Inhibition of Escherichia coli adhesion to human intestinal Caco-2 cells by probiotic candidate Lactobacillus plantarum strain L15

Inhibition of Escherichia coli adhesion to human intestinal Caco-2 cells by probiotic candidate Lactobacillus plantarum strain L15

Microbial Pathogenesis 136 (2019) 103677 Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/...

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Microbial Pathogenesis 136 (2019) 103677

Contents lists available at ScienceDirect

Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath

Inhibition of Escherichia coli adhesion to human intestinal Caco-2 cells by probiotic candidate Lactobacillus plantarum strain L15

T

Behrooz Alizadeh Behbahania,∗, Mohammad Noshada, Fereshteh Falahb a

Department of Food Science and Technology, Faculty of Animal Science and Food Technology, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran b Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

ARTICLE INFO

ABSTRACT

Keywords: Probiotic Escherichia coli Adhesion to Caco-2 Antagonistic activity

Probiotics are microbial strains beneficial to human health if consumed in appropriate amounts. Their potential has recently led to a significant increase in research interest in their effects on the intestine, mainly by reinforcing the intestinal epithelium and modulating the gut microbiota. This study aimed to evaluate the probiotic features of Lactobacillus plantarum strain L15 based on adhesive properties for the inhibition of the adhesion of infectious pathogens. The molecular identification of the strain was performed from the sequencing of 16S ribosomal DNA with 27FYM and 1492R primers, and its probiotic features, including resistance to gastric juices, resistance to bile salts, and hydrophobicity were evaluated. The potential of Lactobacillus plantarum strain L15 to adhere to human adenocarcinoma intestinal cell line, Caco-2, as well as the auto and co-aggregation and anti-adherence activity against Escherichia coli were investigated. The results demonstrated that this strain has a desirable potential for passing through the low pH of the stomach and entering the intestines. Moreover, 54% hydrophobicity, 44% auto-aggregation, and 32% co-aggregation were observed for this strain. The adhesion level of Lactobacillus plantarum strain L15 to Caco-2 cells was 12%, and adhered lactobacilli cells were observed by scanning electron microscopy (SEM). Furthermore, this strain showed appropriate anti-adherence effects, including competition, inhibition, and replacement properties against Escherichia coli. The results indicated that Lactobacillus plantarum strain L15 had good potential for exerting antagonistic effects against E. coli.

1. Introduction

gastrointestinal tract (GIT). Inhibition of the adhesion and the intestinal colonization of pathogenic strains is a relevant feature of probiotic strains. There are some diseases such as obesity, insulin resistance syndrome, type 2 diabetes, and non-alcoholic fatty liver disease which can be treated by probiotics. This bacteria can also improve the body's immunity by increasing its resistance to diseases. Studies have reported that probiotic bacteria also play an important role in the treatment of certain cancers. All the benefits mentioned for probiotics depend on the strain, dose, and components used to produce a given probiotic product [5]. Although several criteria have been proposed for the selection of potential probiotics, the most important feature is their ability to attach to the intestinal cells. Because of this ability to adhere, the persistence of probiotic strains in the intestine can be increased, allowing them to exert their functions. Due to the difficulty of examining the in vivo adhesion of the bacteria to the GIT, in vitro evaluation using adenocarcinoma cell line Caco-2, which express the morphological and functional characteristics of normal enterocytes, has been widely

Lactic acid bacteria (LAB) are Gram-positive, catalase-negative, non-spore forming cocci, coccobacilli, or rods with a DNA base composition of <53 mol% G+C characterized by the ability to convert fermentable carbohydrates into lactic acids as a primary or secondary end-product [1]. LAB are commonly found in traditional fermented foods, e.g., yogurt, cheese, sauerkraut, sausage, and kefir. Among the different genera of LAB, Lactobacillus is the genus including a high number of GRAS (generally recognized as safe) strains. Intestinal Lactobacillus species have been widely used as probiotics [2]. Probiotics are living microorganisms which, when administered in adequate amounts, promote health functions in the human or animal host. Among Lactobacillus strains, L. plantarum, L. fermentum, L. rhamnosus, L. acidophilus, and L. delbrueckii are the most commonly known probiotics [4]. Prevention or reduction of the intestinal infection disease is one of the proposed beneficial effects of probiotics by producing particular substances or preventing the infectious bacteria from adhesion to the



Corresponding author. E-mail address: [email protected] (B. Alizadeh Behbahani).

https://doi.org/10.1016/j.micpath.2019.103677 Received 19 May 2019; Received in revised form 13 August 2019; Accepted 19 August 2019 Available online 19 August 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.

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accepted [6]. Urinary tract infection (UTI) is a common infectious disease which affects bladder, kidneys and ureter, and occurs more often in women. Although UTI can be painful, it could be treated within a few days to several weeks. The clinical symptoms of this infection include feeling generally unwell, need to urinate more often than usual, pain or discomfort when urinating, feeling unable to empty the bladder, and cloudy urine, foul-smelling, or urine containing blood [3,7]. The conventional treatment for this infection is antibiotic therapy. This method has some disadvantages, including the destruction of the intestinal flora and pathogenic microorganisms resistant to antibiotics by gene transition which are a worldwide problem. Probiotic therapy, in contrast to antibiotic treatment, can reduce this infection in humans by acting on the intestine and exerting its beneficial properties. Probiotics can affect UTI thanks to their numerous abilities such as attachment to intestinal cells and production of antimicrobial factors, anti-adhesion of organisms causing UTI, co-aggregation with an infectious agent, and influencing the components of flora and immunomodulation. Probiotics have significant effects on the balance of mucosal immunity and improve the production of cytokines, secretion of IgA, production of inhibitory substances, and phagocytosis. The compounds produced by probiotic bacteria include hydrogen peroxide, bacteriocins, and pH-reducing organic acids such as lactic acid and acetic acid [8]. The aim of this study was to evaluate the probiotic potential of Lactobacillus plantarum strain L15 isolated from Horreh, Iranian fermented cereal-dairy product, analyzing pH and bile resistance, physicochemical properties of the strain (hydrophobicity, auto- and co-aggregation), adhesion ability to Caco-2 cells monolayer, and adhesion competition against pathogenic bacteria (competition, inhibition and replacement assays).

PCR was performed in 20 μL reaction volumes. Amplification conditions were as follows: initial denaturation at 95 °C for 5 min, 28 cycles including denaturation at 98 °C for 15 s, annealing at 54 °C for 35 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min. Next, 3 μl of the PCR product was analyzed by 1.5% agarose gel electrophoresis stained with GelRed in 1 × TBE buffer at 100 V for 35 min, and the PCR amplicons were sent to Macrogene (South Korea) for sequencing. Afterwards, this sequence was compared with the other sequences in the GenBank database using the BLAST program. The result of BLAST indicated that this strain belonged to Lactobacillus plantarum strain L15 [10]. 2.3. Resistance to simulated gastric juices and bile salts The viability of this strain was evaluated using simulated gastric juice, prepared based on the method described by Falah et al. (2019) [3]. Briefly, 0.3% (w/v) pepsin (Sigma-Aldrich, USA) was suspended in saline (0.5%, v/v) solution. Then, its pH was set at 1.5 using 1 mol/L HCl. The solution was filtered through a 0.22 μm syringe filter. L. plantarum strain L15 with concentration of 0.5 McFarland was inoculated to gastric juice and its viability was monitored at 0, 30, 60, 90, and 120 min with the serial dilution method. In order for the probiotic strain to reach the intestinal mucus, it must pass through the specific stress challenge in GIT. One of their most important qualities is their potential for survival at the presence of bile salts in the upper parts of the small intestine. In this study, the survival of the strain against different concentrations of bile salts was evaluated through the plate assay. Overnight L. plantarum strain L15 was centrifuged at 6000 rpm for 10 min at 4 °C; the pellet was washed with sterile PBS (phosphate buffer saline) and then re-suspended in sterile PBS solution so that its density would reach McFarland 1. Next, 10 μL of the strain was spread onto MRS agar plates containing 0.1, 0.2, 1, 0.3, and 1% (w/v) bile-salts and plates were incubated at 37 °C. After 24–48 h, the growth was checked [11].

2. Materials and methods 2.1. Culture conditions of LAB, pathogen and Caco-2 cells

2.4. Determination of cell surface hydrophobicity (CSH)

Lactobacillus strain isolated from dairy product was grown in de Man, Rogosa and Sharpe broth (MRS broth, Merck, Germany) at 37 °C without aeration and maintained in MRS broth with 20% (v/v) glycerol at −80 °C for a long time. Escherichia coli isolated from patient with UTI obtained from Department of Microbiology at Bu-ali institute was grown in Luria Bertani broth (LB, Merck, Germany) at 37 °C for 16 h. The intestinal Caco-2 cell line obtained from The Research Institute of Biotechnology, Ferdowsi University of Mashhad were grown in 75 cm2 cell culture flask containing Dulbecco's modified Eagle's medium (DMEM, Vivacell), supplemented with Penicillin/Streptomycin (1X) and 10% heat-inactivated Fetal Bovine Serum (FBS) (Invitrogen, USA). Preparations were incubated at 37 °C under 5% CO2 -95% air atmosphere for 3 days, until reaching a confluency of 80%.

CSH was determined according to the ability of the LAB to adhere to the non-polar solvent. Actually, the ability of the organisms to adherence to the epithelial cells in the intestine can be estimated by its adherence to hydrocarbons. The overnight cultures of L. plantarum strain L15 in the stationary phase were harvested by centrifugation at 6000 rpm for 10 min at 4 °C. The pellet was washed twice with cold sterile PBS and re-suspended in the same buffer to adjusted the density in OD600 = 0.7 (A1). Afterwards, 3 ml of cell suspension and 1 ml of nhexadecane (Merck, Germany) were mixed and vortexed for 1 min and incubated at 37 °C for 1 h for phase separation. After this period, the aqueous phase was carefully removed and its absorbance value was read at 600 nm (A2). CSH % was calculated as [9,12]:

2.2. Isolation and identification of the strain

Hydrophobicity% =

Five grams of Horreh samples were transferred to 95 ml phosphate buffer saline (Sigma-Aldrich, USA) and homogenized with a stomacher (Bag mixer 400P, INTERSCIENCE, France). Then, 0.1 ml of homogenized samples was spread on MRS agar and plates were incubated at 37 °C for 48 h. The strain was subjected to some primary tests to evaluate morphological characteristics, Gram-positivity, and catalase-negativity. To determine the genus of the isolated strain, biochemical tests, including growth at 10 °C and 45 °C temperatures, salt concentration of 6.5%, and pH values of 4.9 and 9.6 as well as the Durham tube test for CO2 production were performed [9]. Also, the polymerase chain reaction (PCR) was performed for the molecular identification of the strain. Total DNA was extracted, and the primers used for the amplification of 16S rRNA gene were 27FYM (5′-AGAGTTTGATYMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). Furthermore,

A1

A2 A1

× 100

2.5. Auto- and co-aggregation Cell auto-aggregation was determined using the method described by Vasiee et al. (2019) [13]. Overnight grown Lactobacillus cultures were harvested by centrifugation at 6000 rpm for 10 min at 4 °C. The pellet was washed with PBS, re-suspended and the optical density was adjusted to 0.6 at 600 nm (Absinitial). The mixture was kept at 37 °C for 2 h and then the optical density of the supernatant was measured. Autoaggregation (%) was calculated as:

Auto

2

aggregation% =

Absinitial

Absfinal

Absinitial

× 100

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For co-aggregation assay, equal volumes of L. plantarum strain L15 (x) and the pathogen (y) were mixed (x+y), vortexed for 20 s and then incubated at 37 °C for 5 h. The absorbance (A) of the upper suspension was measured at 600 nm. The absorbance of each strain was also measured separately. Co-aggregation (%) was calculated as [13,14]:

Co

ggregation% = 1

Ax + y Ax + Ay

absence of L. plantarum strain L15, respectively. For the replacement assay, the pathogenic strain was firstly added to the wells, and the plate was incubated at 37 °C in the presence of 5% CO2 for 1 h. Unbounded bacteria were washed and L. plantarum strain L15 added to wells. After 1 h of incubation at 37 °C in the presence of 5% CO2, unbounded bacteria were gently washed with PBS and the bacteria and Caco-2 cell were detached with Triton 0.05 %X-100. The plate count method was performed and the degree of replacement was calculated as the percentage of the adhered E. coli in the presence and absence of L. plantarum strain L15 [17].

× 100

2

2.6. Adhesion assay

2.9. Statistical analysis

For adhesion assays, anti-adhesion assays and SEM Caco-2 cells monolayer and polarized cells were prepared in six-well tissue culture plates. Cells were seeded in the wells with the concentration of 25,000 cells/cm2 and the medium was replaced every two days for two weeks to obtain differentiated cells. Afterwards, to determine the adhesion degree, the probiotic strain was grown and harvested by centrifugation at 6000 g for 10 min at 4 °C, the pellet was washed with sterile PBS, and re-suspended in the DMEM to adjust the OD600 = 1. The number of initial cells was investigated by colony counting method on MRS agar plates. The old DMEM medium in the wells was emptied, and the wells were washed with sterile PBS to remove antibiotics. Subsequently, the strain was added to Caco-2 cells. After 1 h incubation, unbounded bacteria were gently washed with sterile PBS, and remaining bacteria and Caco-2 cells were detached with Triton X-100 (0.05%). Bacterial counts were performed on MRS agar [2,13].

All experiments were done in triplicate. Microsoft Windows Excel 2017 and SPSS software (Version 18.0, SPSS Inc. Chicago, USA) were used to analyze the resulting data. Statistical treatment of the data was conducted using ANOVA. Duncan's test was used to compare the means when the overall P-value of the experiment was below the value of significance (P < 0.05). 3. Results and discussion 3.1. Resistance to gastric juice and bile salts Research on Lactobacillus strains has attracted considerable interest in recent years because they affect the host by improving intestinal microbial balance and modulating immune responses. One of the first conditions for selecting bacteria as a probiotic strain is its potential to pass through the stomach conditions and reach the intestines. There are several compounds in gastric juice which may contribute to the bactericidal activity, the most important being the hydrochloric acid which provides the low pH. Most studies have reported that pH < 2.0 could destroy bacteria, and this pH rarely occurs in the stomach. Although this condition is suitable for the inactivation of microorganisms and fighting off infection, it is a barrier to useful bacteria reaching the intestine. Pepsin is a digestive enzyme which is produced in the stomach and breaks down proteins into smaller peptides. Bacterial killing is facilitated by pepsin in the stomach; therefore, it is necessary to investigate the simulated effect of pepsin and pH on the strain [18]. The results of L. plantarum strain L15 resistance to low pH and pepsin are illustrated in Fig. 1. The highest reduction in bacteria population occurred in the first 30 min, due to the sudden exposure of bacteria to low pH and pepsin. The highest reduction in the number of bacteria was observed after 120 min, where about four logarithmic cycles of the strain decreased compared to the control. Zhu et al. (2016) [18] studied the bactericidal effect of low pH and pepsin on some bacteria. They reported that the combination of pepsin and low pH is more effective for killing bacteria. The resistance of the probiotic strain to low pH is attributed to the constant gradient between extracellular and intracellular pH. In Gram-positive microorganisms, F0F1-ATPase is a well-known mechanism for protection against acidic conditions. F0F1-ATPase is induced at low pH and can increase the intracellular pH at a low environmental pH level [19,20]. Bile is a yellow-green aqueous solution produced continuously by the liver and stored in the gallbladder. After eating, bile is discharged into the duodenum and aids the digestion of lipid in the small intestine. Bile can also destroy the lipid in the cell wall of bacteria eliminating them. Therefore, the probiotic strains should be resistant to bile salts. In this study, the resistance of L. plantarum strain L15 was evaluated to different concentrations of bile salts by the plate count method [21]. This strain can grow at all bile salts concentrations (0.1, 0.2, 1, 0.3, and 1% (w/v)), indicating its resistance to bile salts. Bile salts may be hydrolyzed to amino acids by bacterial enzymes known as bile salt hydrolases (BSHs). Several genera of bacteria can express these enzymes, including Lactobacillus, Enterococcus, Clostridium, Bifidobacterium, and Bacteroides. In fact, this enzyme plays an important role in the survival

2.7. Scanning electron microscopy (SEM) SEM was employed to confirm the adhesion of L. plantarum strain L15 to Caco-2 cells. All the steps in the previous section were performed on sterile glass cover slips placed in a 6-well tissue plate. Primary fixation was performed with 2.5% w/v glutaraldehyde (Sigma Aldrich, USA) in 0.1 M phosphate buffer (pH 7.4) for 1 h at ambient temperature. Afterwards, the sample was washed with PBS add post-fixation was conducted with 2% w/v osmium tetroxide (Sigma Aldrich, USA). Eventually, samples were dehydrated in a graded series of ethanol, starting with 30%, followed by 50%, 70%, 80%, and absolute ethanol. Cells were dried in a critical point dryer (E3100, UK.) and coated with gold. The samples were examined using SEM (Zeiss (LEO) 1450 VP model, Germany) [15,16]. 2.8. Anti-adhesion activity of L. plantarum strain L15 against E. coli Three methods were applied for evaluating the anti-adhesion activity of L. plantarum strain L15 against the pathogenic strain: competition, inhibition, and Replacement assays. For the competition assay, equal volumes of each strain with the same concentration were added to the wells. The plate was incubated for 1 h at 37 °C in the presence of 5% CO2. After 1 h, unbounded bacteria (L. plantarum strain L15 and E. coli) were washed, and remaining bacteria and Caco-2 cells were detached with 0.05% Triton X-100. The competition index was calculated as the percentage of the attachment of E. coli added in combination with L. plantarum strain L15 divided by the adhesion of the bacteria in the absence of L. plantarum strain L15. For the inhibition assay, first the L. plantarum strain L15 was added to the wells and incubated for 1 h. The unbounded bacteria were gently washed with sterile PBS. Afterwards, the pathogen strain was added to the wells and the 6-well plate was incubated for another 1 h. The unbounded bacteria were washed and the bacteria and cells were detached with 0.05% Triton X-100. The degree of inhibition was calculated as: Inhibition of pathogen adhesion =

A1

A2

A1

× 100 , where A2 and A1

are the percentage of adhesion by E. coli cells in the presence and 3

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Fig. 1. Effect of gastric juice on the survival of L. plantarum strain L15.

of probiotics in the presence of bile salts [22].

between hydrophobicity and adhesion, Schillinger et al. (2005) [25] reported that L. acidophilus BFE 719 showed good adhesion to HT29 cell while having a poor hydrophobicity of only 2%. These are in agreement with observations reported by Lim et al. (2012) [26] and Ouwehand et al. (1999) [27] who did not report any correlation between the CSH and the adhesion of the probiotic strains to intestinal cells. We also evaluated the cell-binding properties of L. plantarum strain L15, including auto-aggregation and co-aggregation abilities. Both of these properties seem to be important for the adhesion of the probiotic strains as they may orchestrate the bacterial adhesion to GIT (auto-aggregation) and prevent colonization by pathogenic bacteria (co-aggregation) [28]. According to Aslim et al. (2006), the protection of the intestinal environment by lactobacilli strain is performed through two mechanisms: (a) production of antimicrobial compounds by lactobacilli, and (b) attachment to mucus and co-aggregation, which may form a barrier to prevent pathogenic biofilm production and adhesion [29].

3.2. CSH and aggregation An important factor influencing the strength of bacterial adhesion is hydrophobicity. The potential of the microorganism to attach to nonpolar solvents is a measure of its adherence to the epithelial cells in the GIT.Hydrophobicity degree is calculated by mixing the suspension of the bacteria and non-polar solvent and measuring the OD600 of the aqueous phase before and after solvent addition. CHS is suggested to be the screening tool to evaluate the adhesion ability of potential probiotic bacteria. The physical and chemical properties of the outer layer of probiotic bacteria influence cell hydrophobicity [23]. The hydrophobicity degree varies between across microorganism and strains and is affected by the age and surface chemistry charge of bacteria [24]. The results of physicochemical properties in terms of hydrophobicity, autoand co-aggregation of L. plantarum strain L15 are shown in Fig. 2. Although most studies have demonstrated that a correlation exists

Fig. 2. The result of hydrophobicity, auto-aggregation, and co-aggregation of L. plantarum strain L15. 4

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3.3. Adhesion of L. plantarum strain L15 to Caco-2 cells

immunomodulation depend on the age of the host. Moreover, Collado et al. (2007) [36] investigated the adherence properties of some probiotic strains alone and in combination with one another. They reported that probiotics used together can increase the efficiency of adhesion and health effects compared to using individual strains. In fact, the simultaneous use of probiotics can exert synergistic adhesion effects.

In the previous sections, the basic characteristics of the L. plantarum strain L15, including survival at the stomach condition and bile salt, hydrophobicity, and aggregation properties, were discussed. However, the most important feature of any strain to be considered as a probiotic is its ability to adhere. This feature is so important that it is referred to as a golden standard for determining probiotic bacteria. Flow rates are relatively high in the small intestine; therefore, adhesion to mucus is thought to be important. Due to the difficulty of examining the adhesion properties in in vivo conditions, in vitro investigations with adenocarcinoma Caco-2 cells are widely accepted [3,13,30,31]. The adhesion degree (calculated based on the percentage of the bacteria adhered divided by the number of bacteria added) for L. plantarum strain L15 was 12.2%. Duary et al. (2011) assessed the adhesion of putative indigenous probiotic lactobacilli to Caco2 and HT-29 colonic adenocarcinomal human intestinal epithelial cell lines. Based on the results of direct adhesion, the most adhesive strain to HT-29 and Caco2 cell lines was L. plantarum Lp91 with the adhesion value of 12.8 and 10.2%, respectively. Cell adhesion is a complex process involving contact between the probiotic strain and mucus, complicating the interplay of long-range van der Waals and electrostatic forces and various other short-range interactions. Fig. 3 depicts the adhesion of L. plantarum strain L15 to the Caco-2 cells, which is taken by SEM with the magnification of 20,000. Some hydrophobic compounds such as proteins, polysaccharides, teichoic, and fatty acids exist in the cell wall of the bacteria, helping them to adhere to the surface of the intestinal epithelium by covalent bonds. These attachments have been established between the bacteria and mannose, galactose, and fructose of glycoproteins or glycolipids on the intestinal cells. CSH plays a determining important role in the formation of biofilm and adhesion of bacteria to host cells [32,33]. GIT is covered by a mucus layer, which not only protects the epithelial cells from physical and chemical damage and pathogenic bacteria, but also acts as the first barrier between the probiotic strain and the host. Mucin secretion creates a fluid environment in the intestine. Consequently, a critical condition for the colonization of non-motile species in the GIT is their movement in this fluid ecosystem [34]. Kirjavainen et al. (1998) [35] reported that, under probiotic therapy, the potential of probiotic strains to adhere to the intestinal mucosa and colonization and, therefore, their ability to balance the endogenous flora and

3.4. Anti-adhesion effects of L. plantarum strain L15 against E. coli The first step of intestinal infections is the adhesion of the pathogenic strain to the mucosal surface. This process occurs by bacterial adhesions which can distinguish the individual receptors. Therefore, the adhesion of pathogenic bacteria can be inhibited by blocking the receptors on the intestinal cells with specific adhesion analogs. Prevention of adhesion may occur by the probiotic strain which can adhere to the GIT receptors and block them for pathogens [37]. The adhesion degree of E. coli was relatively low (7.1%). The results of antiadhesion properties of L. plantarum strain L15 against E. coli are presented in Fig. 4. Competition between probiotic and pathogen for the same receptors can be a reason for balancing the GI tract microbiota and improve the infection. In this study, after simultaneously adding the L. plantarum strain L15 and E. coli, the adhesion degree of E. coli was reduced by 56%. As mentioned before, this reduction may be due to competition for nutrition and receptors or the production of antimicrobial compounds such as hydrogen peroxide, bacteriocins, organic acids, and polysaccharides [38,39]. Also, inhibition and replacement effects on E. coli equaled 45 and 22%, respectively. Lee et al. (2003) [40] reported that the beneficial effects of probiotic bacteria in the treatment of infections is attributed to non-competition mechanisms, including enhanced healing of the damaged intestinal tissue, modulation of immune responses, and non-specific increase in the proliferation of intestinal anaerobes. When probiotic strains are used in high concentration or their affinity is higher to adhesion receptors than pathogens, they are able to displace adhering pathogens [41]. For both pathogen bacteria and probiotic strains, several factors are likely to influence the adhesion, some of which are related to in vitro conditions, e.g., bacterial concentration, buffer composition, growth medium, and incubation time, and some other factors are related to in vivo conditions, e.g., normal intestinal microbiota and food matrix digestion [42]. Based on Yu et al. (2012) [43] and Intyrri et al. (2016)

Fig. 3. Examination of the adherence of L. plantarum strain L15 to Caco-2 cells by SEM (magnification × 20,000; EHV: 20.00 kV; WD: 10 mm; Signal A: SE1). 5

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Fig. 4. Anti-adhesion assays (competition, inhibition, and replacement %) of E. coli in the presence of L. plantarum strain L15.

[44], the mode of action of a probiotic is complex and includes several mechanisms such as reduction and prevention of the pathogen-adhering bacteria to intestinal cells, production of various types of exopolysaccharides, and improvement of intestinal permeability induced by pathogens.

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4. Conclusion In summary, L. plantarum strain L15 had the ability to survive in the simulated gastric juice condition. It also expressed acceptable resistance to bile salts. The results of physicochemical properties of the strain, including hydrophobicity, auto- and co-aggregation were desirable. The adhesion capabilities of L. plantarum strain L15 were relatively higher compared to other studies determining the adhesion of different L. plantarum strains. However, the higher concentration of this strain during consumption can increase the adhesion ratio. Also, the in vitro results revealed that L. plantarum strain L15 can inhibit E. coli adhesion in the intestine. Although the results suggested that L. plantarum strain L15 can be a candidate probiotic available for human consumption, further in vitro and in vivo studies should be conducted on its potential to induce modulatory effects and immune responses. Acknowledgments The authors wish to express their profound gratitude sincerely to the Research Deputy of Agricultural Sciences and Natural Resources University of Khuzestan for funding this project. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2019.103677. References [1] D. Fayol-Messaoudi, C.N. Berger, M.-H. Coconnier-Polter, V. Lievin-Le Moal, A.L. Servin, pH-, Lactic acid-, and non-lactic acid-dependent activities of probiotic Lactobacilli against Salmonella enterica Serovar Typhimurium, Appl. Environ. Microbiol. 71 (2005) 6008–6013. [2] K. Todoriki, T. Mukai, S. Sato, T. Toba, Inhibition of adhesion of food‐borne pathogens to Caco‐2 cells by Lactobacillus strains, J. Appl. Microbiol. 91 (2001) 154–159. [3] F. Falah, F. Tabatabaee Yazdi, A.R. Vasiee, B. Alizadeh Behbahani, S. Moradi, A. Mortazavi, S. Roshanak, Evaluation of adherence and anti-infective property of probiotic Lactobacillus fermentum strain 4-17 against Escherichia coli causing urinary tract infection in human, Microb. Pathog. 131 (2019) 246–253. [4] W.H. Holzapfel, P. Haberer, J. Snel, U. Schillinger, J.H.H. in't Veld, Overview of gut flora and probiotics, Int. J. Food Microbiol. 41 (1998) 85–101. [5] P. Markowiak, K. Śliżewska, Effects of probiotics, prebiotics, and synbiotics on human health, Nutrients 9 (2017) 1021.

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