Enterococci have evolved over ages as vastly amended members of the intestinal microbiota of a wide range of hosts and environments like water, soil, fermented foods, and dairy products. In dairy and food ecosystems, Enterococci perform dynamic functions resulting in fermentation and preservation of the food (Dinleyici et al., 2013). Due to their enhanced proteolytic, lipolytic, amylolytic, citrate utilization (Foulquié Moreno et al., 2006), and acidification abilities (Giraffa, 2003), they contribute towards the cheese ripening, aroma and flavor generation. Therefore, many strains have been utilized as primary starter or non-starter secondary cultures in some varieties of cheese (Coppola et al., 1990; Hugas et al., 2003). In dairy products, these are important sources of folate production. Mammalian cells are incapable of synthesizing folate, however, folate is vital for numerous essential cell functions, including metabolism, production of vitamins, amino acids, nucleotides, and cell division (Fox et al., 2015). Deficiency of folate leads to various disorders including birth defects in new born, heart diseases, risk of cancer or Alzheimer's disease (Da Silva et al., 2016). The Enterococci are not generally regarded as safe, (GRAS), but are well-known for their unique technological, probiotic, and anti-pathogenic attributes (Jamet et al., 2012). Their presence in fermented foods not only improves the sensory profile but also extends the shelf life through production of various bacteriocins (Martín-Platero et al., 2009). Enterocins are an important class of bacteriocins produced by the genus Enterococcus, and are known to have anti pathogenic properties against emerging pathogens in fermented and non-fermented food products (Callewaert et al., 2000; Franz et al., 2007).

Among Enterococci spp, Enterococcus mundtii is a ubiquitous bacteria, assigned as a member of group Enterococcus faecium on the basis of homology in 16S rDNA sequence (Klein, 2003). It has been associated with raw milk, plants, intestinal tract of humans and dairy cattle (Collins et al., 1986; Giraffa et al., 1997; Giraffa, 2003; Espeche et al., 2009). This bacterium has low GC content ranging between 38 and 39% and lacks catalase and cytochrome-C oxidase enzymes, but can contribute in carbohydrates fermentation to produce lactic acid. It produces enterocins such as Bacteriocin ST15, that are quite active against bacteria such as Pseudomonas, Clostridium, Klebsiella, Lactobacillus, and Acinetobacter etc. (De Kwaadsteniet et al., 2005; Ferreira et al., 2007; Settanni et al., 2008). It is reported to be used for the prevention of mastitis in cows (Espeche et al., 2009).

Due to increasing consumer's demand for safe and consistent quality of products, there is a growing interest toward the exploration of organisms from the diverse population of wild lactic acid bacteria from dairy sources (Wouters et al., 2002). The emerging genome sequencing technologies made it possible to control the genetics and metabolism of microorganisms (Law, 2001), resulting in greater demand of such functional microorganisms in industrial sectors (De Vuyst, 2000).

In the past few decades Enterococci have been considered as opportunistic pathogens, due to the presence of antibiotic resistance and virulence genes within their chromosome or extrachromosomal elements. They are not deemed to be safe, and are considered a sign of fecal contamination and a probable health risk. Hence, studies were restricted only to the clinical isolates of the Enterococci spp. However, in the last decade some studies have suggested the role of specific Enterococcus strains as probiotics for human and animal use. Some of the enterococci strains are supposed to be natural or added starters for fermented products. They are screened for the production of bacteriocin and the ability to act as a starter culture for fermentation (Venema, 2015). Clinical isolates of Enterococci spp. showed resistance against gentamycin, imipenem, penicillin, tetracycline, chloramphenicol, erythromycin, lincomycin, and rifampicin (Chingwaru et al., 2003; Çitak et al., 2004). Some E. faecalis strains are resistant to antibiotics, particularly to vancomycin and such strains are termed as VRE (Vancomycin resistant Enterococci). Antibiotic resistance is of particular interest in food ecosystems as genes for resistance are mainly present on plasmids, which can be transferred horizontally to other bacterial species (Zanella et al., 2006). Since they also contain antibiotic resistance and virulence genes, scientists are concerned about the safety of Enterococcus in food (Giraffa, 2003; Ogier and Serror, 2008).

The virulence of Enterococcus spp. is known to be a specific specie trait (Franz et al., 2001; Rice et al., 2003) and resistance against different antibiotics is not enough to classify a strain as virulent. Other traits which contribute to virulence include cell adherence, attacking factors, ability to release toxic substances, and other harmful factors (Eaton and Gasson, 2001; Dogru et al., 2010). Virulence factors are mostly encoded by, or linked with, transposable genomic elements such as plasmids, IS elements, transposons or phages. Large numbers of such elements are present within the Pathogenicity Associated Islands (PAI) (Schmidt and Hensel, 2004). PAIs are specific coding regions in the genome, which are associated with virulence factors (Hacker and Kaper, 2000). Pathogenicity determinants in clinical samples of Enterococci are quite significant in comparison to those obtained from animal and food matrix (Ben Omar et al., 2004; Khan et al., 2008). Many strains in Enterococcus faecium, including Enterococcus mundtii, are reported to produce biogenic amines in food because of their amino acid decarboxylase activity (Kalhotka et al., 2012). The generation of biogenic amines in food is usually considered an undesirable trait; it is used as a quality indicator for food and dairy products (Aldegunde and Mancebo, 2006). Safety evaluation of microbial strains is necessary for them in order to be incorporated in food and it is linked to their use, which includes the dose response mechanism. If the food microorganism remains viable in the intestinal tract of the host, they could lead to potential infection risks (Sanders et al., 2010). Therefore, the recommendation is to properly test the impact of new strains on laboratory animals' physiology and hematology (Shokryazdan et al., 2016).

Here we present the physiological, technological and safety properties of a strain E. mundtii QAUEM2808 isolated from fermented milk product, Dahi. Its genome was recently reported in genome announcement (Farah et al., 2016). Our results suggest that this strain is safe and has potential to improve the quality of the product as adjunct culture in milk fermentation.

The use of microorganisms for fermentation is well-known since the Neolithic age. Many bacterial strains have been used industrially for hundreds of years for the preservation of food, medication, and other valuable products. Bacteria during fermentation produce a variety of metabolic products which can give the food product several properties related to preservers, texturizers, stabilizers, flavoring and coloring. The enterococci constitute a complex and important group of bacteria occupying multiple ecological niches including human gut microbiome, fermented foods and plants. Usually it is attributed to their capacity to tolerate heat treatments and hostile ecological situations. They also have a significant industrial role in the production of meat and dairy products such as improvement of the aroma or ripening of the cheese. They can also produce bacteriocins against foodborne pathogens. Due to their role in the ripening, flavor refinement and anti-bacterial peptides production, enterococci with desired technical and metabolic characteristics have been proposed to be used as starter culture, adjunct culture or co-cultures in various fermentations. Because of the risks for the involvement of enterococci in spreading resistance to antibacterials and production of toxic agents like biogenic amines, it is compulsory to assess the safety of the strain isolated from foods of various geographical areas, particularly when they are intended to be used as starters or they have related roles in the fermentation and/or ripening of the traditional foods. In this perspective, the initiative of our study was to characterize enterococci strain isolated from the indigenous fermented milk product dahi, due to specific features i.e., occurrence of virulence agents, antibiotics resistance and the production of biogenic amines.

In the present study, Enterococcus mundtii QAUEM2808 showed maximum growth at 37°C. Hence, it was grouped as mesophilic. When two different NaCl concentrations were used, the strain showed little growth at 2% NaCl concentration, while at 4% NaCl concentration the growth rate was maximum for the strain Enterococcus mundtii QAUEM2808 (Fisher and Phillips, 2009). The Enterococcus mundtii QAUEM2808 showed proteolytic, lipolytic, amylolytic and cellulolytic activities in the respective media. Proteolytic and lipolytic activities are responsible for aroma and flavor development by food grade enterococci (Manolopoulou et al., 2003). RAST functional annotation also showed the presence of these genes as shown in Figure 8.

Being a salt tolerant strain, it could be a good candidate for the cheese ripening process. It was found that the Enterococcus mundtii QAUEM2808 was sensitive to commonly used antibiotics including Vancomycin, Ciprofloxacin, Norfloxacin, Tazobactactum, Doxycycline, and Bacitracin except Penicillin for which the strain was found to be resistant. These results are similar to the finding that most of the food reservoir Enterococcus species are less resistant to the commonly used antibiotics while some of the Enterococcus faecium strains were found resistant to the Penicillin in an early report (Kaçmaz and Aksoy, 2005). Resistance to penicillin has first been documented in 1946, while genes responsible for Pencillin resistance are primarily involved in the repair mechanism of the cell wall and are responsible for the stability of the organism (Barber and Rozwadowska-Dowzenko, 1948; Brooks et al., 2006). Unlike clinical isolates of Enterococci, our strain is found to be sensitive to vancomycin (Praharaj et al., 2013; Biswas et al., 2016; Rengaraj et al., 2016) justifying its presence in food matrix. When ResFinder-2.1 was used to search out antibiotic resistance genes present in the genome of E. mundtii QAUEM2808 with minimum 50% and maximum 80% similarity, the server did not detect any putative resistant gene (Zankari et al., 2012).

The E. mundtii QAUEM2808 showed moderate tyrosine decarboxylation activity at 37°C at 1, 2, and 3% NaCl concentration, while activity was negligible at higher NaCl concentrations (4 and 5%). Usually high concentrations of salt are used to reduce spoilage microbes from the fermented foods, during the process of avoiding food intoxication and spoilage (Linares et al., 2012). This reduction of microbes is due to the addition of salt and results in the reduction of biogenic amines producers. In cheese Enterococcus strains are the main cause of biogenic amine production but with the addition of 5% NaCl to the Enterococcus inoculated milk, very low concentrations of tyramine and 2- phenyl ethylamine were reported (Gardini et al., 2001). The folate production was observed in E. mundtii QAUEM2808. Maximum folate production was observed at 37°C in all media which indicated that growth of Enterococcus mundtii QAUEM2808 was optimum at this temperature. Overall in the study, MRS broth was found to be the best among the three media tested for folate accumulated in cultures of E. mundtii QAUEM2808 (Lin and Young, 2000). All the media had shown disparate behavior at other temperatures. Results indicated that Enterococcus mundtii QAUEM2808 grew well in a temperature range of 30–37°C and showed poor growth at extreme of temperatures i.e., 10 and 50°C as reported previously (Barbosa et al., 2016). Cornwell had revealed that lactic acid cultures not only synthesized folate but also consumed it. This could explain why at both maximum and minimum temperatures, Enterococcus mundtii QAUEM2808, instead of producing folate, consumed it for its survival (Patel et al., 2013).

Folate biosynthetic gene cluster of Enterococcus mundtii QAUEM2808 as predicted by KEEG pathway tool integrated with MicroScope (which can be accessed by following the link: http://www.genoscope.cns.fr/agc/microscope/metabolism/keggtabmap.php?Pid=00790&Beg=0&End=1&S_id=9820) was different from organisms reported earlier showing variations from other genus of lactic acid bacteria. The presence of genes folC and folA for enzyme polyglutamyl folate synthetase and dihydrofolate reductase enzymes indicated that folate production pathway is incomplete. It is assumed that this bacterium can grow in association with other indigenous microorganisms and work synergistically with them. The folC is among the common enzymes that have a role in biosynthesis of the folate. As reported in literature, the overexpression of folC increases the production of folate five times in L. lactis indicating the opportunity to enhance folate production in Enterococcus mundtii QAUEM2808. The presence of aminodeoxychorismate lyase pabC gene depicted the formation of PABA from chorismate which is one of the precursors of folate.

Several studies support the hypothesis of the production of folate by lactic acid bacteria. It is reported that industrial starter cultures such as Lactococcus lactis and Streptococcus thermophiles are the native producers of folate. For this reason, some fermented dairy products are supposed to have higher folate level than non-fermented products (Jones and Nixon, 2002; Aryana, 2003; Verwei et al., 2003; Jägerstad et al., 2004; Kariluoto et al., 2006). Different species of Bifidobacteria have the ability to produce folate (Rad et al., 2016). Among the enterococcus group, Enterococcus faciem is reported as a folate producer. It is reported that milk fermented with mixed cultures has high contents of folate (Crittenden et al., 2003).

The safety of microorganisms is an important factor while considering them as consumable food, and besides their beneficial role, they may also have adverse effects when administered live in a complex body metabolism (Pavan et al., 2003). For the last few decades the use of food cultures has increased in a significant manner, followed by the increase in safety guidelines by regulatory authorities ensuring the health and protection of consumers (Laulund et al., 2017). Body immune responses and histological profiles are reliable parameters to investigate the impact and safety of any administered agent. In this regard hematological and histological analysis were carried out, together with the data presented here, follow the safety of Enterococcus mundtii QAUEM2808 in balb/c mice. No harmful effect of this strain was observed on overall health. WBCs being active members of the cellular immune response toward any foreign agent of pathogenic category, are very sensitive to the presence of toxic agents in the body (Hodyl et al., 2008). RBCs and platelets count, along with other parameters in Blood picture, also deviate from the standard when the body gets infected by any pathogenic organism due to its abnormal metabolites and immunogenic substances (Benković et al., 2012). In our study WBCs, along with other immune cellular components, were within recommended reference ranges in the Blood CP of experimental group featuring Enterococcus mundtii EM2808 administration, which is a positive sign for its safety supporting.

Liver and intestine are exposed to all that is present in the gut as well as in the blood and are involved in the detoxification of harmful substances produced by invasive or pathogenic agents in the body, especially when entering through the oral route (Chodorowski et al., 2007). Therefore, the histological analysis of these organs is significant in order to comment responsibly about the compatibility and tolerance of the administered agent in the body. Abnormal Tissue morphology, distorted cellular adhesion, pigmentation and cellular inflammation lead to the loss of tissue function and it depends upon the extent of pathological aspects a tissue faces when encountered with toxic metabolites of any foreign organism (Sherlock and Dooley, 2008). Probiotics, the safe group of microorganisms, are known to prevent and maintain the hepatic functions and physiology in a healthy manner (Ewaschuk et al., 2007); as per the conclusion of our findings, Enterococcus mundtii QAUEM2808, which is emerging as a nonpathogenic food grade strain, also preserved the normal structure and physiology of mice as the tissue structure in the histological slides was preserved without any evidence of pigmentation or inflammation in hepatocytes and in overall liver sections examined.

The results of the present study demonstrated that Enterococcus mundtii QAUEM2808 was safe when close to the dosage of 2 × 10⁹ cfus per mice per day in a 90-days trail. It was well-mounted and no adverse effects were seen on the growth, blood components and vigorous organs of the experimental mice. The normality of parameters inspected above was very sensitive and significant to the safety of any foreign agent, therefore it was successfully concluded that the use of Enterococcus mundtii QAUEM2808 didn't have any harmful effect on the internal organs and systemic functions in treated mice (Shokryazdan et al., 2016). Virulence/resistance gene annotations results predicted that there is no pathogenic gene in Enterococcus mundtti QAUEM2808 genome. These results are comparable with those of (Repizo et al., 2014) who observed that Enterococcus mundtii CRL1656 does not contain the most significant virulence factors present in the clinical isolates of enterococci. Online prophage search and annotation showed that there was only one incomplete prophage of 7.036 kb with GC% 35.42. The size of the intact prophage was too small to be compared to the intact prophages detected in all the bacterial genomes, isolated from bacteria of dairy origin. A comprehensive analysis was done by Bonacina et al. (2016). Majority of the genomes published in NCBI contained prophages (Canchaya et al., 2003). The low number of prophages present in a genome is a sign of safety and stability as the genome of GRAS Lactococcus lactis contained six prophages (Canchaya et al., 2003). Analysis of CRISPER showed that Contig_40 contain a possible CRIPER between sequences 103,287–103,407 of 120 bp with one spacer. Tdim et al. found that resistance to antibiotics and ownership of CRISPR loci are inversely related (Tedim et al., 2015). Both the presence of CRISPR and the sensitivity to daily used antibiotics confirmed the safety of Enterococcus mundtii QAUEM2808. In silico analysis showed that Enterococcus mundtii QAUEM2808 carried class II and III bacteriocins. Class II bacteriocins are produced by streptococcal species to prevent closely related gram positive bacteria. This bacteriocinogenic member of lactic acid bacteria has been proposed as a probiotic strain which can stop mastitis in cows (Espeche et al., 2009). Bacteriocins are antibacterial short peptides which are mostly synthesized by bacteria ribosomally. Bacteriocins are small in size and when no similarity to known proteins is found then these ORFs are omitted from annotations due to their small size (de Jong et al., 2010). The BAGEL is the only available annotation tool which could be used to discover a new bacteriocins as it ensures that no putative bacteriocins is missed (de Jong et al., 2010). BAGEL classifies the bacteriocins based on similarity with identified bacteriocins, presence of motifs, features associated to a specific class and context genE. BAGEL is used for class specific mining of genomes for bacteriocins and it also classifies these putative bacteriocins into their specific classes viz, Class I, Class II, and Class III. Class I consists of lantibiotics which can be further sub classified, and class II are non-lantibiotics which can also be sub classified into groups A, B, C, and D. Class III consists of relatively large antimicrobial proteins (Dong et al., 2010).

Microorganisms present in a particular food are only considered safe and desired when their presence in food does not effect overall quality and quantity of the food or food products but improves the quality, quantity or texture of the food and food products. Their presence increases food demand for consumers and they have no side and adverse effects on the consumer's well-being and health. As the strain Enterococcus mundtii QAUEM 2808 is isolated from the indigenous fermented milk product, dahi is considered natural to this product. The indigenously fermented milk product dahi is known for its antipathogenic activity. That is due to low pH and anti-bacterial peptides. This bacteria, as shown by experiments, have acidification ability along with bacteriocin production and antipathogenic activities. Proteolytic, lipolytic, amylolytic, and cellulytic properties of the bacterium are considered indicators for the improvement in quality of the dairy products in terms of texture, aroma and flavor. In vivo, in vitro and genomic studies of the bacterium confirmed its safety if used in food and food products. The bacterium has the ability to produce folate, hence dahi having E. mundtii QAUEM 2808 can be used as functional food.

This study is approved by the ethics committee of Quaid-i-Azam University Islamabad Pakistan.

FN isolated and characterized the strain. MK did in-silico analysis and drafted the manuscript. AJ checked the strain for cytotoxic effect in mice. SB did genomic analysis. IA, NA, and MA helped in the interpretation of the results. MI supervised the study, critically reviewed and finalized the manuscript.