Untargeted metabolite profiling of Enterococcus villorum SB2, isolated from the vagina of pregnant women, by HR-LCMS

Keywords: Enterococcus; LC-HRMS; Secondary metabolites; Metabolome; Food; Therapeutics

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Enterococcus belong to the class of lactic acid bacteria. They are the inhabitant of the gastrointestinal tract of humans and animals. They have been reported to play an important role in the food industry due to their role in flavor development in cheese and fermented milk products, antimicrobial properties, and probiotic potential. Enterococcus spp. contribute to flavor development due to citrate metabolism, proteolysis, and lipolysis (Moreno et al. [43]). They also produce enterocins with the anti-Listeria activity that shows inhibition towards other pathogenic microbes like Clostridium spp. and gram-negative bacteria like E.coli, Vibrio.cholerae, S.aureus etc. (Kang et al. [33]; Cotter et al. [13]; Phumisantiphong et al. [54]; El-Gendy et al. [22]). Their antimicrobial property enables their potential use as a starter culture to produce cheese varieties like mozzarella, feta, venaco, and cebeiro (Hadji-Sfaxi et al. [26]; Hayaloglu [29]).

Enterococcus spp. has also been studied for its potential probiotic properties, apart from the above industrial applications (Nami et al. [45]; Hanchi et al. [28]; Dinçer and Kıvanç [18]; Anjum et al. [2]). Two probiotic capsules, one containing combination of Enterococcus strains with other probiotic strains such as L.acidophilus and Bifidobacterium and the other containing E.faecium in combination with B.subtilis has been used in liver cirrhosis to prevent gastrointestinal dysfunction (Zhao et al. [69]). Enterococcus like E.faecium strain SF68, E.faecium CRL83, and E.faecium PR88 have been approved and are currently widely used to treat diarrhea, irritable bowel syndrome, developing cheese flavour, as well as in feed probiotics (Giraffa [23]; Motarjemi and Adams [44]). Although Enterococcus has been explored a lot for its different applications in the dairy, medicine, and other industrial uses, very little research has been targeted to explore the industrially important biomolecules, such as its secondary metabolites and possible industrial application. Since Enterococcus use in the food industry is limited due to its possible pathogenic behavior, the study of these secondary metabolites and their subsequent purification can reveal new applications of Enterococcus in the food and pharma industry.

The microbiome of the vagina during pregnancy has not been explored much. The metabolites secreted by the vaginal microbiome play an important role in maintaining maternal health and the development of the fetus. The gut microbiota dysbiosis among the infants results in metabolic dysfunction, which leads to different health problems, especially asthma or lung disorders as they are not exposed to many anti-inflammatory metabolites (Smith et al. [61]; Arrieta et al. [3]; Durack et al. [20]). The maternal microbiome and its metabolites play a huge role in developing the immune system of the infant. The vaginal microbiota is known to be dominated by lactic acid bacteria, including Lactobacillus and Enterococcus species, which secrete different bactericidal compounds and get transferred to the neonate, developing the microbiome of the infant. The lactic acid produced by these bacteria also helps fight various vaginal infections and maintains health (Ravel et al. [55]; O'Hanlon et al. [49]; Petrova et al. [53]; Bhagwat and Annapure [9]). There has not been much research on the untargeted global metabolites produced by vaginal bacteria during pregnancy. This research gives an insight into the Enterococcus species isolated from the vagina of pregnant women. From limited reports available on the vaginal metabolites, it is known that the vaginal microbes secrete metabolites, including mainly short-chain fatty acids, amine, organic acids, nitrogenous bases, amino acids, and carbohydrates (Oligosaccharides or monosaccharides)(Cunha et al. [15]; Tachedjian et al. [62]).

In a previous study done by Bhagwat et al. (Bhagwat and Annapure [8], [10]), the Enterococcus strains were isolated from the vagina of pregnant women. In earlier studies, it was revealed that few of these isolated Enterococcus strains did not show any virulence factors and exhibited probiotic properties indicating the possibility of their application in food and therapeutics. Thus, the previously isolated and reported potential probiotic strain of Enterococcus villorum SB2 was selected in this study for global metabolite profiling to study the important metabolites for possible industrial application.

Three types of media M17 (Tryptone, 2.5 g/l; peptone, 2.5 g/l; soya peptone, 5 g/l; yeast extract, 2.5 g/l; HM peptone; Lactose, 5 g/l; ascorbic acid, 0.5 g/l; Disodium-β-glycerophosphate, 19 g/l and Magnesium sulphate, 0.25 g/l), MRS broth (Proteose peptone, 10 g/l; Peptone, 10 g/l; yeast extract, 5 g/l; Dextrose, 20 g/l; Tween 80, 1 g/l; Ammonium citrate 2 g/l; Sodium citrate, 5 g/l; Magnesium sulphate 0.1 g/l; Manganese sulphate 0.05 g/l; and dipotassium hydrogen phosphate (K2HPO4), 2 g/l) and LAPTg media (Tryptone, 12.5 g/l; Yeast extract 7.5 g/l; dextrose, 10 g/l; sodium citrate, 5 g/l; sodium chloride, 5 g/l; K2HPO4, 5 g/l; Magnesium Sulphate, 0.8 g/l; Manganese chloride, 0.14 g/l; ferrous sulphate, 0.40 g/l; tween 80, 0.2 g/l and thiamine hydrochloride, 0.001 g/l) were purchased from Himedia. All other chemical and solvents were also purchased from Himedia. The potential probiotic bacteria Enterococcus villorum SB-2, previously isolated by Bhagwat and Annapure ([9]) from vaginal source of pregnant women was used for this research.

Glycerol stock of E.villorum SB2 was revived by inoculating 100 µl of stock into 2 ml MRS broth and incubated at 37 ℃ overnight in a rotary shaker at 150 rpm. After revival, the culture was plated and stored for further experiments. Fresh Glycerol stocks (50%) were prepared, stored at – 20 ℃ and subcultured whenever required.

For the growth curve study, a single colony was inoculated into the 10 ml M17, MRS, and LAPTg broth in different tubes and incubated overnight in a rotary shaker at 150 rpm maintained at 37 ℃. After overnight incubation, 5 ml of inoculum at OD600 = 1 was inoculated into 100 ml of M17, MRS, and LAPTg broth and again incubated at 37 ℃ for 24 h at 150 rpm. The OD was taken at regular intervals of 1 h at 600 nm for 24 h to record the growth curve. The growth studies in all the three media were done in triplicates and the average of the readings were used to plot the graph. For evaluation of Colony Forming Units (CFU), the 1 ml culture was taken from all the three broths at every 1 h interval and serially diluted 7 folds using sterile saline (0.9%). The cells were enumerated by plating the 100 µl of the dilutions (5th,6th and 7th) onto agar plates. Plates with 30-300 CFUs were considered, and the growth curve was then plotted using the data.

The bacterial growth is considered as a function of ln(OD). The doubling time and growth rate were calculated using the OD values as per Hall et. al. ([27]) using following formula (Eqs. 1&2). Growth rate here is estimated as change in OD per minute.

1 $$ln\left(\frac{\mathit{Nt}}{\mathit{No}}\right)=k\left(t-to\right),$$

Graph

2 $$Td=ln\left(2\right)/k,$$

Graph

where, Nt = No. of cells at time t, No = Initial number of cells, to = initial time, k = growth rate constant, Td = doubling time

Growth rate constant (k) is taken as the slope of log OD600 vs. time. Three growth media were tested to select the production media that gave maximum culture growth. The media giving the highest OD and CFU was selected as production media for the secondary metabolites.

For hemolysin activity, the protocol by De Vyust et al. ([16]) was followed. The hemolysin production by the E.villorum SB2 was analyzed by growing the bacterial culture overnight in an MRS medium at 37 ℃. The culture was then streaked onto the blood agar base plates supplemented with 5% sheep blood. Plates were incubated for 24 h at 37 ℃. The plates were then examined for hemolysin activity by observing the presence of clear zones around the colonies, green zone, or partial hemolysin activity or no reaction.

For metabolite production, 5% inoculum with approx. 10–10 CFU/ml was used to reduce the lag phase and ensure higher cell density. A single colony of E.villorum SB2 was taken with the help of inoculating loop and inoculated into 5 ml fermentation media broth and incubated in a rotary shaker at 37 ℃ overnight under aerobic condition. The 5 ml overnight culture at OD600 = 1 was then inoculated into the 100 ml media broth and incubated for 24 h at 150 rpm. The fermentation was carried out at a pH of 5.5. After 24 h, 10 ml of the cell culture was filtered using a 0.2 µ syringe filter to remove the cells. The filtrate was stored at − 20 ℃ as the extracellular fraction. Media without any microbe was used as control.

To extract intracellular metabolites, the cells were first quenched using the cold Glycerol-saline method described by (Villas-Bôas and Bruheim [65]). Briefly, 10 ml of the culture was transferred to Cold Glycerol-saline solution (3:2) at − 20 ℃. The mixture was vortexed and incubated in the ice bath for 5 min. It was then centrifuged at 10,000 RPM for 30 min at − 20 ℃. The supernatant was discarded, and 2 ml of wash solution (1:1 Cold Glycerol solution) was added to the cell pellet and again centrifuged at 10,000 RPM at − 20 ℃. The supernatant was again discarded, and the cell pellet was used to extract intracellular metabolites. 2.5 ml of 50% cold methanol was added to the cell pellet, vortexed, then kept at − 80 ℃ for 30 min, and then thawed on an ice bath. Three freeze–thaw cycles were done to ensure the breakage of cell wall and release of intracellular metabolites. After the freeze–thaw cycle, the tubes were centrifuged at 10,000 RPM for 30 min at − 20 ℃. The supernatant was stored at -80℃ as the intracellular fraction.

For concentration of the samples, the extracellular fraction and the extracted intracellular fraction were subjected to lyophilization, and the sample was stored for analysis by HR-LCMS.

The samples were analyzed using Agilent, G6550 iFunnel Q-TOF equipped with Hypesil GOLD C18 column (100 × 2.1 mm- 3micron). The acquisition was performed in both positive and negative ionization mode. The mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in Acetonitrile (B) was carried with an elution gradient as follows: 1 min, 5% B; 20 min, 100% B; 25 min, 100% B; 26 min 5% B; 30 min, 5%B, which was delivered at 0.3 ml/min. The total run time was 30 min. The column temperature was maintained at 40 ℃. The acquisition software used to evaluate MS data was 6200 series TOF/6500 series Q-TOF B.05.01(B5125.3), which collects and does the MS/MS spectra acquisition depending on pre-set criteria. A dual electron ionization source was operated in positive and negative mode to obtain full scan mass spectra from m/z 120-1100 with a scan rate of 5 spectra/sec. Source parameters were: Gas temperature, 250 ℃; Gas flow rate, 11–12 l/min; nebuliser, 35 V. Additional scan source parameters were: Vcap, 3500 V; Fragmentor, 175 V; Skimmer 1, 65 V; Octopole RF peak, 750 V. The sample injection volume was 5 µl. All the compounds detected were identified using Metlin database.

The absorbance of E.villorum SB2 culture was taken at 600 nm for 24 h at every 1-h interval. The graph of OD against time was plotted to obtain the growth curve. The growth curve depicted three phases, lag phase, log phase, and stationary phase. As can be seen from the graph (Fig. 1), usage of 5% inoculum decreased the lag phase, and the exponential phase started within the first hour of fermentation. The growth of E.villorum SB2 became stationary after 18–20 h of fermentation. The highest growth was observed in M17 media compared to MRS and LAPTg media. This can be seen in Fig. 1a, b and c.

Graph: Fig. 1 Growth curve of E.villorum SB2 in different media (M17, MRS and LAPTg) showing highest growth rate (k) in the M17 media a Growth curve based on OD600 values in different media (Purple- M17 media, Green MRS media and Yellow- LAPTg media), b Doubling time (Td) and Specific grpwth rate (k) of the E.villorum SB2 (Green – M17 media; Blue MRS; Yellow- LAPTg) based on the OD600 values with standard deviation c Growth curve based on CFU values in different media (Purple- M17 media, Green MRS media and Yellow- LAPTg media)

The growth rate was highest for M17 media (Td = 1.6 h & k = 0.4 h−1) as seen in Fig. 1b. This implies higher bacterial growth in M17 media in comparison to MRS and LAPTg and thus, was selected as the production media for metabolite profiling of E.villorum SB2. Selection of M17 media is solely based on healthier bacterial colonies and better bacterial growth as compared to other media. However the difference in the metabolite production in different media needs to be studied in future studies.

The tested strain of E.villorum SB2 did not show any zone around the colonies, indicating that they do not produce hemolysin and hence are called γ hemolytic as there is no lysis of hemoglobin. This lack of hemolysin activity makes them safe for different industrial applications, including food industries. This result is also supported by the previous report of Bhagwat et al. (Bhagwat and Annapure [8]), who found the absence of cytolysin gene (Cyl A) in the E.villorum SB2 strain, confirming the absence of virulence factor.

The detected compounds were identified using Metlin database. The compounds were identified by Auto MS/MS search. As per their software the search was performed based on the mass with a tolerance of 20 ppm and was limited to 10 best searches. The exclusion criteria included compounds in range of ± 10 ppm error and with minimum hits. The LC-HRMS analysis of the extracellular and intracellular fraction of E.villorum SB2 revealed various important compounds that have not been reported earlier. A total of 58 extracellular metabolites and 49 intracellular metabolites were identified. Out of the total metabolites detected, 20 extracellular and 12 intracellular metabolites could not be identified. The extracellular and intracellular metabolites revealed the presence of various aromatic and aliphatic compounds that included fatty acyl glucosides, organic acids, amino acid derivatives, carbohydrates, phenyl compounds, ketones, indole derivatives, lipid derivatives, and flavonoids.

Some extracellular metabolites produced by E.villorum SB2 (Table 1) include amino acids derivatives, phenylacetaldehyde, aminoglycosides (Fortimicin A), terpenoids (Austinol), Tryptophan derivative (Indoleacrylic acid,), ribonucleosides (Adenosine), aromatic compounds (p-mentha-1,3,5,8-tatraene), amino acids, Lactic acid, peptides, ketones (6-hydroxypseudooxynicotine), flavonoids (Hordatine B, Quercitin 3-O-manoglycoside), glycerol derivatives (DL—Glycerol 1-Phosphate), and also soluble fibers which are secreted by the bacterial cell wall (4-beta-D-glucan). Intracellular extract (Table 1) depicted metabolites like Glutamate amino acid analog (L-Theanine), Galactose, Lactate, ketohexose deoxy sugar (L-Fuculose), acetylated glycerols (Glycerol-1-Propanoate), dipeptides (Threoninyl-Proline, Valyl-Glycine), Butyrolactones (5-Butyltttrahydro-2-oxo-3-furancarboxylic acid) and many others. A list of detected metabolites is mentioned in Table 1 and the obtained chromatograms are shown in Figs. 2 and 3.

Table 1 List of Extracellular and Intracellular metabolites produced by E.villorum SB2, detected by LC-HRMS

Graph: Fig. 2 LC-HRMS chromatogram of extracellular metabolites of E.villorum SB2 in a positive ionisation mode and b negative ionisation mode showing peaks of different metabolites

Graph: Fig. 3 LC-HRMS chromatogram of intracellular metabolites of E.villorum SB2 in a positive ionisation mode and b negative ionisation mode showing peaks of different metabolites

Microorganisms produce a wide range of metabolites and exert their effect when they live in the human body as microbiota. Gastrointestinal microorganisms secrete various metabolites that play a role in the physiology and immune response of the body. A detailed study of these metabolites produced by the microorganisms present in the human body can help us understand their role in human health and discover new applications.

In the present study the growth of E.villorum SB2 was studied in three media, which resulted in the selection of M17 media for metabolite production as it gave maximum cell density. Glycerophosphate in M17 media seems to enhance the bacterial growth (Terzaghi and Sandine [63]). Enterococcus faecalis has also been reported to have better growth and survival rate when grown on M17 media than MRS (Carvalho et al. [12]). Owing to the reports available on the higher production capacity of bacteria in M17 media and higher E.villorum SB2 growth observed, this media was selected as production media for metabolites in our study. However, the effect of different media on the metabolite production can be studied in future studies to have a better understanding of the metabolite production in different media composition.

Hemolysin is a potent virulence factor found in pathogenic bacteria. It is responsible for increasing the severity of enterococcal infections in humans. Various researches have been done to determine the presence of this activity to examine the enterococcal pathogenicity (Ike et al. [31]; Libertin et al. [38]; Jett et al. [32]; Dupont et al. [19]). In a few reports, Enterococcus species, especially those isolated from food sources, e.g., E.faecium, have been found to be non-hemolytic (Eaton and Gasson [21]; De Vuyst et al. [16]). Production of hemolysin is associated with the presence or absence of a virulence determinant called the cytolysin (cyl) gene. The regulation of this gene product is also responsible for hemolysin activity. However, the hemolysis activity could also be seen even in the absence of the cytolysin gene, resulting from other cytotoxic components that cause the lysis of hemoglobin (De Vuyst et al. [16]).

The HR-LCMS of E.villorum SB2 extracts revealed some antimicrobial compounds secreted extracellularly into the fermented media like glycosides, including Fortmicin A, which is an aminoglycoside antibiotic of the astromycin class. Antimicrobial compounds like istamycins and fortimicins have been reported to be produced by many actinobacteria including Actinomycetes and Streptomyces species (Ossai et al. [52]). Fortimicin has broad-spectrum antimicrobial activity and can be used as an antibiotic (Girolami and Stamm [24]; Yamamoto et al. [68]; Nara et al. [46]; Huong et al. [30]). In the present study, other aminoglycosides have also been produced intracellularly, which include Istamycin C1, which is a bioactive molecule belonging to the 2,4,diaminocyclohexanol class. It is also reported to be produced naturally by the bacteria Streptomyces tenjimariensis, as a secondary metabolite that inhibits the ribosomal subunit (Nguyen et al. [48])..

From the limited reports available, it is found that the vaginal microbiota consisted mainly of Lactobacillus and Bafidiobacteriaceae. They detected the presence of indole-3-lactate, which is a tryptophan metabolite and has antimicrobial properties. The vaginal environment of a healthy pregnant women may be dominated by the different microbial communities that produce different metabolites to allow them to survive while maintaining vaginal health (Oliver et al. [50]). In the present report, the indole derivative of the beta carboline-alkaloid has also been found to be produced by E.villorum SB2. Many bacterial species have also reported these indole derivatives earlier, including Kliebsiella oxytoca, Shigella dysenteriase, Vibrio cholerae, and Enterococcus faecalis. Indole derivatives help in the bacterial process of biofilm formation. They can also be used for anti-viral, anti-inflammatory, antioxidant, and anti-malarial activities (Melander et al. [40]; Kumar and Ritika [35]). Some other tryptophan metabolites produced by E.villorum SB2 include indoleacrylic acid and 5-hydroxykinurenamine. 5-hydroxykinurenamine plays a major role as a neurotransmitter. Based on reports (Wlodarska et al. [66]), extracellular production of indole acrylic acid by E.villorum SB2 is hypothesized to be responsible for maintaining the epithelial lining of the vagina and preventing infection and inflammation.

Gut microbes have also been related to the production of neurotransmitters from tryptophan metabolism, as gut microbes can affect a person's behavior, mood, and anxiety. Probiotics and gut microbes including Enterococcus have the ability to affect tryptophan metabolism and regulate tryptophan availability, thereby resulting in the prevention of neurodegenerative diseases by reducing neurotoxins (Cryan and Dinan [14]; Agus et al. [1]; Dehhaghi et al. [17]). LC-HRMS of E.villorum SB2 metabolites has revealed the production of aminobutyric acid by this strain.

We have also found the presence of fatty acids like 2-Oxo-4-methylthiobutanoic acid and fatty alcohol Avocadyne 4-acetate. Lipids such as triterpenoids and terpene compounds (Table 1) have also been detected in the extracellular fraction. Terpenoids are synthesized by mevalonate pathway and reported to be produced by Streptomyces and Actinomycetales species. These terpenes also help in the flavor development of fermented wines. Production of terpenes has also been reported by lactic acid bacteria in cheese samples (Carrau et al. [11]; Belviso et al. [7]; Yamada et al. [67]). 2-Oxo-4-methylthiobutanoic acid is a fatty acid derivative with a sulfur group. It is synthesized from methionine and butanoic acid using the Methionine transaminase enzyme. Lactic acid bacteria produce both short-chain and long-chain fatty acids, where the fatty acids help these bacteria adapt to different environmental changes such as changes in pH, temperature, medium, etc. (Baldewijns et al. [6]). Fatty acids have been associated with the probiotic and postbiotic properties of many lactic acid bacteria. They play an important role in maintaining a healthy vaginal environment (Baldewijns et al. [6]). A meroterpenoid, Austinol detected in this report as the intracellular metabolite, has been earlier reported to be produced as a secondary metabolite by bacteria like Streptomyces from the marine environment, fungi like Aspergillus nidulans, animals as well as plants (Lo et al. [39]; Russo and Milella [58]). These meroterpenoids have anti-cancer, anti-bacterial, anti-inflammatory, and anti-malarial properties, making them suitable for therapeutic applications. Terpenoids are also produced by bacteria under stress conditions or as defense mechanisms (Avalos et al. [5]).

In the present report, by LC-HRMS analysis of extracellular and intracellular metabolites produced by E.villorum SB2, we have reported several new metabolites, including some plant metabolites that were not reported earlier from Enterococcus species. However, the production of various plant metabolites has been reported from other bacterial sources, including gut microbes and some fungal sources. The deoxycytidine derivates have been reported to be produced by bacterial sources. They are used as an antiretroviral drug for the treatment of HIV (Asahi et al. [4]). Moving forward to the production of flavonoid compounds, flavonoid synthesis occurs through the flavonol biosynthesis pathway and has been detected mainly in plants. It is interesting to know that Flavanol synthesis has been reported by many human intestinal bacteria involved in converting rutin to quercetin. Enterococcus avium from the human intestine was shown to convert rutin to isoquercetin and finally to quercetin, reporting that Enterococcus bacteria have the potential to convert flavonoid glycosides to flavanol (Shin et al. [60]). In a report by Kim et al. ([34]) The isoflavone metabolizing bacteria isolated from the human intestine belonged to the Enterococccus and Lactobacillus species. Lactic acid bacteria play a crucial role in improving the flavonoid and phenolic content in food products and help in improving the taste, aroma, and health benefits of the product.

Detection of soluble fiber (1,4-beta-D-glucan) in the extracellular fraction of E.villorum SB2 indicates that this strain can also be used in prebiotic fiber. Soluble fibers come from the cell wall of bacteria, fungi, and some plants. They seem to improve digestion, cholesterol metabolism, and glucose metabolism in the body and improve growth viability and colonization of probiotic microbes. Soluble fibers are also reported to have a prebiotic effect on bacterial species of Bifidiobacteria, Lactobacillus, and Streptococcus bacteria (Russo et al. [57]).

Some steroidal alkaloids were also found to be produced in the present study. Although their production from Enterococcus species has not been reported, there is evidence of the production of steroidal alkaloids by Lactic acid bacterial species, e.g., L.plantarum 976H (Veselá et al. [64]). Sphinganine was also detected in the intracellular fraction of E.villorum SB2. It has a role as a protein kinase C inhibitor and acts as the precursor for ceramide. Lactic acid bacteria were found to reduce the mycotoxin levels in maize fermentation by sphinganine production (MOKOENA et al. [41]). Sphingolipids also find application in foods and are reported to be produced by gut microbes. The reports mention that assimilation of sphinganine alkyl is done primarily by the gut microbe, Fusobacterium, Mycoplasma, Cystobacter, Acetobacter, etc. (Olsen and Jantzen [51]; Lee et al. [36]; Rohrhofer et al. [56]).

Additionally one important thing to notice here is the intracellular secretion of Chloramphenicol-3-acetate, an acetate ester, and a co-metabolite of the Chloramphenicol. It is seen that the bacteria possessing the Chloramphenicol acetyltransferase enzyme are found to be resistant to Chloramphenicol. E.faecalis has been reported to possess this enzyme. Acetylation of Chloramphenicol makes the bacteria resistant to the antibiotic. Also, this activity can be found in antibiotic-producing bacteria Streptomyces (Gross et al. [25]). The secretion of this metabolite can account for the resistance of E.villorum SB2 to Chloramphenicol.

Pieces of evidence and reports on the occurrence of plant metabolic pathways in bacteria (Moore et al. [42]; Seshime et al. [59]) support the current report, where many plant metabolites (Phenols, Phenylpropanoids, terpenoids, and flavonoids) are secreted by E.villorum SB2. Many metabolites could not be identified.

As there are very limited reports on the metabolite profile of the Enterococcus species from the human origin, we have tried to explain the production of detected metabolites using researches on other related lactic acid bacteria to support our findings. The production of unreported new metabolites by E.villorum SB2, might be due to the source of the strain from where it originated, i.e., the vagina of pregnant women, where Enterococcus have survived and adapted to the environment by producing these metabolites and have maintained vaginal health at the same time. Production of the detected metabolites helps maintaining a healthy vaginal environment for fetal growth and maintains the immune system of the infant and mother. The probiotic potential of the studied E.villorum SB2 has already been reported (Bhagwat and Annapure [8], [10]), where it lacked any antimicrobial resistance gene and also displayed other probiotic properties.

The LC-HRMS analysis of metabolites produced by E.villorum SB2 from the vagina of pregnant women reveals some new metabolites that are reported for the first time from Enterococcus specie. The secretion of antimicrobial compounds, flavonoids, esters, phenols, fatty acids, and soluble fibers indicates the probiotic and therapeutic properties of the strain. During pregnancy, the vaginal environment can influence the microbial flora and their metabolite production to ensure the healthy growth of the fetus and transfer the healthy microbiome to the infant. This is only a preliminary study on the metabolite profiling of a new strain of bacteria E.villorum SB2, from vaginal source. The relevance of the metabolites can be studied further by doing the quantitative analysis in the further studies. A more detailed study of vaginal microflora and their metabolites and complete genome sequencing of E.villorum SB2 can be helpful to understand the bacteria in more detail and discover some new industrial applications.

We want to express our special thanks to Bhagwat et al. (Bhagwat and Annapure 2019b) for providing us the isolated Enterococcus strains for the current study. We would like to express our gratitude towards DST and SAIF/CRNTS, IIT Bombay, for providing HR-LCMS analytical facility for the research work.

All authors contributed to the study conception and design. Material preparation, data collection, analysis and manuscript drafting was performed by SSG. The guidance, comments on the manuscript draft, and approval was provided by Prof. USA. Both the authors read and approved the final manuscript.

The authors declare that no funds, grants or other support was received during the preparation of the manuscript.

The authors have no relevant financial or non-financial interests to disclose.

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