Rep PCR Characterization of Lactic Acid Bacteria Isolated From Raw Cows’ Milk in Algeria.

Milk and indigenous fermented, milk based products are a rich source of lactic acid bacteria (LAB) and may serve as a source of potential probiotics. In the present study LAB were isolated from raw cows’ milk sampled at ve different Algerian farms. A total of 24 Gram positive, catalase negative isolates where identied to species level using a combination of (GTG) 5 -based rep-PCR ngerprinting and 16S rRNA gene sequencing. All isolates were identied as Enterococcus faecium and all isolates had highly similar rep-PCR proles. Four representative isolates were screened for acid tolerance, bile salts tolerance, antimicrobial susceptibility, antibacterial activity and haemolysis. The four selected strains all exhibited good tolerance to low pH (2, 3, and 4), and to bile salts (concentrations of 0.5%, 1%, and 2%) and were sensitive to chloramphenicol, vancomycin, tetracycline, gentamicine and peniciline G, but were resistant to oxaciline. Cell-free supernatants of the four tested strains all inhibited Staphylococcus aureus, Escherichia coli and Listeria monocytogenes but not Salmonella Typhi. No haemolytic activity was observed.


Introduction
Lactic acid bacteria (LAB) are contributing to texture and avour development of a wide range of fermented foods in addition to enhancing microbial safety and shelf-life [1], due to their production of lactic acid, acetic acid, H 2 O 2 , bacteriocins and diacetyl [2].
Probiotics are de ned as live microorganisms that, when administered in adequate amounts, confer a health bene t on the host. Desired characteristics include e.g. tolerance to acid and bile, cholesterollowering potential, ability to hydrolyze bile salt, being non-hemolytic, ability to possess antimicrobial properties and lack of antibiotic resistance [3].Cow's milk is widely consumed by Algerians (also more than goat and sheep milk) either as raw milk products or as traditional fermented types. Both raw cow's milk and indigenous fermented milk products are good sources of LAB [4].
Therefore, the objectives of this study were to isolate LAB from raw cow milk and investigate potentially important characteristics such as acid and bile tolerance, antibiotic resistance pro le, [5], and inhibition of known pathogens as a rst step towards developing probiotics based on Algerian, indigenous milkrelated isolates.

Sample Collection
Five raw cow milk samples were collected in sterilized bottles from different cow farms in Algeria. The samples were kept in ice boxes transported to the laboratory of Food and Industrial Microbiology, University of Oran Ahmed Ben Bella, Algeria.
Isolation of lactic acid bacteria Milk samples were incubated at 37° C until coagulation. Coagulated samples were 10-fold diluted, plated on MRS agar [6], and incubated anaerobically at 37°C for 48 h. Sixty colonies with different morphologies were subjected to Gram stain and catalase test. Twenty-four Gram-positive and catalase-negative isolates were subjected to further studies. Glycerol stock of LAB isolates were prepared and stored at -80°C. Prior to use ,the puri ed cultures were activated by sub-culturing twice (37°C, 24 hours) in MRS broth before use [7].

Identi cation of strains Phenotypic Characterization
Phenotypic characterisation of the LAB strains were performed as described previously [8].Provisional, tentative identi cation was made by Gram staining, cell morphology and catalase reaction. Further characterization such as production of gas from glucose, growth at different temperature (10, 15, and 45°C) and pH 9,6 as well as the ability to grow in different concentrations of NaCl (2%, 4% and 6.5%.w/v), Shermans test and survival after heating to 60°C for 30 min were carried out [9]. Further, hydrolysis of arginine and esculine [10], and production of acetoin from glucose, as determined by using the Voges-Prokauer test and the ability to ferment cellebiose, galactose, mannitol, melizitose, melibiose, ribose, trehalose, xylose, glucose, lactose, saccharose, fructose and arabinose were carried out [11].

DNA Extraction for PCR and sequencing Reactions
Overnight-cultures of each strain were grown at 37°C in MRS broth. Cells were harvested by centrifugation (12,000 rpm, 1 min). Genomic DNA was extracted using the Instagene Matrix Kit (Bio-Rad Laboratories), following the instructions of the manufacturer. Extracted DNA was stored at -20°C until use.
16S rRNA gene sequencing The 16S rRNA gene was ampli ed using the universal primers 27F and 1540R (Nielsen et al., 2007) under the following conditions: initial denaturation at 95°C for 5 min, 35 cycles at 95°C for 30s, 60°C for 30s and 72°C for 120s, followed by a nal extension at 72°C for 10 min. PCR products were sent to a commercial sequencing facility (Macrogen, South Korea). Sequences were manually corrected and assembled using CLC Genomics Workbench 8.0 (Aarhus, Denmark). Subsequently, the corrected sequences were aligned to 16S rRNA gene sequences in the GenBank database using the BLAST algorithm [14]. The nucleotide sequences obtained in this study have been assigned GenBank Accession Nos.

Technological charcterisation
Acid tolerance Resistance to acid conditions was determined according to [8]. Isolates were cultivated anaerobically in MRS at 37°C, the cells were centrifuged (3000 x g, 10 min) and washed twice with sterile saline (0.85% NaCl), and re-suspended in 10 ml of MRS broth. Cell suspension (1% of total volume) was inoculated into 10 ml of MRS with pH adjusted to 2.0, 3.0 or 4.0 using 6 N hydrochloric acid (HCl). The cultures were then anaerobically incubated at 37°C for 3 hours. At the end of incubation, the viable cells were enumerated by pour plate counts on MRS agar.

Bile tolerance
The ability of the isolates to grow in the presence of bile was determined according to the method of [15], with some modi cations. A suspension of 100 ml of cells was collected by centrifugation (3400×g, 10 min), washed twice in saline (8.5 g NaCl/L) and resuspended in 10 ml MRS broth with 0.5, 1% or 2% of oxgall (Sigma-Aldrich, MD, USA). At the end of incubation at 4 hours, viable cell counts were examined on a MRS agar plate using the spread plate technique. Experiments of acid and bile tolerance were repeated in triplicate.

Antimicrobial activity
The ability of the isolates to inhibit Listeria monocytogenes (ATCC 7659), Escherichia coli (ATCC 25955), Staphylococcus aureus (ATCC 7153) and Salmonella Typhi (ATCC 25925) was examined by the agar disc diffusion test [11]. 100µl of each pathogen was suspended in 4ml sterile water, approximately (10 8 CFU/mL) and applied onto PCA medium until absorption, after that, a sterile paper disc (5 mm) moistened with 20 µl of cell free supernatant (obtained by centrifugation (2500×g/10 min and neutralized to pH 6.5 ± 0.1 ) of each strain tested was added. Susceptibility of the indicator pathogens to the cell free supernatants was assessed by measuring the zone of inhibition of bacterial growth around the discs (radius -mm) after incubation for 24 h at 37 ºC. A clear zone of inhibition of at least 1 mm radius was recorded as positive [16]. The experiment was performed in triplicate.

Antibiotic susceptibility
The sensitivity to antibiotics was determined as described by (de Almeida Júnior et al. 2015) by the use of discs of antibiotics containing either chloramphenicol (30 mg/disc), oxacillin (1 mg/disc), vancomycin (30 mg/disc), tetracycline (30 mg/disc), cipro oxacin (5 mg/disc) or penicillin G (10 µg UI/disc). The tested isolates were propagated as lined out above and suspended in 4 ml sterile distillate water in order to achieve 0.5 McFarland turbidity standard, then the inoculums was spread onto MRS, after which the antibiotic disks was applied to the plates. After incubation for 24 h at 37°C, the zone of inhibition was measured. The experiments were performed in triplicate.

Hemolytic activity
The tested isolates were examined on Colombia blood agar after incubating in MRS broth at 37°C for 15 h. The hemolytic reaction was evaluated by observing both partial hydrolysis of red blood cells and the production of a green zone (α-hemolysis), as well as the total hydrolysis of red blood cells producing a clear zone around the bacterial colony (γ-hemolysis) or no reaction (β-hemolysis)

Results
A total of Twenty-four gram-positive, catalase negative coccoid isolates obtained from MRS were presumptively considered as LAB (Gram positive, catalase negative coccoid cells). The presumptive LAB isolates were clustered by (GTG) 5 -based rep-PCR ngerprinting, resulting in only one cluster, as shown in Representative isolates were selected for sequencing of the 16S rRNA gene followed by BLAST search at GenBank database. High similarities (99 − 100 %) between the isolates obtained during the present study and sequences deposited in GenBank were found. All isolates were identi ed as Enterococcus faecium. All isolates belonging to this group grew at 45°C as well as 10°C, and in media containing 6.5 % of NaCl, or with pH 9.6 and did not hydrolyze arginine and esculine. All had a broad carbohydrate fermentation pattern fermenting fructose, glucose, lactose, and mannitol, trehalose, amidon, sucrose and melibiose while not fermenting sorbitol, glycogen, rhamnose and melezitose.
Four isolates of E. faecium were subjected to a range of tests with the aim of assessing traits of potential importance for further investigations into probiotic properties. The isolates exhibited tolerance to acidic conditions (Table 1), though pH 2 were detrimental to all 4 strains. Isolates L11 and L13 retained the same level of viability after 3 hours at pH 3. When exposed to bile salts all isolates exhibited reduction in viability, with the exception of Isolate L9, which demonstrated high tolerance ( Table 2). The tested Enterococcus faecium strains were sensitive to penicillin G, chloramphenicol, gentamicin, tetracycline, and vancomycin and were resistant to oxacillin (Table 3). Furthermore, the selected isolates were screened for their antimicrobial activity using agar disc diffusion. All 4 tested isolates showed strong inhibitory activity against E.coli and S. aureus while only L11 and L13 were active against both L. monocytogens and S. Typhi. Isolate 9 also showed modest activity against L. monocytogenes ( Table 4). The four tested isolates showed no hemolysis of sheep blood.    Ch: chloramphenicol (30 mg/disc); Ox oxacillin (1 mg/disc); Tetra: tetracycline (30 mg/disc); Van: vancomycin (30 mg/disc); Gent: cipro oxacin (5 mg/disc); P: penicillin G (10 µg UI/disc).

Discussion
This study focused on isolation and identi cation of lactic acid bacteria from spontaneously fermented raw cow's milk. All isolates were identi ed as Enterococcus faecium with high certainty. Interestingly, all isolates had highly similar rep-PCR-pro les indicating high interrelatedness between the isolates even though they were obtained from milk from 5 different farms. E. faecium has many traits that makes it suitable as probiotic. Especially the rather broad-spectrum antimicrobial activity against pathogens makes them interesting. However, many E. faecium strains are resistant to a wide range of antibiotics and some strains have been implicated as opportunistic pathogens, making it essential to properly characterize the isolates, if they are to have potential use a probiotics in the future [17]. Consequently, a range of tests to establish fundamental traits important for putative probiotics were investigated. Further, acid and bile salts tolerance are important traits if a given putative probiotic strain is to survive stomach, which has pH varying between 2 and 4 and small intestine passage with a concentration between 0.

Conclusion
A total of 24 LAB isolated from row cow milk were characterized by phenotypic techniques and genotypic techniques using rep-PCR and 16S rRNA gene sequencing. All isolates were identi ed as Enterococcus faecium. Further, LAB isolated from cow milk exhibited some desirable probiotic properties in vitro, such as antimicrobial activity, bile and acid tolerance. More investigations may be warranted to elucidate its potential health bene t and its application as promising probiotic strain in fermented food and in the feed industry.