Spiegel and colleagues 
previously compared VFA profiles between BV-positive and asymptomatic women, finding an increase in succinate, acetate, butyrate and propionate with a concomitant decrease in lactate occurring with disease state. Consistent with this, our metabolomic analyses revealed that the most distinguishing determinant among samples from women with a high Nugent score and those with a low-moderate Nugent score was a marked decrease in lactic acid. Although, the relative proportion of lactic acid was not found to be significantly different among women delineated by the Amsel criteria (p>0.05). We also observed an increase in acetate with SBVI and propionate with SBVII. However, we did not observe significant changes in succinate or butyrate for women with BV-positive Amsel criteria, or those with a high Nugent score (p>0.05).
One rationalization for these observations could be a reduction in lactic acid-producing lactobacilli along with increases in other microbes capable of producing acetate and propionate in the vaginal tracts of BV-positive women. Decreases in lactobacilli have been described previously in women with high Nugent scores 
, as have increases in G. vaginalis
, which are known to produce acetate and succinate 
. The relative proportions of lactobacilli and Gardnerella
spp. are generally considered hallmarks of the Nugent criteria 
. Consistent with this, the relative proportions of Gardnerella
spp. and lactobacilli 16S rRNA reads were highly discriminatory among samples with high and low Nugent scores. Gardnerella
spp. were significantly more abundant in samples from women with high Nugent scores. However, while the abundance of lactobacilli was clearly reduced in several samples with high Nugent scores, our results did not indicate a significant reduction in overall representation of lactobacilli consistent with Nugent classifications. This finding may have been confounded by the inability of our sequencing primers to detect certain lactobacilli, such as L. iners
. However, the addition of qPCR enumerations of L. iners
did not affect these findings. Dialister
spp. were also observed to be significantly more abundant in samples with high Nugent scores relative to those with low or moderate scores. Human isolates of Dialister
spp. are reported to produce propionate 
, presumably through the decarboxylation of succinate 
, which may influence the relative abundances of these metabolites in Amsel-positive samples.
The relative abundances of 16S rRNA reads from lactobacilli, Gardnerella
spp., or Dialister
spp. were not significantly different among Amsel-positive or -negative samples, suggesting a limited relationship between the relative abundance of these bacterial genera and disease symptomology. 16S rRNA reads belonging to Gardnerella
spp. were detected in 50% of Amsel-negative samples, which is consistent with historical reports, where G. vaginalis
could be cultured from 58%–68% of healthy samples 
. Yet, 16S rRNA reads pertaining to Gardnerella
spp. were more commonly detected in women distinguished by either a high Nugent score or by positive Amsel criteria. These observations may reflect recent findings that different strains of G. vaginalis
have different metabolic or virulence potentials 
, with only a subset of strains being important to the onset of BV or defining the BV state.
In contrast to our diagnostic capabilities using 16S rRNA gene profiles, we were able to clearly delineate Amsel-defined BV-positive samples from BV-negative samples using metabolite profiling. Our analysis revealed the presence of two distinct metabotypes that are delineated by significant differences in 48 discrete metabolites. It was unclear if these metabotypes were affected by microbial or host metabolisms, but we did observe negative correlations between metabotype I and Ravel's biotype IV vaginal microbiomes 
, as well as biotype IV microbial genera including Aerococcus
. Amsel-defined BV-positive samples formed distinct groups within each metabotype, which we denoted as SBVI and SBVII.
We observed considerable overlap between the significantly affected metabolites of women with a high Nugent score and those with SBVI, suggesting that Nugent criteria were highly diagnostic for this diseased state. Consistent with this, all SBVIs had a Nugent score≥7. In contrast, no overlap was observed between the vaginal metabolomes of women with a high Nugent score and those with SBVII. In these samples, more than half had a Nugent score<7.
Women defined as BV-negative based on Amsel criteria, but having a high Nugent score, were observed in both metabotypes I and II. Our networking approach suggested that a high Nugent score was only indirectly connected to Amsel criteria, via a negative connection to sedoheptulose, a common urinary metabolite 
The most determinate metabolite for both SBVI and SBVII was a marked reduction in 1,2 propanediol, a hydrogenated derivative of lactic acid. This reduction reaction is carried out by some lactobacilli under pH conditions <5.8 
. Therefore, it is unclear whether 1,2-propanediol, and by extension the subset of lactobacilli capable of reducing lactic acid, is critical to vaginal health or if this merely reflects an elevated pH, as is characteristic of a BV state. The reduction of lactic acid also typically results in acetic acid at a 1
1 ratio with 1,2-propanediol 
. In agreement with this, we observed a marked reduction in acetic acid, but only in SBVI, which may reflect additional metabolic pathways that utilize acetic acid in the vaginal microbiomes of metabotype II women.
The second most determinate metabolite for each symptomatic BV type was 1-acetoxy-2-propanol, which can be resolved from 1,2-propanediol in the presence of lipase activity 
. Many of the metabolites significantly affected in samples from women with a high Nugent score or displaying symptoms of BV types were fatty acids, including both SCFA and those with longer chain moieties. These and other metabolites found to be reduced in SBVI and SBVII (such as ethanolamine, glycerol, serine, phosphate) are related to glycerophospholipid metabolism and are highly suggestive of either reduced glycerophosphodiester phosphodiesterase-activity 
or rapid utilization of these and other host cell membrane degradation bi-products (such as cholesterol; 2-aminoethylphosphate which were also significantly decreased) in the symptomatic BV state. Consistent with the latter hypothesis, increases in the biomembrane lipid anchor, tetradecanoic acid, an essential structural component of host cell membranes and their derivatives, was observed in the vaginal metabolomes of SBVI patients (). We also noticed significant differences in amino acids characteristic of integral membrane proteins. Amino acids with aliphatic side-chains, predominant in the transmembrane regions spanning the middle of the phospholipid bilayer (leucine, isoleucine, alanine and valine) were all depleted in metabolome samples from women with SBVI and/or those with a high Nugent score. While the amino acids tyrosine and tryptophan, which are typically located at the polar/non-polar interface of transmembrane protein regions were depleted in samples from women with high Nugent scores. The finding that these observations overlap with Nugent scores implies an important role for G. vaginalis
and other Nugent-defined morphotypes in the reduction of cell wall integrity. In accord with this, the genomes of two G. vaginalis
strains isolated from women with symptomatic BV harbor mucinolytic enzymes 
We tested the interrelationships among microbial genera, metabolites, host behaviors, disease symptoms, Nugent score, and clinician-determined disease state using a systems-based networking approach. Although the nature of these analyses precludes conclusive evidence, they potentiate significant new insight into the underlying mechanisms driving the disease process. Features uncovered by systems-biology approaches can then be explored using more conclusive analyses.
Our networking approach indicated connections between odor and the biogenic amines putrescine and cadaverine. Putrescine and cadaverine are both formed in fish and shellfish by bacteria during decomposition 
and have been strongly correlated with the characteristic odor exhibited by spoiled fish 
. These matched the profiles of the characteristic odor of BV, described as having an amine or “fishy” smell 
. Coordinated with increases in these metabolites, we observed decreases in volatile compounds reported to exhibit more pleasant odors, such as 2(5H)-furanone 
and 2-ethyl-4-methyl-1,3-dioxolane 
. Putrescine and cadaverine were not universally increased in SBVII metabolome profiles; however, odor was only clinically ascribed to one of these subjects, compared to all SBVI subjects. In this SBVII individual, levels of putrescine and cadaverine were higher than in any asymptomatic individual. The lack of correlation between odor and SBVII was an interesting observation and may partially explain the linkage of douching to this group. Two recent papers have described the ability of douching to reduce or eliminate vaginal odor 
. Douching was not inversely correlated with either putrescine or cadaverine; however, such a correlation would only be expected if these metabolites were found more universally in the absence of douching (i.e., if not douching would equally lead to an increase in these metabolites). Although douching had a significant affect on the compositions of the microbial community and the metabolome, it did not alone explain the delineation between metabotypes I and II.
We observed links between 2-methyl-2-hydroxybutanoic acid and discharge, as well as diethylene glycol and pain. The nature of these linkages is less clear, but it is worth noting that interpretation of metabolomic data can be difficult. Being only privy to metabolites above an appreciable level (typically metabolic end-products or those produced faster than they are utilized) means pathway intermediates, and consequently the pathways leading to these metabolites are not "observed". This makes a strong case for combining metabolomics approaches with metagenomics and metatranscriptomics.
We identified links between four metabolites that appeared important to symptomology (putrescine, cadaverine, 2-methyl-2-hydroxybutanoic acid, and diethylene glycol) and several microbial genera. For example, Dialister spp., detected in all SBVI samples, were linked to increased levels of both putrescine and cadaverine and Gardnerella spp. were linked to increased diethelene glycol levels, while Mobiluncus spp. were linked to increases in 2-methyl-2-hydroxybutanoic acid, supporting roles for these bacteria in defining the BV state.
With the exception of douching, most factors relating to subject behavior were many steps removed from BV-positive Amsel criteria or from a high Nugent score within the resulting positive association network (). We therefore hypothesize that, while personal habits and sexual practices may be influential, or even be essential precursors, additional factors contribute to the onset of BV. Marriage had a strong negative correlation with Amsel-determined BV-positive women, but we did not observe similar associations with promiscuity or sexual frequency, suggesting changes in these behaviors did not account for this observation. Many speculations could be posited for this, but we hypothesize it relates to findings that couples share more similar microbial types 
, such that intercourse between stable sexual partners results in less perturbation of the vaginal microbiome.
Overall we have shown clear delineations between the vaginal metabolomic profiles of women displaying BV-positive Amsel criteria and those from asymptomatic women. Our results hint at the possibility of two different paths to the symptomatic BV state, with each sharing a common disruption of host epithelial cell integrity, but differing in some symptoms, implicative microbial taxa and their detectability by the Nugent method (). Based on these observations and an accumulation of evidence over the years, it is becoming increasingly clear that the break down of a typically lactobacilli-dominated vaginal microbiome only increases susceptibility to BV. The onset of disease symptoms requires the presence or absence of certain metabolites that may be positively or negatively affected by certain microbial metabolisms. Furthermore, symptoms of BV (discharge, odor, itching and pain) may not have a common origin, but instead may each be related to separate metabolic processes elicited by the presence of Dialister
spp., or other bacteria. Findings that horizontal gene transfer is extensive within the vaginal microbiome 
and that such genetic exchange has led to dramatic disparities in the virulence potentials of Gardnerella vaginalis
strains highlight the murky relationship between BV and specific microbial taxa. Metabolomic profiling may offer a fast and cost-effective alternative to other methods enabling definitive diagnoses.