For over three decades, it is known that the human body comprises of ten-fold more microbial cells than the human cells. These microorganisms colonize every surface of human body whose exposure is to external environment such as skin, respiratory tract, oral cavity, urogenital and gastrointestinal tract. Of all these body sites, the most compactly colonized organ is gastrointestinal tract (GIT) (14). The human gut flora is a complex community of about thousands of different types of microorganisms including a great majority of bacteria while others are some yeasts and viruses. The bacteria of the gut flora are beneficial microorganisms, suppress the growth of the disease-causing bacteria and yeasts and thus have a very important role in human health. Probiotics are also a group of beneficial bacteria that stop pathogenic bacterial growth by competition, growth inhibition and attachment to gut epithelium. Studies have shown the beneficial effects of probiotics on immune system, destruction of pathogenic bacteria, inflammations, gastrointestinal diseases, colorectal carcinomas, anxiety, depressions, post antibiotics diarrhea and some kind of skin disorders (15).
Nowadays, probiotics are consumed as fermented dairy products such as yogurt and freeze-dried cultures. When they pass through stomach and small intestine, some probiotics survive and establish rapidly in the large bowel. The fermentation capability of colon is modified after the uptake of probiotics and the number of lactic acid bacteria will increase in the human feces (16). For the maintenance of health and disease control, dietary bacterial products along with carbohydrate supplements are preferred which help in building a barrier in the host against pathogens (17).
The enterococcal strains isolated in this study were screened for their ability to produce bioactive compounds. The antimicrobial activity of the methanolic crude extracts as well as supernatants was checked against Klebsiella, Staphylococcus, Pseudomonas, and Enterobacter. Almost all strains showed maximum growth inhibition of Staphylococcus and Pseudomonas while some strains also showed growth inhibition of Klebsiella and Enterobacter. The peptides i.e., enterococcins or enterocins produced by Enterococcus faecalis and Enterococcus faecium revealed the activity against Salmonella pullorum and Escherichia coli (18). Two bacteriocins which are produced by Enterococcus mundtii showed their antimicrobial activity against Listeria monocytogenes (19).
In addition to the production of bioactive compounds, the strains were also screened for their resistance against certain antibiotics by antimicrobial susceptibility testing. Majority of the strains were sensitive towards norfloxacin, streptomycin, vancomycin, nalidixic acid, and amoxicillin. Furthermore, the crude extracts of the selected strains of Enterococcus showed good cytotoxicity. Some strains such as AE2, AE12, WT8, and WT9 showed high cytotoxicity towards Artemia salina and the percentage was more than 50%.
The human colon, the most metabolically active organ because of the presence of diverse microbiota. The main role of these bacteria is to ferment the undigested food materials. The selected isolates were also screened for their antitumor activity against HCT-116 carcinoma cell line. The anti-tumor activity was checked at different concentrations and graphs were plotted where different concentrations were at x-axis while percentage cytotoxicity at y-axis. The gut microflora affects the GIT by showing resistance to pathogens, decreases gut tumors and blood lipids (14). The use of TLC with certain staining reagents and HPLC-MS/MS for chemical screening is a useful source for visualizing almost complete picture of microbial secondary metabolite pattern. So, new strains could be detected and microbial strain collection can also be modified using these techniques especially as a fundamental step of efficiently applied biological high through-put assays. The crude extracts of selected Enterococcus strains were examined by thin layer chromatography (TLC) using Anisaldehyde/HSO and Ehrlisch’s reagent as staining reagents. The bands of fractions were detected under short and long UV (254 nm and 366 nm). The crude extracts constituents displayed UV absorbance. After developing the TLC plate, it was stained with two different staining reagents. A pattern of secondary metabolites specific for each strain was observed and recorded. Results of TLC demonstrated that each strain exhibits a unique pattern of secondary metabolites.
The crude extracts of selected Enterococcus strains were analyzed through HPLC-UV. The HPLC chromatogram of the bioactive Enterococcus showed major peaks at shorter retention time and mostly there were two to three peaks which showed that there is more than one type of secondary metabolites present in the crude extract of the strains and the shorter retention time of peaks indicated that metabolites were of low molecular weight and potentially less harmful. Form the present study, it is concluded that the Enterococcus isolates are a useful source of many bioactive compounds having antimicrobial activity against many pathogens and could inhibit the growth of Klebsiella, Staphylococcus, Enterobacter and Pseudomonas strains. Cytotoxicity test of selected strains also revealed that they have high toxicity of more than 50% against brine shrimp. They could also be used against tumor cells as demonstrated by the in vitro antitumor activity against HCT 116 colorectal carcinoma cell lines. So, they can be used in the form of probiotics to treat many diseases particularly of gastrointestinal tract. They can also be used to enhance the indigenous gut flora because of their probiotic activity. TLC and HPLC also showed that these isolates produce secondary metabolites which can be used in combination to inhibit the growth and to provide safety against certain pathogens.