Sampling
During the honey harvesting season starting, between May and August 2019, a total of eight (n=8) raw honey samples were collected, under aseptic conditions, from different localities of Mostaganem region situated in north west of Algeria. All honey samples, were directly obtained from beekeepers and then were stored in sterile bottles at -20°C. Floral origins of these samples were provided by the beekeepers’ in relation to the locations where the beehives were situated (Table 1).
Isolation of Lactobacillus
Approximately ten grams (10 g) of fresh honey samples were aseptically weighed into a sterile stomacher bag and mixed with 90 mL of sterile 0.1% (w/v) peptone water for 2 min using a stomacher homogenizer. Then, one milliliter (1 ml) of the resulting solution was added to 9 ml of MRS broth and incubated at 30°C for 48 h, followed by serial dilutions with sterile peptone water (0.1% w/v). A volume of 0.1 ml was spread on several specific modified media, namely MRS agar (de Man, Rogosa, Sharpe), MRS agar with (0.8% CaCO
3), MRS agar with (0.1% L-cysteine and 2% fructose) and Rogosa agar. These specific seeded isolation media were incubated at 37°C, under anaerobic conditions for 72 h
(Feizabadi et al., 2020; Homrani et al., 2019).
Biochemical screening of Lactobacillus
Pure colonies grown on microbiological agar medium were tested biochemically for catalase activity by adding a drop of 3% hydrogen peroxide (H
2O
2). Only catalase-negative colonies (not releasing gas bubbles in the presence of H
2O
2) were retained for Gram staining and cell morphology tests by light microscopy. All catalase-negative, Gram-positive and non spore-forming bacteria were selected and stored in MRS broth supplemented with 15% glycerol and stored at -20°C (
Mahmoud Elzeini et al., 2020).
Molecular identification
Bacterial identification by the repetitive element palindromic-polymerase chain reaction (REP-PCR) was done as follows:
DNA extraction
The total bacterial DNA of selected isolates and reference strains used was checked and extracted using the method developed by BIO-RAD laboratories and adopted by (
Cholet, 2006;
Gevers et al., 2001; Koenraad et al., 2008;
Dahou et al., 2021).
From a well individual colony, a 1 ml suspension is prepared in 1 ml of sterile distilled water, then centrifuging for 10 min at 3000 rpm. The bacterial pellet is stored at -20°C for 1 hour, the pellet obtained is washed with 1 ml of TES buffer with its resuspensed in 300 µl of STET buffer (1 litter= 80 g Sucrose, 50 ml Triton, 18.6 g EDTA, 6.05 g Tris). An addition of 40µl of an SDS solution (sodium dodecyl sulphate solution) at 20% of a TE buffer composed of 10 Mm Tris-HCL and 1 mM EDTA, pH=8.0, the extraction of the lysate was carried out with an equal distributed between phenol, chloroform and iso-amyl alcohol, then phase separation is carried by centrifugation for 20 min at 3000 rpm. After mixing of the aqueous phase with 70 µl of 5 M NaCl and 1 ml of isopropanol, by the following the DNA precipitate is collected by centrifugation at 3000 rpm for 20 min. Concentration and purity of DNA extract was determined spectrophotometrically by weigh 260 nm.
DNA amplification by REP-PCR
The amplification of the DNA fragments obtained from the extraction was performed on a cycler from BIORAD (BIORAD, USA) by the use of specific primers wich will amplify the DNA fragment encoding the 16 S region.
Agarose gel electrophoresis
Visualization of the REP-PCR amplicons was performed by electrophoresis on 1.2% agarose electrophoresis was achieved using a fluorescent dye ethidium bromide (BET).
Comparison of DNA sequences obtained
The acquisitions obtained by the REP-PCR are compared with reference species
Lactobacillus plantarum subsp. plantarum ATCC 14917 and
Lactobacillus pentosus ATCC 8041 and with those from the Gene Bank data base using the program blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) from NCBI.
Pathogenic bacterial strains
Four pathogenic strains (n=4) responsible for bovine subclinical mastitis which are
Staphylococcus xylosus,
Staphylococcus simulans,
Klebsiella pneumonea and
Enterobacter spp, provided by the Laboratory of Sciences and Technics of Animal Production, University of Mostaganem were used as indicator strains for assessment of antimicrobial activity. These strains were identified by standard biochemical test (Bio-Merieux, France), API Staph and API 20E.
Antibiotic susceptibility test of bacterial strains
Antibiotic susceptibility, for the four pathogen strains, was performed using the disc diffusion method, according to the national committee of clinical laboratory standards (NCCLS) (CLSI. 2018). Broth cultures, of each pathogen strains were diluted in saline solution to about 10
7 CFU/ml (0.5 Units of McFarland turbidity standard). Subsequently, 100 μL of these growing cultures was spread on Mueller-Hinton (MH) agar. The antibiotic discs were then, placed on the surface of plates. After incubation at 37°C for 24 h, the diameters of the zone of inhibition around the discs were measured in millimeter using a ruler. The tested antibiotics and their concentrations were as follows: Amoxicillin + Acid clavulanic 20+10 µg, Penicillin 6 µg, Oxacillin 1 µg, Bacitracine 130 µg, Ampicillin 10 µg, Streptomycin 300 µg, Trimetoprim + Sulfametoxazol 1.25+23.75 µg.
Antibacterial activity of Lactobacillus isolated from honey
The overlay method, described by
Fleming et al., (1975) was used to evaluate the antibacterial potential of Lactobacillus isolates on selected pathogenic bacteria. Fresh culture of
Lactobacillus realized in MRS broth was inoculated in spot on MRS agar plates and grown at 30°C for 18 h under anaerobic conditions. The plates were then, overlaid with 8 mL of MRS soft agar (0.7% agar) seeded by 100 µl of indicator strain cultivated overnight. After 24 h of aerobic incubation at 30°C the diameter of inhibition zone was measured in millimeters (mm).
