samples. Analyzing shared OTUs achieving 0.001 % in any sample, we found that 2197 OTUs were recovered in the oral samples, 16 in the maternal side placental samples, and 5 in the fetal side placental samples. Two OTUs appeared in both fetal and maternal side samples, leaving 19 unique placental OTUs. Of these OTUs, 16 OTUs were seen in one placenta only and three were seen in multiple placentas. The three OTUs found in multiple placentas could not be assigned to any bacterial taxa, and further analysis showed the sequences were 100 % identical to human chromosomal sequences. Thus, focusing on the more abundant OTUs, there were no OTUs that appear reproducibly in the placental samples with proportional abundance 1 % or higher that were not also present at the same or higher levels in the contamination controls.