Bacterial isolates
Enterococcus faecium Smr18 was received from Dr. Sukhraj Kaur's laboratory. It was isolated from the swab samples of healthy vaginal microflora of woman after obtaining her written informed consent. The study was approved by the Human Ethics Committee of Guru Nanak Dev University, Amritsar, India. E. faecium was cultured in de Man Rogosa and Sharpe (MRS, Himedia Laboratories Pvt. Ltd., Mumbai, India) broth at 37 °C in anaerobic jars under stationary conditions. For conducting the experiments, E. faecium was propagated twice in MRS medium at 37 °C. All the chemicals used in the study were purchased from Himedia, except where specifically mentioned.
The strain was identified by using partial sequencing of 16sRNA done at National Centre for Cell Science, Pune, India. The sequence so obtained was compared with the known sequences of other Enterococcal spp. aligned by using National Center for Biotechnology Information—Basic Local Alignment Search Tool (NCBI-BLAST) database. Phylogenetic tree was constructed by using MEGA 6 software following Neighbourhood Joining method and Kimura2 Gamma I model. The strain was deposited to Microbial Type Culture Collection (MTCC), Institute of Microbial technology, Chandigarh, India with MTCC number 13248.
The pathogenic bacterial strains used in the study and procured from National Collection of Industrial Microorganisms (NCIM), Pune, India were Listeria monocytogenes NCIM 5277, Staphylococcus aureus NCIM 5718, Pseudomonas aeruginosa NCIM 2862, Shigella flexneri NCIM 5265, Klebsiella pneumoniae NCIM 5215 and Escherichia coli NCIM 5662. S. enterica serotype Typhi MTCC 733, S. enterica serotype Typhimurium MTCC 1251, S. enterica serotype Typhimurium MTCC 1252 and Streptococcus pyogenes MTCC 1927 were procured from MTCC. The pathogenic indicator bacteria were propagated at 37 °C under aerobic conditions in Brain heart infusion (BHI) broth.
Preparation of enterocin and its susceptibility to various enzymes
Purification of the enterocin ESmr18 was done by ammonium sulphate precipitation of cell-free culture supernatant (CS) of E. faecium followed by cation exchange chromatography. The proteins were precipitated from the CS by adding ammonium sulphate at 60% saturation (w/v) and mixing it on magnetic stirrer at 4 °C, for overnight. The precipitated proteins were separated by centrifugation (8000g; 10 min) and dissolved in sodium acetate buffer (20 mM; pH 4.5). The desalting of the precipitates was done by using Biogel PD-10 column (GE Health Care, USA) and the active fractions from the PD-10 column were pooled and referred as crude ESmr18.
For preparation of the purified ESmr18, the pooled fractions from PD-10 column were loaded onto SP-Sepharose Fast Flow cation-exchange column (5010 mm; GE Health Care) and the bound proteins were eluted by using linear gradient of 0.1 to 1 M NaCl. The active fractions were lyophilized and dissolved in distilled water. The purity of the protein was evaluated by electrophoresis on a 17% denaturing polyacrylamide gel and the protein concentration was evaluated by Bradford’s method (Bradford 1976).
Further, the susceptibility of various enzymes on the antimicrobial activities of crude and purified ESmr18 was determined. CS and ESmr18 were treated with enzymes proteinase K, trypsin, pepsin, and lipase (Sigma Aldrich, India) at the concentration of 1 mg/ml for 1 h at 37 °C, followed by heat inactivation at 60 °C for 10 min. The residual antimicrobial activity was determined by agar gel diffusion assay.
Antimicrobial activity
The antimicrobial activity of the CS of E. faecium, and the purified enterocin ESmr18 was determined against various pathogenic bacterial strains by using agar gel diffusion assay (Geis et al. 1983). CS was prepared by centrifuging the overnight culture of E. faecium Smr18 at 8000g for 10 min at 4 °C and then passed through syringe filters (0.22 µm) and kept at 4 °C till further use. For conducting agar gel diffusion assay the optical density (OD; at 550 nm) of pathogenic bacteria in log phase was adjusted to 0.1 and 100 µl of the culture was distributed onto BHI agar medium plates. A cork borer was used to cut wells in the agar plates with a diameter of 6.0 mm. Thereafter, 100 µl of CS (pH 6.5) crude extract and purified ESmr18 were added to the wells, and the plates were incubated at 4 °C for 4 h to allow the samples to diffuse. The plates were then incubated at 37 °C under aerobic conditions. After 24 h, the zones of inhibition were measured in millimetres.
Immobilization of E. faecium in films and antimicrobial activity of the films
Viable E. faecium Smr18 cells were immobilised in a sodium alginate film. The film was prepared by mixing sodium alginate (4% w/v), agar (3% w/v) and glycerol (20% v/v) in distilled water for 15 min on a magnetic stirrer at ambient temperature. The mixture was then sterilised by boiling for 20 min in a water bath. The solution so formed was mixed with autoclaved MRS medium in the ratio 1:1 under sterile conditions. 10 ml of the mixture was poured in Petri plates and allowed to cool down to semi-solid state before adding viable E. faecium cells (5 × 108 CFU). The plates were left undisturbed for 20 min. After 20 min, 20 ml of 2% calcium chloride solution was added for the polymerisation of sodium alginate film and the plates were again left undisturbed for 15 min. The extra calcium chloride was discarded, and the films were allowed to dry. Another film prepared by following similar process but without E. faecium cells was used as negative control. For determining the antimicrobial activity, films were cut with the help of well borer and placed on BHI agar plates inoculated with 100 µl of the overnight grown culture of S. enterica (OD set at 0.1). The plate was kept at 37 °C for 24 h and clear zones were measured.
The bio preservative efficacy of the film was tested against S. enterica-inoculated chicken model. Fresh boneless chicken (500 g) was procured from the local market and autoclaved for 10 min for complete sterilization. Overnight cultured S. enterica cell suspension containing 6 × 107 CFUs/g was added to the pieces. The chicken pieces were covered with E. faecium-immobilised film and film without E. faecium in separate Petri dishes and stored at 7–8 °C. The pieces (1 g) were removed at different time intervals and plated on Salmonella Shigella agar (SS agar) containing plates for CFU counting.
Efflux of potassium ions
To determine the mechanism of action of enterocin, we evaluated the effect of ESmr18 on the stability of the cell membrane of S. enterica MTCC 733 and L. monocytogenes NCIM 5277. Disruption of the cell membrane by the action of enterocin may result in efflux of small ions from the cell. Therefore, we evaluated the effect of ESmr18 treatment of pathogens at minimum inhibitory concentration (MIC) values on the extracellular potassium ion concentration (McAuliffe et al. 1998). The bacterial cells in mid-log phase were harvested by centrifugation at 8000g for 5 min to obtain cell pellet. The pellet was washed twice and re-suspended in 2.5 mM sodium HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (pH 7.0) at OD600 1.0. Purified ESmr18 was added to the cell pellets of S. enterica and L. monocytogenes in two separate tubes to obtain final concentrations 3.2 µg/ml. Samples (1 ml) were taken at different time intervals and immediately chilled on ice. S. enterica and L. monocytogenes cells in HEPES buffer without ESmr18 was used as controls. The samples were filter sterilised (0.2 µ) to separate the cells and the potassium ion concentration in the supernatants was determined by flame photometry (Systronics 128, Gujarat, India). The experiment was performed thrice in triplicates.
Shelf life, stability, and bio preservative effect of crude ESmr18
Before the bio preservative effects of the crude preparation of ESmr18 was tested in chicken samples, it is important to study the shelf life of ESmr18 dissolved in water and sodium acetate buffer. The crude ESmr18 dissolved in distilled water and sodium acetate buffer (pH 4.5) were stored at refrigeration conditions (7–8 °C) for 6 months. At different time points twofold dilutions of the two samples were tested for its antimicrobial activity against S. enterica by agar gel diffusion assay in terms of arbitrary units (AU)/ml. AU is defined as the reciprocal of the highest dilution that showed zone of inhibition.
The stability of CS and crude Esmr18 at different pH and temperature treatments were evaluated. CS and crude ESmr18 were exposed to different temperatures (60, 80 and 100 °C) for upto 90 min and autoclaving for 40 min. The residual antimicrobial activity was determined by using agar gel diffusion assay. To determine the effect of pH, the pH of CS and crude ESmr18 was adjusted to different values ranging from 2 to 10 and incubated at 37 °C for 1 h. Thereafter, the pH was reset to 6.5 and the residual antimicrobial activity was determined by using agar gel diffusion assay.
The bio preservative effect of crude ESmr18 was determined on the chicken meat inoculated with S. enterica. Fresh boneless chicken meat was purchased from a local vendor in Amritsar, India. Fresh boneless chicken (500 g) was procured from the local market and autoclaved for 10 min for complete sterilization. Overnight cultured S. enterica cell suspension containing 6 × 107 CFUs/g was added to the pieces and then crude ESmr18 (15 µg/g). For vehicle control, sodium acetate buffer was used. The counts of Salmonella in different samples were quantified at different time points over a period of 35 days by plating on SS agar plates.
Hemolysis assay
Some bacteriocins are known to have toxicity against host cells. Therefore, we tested the hemolytic activity of ESmr18 against human red blood cells (RBCs) by using haemoglobin release assay (Paiva et al. 2012). For the preparation of RBCs, blood was drawn from persons over the age of 18 after obtaining their written informed consent. The protocol was approved by the Institutional Human Ethics Committee, Guru Nanak Dev University, Amritsar, and the study was carried out as per the guidelines of the Ethical Committee. Defibrinated human blood was centrifuged at 135g for 15 min at 37 °C and the RBC-containing pellet was suspended in 10 ml of phosphate-buffered saline (PBS; pH 7.2). RBC suspensions (500 µl) were treated for 1 h at 37 °C with 100 µl of various concentrations of ESmr18. The suspensions were then centrifuged for 5 min at 825g, and the haemoglobin release in the supernatant was measured at OD 415 nm. TritonX-100 (1%)-treated RBCs and PBS-treated RBCs were used as positive and negative controls, respectively. The percentage RBC lysis was calculated by using equation:
$$\left( {{\text{OD}}_{{\text{T}}} {-}{\text{OD}}_{{\text{C}}} } \right)/\left( {{\text{OD}}_{{\text{X}}} {-}{\text{OD}}_{{\text{C}}} } \right) \, \times {1}00.$$
ODT is OD415 of ESmr18-treated RBCs; ODC is OD415 of PBS-treated RBCs and ODX is OD415 of 1% triton-treated RBCs.
Safety evaluation of ESmr18 in fish
The use of ESmr18, warrants oral consumption, therefore it is important to determine the in vivo effects of the orally administered ESmr18. The in vivo effects of crude ESmr18 were evaluated in healthy Cirrhinus mrigala. The fishes having average length of 15–18 cm and average weight of 90–100 g were acquired from the Government Fish Farm, Rajasansi, Amritsar. They were transported to the lab and placed directly in acclimation tanks with tap water temperature at 24.8 ± 0.32 °C, dissolved oxygen 6.4 ± 0.09 mg/L, total dissolved solids 133.3 ± 2.33 mg/L, electrical conductivity 457 ± 1.15 S/cm and pH 7.01. During the acclimatisation and testing phases, the photoperiod was kept at a regular 12 h light–dark cycle. Throughout the trial, fish were given commercial fish food (fishmeal, vegetable proteins, and binding agents such as wheat) ad libitum at a rate of 2% of body weight. The test water was changed daily 1 h after feeding the fish.
To study the biosafety of crude ESmr18, 700 µg of crude ESmr18 was orally administered to a group of 6 fishes. The vehicle treated (VC) group was administered 200 µl of sodium acetate buffer (pH 4.5). The third group was left untreated (UT). The fishes were monitored for any behavioural change before and during the experiment. After 96 h fishes were sacrificed, the liver, kidney, and blood were taken and used in the comet assay. Blood was taken through cardiac puncture.
Comet assay
DNA damage in the blood, liver, and kidney of the fishes in the treated, VC and UC groups was determined by using comet test (Yun et al. 2014) with minor changes. Slides covered with 1% normal melting point agarose were layered with 0.75% low melting point agarose containing blood, liver, and kidney cells and allowed to settle at 4 °C. The slides were subsequently submerged for 2 h at 4 °C in cold lysing buffer (2.5 M NaCl, 100 mM EDTA, 0.25 M tris aminomethane, 0.25 M NaOH, 1% triton X-100, 10% DMSO, pH 10.0). The slides were then coated again with 0.5% normal melting point agarose and allowed to solidify. Electrophoresis was carried out for 20 min at 25 V and 300 mA after the slides were coated with electrophoresis buffer (1 mM EDTA and 300 mM NaOH; pH 13). The slides were neutralised with 0.4 M Tris amino methane (pH 7.5) for 15 min, dried and stained with 20 μg/ml ethidium bromide. Analysis of the slides were done by fluorescence microscope (Nikon ECLIPSE E200) and images shot with Nikon D5300 camera. For each treatment group, 100 cells per sample were scored in triplicate. Various parameters like tail length (TL), tail moment (TM), and % tail DNA were calculated using Casplab Software.
Micronucleus test
Homogenous blood smear of fish was prepared on a clean glass slide and air-dried for half an hour at room temperature. The slides were fixed in methanol, stained with 5% Giemsa dye for 15–20 min, and 1000cells/group were scanned at 100× by using light microscope (Olympus scanner; CX31) for evaluating any nuclear or cellular abnormalities.
Statistical analysis
All the experiments were carried out in triplicates, and bars depict means ± SD standard deviation. To determine differences between mean values of different groups, a one-way analysis of variance (ANOVA) was used. The different treatment groups were compared by using the Tukey's test and the level of significance was set at 5% (p < 0.05). The software SPSS version 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.