Vaginal samples used in this study were identified as containing a high relative abundance (> 60%) of BVAB1 using 16S rRNA gene amplicon sequencing of the V3–V4 regions as previously reported (Holm et al., 2019). Cervicovaginal lavages from six participants that were collected as part of the NIH Longitudinal Study for Vaginal Flora (LSVF) (Klebanoff et al., 2004) by washing the vaginal walls with 3 mL sterile, deionized water, aspiration from the vaginal vault via pipette and stored at −80°C, were included in this study. Gram stain smears were prepared for Nugent scoring as previously described (Nugent et al., 1991). DNA was extracted from 200 μL of lavage fluid using the MagAttract Microbial DNA Kit (QIAGEN Inc., Germantown MD) automated on a Hamilton Star robotic platform according the manufacturer recommendations. DNA was eluted in a final volume of 110 μL nuclease-free water.

An additional swab sample collected as part of the UMB-HMP study was used in this study (Ravel et al., 2013). The swab was self-collected by a participant using a Copan Eswab re-suspended into 1 mL Amies transport medium (ESwab, Copan Diagnostics Inc.), frozen at −20°C for no more than a week, and then transferred to −80°C until analyzed. High molecular weight DNA was extracted from this sample using the MasterPure DNA purification kit (Lucigen) with two phenol/chloroform cleanups prior to DNA precipitation. DNA extraction was quantified on a TapeStation 2200 instrument run with a Genomic DNA tape (Agilent).

The extracted DNA from the swab collected as part of the UMB-HMP study (Ravel et al., 2013) was found to be of sufficient concentration (5.49 ng/μL in 200 μL) for long-read sequencing using the Pacific Biosciences Sequel II platform (Pacific Biosciences). The sequencing library was prepared with SMRTBell Template Prep Kit 1.0 and was size-selected on a BluePippen (Sage Science) with a cutoff of 5 kb. The library was barcoded and sequenced as part of a multiplexed run with four other unrelated samples. Sequencing was performed on a PacBio Sequel II instrument with an 8M cell loaded at 60 pM at the Genomic Resource Center of the University Maryland School of Medicine.

Raw reads were demultiplexed with lima (version 1.9.0) using default parameters except for minimum barcode score set at 26 and a minimum read length of 50 bp after clipping of the barcode was enforced. Both tools are part of the SMRTLink 6.0.1 software package with updated CCS version 3.4.1. Human reads were detected using pbalign v0.4.1 (Pacific Biosciences) and the human genome build 38 (GRCh38.p12). Remaining reads were corrected and assembled via Canu v1.7 and the “-pacbio-raw” protocol (Koren et al., 2017). The largest resulting contig was ca. 1.6 Mb in length, much larger than the second longest contig (ca. 600 kb). Four copies of the 16S rRNA gene were detected on this contig and were identical to the existing BVAB1 16S rRNA gene reference AY724739 Fredricks et al., 2005 (NEJM). The contig had 6.5 kb of overlapping ends and was determined to be circular by Canu. This contig was then manually circularized using Geneious version 2019.2.1 (Galens et al., 2011), searched for the dnaA gene, and rotated so that dnaA was the first gene (Kearse et al., 2012). The circularized metagenome-assembled genome (cMAG) was annotated using the IGS Prokaryotic Annotation Pipeline (Galens et al., 2011). Translated gene sequences were assessed against KEGG using the BlastKOALA algorithm for insights into “Candidatus Lachnocurva vaginae” metabolism (Kanehisa et al., 2016). Bacteriophages were detected using PHASTER (Zhou et al., 2011; Arndt et al., 2016). Completeness and contamination of the cMAG was analyzed using CheckM version 1.0.18 (Parks et al., 2015) and the taxonomy_wf flag specifying Order Clostridiales and rerun specifying Family Lachnospiraceae. Metabolic reconstruction was examined with the cMAG using the metabolic modeling function in KBase (Arkin et al., 2018). Complete media for gapfilling and a Gram-positive template were used.

Metagenomic libraries for the six samples from the LSVF study were prepared using the KAPA HyperPlus Kit (Kapa Biosystems) with KAPA Single-Indexed Adapter Kit Set B. A fixed volume (35 μL) of genomic DNA was used as input, and libraries were prepared following the manufacturer's protocol with modifications based on their amount of input DNA as in Supplemental Data Sheet 1. For samples with 0.5 or 0.2 ng input DNA, the fragmentation enzyme was diluted 1:2 or 1:5 with water. All samples were fragmented at 37°C for 5 min. Adapter concentrations varied according to the input DNA as listed in Supplemental Data Sheet 1, and the adapter ligation was carried out overnight at 4°C for all samples. The post-ligation cleanup was performed with 0.8x Ampure XP beads (Beckman Coulter, Indianapolis IN) and 20 μL of sample was used in library amplification. Amplification library cycles varied by input DNA as listed in Supplemental Data Sheet 1. Post-amplification cleanup was performed with 1x Ampure XP beads; libraries with remaining adapter dimer peaks were cleaned a second time. The final elution was in 25 μL of nuclease-free water. Libraries were run on a TapeStation instrument with a D1000 tape (Agilent) to assess quality and concentration. Libraries were sequenced (8 libraries/lane) on an Illumina HiSeq 4000 instrument using the 150 bp paired-end protocol.

Metagenomic reads were quality filtered using Trimmomatic v0.36 (Bolger et al., 2014) to remove sequencing adapters allowing for 2 mismatches, a palindromic clip threshold of 30, and a simple clip threshold of 10. Bases with quality scores < 3 were removed from the beginning and end of reads combined with a 4 bp sliding window which trimmed a read if the average quality score within that window fell below 15. Reads < 75 bp in length were removed. Human reads were detected by mapping to the human genome build 38 (GRCh38.p12) with Bowtie 2 v2.3.4.1 and default settings (Langmead and Salzberg, 2012), and removed using samtools v1.9 (Li et al., 2009) and bedtools v2.27.1 (Quinlan and Hall, 2010; Quinlan, 2014) (see Supplemental Data Sheet 2 for specific scripts). Metagenomic assemblies were produced using SPAdes genome assembler v3.13.0 (Bankevich et al., 2012) with the careful setting. Resulting contigs were aligned to the cMAG using NUCmer with the –mum setting and allowing for 5,000 bp breaklength, filtered to remove aligned contigs < 100 bp, and minimum contig coverage of 25%. MAGs were annotated with the Live Annotate & Predict tool from Geneious version 2019.2.1 (Kearse et al., 2012) using the “Ca. Lachnocurva vaginae” cMAG as annotation source (sequence similarity of ≥ 95% required). Genes with no similarity to the cMAG were annotated with Prokka v1.13 (Seemann, 2014). Phages were detected using PHASTER (Zhou et al., 2011). A circle plot was constructed with the BLAST Ring Image Generator v0.95 (Alikhan et al., 2011), and the annotated cMAG.

Metabolomics analysis was conducted as previously described (Nelson et al., 2018) by Metabolon Inc. using 200 μL of each LSVF lavage sample used in this study or 200 μL of a frozen dry swab eluted in 1 ml of PBS for the sample from the UMB-HMP study (Ravel et al., 2013). The abundances of 561 compounds were quantified using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS). Quantities were corrected for instrument block variability and reported as normalized area-under-the-curve estimates. Figures were generated using ggplot2 v3.2.0 (Wickham, 2009).

Average nucleotide identities (ANI) were calculated using FastANI v1.1 with fragment lengths of 1,000 bp, which is about the mean length of coding sequences (Jain et al., 2018). Maximum-likelihood phylogenetic trees of full-length 16S rRNA gene sequences alignments were generated with MUSCLE v3.8.425 using default parameters (Edgar, 2004) and FastTree v2.1.11 using the Generalized Time Reversible model and default parameters (Price et al., 2009, 2010) in Geneious v11.0.3 (Kearse et al., 2012). The tree was rooted with Fusobacterium nucleatum (AJ133496) and Propionigenium modestum (X54275) (Domingo et al., 2009), and members of the genus Lachnoclostridium were included as neighbors (Yutin and Galperin, 2013) as well as 16S rRNA genes from Shuttleworthia satelles (NR_028827), and Lachnospiraceae bacterium 2_1_46FAA (NR_025127). In addition, the cMAG was submitted for taxonomic placement using the TrueBacID tool from EZBioCloud (Yoon et al., 2017). cMAG and MAG pseudomolecules were aligned and visualized using AliTV v1.0.6 (Ankenbrand et al., 2017). Genes were ordered by gene synteny of the cMAG. To examine homology to other reference sequences, the cMAG was aligned to the NCBI protein reference database (downloaded 1/28/2019), as well as the genomes of Shuttleworthia satelles (NZ_ACIP00000000), Lachnobacterium bovis (GCF_900107245.1), and Lachnospiraceae bacterium 2_1_46FAA (ADLB02000001.1) using BLAST v2.8.1+. The top 3 hits were chosen by lowest e-value, highest percent identity, and longest alignment length, in that order. Genomic and pathogenicity islands were explored using IslandViewer4 (Bertelli et al., 2017).

All six participants in the LSVF study were of reproductive age and had high Nugent scores (6–10) (Table 1). Four were diagnosed with asymptomatic Amsel-BV, one with symptomatic Amsel-BV, and one was negative for Amsel-BV (Mckinnon et al., 2019). The woman who participated in the UMB-HMP study (Ravel et al., 2013) had a Nugent score of 8 and was diagnosed with asymptomatic Nugent-BV. The vaginal microbiota of these 7 samples had >60% BVAB1 as defined by 16S rRNA gene V3-V4 amplicon sequencing (Ravel et al., 2013).

A phylogenetic analysis using the full-length 16S rRNA genes extracted from all MAGs revealed BVAB1 belongs in the Clostridales family Lachnospiraceae, and that S. satelles is the closest known relative (Figure 1), though nucleotide identity is only 89.2%. Similar results were obtained from EZBioCloud's TrueBacID (Supplemental Data Sheet 3). We therefore propose a new candidate species for BVAB1: “Candidatus Lachnocurva vaginae”, which represents the phylogenetic placement, the curved morphology of the cells, and the source of this cMAG. Considering the entire cMAG, the ANI between “Ca. Lachnocurva vaginae” and the S. satelles draft genome (NZ_ACIP00000000) was 81.85%. Relative to each other, “Ca. Lachnocurva vaginae” 16S rRNA genes were >99.7% identical and the cMAG and MAG average nucleotide identities were also high (98.6–99.2%, Table 2).

The cMAG of “Ca. Lachnocurva vaginae” is 1,649,642 bp in size with 31.8% GC content, encodes 1,578 genes and was estimated to be 99.1% complete and 0% contaminated at the order level and 90.9% complete and 0% contaminated at the family level. The relatively low completion estimate at the family level was due to 22 missing marker genes out of 312 (Supplemental Table 2). Mean coverage of the cMAG assembly by long reads was 124X (range: [26–285]). The “Ca. Lachnocurva vaginae” MAGs constructed using the short-read Illumina HiSeq 4000 metagenomic sequence data were 1.48–1.62 Mb in size (mean: 1.57 Mb) with a number of contigs ranging from 26 to 152 (mean N50: 99,943 bp) and 31.6% GC (Table 3). Genome annotation indicated the presence of four complete rRNA operon copies (Figure 2) and 42 tRNA genes in the cMAG. As expected, partial rRNA operons were identified in the Illumina-based MAGs (Supplemental Image 1).

Metabolic modeling and reconstruction of the “Ca. Lachnocurva vaginae” cMAG contained 122 genes, 480 reactions, 581 metabolites, and no mass imbalance was found. Of the 1,578 genes detected, 255 genes had best blast matches of 80% sequence identity covering > 80% of the query gene. Of these, 165 matches were within the order Clostridiales, and 90 were not (Supplemental Image 2). Genes encoding transporters for mannose, fructose, and L-ascorbate were identified in all MAGs (Figure 3 and Supplemental Table 1, LCVA_199-201, LCVA_1491-1494), as were complete pathways for glycolysis, pyruvate oxidation, and the non-oxidative phase of the pentose phosphate pathway. Mannose was noticeably absent, or below the limit of detection, in the metabolome of all the samples, but present in other BV-like samples that did not contain a high relative abundance of the candidate species (Figure 4). We found “Ca. Lachnocurva vaginae” to have the genetic capability to uptake and metabolize mannose (LCVA_1184), suggesting mannose could also be a carbohydrate source for the candidate species (Figure 3).

The D-lactate dehydrogenase gene (LCVA_41), but not an L-lactate dehydrogenase gene was also observed in all MAGs. This result was unexpected, as the production of D-lactate in the vaginal environment is thought to be a key and somewhat unique feature of certain Lactobacillus spp. (Witkin et al., 2013; France et al., 2016). Metabolomic data for each sample were explored for the presence of lactate to provide evidence supporting this genomic finding. While lactate was observed in most samples, its abundance was substantially lower than that from representative samples that were dominated by Lactobacillus crispatus, a known D-lactate producer (Figure 4). Thus, either the enzyme exhibits lower activity in “Ca. Lachnocurva vaginae” or is instead used in the reverse reaction to consume Lactobacillus spp.-produced D-lactate to produce pyruvate. This is a strategy described for another vaginal anaerobic Gram-negative coccus, Veillonella parvula, which is able to grow on lactate as the sole source of carbon (Gronow et al., 2010), and would possibly contribute to an increase in vaginal pH, thereby creating a more favorable growth environment. However, for lactate to convert to pyruvate in anaerobic bacteria, an electron-bifurcating complex with an electron transfer flavoprotein alpha and beta subunit (EtfAB) would be necessary to make the reaction favorable (Weghoff et al., 2015). An EtfAB homolog was not observed in the “Ca. Lachnocurva vaginae” cMAG, suggesting that another mechanism may be involved. Instead, succinate was the most abundant short chain fatty acid in the communities from which “Ca. Lachnocurva vaginae” MAGs originated (Figure 4). Not surprisingly, succinate has been associated with bacterial vaginosis, a condition in which “Ca. Lachnocurva vaginae” is often found (Srinivasan et al., 2015). Interestingly, all “Ca. Lachnocurva vaginae” MAGs lacked genes for all steps of the tricarboxylic acid cycle, indicating it is unable to produce succinate via this pathway. It is possible that “Ca. Lachnocurva vaginae” produces metabolite(s) other species in the community can convert into succinate or produces it via a yet to be determined pathway. While “Ca. Lachnocurva vaginae” does not seem to be able to produce succinate, it has the genetic machinery to produce acetate from pyruvate (i.e., ackA and pta LCVA_939 and LCVA_1006). We were, however, unable to detect acetate in the metabolome through the methods used, as it is too small of a molecule.

A full suite of genes required for flagella assembly was observed in all MAGs, as well as genes required for methyl-accepting chemotaxis (LCVA_205, LCVA_992, LCVA_1053, LCVA_1122) and for the downstream signal process that mediates flagellar response, cheY (LCVA_362, LCVA_688) (Bren and Eisenbach, 2000). Flagella have yet to be visually observed on “Ca. Lachnocurva vaginae” and were not observed in the source samples used for this study (Figure 5).

We found the cMAG and some MAGs to also include genes that encode protection against antibiotics including the tetO gene (LCVA_1310, also observed in MAG Y3255) and drug efflux pumps for macrolides (macB LCVA_500, also observed in MAGs Y2337 and Y2266) and fluoroquinolones (LCVA_1202, present in all MAGs). A gene for hemolysin, tlyA, was also observed in all MAGs (LCVA_1031), suggesting direct interaction between “Ca. Lachnocurva vaginae” and host tissues.

Additionally, choline transporters, as well as the cutC and cutD genes were detected in all MAGs (LCVA_1194 and LCVA_1193, respectively). The cut genes metabolize choline and produce trimethylamine (TMA) (Martinez-Del Campo et al., 2015). Aside from transport of exogenous choline, another potential source of choline would be via phospholipase D hydrolysis of phosphotidyl choline into choline as seen in gut bacteria (Chittim et al., 2019), however no genes encoding such activity were found in the cMAG of “Ca. Lachnocurva vaginae”. Comparing the translated cutC amino acid sequence from “Ca. Lachnocurva vaginae” to those reported by Martinez-Del Campo et al. (2015), we observed three of the five active sites conserved in “Ca. Lachnocurva vaginae” (Cys489, Glu491, Gly821, data not shown) indicating that it is likely functional. TMA is one of the substances believed to be responsible for the fishy odor associated with BV (Brand and Galask, 1986; Wolrath et al., 2002), however it is rarely detected in metabolomics analyses as it is highly volatile, unless it is performed on freshly collected samples (Wolrath et al., 2002).

Two intact bacteriophages were detected in the cMAG (Figure 2). Partial matches to the same phages were observed in all MAGs (Supplemental Image 1). Best BLAST hits indicated that both bacteriophages belong to the Siphoviridae family of double-stranded viruses (NC_009552, NC_011167) which can exhibit both lytic and lysogenic phases. Bacteriophages of this family have previously been reported in the vaginal species Lactobacillus jensenii (Martin et al., 2010). Genomic islands were detected at multiple sites (Supplemental Table 1 and Figure 2). Analysis of proteins encoded in the largest island (65.5 kb, coordinates 1,331,959–1,391,037, LCVA_1280-1330) showed similarity to proteins from Shuttleworthia satelles, and other taxa from the Clostridiales (Supplemental Table 3 and Supplemental Image 2) and several transposons and integrases, as well as tetracycline resistance proteins (TetO, LCVA_1310), and ABC transporters. We ruled out the presence of genomic islands due to mis-assembly by analyzing the long-read coverage spanning the 5′ and 3′ ends of genomic islands. More than 100 long PacBio reads of a means size of 9 kb span the junctions of all detected genomic islands. Further, mean coverage of the genomic islands was similar to that of the mean cMAG coverage of 124X. Portions of these islands were observed in the other MAGs assembled in this study (Supplemental Image 1 and Supplemental Table 1). The stringency of the mapping would not recover reads to regions of sequence diversity, thus these results may indicate this “Ca. Lachnocurva vaginae” likely contains multiple regions of genetic fluidity or diversity. However, the missing regions in MAGs may also be an artifact of metagenomic assembly.

JR is co-founder of LUCA Biologics, a biotechnology company focusing on translating microbiome research into live biotherapeutics drugs for women's health. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.