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A novel probiotic strain of Lactobacillus fermentum TIU19 isolated from Haria beer showing both in vitro antibacterial and antibiofilm properties upon two multi resistant uro-pathogen strains

Highlights • L. fermentum TIU19 was isolated from the ethnic traditional rice beverage, Haria.• L. fermentum TIU19 showed antagonistic activity against uro-pathogens in both planktonic and biofilm conditions.• L. fermentum TIU19 had potential probiotic traits.


Introduction
Urinary tract infections (UTIs) affect people of all ages around the world, especially young women, children, and the elderly (Magill et al., 2014). According to previous studies, almost 40% of women have suffered from UTIs at some point in their lives (Foxman, 2014). Earlier studies have reported that Gram-positive Enterococcus faecalis (Kline and Lewis, 2016) and Gram-negative Escherichia coli (Flores-Mireles et al., 2015) are the most prevalent organisms among the UTIs pathogens. Moreover, uro-pathogens are known to form a biofilm that protects the bacteria from host immune response and antimicrobial therapy. As a result, uro-pathogens that are biofilm producers, create obstacles to the treatment of UTIs. Bacteria are attached to the biofilm, helping to share antimicrobial resistance genetic material (Schroeder et al., 2017). Both E. coli and E. faecalis have also been known to form biofilms in the urogenital tract (Anderson et al., 2003). Furthermore, antibiotic therapy disrupts the normal microbial flora of the urinary tract, which can lead to more severe conditions (Flores-Mireles et al., 2015).
Conventional antibiotic treatment for the management of infections associated with multiple antibiotic-resistant E. coli and E. faecalis are difficult. In recent days, the continuous rise in multidrug resistance in both E. coli and E. faecalis has also created a challenge to effective treatment. Antibacterial compounds from natural sources may be an alternative to the treatment of multidrug-resistant bacteria (Vaseeharan et al., 2011). However, they still have some restrictions, such as natural antibacterial agents being less effective than antibiotic therapy (Gavarić et al., 2015). Since both E. coli and E. faecalis are normal microflora of the urinary tract, they cannot be easily eliminated with antimicrobial compounds. Therefore, more clinical studies need to be carried out to determine their role in UTI prevention (Al-Badr and Al-Shaikh, 2013).
Functional and safety properties should be considered during probiotic selection. Lack of virulence factors, multiple antibiotic resistance, survival under gastrointestinal tract (GI) conditions, biofilm-forming ability, and antimicrobial activity against pathogens are all important criteria for the selection of probiotic cultures (Silva et al., 2020). However, there are many commercial probiotics with a narrow spectrum of activity. Therefore, it is important to isolate and characterize novel strains that could have a broad spectrum of antibacterial properties. Among probiotic bacteria, Lactobacillus spp. has been shown to have beneficial effects on host health through a variety of mechanisms. (Didari et al., 2014).
In central and eastern India, Haria is the most popular traditional rice beer consumed by the ethnic people of the tribal community of that region. Previously, it has been reported that Haria has many ethnomedicinal properties (Ray et al., 2016), which have not been properly explored. According to a previous study, lactic acid bacteria (LAB) were found to be the dominant microbial community in Haria. (Ghosh et al., 2015a). The presence of probiotic bacteria in Haria, along with their therapeutic potential against pathogenic bacteria, is still limited. To our knowledge, only one study reported the probiotic potential of the L. fermentum KKL1 strain from Haria without revealing antimicrobial work using pathogenic bacteria (Ghosh et al., 2015). Probiotics such as lactobacilli are the dominant bacteria of the vaginal flora and possess antimicrobial properties that regulate other urogenital microbiota (Barrons and Tassone, 2008). Lactobacillus spp can involve acidification of the mucosal surface, inhibition of adhesion of pathogens, production of substances such as vitamins and immunomodulators, and synergistic activity with the host's immune system (Kaur et al., 2002). Clinical studies have established that oral administration of Lactobacillus can confirm its effects after reaching the vagina (Reid et al., 2001a). Previous studies stated that probiotic capsules containing Lactobacillus spp. were administered orally at a dose to prevent UTIs (Kontiokari et al., 2003;Reid et al., 2001b).
As a result, the study was designed and carried out in the following ways: (i) to isolate a probiotic strain from Haria, showing antibacterial activity upon uro-pathongens; (ii) to assess the characterization of probiotic properties and antibiofilm activity of the isolated strain; and (iii) to investigate the antagonistic efficiency of the isolated probiotic strain against two uro-pathogen strains.

Isolation and identification of the L. fermentum strain from Haria
To isolate a single probiotic strain, 100 µl of Haria was placed in De Man, Rogosa, and Sharpe (MRS) broth (Himedia, India) and incubated (CO 2 incubator) at 37 • C for 12 h in the presence of 5% CO 2 . After incubation, a 100 µl MRS broth culture was transferred to fresh MRS broth (pH 3, acidified with 5 N HCl) and incubated for 3 h more prior to be spread on MRS agar and further incubated for both verifying its viability and isolate their anti-uropathogenic activity in vitro.
The antagonistic activity of the isolated probiotic strains was determined by an agar overlaid assay, using E. coli MTCC41 as indicator strain. Briefly, a loop full of pure lactobacilli broth culture was inoculated in spot (5 mm in diameter) on MRS agar and incubated at 37 • C for 24 h as described further. (Halder et al., 2017).
Selected colonies were subjected to Gram staining and catalase test, and further identified as Lactobacillus spp. by analytical profile index (API) analysis using the HiLacto Identification Kit (Himedia).
The Gram-positive and catalase-negative pure culture of Lactobacillus sp. with the highest antagonistic activity against E. coli MTCC41 was identified by 16 s rRNA gene sequencing. Initially, genomic DNA was isolated using a QIAamp DNA Kit (Qiagen) and electrophoresis on a 1.2% agarose gel. 16 s rDNA gene was amplified by PCR (Thermo Fisher SCIENTIFIC) (initial denaturation at 95 • C for 5 min; Thirty-five cycles of denaturation at 95 • C for 30 s, annealing at 60 • C for 1 min, extension at 72 • C for 1 min; final extension at 72 • C for 7 min) from isolated DNA using the universal primers (forward primer 27F 5 ′ -AGAGTTT-GATCCTGGCTCAG-3 ′ , and reverse primer 1492R 5 ′ -GGTTACCTTGTTACGACTT-3 ′ ) (Srinivasan et al., 2015). The PCR products were analyzed using 1.2% (w/v) agarose gel electrophoresis, and contaminants were removed using a Qiagen Mini Elute Gel extraction kit (Qiagen). The forward and reverse DNA sequencing reactions of the pure PCR amplicon were performed by an ABI genetic analyzer. Nucleotide sequences were obtained and homology was analyzed using the Basic Local Alignment Search Tool (BLAST) (Pearson, 2013). The reference-type bacterial sequences were retrieved from the NCBI database and aligned with the sequences isolated by Clustal W. The CLC sequence viewer has constructed a phylogenetic tree.

Multiple antibiotic resistance index (MAR)
The Multiple Antibiotic Resistance Index (MAR) was determined using the standard protocol developed by the Clinical and Laboratory Standards Institute (CLSI), USA. The isolated bacteria were assayed for resistance or sensitivity (S-Sensitive and R-Resistant) against multiple antibiotics. The antibiotic sensitivity pattern of the selected strain was examined by using standard antibiotic discs (Ampicillin 10 µg, Vancomycin 30 µg, Gentamycin 10 µg, Kanamycin 30 µg, Streptomycin 10 µg, Erythromycin 15 µg, Chloramphenicol 30 µg, Tetracycline 30 µg) (Himedia, India) placed aseptically over the homogenous bacterial lawn on MRS plates. The plates were incubated at 37 • C for 48 h. After incubation, the diameter of the zone of inhibition (ZOI) was measured (Ayandele et al., 2020). All tests were performed in triplicate. The MAR index was calculated as follows

MARIndex =
Number of antibiotics ineffective Number of antibiotics used

Haemolytic activity and gelatinase activity
Haemolytic activity was investigated as described by Kaktcham et al. (2018). The overnight grown cells in MRS broth were streaked on a blood agar plate (Himedia, India) and incubated at 37 • C for 72 h. After incubation, a clear zone around the bacterial growth (Red blood cells were haemolyzed completely) demonstrated positive hemolysin activity, whereas no change around the growth (Red blood cells were not haemolyzed) indicated negative results. Experiments were performed in triplicate.
For gelatinase activity, 10 μl of a 6-hour culture was spotted on the nutrient gelatine agar (Himedia, India). The plates were incubated at 37 • C for 48 h. Positive results were indicated by clear zones around the colonies, while negative results were indicated by no zones around the colonies. Experiments were performed in triplicate.

Survival under conditions that simulated the human gastrointestinal tract
The log culture of bacterial cells (10 8 CFU ml − 1 ) was centrifuged at 5000 rpm for 15 min, and the pellet was washed twice with cold and sterile PBS (phosphate-buffered saline). Then the pellet was resuspended in PBS, and the obtained bacterial suspension was used to evaluate the survival of bacteria under human GI tract conditions. For the test of resistance to low pH conditions, cells were incubated in low pH solutions (pH: 2, 3, and 4) adjusted with HCl (1 N) for 0, 1, 2, 3 h at 37 • C. To assess resistance to bile salts, cells were incubated in PBS at pH 7.4, supplemented with 0.1, 0.2, 0.3 and 0.4% (w/v) oxgall (Sigma Aldrich, USA) for 0, 1, 2, 3 h at 37 • C. Simulated gastric fluid (SGF) was prepared according to Lian et al. (2003). At first, pepsin (Sigma Aldrich, USA) (3 g/lit) was suspended in sterile PBS. The solution was adjusted to pH 2, 3, 4 with 12 M HCl, and then filter sterilized through a 0.22 μm filter membrane (Tarson, India). Bacterial cells were re-suspended in simulated gastric fluid (SGF) solution and incubated for 0, 1, 2, 3 h at 37 • C. The simulation of intestinal fluid (SIF) was prepared according to Amakiri et al. (Amakiri and Thantsha, 2016). Briefly, pancreatin (Sigma Aldrich, USA) (10 g/lit) was suspended in sterile PBS. The solution was adjusted to pH 6.8 and 8 with 0.2 M NaOH or 0.2 M HCl and then sterilized by filter through a 0.22 μm filter membrane (Tarson, India).
Bacterial cells were resuspended in a simulated intestinal fluid (SIF) solution and incubated for 0, 1, 2, 3 h at 37 • C.
The resistance of the selected strain in these conditions was assessed in terms of viable colony count on MRS agar after the treatment (Son et al., 2018). Experiments were performed in triplicate. Survival rates were calculated according to the following equation.
% of survival = (N 1 / N 0 ) × 100 N 1 : The total viable count of bacterial cells after treatment. N 0 : The total viable count of bacterial cells before treatment.

Cell surface hydrophobicity
The cell surface hydrophobicity of the selected strain was assessed according to Kaktcham et al. (2017). The bacterial culture was centrifuged at 5000 rpm for 15 mins. The pellet was washed twice with cold and sterile PBS and resuspended in the same buffer to obtain an OD of 600 nm of approximately 1.0 (OD Initial ). Then, 3 ml of the bacterial cell suspension was mixed with 1 ml of xylene (Sigma-Aldrich, USA) and vortexed for 2 mins. The mixture was then incubated at room temperature for 60 min to separate the two phases (water and xylene). The aqueous phase was collected and its absorbance at 600 nm (OD Time ) was measured. Experiments were performed in triplicate.

Auto-aggregation
The overnight bacteria culture was centrifuged at 5000 rpm for 15 mins. The pellet was washed twice with cold and sterile PBS and resuspended in the same buffer to obtain an OD of 660 nm of approximately 1.0 (OD 0min ). After that, this cell suspension was mixed by vortexing for 2 mins, incubated at room temperature for 60 mins, and the optical density (OD 60min ) of the supernatant was determined at 660 nm. All experiments were performed in triplicate. Auto-aggregation was determined using the following equation:

Adhesion ability
HeLa cells were purchased from the National centre for Cell Science (NCCS) in Pune, India. HeLa cells were grown and maintained in a 24well tissue culture plate (Tarson, India) in 1 ml of Dulbecco's modified Eagle medium (DMEM) containing 0.11 g/l sodium pyruvate, 2 mM L-glutamine, 4.5 g/l glucose, 10% foetal bovine serum (FBS), 100 µg/ml penicillin, and 100 µg/ml streptomycin at 37C in an atmosphere of 5% CO 2 at constant humidity. For the adhesion ability of the selected strain, HeLa cells were seeded at a concentration of 1 × 10 5 cells/ml in a 24well tissue culture plate (Tarson, India). After incubation for 24 h at 37 • C, 10 µl of L. fermentum strain was diluted in sterile PBS at a concentration of 1 × 10 8 cells/ml and added to each well. After incubation for 2 h at 37 • C, the monolayers were washed three times with 500 µl of PBS and lysed with 0.1% (v/v) Triton X-100 (Sigma-Aldrich, USA). Cell lysates were serially diluted, spread on MRS agar, and incubated at 37 • C for 24 h. The adhesion ability (%) of the L. fermentum strains to HeLa cells was calculated as the percentage of adhered bacterial cells after 2 h (N 2h ) divided by the initial number of bacterial cells at 0 h (N 0h ) (Son et al., 2018). Experiments were performed in triplicate.

Biofilm forming ability
With some modifications, quantification of biofilm production was performed as previously described by Borges et al. (2011) with minor modifications. The 2 ml of MRS broth and bacterial cells of the selected strain (10 7 -10 8 CFU/ml) were added to each well of a sterile 12-well polystyrene microtiter plate (Tarson, India). The cells were then aerobically incubated at 37 • C for 0, 24, 48, and 72 h. Time 0 hour was used as control. As tests, time intervals of 24, 48, and 72 h are used. After incubation, planktonic cells were removed, and bacterial sessile cells were fixed with 2 ml of methanol (Finar Limited, India) for 15 mins. Subsequently, 2 ml of 2% crystal violet solution (Sigma-Aldrich, USA) was used for bacterial sessile cell staining and kept at room temperature for 10 min, and the excess amount of crystal violet was rinsed off using distilled water. After the dissolution with 2 ml of 30% glacial acetic acid (v / v), biofilm biomass was determined by measuring OD595.
The percentage of biomass formation was determined using the following equation:

Study of β-galactosidase production
For determination of β-galactosidase production, a colony of the selected strain was removed from MRS agar plates and thrown into tubes containing Ortho-Nitrophenyl-β-galactoside (ONPG) discs (Sigma-Aldrich, USA) and 100 μl of sterile 0.85% saline solution. Tubes were incubated at 37 • C and observed at an interval of 1 hour to 6 h. The color transition to yellow indicates a positive result in the production of β-galactosidase by releasing the chromogenic compound, o-nitrophenol (Vinderola et al., 2011).

Antioxidant assay
Quantification of the antioxidant assay was performed as previously described by Talib et al. (Talib et al., 2019). 1 ml of 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution (0.05 mM) was added to the different numbers of freshly prepared cells from 1.0 to 6.0-log CFU/ml in PBS. The reaction mixture was kept in the dark for 30 mins at room temperature. The DPPH solution in PBS and the different numbers of cells 1.0 to 6.0 log CFU/ml in PBS were used as control and blank, respectively, during this assay. The spectrophotometer (UV-visible Spectrophotometer 2206, SYSTRONICS) was used to measure the optical density (OD) at 517 nm to assess the DPPH scavenging capability. All tests were performed in triplicate.
The percentage of scavenging potential was estimated as follows: 2.4. In vitro tests to determine the antagonistic activity of L. fermentum against uro-pathogens 2.4.1. Antagonistic activity of probiotic bacteria against uro-pathogens Multidrug-resistant uropathogenic E. coli (amp R , tet R , str R ) and E. faecalis (amp R , tet R , str R , chl R ) were used as marker strains to check the antagonistic activity of the isolated strain. The antagonistic activity of an isolated strain was determined by an agar overlay assay (Halder et al., 2017). For this assay, multidrug-resistant pathogenic strains, such as E. coli and E. faecalis, were used. An aliquot of the selected L. fermentum strain (1 × 10 8 CFU/ml) was spotted on MRS agar and incubated at 37 • C for 24 h. The MRS agar plate was then overlayed with 9 ml of Tryptone soya agar (0.8% agar) mixed with 1 ml of each pathogenic strain (1 × 10 6 CFU/ml) and incubated at 37 • C for 24 h. The diameter (mm) of clear zones on the agar plate was measured. All tests were performed in triplicate.

Co-aggregation assay
The experimental protocol for the study of the co-aggregation assay was similar to the auto-aggregation assay. Probiotic bacteria (L. fermentum TIU19) and bacterial pathogens (E. coli or E. faecalis) were inoculated into the respective growth medium and incubated at 37 • C for 18 h. The cells were centrifuged at 5000 g for 15 min, washed twice, and resuspended in phosphate-buffered saline (PBS). Equal volumes (2 ml) of each cell suspension of single species pathogens (E. coli or E. faecalis) were mixed individually with L. fermentum TIU19 by vortexing for 10 s. Control tubes were set up at the same time, containing 4 ml of each bacterial suspension on its own, and the upper cell suspension was measured at OD 660 nm at 0 h. After 37 • C for 60 min of incubation, the optical density (OD60min) of the upper cell suspension was determined at 660 nm. Samples were taken in the same way as in the autoaggregation assay (Kos et al., 2003). All tests were performed in triplicate. The percentage of coaggregation was calculated using the equation: × 100 Where x represents L. fermentum TIU19 and y represents (E. coli/E. faecalis) each of the two strains in the control tubes and (x + y) the mixture.

Anti-adhesion assay
HeLa cells were seeded in a 24-well tissue culture plate (Tarson, India). After a 24-hour incubation at 37 • C, single species pathogens (E. coli, E. faecalis) and co-culture [(L. fermentum TIU19+E. coli), (L. fermentum TIU19+E. faecalis)] (at a ratio of 1:1 diluted in DMEM medium containing L-glutamine) were added to the 24-well tissue culture plate to a final concentration of 1 × 10 8 CFU/ml. After incubation for 2 h at 37 • C, the monolayers were washed three times with PBS and lysed with 0.1% (v/v) Triton X-100 (Sigma-Aldrich, USA). Cell lysates were serially diluted and spread in selective media, MacConkey sorbitol agar, and Pfizer selective Enterococcus agar for E. coli and E. faecalis, respectively, and incubated at 37 • C for 24 h. Each experiment was repeated three times. The ability of a given pathogenic strain to adhere to HeLa cells in the absence (N P ) and presence (N Mix ) of the L. fermentum strain was compared as follows (Son et al., 2017) Anti − adhesionability(%) = [(N P − N Mix ) / N P ] × 100 N P = number of bacterial cells, when the wells were seeded with only pathogenic bacteria. N Mix = number of bacterial cells when wells were seeded with L. fermentum -pathogenic bacteria mixture.

Biofilm inhibition assay
The isolated strain of L. fermentum TIU19 and uro-pathogens (E. coli, and E. faecalis) were grown overnight in LB media individually. The coculture [(L. fermentum TIU19+E. coli), (L. fermentum TIU19+E. faecalis)] was prepared by an equal volume of the overnight grown single-species culture in a 96-well microtiter plate (Tarson, India) (8.0-log CFU/ml) at the ratio of 1:100 dilution under aerobic conditions at 30 • C for 72 h. Here, single-species E. coli, E. faecalis were used as the control, and coculture [(L. fermentum TIU19+E. coli), (L. fermentum TIU19+E. faecalis)] used as a test. After incubation for 72 h, planktonic cells were collected from each well and adherent bacteria sessile cells (biofilm) in each well were resuspended with PBS by vigorous pipetting and vortexing followed by 30-s sonication. For the determination of E. coli and E. faecalis bacterial counts, cultures were seeded on Mac-Conkey sorbitol agar and Pfizer selective Enterococcus agar medium, respectively, and incubated at 37 • C for 24 h (Kaur et al., 2018).

Isolation, identification, and biochemical characterization of L. fermentum
There are only a few reports of the isolation of probiotic bacteria from Haria. In this study, from Haria, we are therefore attempting to isolate and establish a novel probiotic L. fermentum strain with antibacterial activity. Based on our initial screening, we selected bacteria with Gram-positive, catalase-negative, low pH (pH -3) tolerance, and antagonistic activity against the E. coli MTCC41 strain, based on our initial screening. API analysis allows us to select the strain belonging to the genus Lactobacillus (data not shown). Analyses of 16S rRNA gene sequence show that the strain has 99.3% identity with the type strain of Lactobacillus fermentum LMEM37. The strain was designated as Lactobacillus fermentum TIU19 (GenBank: MK752864.1) (Fig. 1A). L. fermentum has been previously reported to be a dominant non-pathogenic fermentable species of Haria with probiotic potential (Ghosh et al., 2015).

Assessment of safety and virulence factors of selected L. fermentum strain
According to the guidelines of FAO and WHO, before defining a strain as a probiotic, it is important to analyze both the functional properties and its safety for public health. We first determined the safety of L. fermentum TIU19 by determining the MAR index and assessing virulence factors such as gelatinase and hemolytic activity.

MAR index
The MAR index is a key parameter for understanding the drugresistant properties of microorganisms. The L. fermentum TIU19 strain was found to be sensitive to all antibiotics used, such as ampicillin, gentamycin, kanamycin, streptomycin, erythromycin, and chloramphenicol, tetracycline, and resistant to vancomycin. The MAR index was 0.125 and is shown in Table 1. If the MAR index value was less than 2, the isolate was not considered to be resistant to multiple antibiotics. Hence, the strain was not considered antibiotic-resistant (Resende et al., 2014).

Assay for haemolytic and gelatinase activity
The absence of virulence factors, for example, haemolytic and gelatinase activity, are crucial safety criteria for a probiotic strain. The L. fermentum TIU19 strain was tested for both haemolytic and gelatinase activity. The strain showed a lack of these virulence factors (Fig. 1B  and1C). This is very important as previously different Lactobacillus probiotic strains had similar results and were therefore considered safe for applications as probiotics (Kaktcham et al., 2018).

Survival under conditions simulating the human GI tract
In the gastrointestinal tract, the entry of bacteria is restricted due to the acidic pH of the stomach and the antibacterial action of pepsin. These are important criteria for the selection of probiotic bacteria. At the time point of 2 h, there were almost no viable cells left at pH 2, but there was a slight change and no change in numbers at pH 3 and pH 4, respectively. At the time point of 3 h, the percentage of cell survival decreased to 97.2 ± 1.3% at pH 3, again without change at pH 4 ( Fig. 2A).
A probiotic bacterium in the small intestine is expected to survive in the presence of bile salt and pancreatin. A thick defensive layer of extracellular polysaccharides and secretion of bile salt hydrolase protect Lactobacillus from bile salts (Nawaz et al., 2017). In our study, the bile salt tolerance profile of the L. fermentum TIU19 strain showed that nearly 10% of viable cells were detected at 0.3% and 0.4% oxgall after 3 h of exposure. At the time point of 4 h of treatment with 0.2% and 0.1% oxgall, the number of cells of L. fermentum TIU19 decreased by approximately 25% and 3%, respectively (Fig. 2B).
Both the pepsin and pancreatin resistance of the L. fermentum TIU19 strain have been shown in Fig. 2C and 2D. At pH 3 and pH 4, when L. fermentum TIU19 was treated with pepsin for 3 h, 99% of the bacterial cells survived, but that decreased to 75 ± 3.1% at pH 2. Not only that, viable cells were not completely diminished in the presence of pepsin. Exposure to pancreatin (SIF) at pH 6.8 or pH 8 resulted in nearly 99% of the bacterial cells surviving, indicating that pancreatin had almost no effect on the number of viable cells. The results of the survivability of L. fermentum TIU19 under simulated gastric-intestinal juice and bile salt tolerance are quite different from the previously reported probiotic L. fermentum KKL1 strain isolated from Haria (Ghosh et al., 2015, p. 1). The L. fermentum KKL1 was able to survive in the simulated gastric juice at pH 2 (12.19%) and pH 3 (57.56%) and had strong resistance to bile salt and could tolerate 2% of oxgall for 4 h of incubation periods.

Cell surface hydrophobicity
For any good probiotic strain, the nonspecific hydrophobic surface interaction between the microorganism and the host cell acts as a key adhesion mechanism (Ramos et al., 2013). In our study, the cell surface hydrophobicity of L. fermentum TIU19 was 61.53%. This indicates a significantly high surface hydrophobicity property. As previously isolated L. fermentum KKL1 from Haria was reported to exhibit only moderate hydrophobicity (31.03%), it can be hypothesized that newly isolated L. fermentum TIU19 significantly improves the probiotic potential, at least based on hydrophobicity.

Auto-aggregation and adhesion ability
The probiotic strain should have the capacity to adhere to the mucosal surface (Muryany et al., 2018). In general, the higher hydrophobicity of the probiotic strain shows its auto-aggregation and high adhesion capability. In our study, L. fermentum TIU19 demonstrated a good autoaggregation ability of approximately 70.23 ± 4.2% at 37 • C, which is very similar to the previously reported probiotic strains Lactobacillus pentosus ST712BZ1 and Lactobacillus plantarum ST284BZ2, isolated from traditional Bulgarian milk products. The auto-aggregation ability of Lactobacillus pentosus ST712BZ1 and Lactobacillus plantarum ST284BZ2 was found to be approximately 67% and 99% (Todorov et al., 2007). Aggregation capacity is an important property for probiotic  strains; it leads to the formation of biofilms to protect the host from pathogen invasion (Grigoryan et al., 2018). This property of lactobacilli gives them the ability to adhere to the surface of intestinal cells. The adhesion ability of the selected strain was determined by using the HeLa cell line. For the adherence study of various pathogens, including uro-pathogens, the use of Hela cells has already been reported (Leccese Terraf et al., 2017). In addition to this, the morphology and characteristics of urethral epithelial cells are very similar to Hela cells (Zuiverloon et al., 2018).
The adhesion ability of the isolated strain to HeLa cells was 77.42 ± 1.7%. It is very similar to the previous report where four LAB strains (Leu. mesenteroides H40, L. plantarum FI10604, L. brevis FI10700, and L. perolens FI10842) showed high adhesion ability (Son et al., 2018).
Therefore, it can be hypothesized that; the adhesion ability of the isolated strain of L. fermentum could play a role in its function as probiotics and may allow bacteria to grow on the mucosal surface.

Biofilm forming ability
Biofilm formation is another important factor in the selection of probiotic strains. The biofilm biomass of the selected L. fermentum TIU19 strain was increased in a time-dependent manner, as illustrated in Figs. 3A and 3B. After 72 h of incubation, the biofilm biomass of the isolated strain was found to be 84.29 ± 1.7%. It is very similar to the previous report, where Lactobacillus strains were found to be good biofilm producers (Terraf et al., 2012).

β-galactosidase production
From a technological point of view, the ability of the probiotic strain to produce β-galactosidase enzyme production is an important factor. Bacterial cells were incubated with a reaction buffer and ONPG substrate. β-galactosidase converts the colourless ONPG substrate into galactose and the chromophore o-nitrophenol, producing a bright yellow solution. The L. fermentum TIU19 probiotic strain was found to have positive β-galactosidase activity, which should provide an additional benefit, as reported earlier (Zárate and Chaia, 2012).

Antioxidant property
The DPPH radical scavenging activity of L. fermentum TIU19 is shown in Fig. 4A. 4-log to 6-log CFU/ml bacterial cells showed free radical scavenging activity ranging from 46.7 ± 2.10% to 86.58 ± 1.06%. In the present study, L. fermentum TIU19 showed significant concentrationdependent free radical scavenging activity, which is very similar to the previously reported probiotic strain L. plantarum KCC-24, isolated from Italian ryegrass. The scavenging activity of L. plantarum KCC-24 was found to be approximately 70%.

Antagonistic activity against uro-pathogens
The antagonistic activity of L. fermentum TIU19 against uropathogens shows that L. fermentum TIU19 inhibited the multi resistant E. coli and E. faecalis strains. The inhibition zones observed were 11 ± 1.2 mm and 19 ± 0.75 mm, respectively. Various probiotic Lactobacillus strains have been reported to exhibit antibacterial activity against a variety of pathogenic bacteria, including E. coli and E. faecalis (Prabhurajeshwar and Chandrakanth, 2017).

Effects of L. fermentum TIU19 on the co-aggregation
Co-aggregation ability may form a barrier that prevents colonization by pathogenic microorganisms (Kos et al., 2003, p. 92). To understand the co-aggregation activity of the probiotic bacteria with both uro-pathogens, single species pathogens were mixed individually with L. fermentum TIU19 (E. coli + L. fermentum TIU19 and E. faecalis + L. fermentum TIU19) and co-incubated for 60 mins. We found that the co-aggregation activity of L. fermentum TIU19 with uro-pathogenic E. coli was 61 ± 2.9% and 52 ± 1.8% for E. faecalis. These findings are comparable to earlier reports, where a high coaggregation percentage of lactobacilli was obtained with E. coli ATCC 11,229 (Ekmekci et al., 2009). In this study, L. fermentum TIU19 demonstrated strong co-aggregative behavior with both uro-pathogenic strains.

Effects of L. fermentum TIU19 on the adhesion of uropathogens to Hela cells
Probiotic bacteria may establish a defensive barrier that prevents pathogenic bacteria from colonizing the epithelial cell lining (Odenwald and Turner, 2017;Takiishi et al., 2017). For the adherence study of various pathogens, including uro-pathogens, Hela cells have already been reported (Leccese Terraf et al., 2017). In addition to this, the morphology and characteristics of urethral epithelial cells are very similar to Hela cells (Zuiverloon et al., 2018). To investigate the effect of L. fermentum TIU19 on the adherence capacity of the two uro-pathogenic strains, L. fermentum TIU19 was co-inoculated into HeLa cells by each uro-pathogen (Fig. 5A). We found that L. fermentum TIU19 was found to be competitive and significantly reduced the adhesion of uro-pathogens E. coli and E. faecalis to HeLa cells by 69.93 ± 2.2% and 71.62 ± 3.1% respectively. In this study, L. fermentum TIU19 demonstrated strong inhibition of bacterial uro-pathogenic adhesion to HeLa cells.

Biofilm inhibition assay
Lactobacillus species have been considered beneficial microflora since they are capable of inhibiting the growth and/or colonization of numerous pathogenic bacteria (Di Cerbo et al., 2016).To evaluate the isolated L. fermentum's ability to prevent biofilm formation of uro-pathogens, single species of uro-pathogens were mixed separately with it and co-incubated to form a dual-species biofilm. After incubation, we determined the number of E. coli and E. faecalis cells. Planktonic and sessile cells of E. coli decreased by 4-log and 2.1-log (Fig. 5B), and E. faecalis was reduced by 3.8-log and 4.1-log, respectively, compared to the control (Fig. 5C). From these results, we assumed that the L. fermentum TIU19 strain may inhibit the formation of E. coli and E. faecalis biofilms. Our findings are consistent with previous research, which found that different Lactobacillus species decreased biofilm-associated cells of E. coli O157:H7, L. monocytogenes, and S. Typhimurium by 5-log, 4-log, and 7-log, respectively (Gómez et al., 2016).

Conclusions
The results presented show that L. fermentum TIU19 has safety properties in vitro and inhibitory activity upon multi resistant uropathogens E. coli and E. faecium. Thus, this strain harbor promising probiotic traits, but further analyses are needed to verify in vivo the probiotic behavior of L. fermentum TIU19 too.

Funding information
The author(s) received no financial support for the research, authorship, and/or publication of this article.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on a reasonable request.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that would have appeared to influence the work reported in this paper.