In-Vitro Characterization of Potentially Probiotic Lactobacillus Strains Isolated from Human Microbiota

Annals of Microbiology (2019) 69:61–72 https://doi.org/10.1007/s13213-018-1396-1

ORIGINAL ARTICLE

In-vitro characterization of potentially probiotic Lactobacillus strains isolated from human microbiota: interaction with pathogenic bacteria and the enteric cell line HT29

Yosra Gharbi1 & Imene Fhoula1 & Patricia Ruas-Madiedo2 & Najjari Afef1 & Abdellatif Boudabous1 & Miguel Gueimonde2 & Hadda-Imene Ouzari1

Received: 4 January 2018 /Accepted: 24 October 2018 /Published online: 8 November 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Abstract Among the various tests commonly used for selecting probiotic microorganisms, the tolerance to gastrointestinal transit conditions remains being commonly used to evaluate the probiotic potential of the strains. Besides, the adhesion to epithelial cells and the competition with pathogens constitute significant traits for evaluating the colonization ability and functional performance of candidate strains. In this study, a total of 13 lactic acid bacteria strains isolated from human feces were first identified by biochemical tests and 16S rRNA gene sequencing, and then characterized in vitro for their tolerance to gastrointestinal conditions, hemolytic activity, and antibiotics sensibility. The isolates were identified as Lactobacillus fermentum (06), Lactobacillus rhamnosus (04), Lactobacillus plantarum (02), and Lactobacillus salivarius (01). The adhesion to epithelial cells HT29 was shown to be a strain-dependent character. L. fermentum 88 and L. plantarum 9, being the ones showing higher adhesion values. They were further characterized by determining their antimicrobial activity, hydrophobicity, co-aggregation, antioxidant activity, as well as the ability to inhibit the adhesion of pathogens to the human epithelial cell line HT29. Moreover, these two strains were able to reduce the adhesion of Escherichia coli to HT29 cells, although they failed for inhibiting the adhesion of other pathogens such as Cronobacter sakazaki or Salmonella enterica. These results point out the importance of considering the ecological fitness of the strains in selecting probiotic bacteria and the potential of some of the analyzed strains for the development of food products.

Keywords Lactobacillus . Feces . Probiotic . HT29 . Adhesion . Inhibition of pathogens

Introduction bacteria, and alleviation or prevention of intestinal disorders (Liong and Shah 2005). Many different criteria have been Probiotics are defined as live microorganisms which when used in the selection of strains to be used as probiotics. administered in adequate amounts confer a benefit to the host These include lack of pathogenicity, tolerance to gastrointes- (FAO/WHO 2006). They have been proposed to confer mul- tinal conditions (acid and bile), ability to adhere to the gastro- tiple health benefits, such as the intestinal microbiota balance intestinal mucosa, and competitive exclusion of pathogens improvement, modulation of the immune system, lowering (Shillinger et al. 2005). Therefore, the selection of a promising serum cholesterol levels, growth inhibition of pathogenic probiotics for the development of functional foods requires assessing properties such as strain identity, non-pathogenicity, tolerance to gastrointestinal tract (GIT) conditions, sensitivity * Hadda-Imene Ouzari to antibiotics, adhesion, aggregation, or antagonism against [email protected] pathogens. In fact, the resistance to low pHs and bile salts have often been considered as essential parameters that the 1 Faculté des Science de Tunis, LR03ES03 Laboratoire probiotic strain should possess in order to survive during Microorganismes et Biomolécules Actives, Université de Tunis El Manar, 2092, Campus Universitaire, Tunis, Tunisie the passage through stomach and intestines (FAO/WHO 2006). Moreover, the adhesion ability has traditionally 2 Departamento de Microbiología y Bioquímica de Productos Lácteos, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior been considered important for the bacteria to resist and de Investigaciones Científicas (CSIC), 33300 Villaviciosa, Asturias, to establish, at least transiently, through the gastrointesti- Spain nal tract (Ruiz et al. 2013). Although no definitive proof 62 Ann Microbiol (2019) 69:61–72 is available, the relationship between in vitro adhesion Plates were incubated under anaerobic conditions at and in vivo colonization has been repeatedly proposed 37 °C for 72 h. After incubation, colonies were picked up (Castagliuolo et al. 2005; Cesena et al. 2001). andculturedinMRS,andtheisolateswerepresumptively Many early reports have shown that lactic acid bacteria identified as LAB by culturing and examining cell mor- (LAB), such as strains of lactobacilli, are appreciated for their phology (size, shape, and color), catalase activity, Gram probiotic properties and constitute a potential source of strains stain, and motility of each colony. Only Gram-positive, for the design of starter cultures for manufacturing and devel- catalase-negative strains with rod shape were selected for oping fermented functional foods (Mannu et al. 2000; further studies. The isolates were stored at − 80 °C in MRS Woutersetal.2002). supplemented with a 50% (v/v) glycerol. Lactobacillus is, together with Bifidobacterium, the bacterial genus more deeply studied and used as probiotic, mainly Identification of the isolates including strains for the species Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus Biochemical characterization paracasei, Lactobacillus gasserii, Lactobacillus reuteri,and Lactobacillus plantarum (Mannu et al. 2000; Wouters et al. The identification of the isolates at genus level was carried out 2002;Lemaetal.2001;Martinetal.2006). During the last according to the criteria of (Sharpe 1979) using morphologi- years, many studies have been focused on the use of probiotic cal, phenotypic, and biochemical methods. The growth of bacteria from intestinal origin as starters in the creation of new strains at different temperatures (10 °C, 15 °C, 42 °C, and products and this is a trend that still continues in this area of 45 °C) and in the presence of NaCl (0.2, 3.0, and 6.5%) were research. However, attention should be also paid to the safety considered for the identification. All strains were also tested of the selected strains, since different studies have linked the for the fermentation of glucose, lactose, galactose, xylose, use of probiotic Lactobacillus strains with cases of infection or raffinose, maltose, starch, and melibiose. sepsis humans (Kochan et al. 2011). Therefore, the isolation of Lactobacillus strains from human feces and the characteriza- DNA extraction and PCR amplification of 16S DNA tion of the properties of the isolates constitute a very encour- gene aging part for the development of new products targeting the modulation of the human gut microbiota. Moreover, compet- DNA was extracted by using a CTAB/NaCl method as de- itive behavior of the probiotics is one of the important traits to scribed by Wilson and modified by using 1 mg/mL lysozyme be considered as it reflects the ecological fitness of the strains (BIOMATIK) for cell wall digestion. Amplification of the 16S and their persistence in a given environment. rRNA gene V3-V5 was performed by PCR using the primers The aim of this study was to evaluate the ability of a num- s-d-bact-0008-a-S-20 and s-d-bact-1495-a-A20 (Daffonchio ber of potential probiotic Lactobacillus strains isolated from et al. 1998), and the amplified products were analyzed by human fecal samples, to colonize the intestinal tract and to 0.75% (w/v) agarose gel electrophoresis at constant voltage compete with pathogens and that may be considered as prom- and visualized with BET under UV light. Partial 16S ising for the development of new functional products. ribosomal DNA (rDNA) sequencing was performed. They were determined using a BigDye Terminator cycle sequencing kit V3.1 (Applied Biosystems) and an Applied Biosystems Materials and methods 3130XL Capillary DNA Sequencer machine. The sequences were analyzed by chromas pro 1.7, and research for DNA Isolation of lactic acid bacteria similarity was performed with the BLAST suite.

Human fecal samples were obtained from four adult females In vitro screening for probiotic properties (24 to 27 years old), two males (12 years old) and two infants (≤ 3 years old), affiliated to North and South of Tunisia. The Acid tolerance donors were healthy, using a normal diet, and did not utilize antibiotics during the last 3 months. The samples were imme- The tolerance of the Lactobacillus strains to low pH was tested diately stored at + 4 °C and processed within 6 h. Two milli- according to Klayraung et al. (2008). Cells were grown over- liters of peptone water (Oxoid) (containing 0.1% Tween 80 night on MRS broth at 37 °C. The cultures were centrifuged and 0.05% of L-cystein monohydrate) were then added to 1 g (12,000 rpm, 5 min at 4 °C), washed, then resuspended in 5 ml of fecal material, and the samples were homogenized. Fecal of PBS [54 mM KH2PO4, 0.1 mM NaCl, 2.71 mM Na2 HPO4 preparations were then serially diluted (10 fold) in phosphate 7H2O] adjusted to three low pH values (2.0, 2.5, and 3.0) buffer (0.1 M, pH 6.2), and 0.1 ml of each dilution were using 1 mol l−1 HCl, and incubated for 2 h at 37 °C. The initial spread on MRS (Man Rogosa Sharpe Merck, Germany). bacterial concentration (108 CFU/ml), as well as those Ann Microbiol (2019) 69:61–72 63 obtained after incubation, was determined by plate counting Standard protocols were used for maintaining the cells on MRS agar after 48 h of incubation under anaerobic condi- line; the culture medium was changed every 2 days; and tions at 37 °C. The survival rate was calculated as the percent- when appropriate, the cell line was trypsinized with age of LAB colonies grown on MRS agar after the 24-h incu- 0.25% trypsin-EDTA solution (Sigma). For the adhesion bation at low pH as compared to the initial bacterial concen- assay, HT29 cells were seeded at a concentration of 105 tration, which was arbitrarily set to 100%. cells/ml in 24-well plates and were grown to confluence

(11 ± 1 days) at 37 °C with a 5% CO2 in an SL water Bile tolerance jacketed CO2 incubator (Sheldon Inc., Corneluis, Oregon, USA). At the day of the experiment, a bacterial The bile tolerance was performed using the method described suspension was prepared by harvesting (3000 rpm, by Klingbery et al. (2005). The bile salt solution was prepared 10 min) an overnight culture of the strains grown in using oxgall (Sigma-Aldrich) powder at the final concentra- MRSat37°CandwashingtwicewithPBSbuffer tion of 0.3% (w/v). Lactobacillus cells were harvested by cen- (Sigma). The harvested bacteria were resuspended in trifugation (12,000 rpm, 5 min) from freshly grown culture McCoy’s medium without antimicrobials at a concentra- (24 h, MRS broth) and resuspended into 5 ml of MRS broth tion of 108 CFU/ml. The HT29 monolayers were then with or without 0.3% bile salts at a bacterial concentration of washed twice with Dulbecco’sPBS;1mlofthebacterial 106 CFU/ml. All samples were incubated for 3 h at 37 °C, and suspension was added to each well, and the plates were then cells count were determined as indicated above. incubatedfor1hat37°Cwitha5%CO2 in a Heracell® 240 incubator (Thermo Electron Gmbt, Langenselbold, Antibiotic susceptibility assay and hemolytic activity Germany). After incubation, supernatants were discarded, and the wells were gently washed two times with PBS Susceptibility of the isolates to seven different antibiotics was Dubelcco’s buffer to remove the non-adhered bacteria. carried out by using the disc diffusion method according to the Afterwards, the monolayers were trypsinated, and bacte- criteria of the National Committee of Clinical Laboratory rial counts were carried out in MRS agar to obtain the Standards (CLSI 2010) on Muller Hinton agar (MHA) sup- number of adhered bacteria. L. rhamnosus GG plemented with 0.4% of yeast extract and 2% of glucose. (ATCC53103) was used as control following the same Commercially available antibiotics disc (Oxide) containing protocol. The adhesion percentage was calculated by com- penicillin G (10 μg; S ≥ 15; R ≤ 14), erythromycin (15 μg; S paring the number of adhered cells to the amount of bac- ≥ 23; I 11–13; R ≤ 10), streptomycin (10 μg; S ≥ 10; I 7–9; R teria added (% CFU bacteria adhered/CFU bacteria ≤ 6), rifamipicin (5 μg; S ≥ 20; I 17–19; R ≤ 16), lincomycin added). The adhesion assay was conducted in triplicate (2 μg; S ≥ 19; I16–18; R ≤ 15), vancomycin (30 μg; S ≥ 17; R for each strain. ≤ 14), and chloramphenicol (30 μg; S ≥ 18; I 13–17; R ≤ 12) were used, and inhibition zone diameters were measured after Inhibition of pathogens adhesion to HT29 by lactobacilli incubation at 37 °C for 24 h. Results were expressed as sensitive (S), intermediate (I), and resistant (R). To test the ability of the Lactobacillus strains to inhibit the To test the hemolytic activity, fresh lactobacilli cultures adhesion of pathogens to HT29 cells, the pathogens were streaked on colombia agar plates containing 5% (w/v) Escherichia coli LMG 2092, Salmonella enterica subsp. horse blood (Pasteur Institute, Tunisia) and incubated for 48 h enterica serovar typhimurium LMG 15860, and at 37 °C. Blood agar plates were examined for signs of β– Cronobacter sakazakii LMG 5740 were grown in GAM broth haemolysis (clear zones around colonies) or α-hemolysis (Nissui Pharmaceutical CO., Tokyo, Japan) incubated at (green-hued zones around colonies). 37 °C, resuspended in McCoy’s medium without antimicrobials containing 15 nM SYTO9 green fluorescent nucleic acid Adhesion to HT29 cell line stain (Molecular Probes, Madrid, Spain), and incubated for 2 h at 37 °C under continuous stirring in darkness as previously The adhesion of Lactobacillus strains was studied using described by Zivkovic et al. (2015). After incubation, the intestinal epithelial cell line HT29 (ECACC No. 108 CFU of dyed pathogens were mixed with non labelled 91072201, European Collection of Cell Cultures, lactobacilli strains at a ratio 1:1. All the combinations of Salisbury, UK). HT29 cell line was grown routinely in lactobacilli-pathogens, and the pathogens alone, were added McCoy’s medium supplemented with 10% (v/v) heat- into wells containing HT29 monolayers and incubated at inactivated bovine fetal serum and a mixture of antimicro- 37 °C for 1 h. Then, wells were washed twice with bials (50 μg/ml penicillin, 50 μg/ml streptomycin, Dulbecco’s PBS and trypsinized, and the fluorescence emitted 50 μg/ml gentamicin, and 1.25 μg/ml amphotericin B). by the adhered pathogens was measured using a fluores- All media and supplements were obtained from Sigma. cence spectrophotometer (Varian Ibérica, S.A. Madrid, 64 Ann Microbiol (2019) 69:61–72

Spain; Excitation 470 nm, Emission 512 nm). The per- hydrophobicity assay determinations were performed in centage of adhesion was calculated as follows: fluores- triplicate and repeated twice. cence emitted by bacteria adhered/fluorescence emitted by bacteria added. Experiments were carried out in dupli- Antibacterial activity assay cate, using two duplicate wells in each of plate. Antibacterial activity of the culture supernatant of the lactobacilli strains was evaluated by the well diffusion Autoaggregation and coaggregation assays assay method (Tagg and McGiven 1971). The strains were grown overnight in MRS broth at 37 °C and Autoaggregation and coaggregation were performed ac- centrifugated (7000g, 5 min). The obtained cell free su- cordingtoKosetal.(2003) with some modifications. pernatants were previously filtered, and then used to carry For autoaggregation, 5 ml of MRS broth for lactobacilli, out the antimicrobial activity tests. The pathogenic bacte- or BHI broth for E. coli DH5α, S. typhimurium IPT13, riausedwereprovidedbytheLaboratoryMicroorganisms and Staphylococcus aureus ATCC 6538, were incubated and Biomolecules (University El Manar, Tunisia) and in- at 37 °C for 18 h. Then, cells were harvested by centrifu- cluded Listeria monocytogenes L15, St. aureus ATCC gation (9000 rpm, 4 min), washed three times in phos- 6538, Bacillus cereus 49, Pseudomonas aeruginosa phate buffer saline (PBS; pH7.0), and suspended in the ATCC 27853, S. typhimurium IPT13, E. coli DH5α, same solution. The OD of the cell suspension was then 660 and Enterococcus faecalis ATCC 29212. The pathogens adjusted to OD of 0.3. Samples were kept to stand for were cultured in BHI broth for 24 h, and then diluted with 60 min. Then, the OD 600 nm of supernatants were mea- sterile peptone water. After dilution, cells were mixed sured and used to determine the autoaggregation index with 5 ml of soft agar (agar 0.7% w/v) at the concentra- (%) as: (OD − OD /OD ) × 100. For total supernatant total tion of 105 CFU/ml. Then, 100 μl of the supernatants coaggregation assay, an equal volume of each cell suspen- from the Lactobacillus culture was applied in wells sions of co-aggregation partners (OD600 of 0.3; PBS, pH (5 mm of diameter) cutted into the solidified tryptone 7.0) were mixed together. Control tubes containing pure soyagarseededwithtestedpathogen.Theplateswere bacterial suspensions were set up at the same time, to kept at room temperature for 1 h to allow the radial dif- check self-floculation. Samples were kept to stand for fusion, incubated for 24 h at 37 °C, and checked for the 60 min. Then, the OD 600 nm of supernatants were de- appearance of inhibition zones. According to the diameter termined, and the percentage of coaggregation was calcu- of inhibition (mm), the Lactobacillus strains were classi- lated as: Coaggregation % = ((A + A )/2) − A(x + y)/(A + x y x fied as strains with weak inhibitor activity with d ≤ 12, A /2) × 100. Where x and y represent each of the two y medium activity with diameter 12 < d ≤ 15, and strong strains in the control tubes, and (x + y) the mixture. inhibitor activity (diameter d > 15). Experiments were performed in triplicate. Determination of antioxidant activity Hydrophobicity assay For preparation of cells and intracellular extracts, overnight Bacterial cell surface hydrophobicity was assessed by cultures of lactobacilli were centrifuged (12,000 rpm, 3 min measuring microbial adhesion to hydrocarbons (MATH) at 4 °C) and used to prepare the intracellular extract. The cell as described by Perez et al. (1998). Strains were grown pellet was washed twice, resuspendedinultrapurewater,and in MRS broth at 37 °C for 18 h and centrifuged at 9000g subjected to ultrasonication at 30 KHZ during 3 min, with for 5 min at 4 °C. The obtained pellet was washed twice three intervals of 1 min in an ice bath. After sonication, the in 50 mmol/L phosphate buffer saline pH 7.0 and cellular debris was removed by centrifugation at 200×g for suspended in the same solution to an initial OD600 of 15 min at 4 °C. The cell free extract was used as intracellular 0.4, and the absorbance was measured (A0). One milliliter extract to evaluate the antioxidant activity. The antioxidant of the bacterial suspension was then mixed with 0.2 ml of activity of intact cells and intracellular extracts were calcu- n-hexadecane and stirred for 2 min. After incubation for lated using the DPPH free radical scavenging assay as de- 1 h at room temperature, the two phases were separated, scribedby(LinandChang2000) which was based on the and the aqueous phase was carefully removed, and its capture of the DPPH (2, 2-diphenyl-1-Picryl hydrazyl) rad- absorbance at 600 nm was measured (A1). The hydropho- ical by antioxidant, leading to decrease in absorbance. The bicity percentage was calculated using the following for- control was performed using PBS (pH 7.4). The absorbance mula: Hydrophobicity % = [(A0 − A1)/A0] × 100. A0and was measured at 517 nm using UV visible spectrophotome- A1 are the absorbance values of the aqueous phase before ter, and the readings were recorded in duplicate and the av- and after contact with n-hexadecane, respectively. The erage absorbance value was calculated. The antioxidant Ann Microbiol (2019) 69:61–72 65 activity of the samples was expressed as the percentage of tested Lactobacillus strains, likely suggesting a high adap- radical scavenging as follows: (%) = [(A517 control − A517 sam- tation to the use of niche-specific carbohydrates. ple)/A517 control] × 100. Screening of probiotic properties Statistical analysis Tolerance to low pH and bile salts Data were statistically analyzed using the SPSS 11.0 software for Windows (SPSS Inc., Chicago, IL, USA). One-way In order to exert their beneficial effects in the host, orally ANOVA tests were performed to determine differences ingested bacteria must remain alive under unfavorable among strains. Data were considered significantly different conditions during ingestion and their transit prior to at p < 0.05. When appropriate, the post-hoc mean comparison reaching the large intestines. To this end, the strains must LSD test was additionally used. survive the acidic stomach conditions and the bile salts secreted into the luminal content in the proximal small intestine (Owehand et al. 2005). Results and discussion To assure their resistance against gastric stress, lactobacilli strains were screened for their ability to survive at low pH (pH 2.0, pH 2.5, and pH 3.0) (Fig. 1). All the strains revealed high Isolation and identification of lactic acid bacteria survival rates (> 50%), even at pH 2, the values being compa- rable or even higher than those observed for the commonly Thirteen lactobacilli strains were isolated from feces of used L. rhamnosus strain GG (which was included as positive healthy humans. The isolates were firstly selected based control). This observation is in agreement with the on being Gram-positive, catalase-negative rods. Then, results reported by (Liong 2011) revealing that several identified based on the 16S rRNA genes. The obtained Lactobacillus strains were in general very resistant to low sequences showed similarity with those of known species pH. (Kailasapathy and chin 2000), reported that many available in the NCBI database (Table 1). The iso- Lactobacillus strains exhibited a tolerance to acidic conditions lates were identified as L. fermentum (06 strains), due to their capacity of cytoplasmic buffering at low pH, en- L. plantarum (02 strains), L. salivarius (01 strains), and abling them to tolerate pH changes and to maintain stability L. rhamnosus (04 strains). This finding is in agreement (Krulwich et al. 2001). with several studies showing the presence of these The lactobacilli strains were also tested for their ability Lactobacillus species in human fecal samples (Hammes to survive for 3 h in MRS broth supplemented with 0.3% and Vogel 1995;Bird2002). bile (Fig. 2). All strains, but L. fermentum 10, were able to tolerate 0.3% bile salts after 3 h of exposure with sur- Biochemical and physiological properties of LAB vival rate ranged between 53 and 108%. However, we observed a significant variability in resistance to bile salts The results of the biochemical characterization of the among the Lactobacillus strains, being L. rhamnosus 5, strains are shown in Table 1. All the isolates showed which showed a high survival rate. The concentration of homofermentative metabolism without CO2 production. bile used in this study is considered physiological (Dunne All but the strains of L. plantarum were able to grow at et al. 2001) and often chosen as critical for the evaluation 42 °C. Moreover, these two L. plantarum strains (9 and 48) of strain resistance to bile (Gilliand 1989). In general, our were able to develop at 10 and 15 °C. Besides, strains of results confirm previous reports showing that the toler- L. fermentum 15 and 88, and L. plantarum 48 showed a ance is not necessarily related to the species, but strain tolerancetoNaCluptoa6%,whilethemajorityofthe specific (Maldonado et al. 2012). In the view of its poor strains were tolerant to a 3% NaCl. The sugar fermentation tolerance to bile, L. fermentum 10 was eliminated from profiles showed that all the strains fermented lactose and the further screening procedures. glucose, while variability was found for the other analyzed carbohydrates (Table 1). Interestingly, L. fermentum 32 and Antibiotic succeptibility and hemolytic activity 10 were able to ferment raffinose and melibiose. Besides, L. fermentum 88 and L. plantarum 9wereabletousexy- Excepting L. rhamnosus 36, all the strains were sensitive to lose and melibiose. These sugars are short-chain carbohy- chloramphenicol and penicillin, (Table 2). Besides, all the drates, non-digestible by humans and that selectively stim- strains showed sensitivity towards erythromycin, a part of ulate the growth of a limited number of beneficial bacteria L. rhamnosus 36, and L. salivarius 44. Lactobacillus spe- (Velikova et al. 2014). The current findings perceptibly cies should be susceptible to chloramphenicol and erythro- suggest that the metabolic activity differs between the mycin, antibiotics that inhibit protein synthesis (Klare et al. 66

Table 1 Phenotype and biochemical characterization of lactobacilli isolates from human feces

L. rhamnosus L. plantarum L. fermentum L. salivarius

5 12 36 54 9 48 15 19 32 10 88 89 44 MF066923 MF066924 MF066935 MF066929 MF066932 MF066934 MF066925 MF066926 MF066927 MF066936 MF066930 MF066931 MF066933

Growth at temperature 10 °C –– – – ++ –––– ––– 15 °C –– – – ++ –––– ––– 42 °C ±+ + + ±± + + + + +++ 60 °C –––––––––– +++ Growth in NaCl 0.2% +++++++++++++ 3% ++++++++±++++ 6% –– – – –++–––––+ Carbohydrates fermentation Lactose ++++++++++ +++ Glucose ++++++++++ ++– Galactose + – ++++++++ ––+ Xylose –– + – + ––––– ++– Maltose ++ + + +– +++– +++ Raffinose – + – + –– – +++ ––+ Melibiose –– – – ++ + – + + +++ Amidon ++ + + +––+++ + + – n irbo 21)69:61 (2019) Microbiol Ann L, Lactobacillus – 72 Ann Microbiol (2019) 69:61–72 67

Fig. 1 Survival rate of Lactobacillus strains to low pH. Each value represents the mean value ±standard deviation (SD) from three trials

2007). Most of the strains resulted sensitive to rifampicin Adhesion assay to intestinal epithelial cell HT29 and streptomycin, while the contrary was true for vancomycin. High rate of resistance to vancomycin was ob- The adhesion and transitory colonization of probiotic served, since lactobacilli are known to be naturally resis- bacteria in the gastrointestinal tract of the host is be- tant towards vancomycin by a non-transferable resistance lieved to be one of the desired features required for the mechanisms (Zhou et al. 2005),basedonthepresenceof delivery of their health benefits (Reid 1999). Among D-alanine ligase-related enzymes which renders the cell our isolates L. fermentum 88 showed the highest value wall not susceptible to the antibiotic (Bernardeau et al. of adhesion (Fig. 3) to the human cell line HT29 2008). The strain L. salivarius 44 showed a resistance to (17.5%), followed by L. plantarum 9 (9.5%), with both several antibiotics and a β-hemolytic activity (Table 2). It strains showing significantly better adhesion values than is desirable that the strains for probiotic or starter cultures the reference strain L. rhamnosus GG (7%). This high do not present a pathogenic feature and a risk of transmis- adhesion may be indicative of a good colonization abil- sible antibiotic resistance genes (Gueimonde et al. 2013). ity of these strains, likely related to their surface prop- For this reason, L. rhamnosus 36 and L. salivarius 44 were erties. Different surface determinants have been reported excluded from the following tests. to be involved in the interaction of lactobacilli with

Fig. 2 Survival rate of Lactobacillus strains at 0.3% bile. Each value represents the mean value ±standard deviation (SD) from three trials 68 Ann Microbiol (2019) 69:61–72

Table 2 Antibiotic susceptibility profiles and hemolytic activity of tested Lactobacilli strains

Chloramphenicol Lincomycin Penicillin Rifampicin Streptomycin Vancomycin Erythromycin Hemolytic (30 μg) (2 μg) (10 μg) (5 μg) (10 μg) (30 μg) (15 μg) activity

L. rhamnosus 5 SSSRSRSα-hemolytic L. rhamnosus 12SSSSRRS α-hemolytic L. rhamnosus 36 R R R S S R R α-hemolytic L. rhamnosus 54 S S S S S R S α-hemolytic L. plantarum 9SSSSSR S α-hemolytic L. plantarum 48 S R S S S R S α-hemolytic L. fermentum 15 S S S S S R S α-hemolytic L. fermentum 19 S S S S S R S α-hemolytic L. fermentum 32SSSSRRS α-hemolytic L. fermentum 88 S S S R S R S α-hemolytic L. fermentum 89 S R S S S R S α-hemolytic L. salivarius 44 S R S R S R R β-hemolytic

S,sensitive;I, intermediate;R,resistant;L, Lactobacillus

intestinal epithelial cells including passive forces, elec- been repeatedly reported by other authors. On the basis trostatic interactions, hydrophobic, steric forces, and sur- of the adhesion assay to epithelial cells, which is con- face molecules such as Pili and lipoteichoic acids sidered as one of the most important criteria for the (Granato et al. 1999). Therefore, the adhesion to epithe- bacteria to resist and to establish, at least transiently, lial cell lines seems to be a multifactorial process with through the gastrointestinal tract (Ruiz et al. 2013), the implication of many different potential mechanisms. L. plantarum 9andL. fermentum 88 were selected for Our data, however, do not allow establishing conclu- further analysis. sions on the potential mechanisms involved in the high adhesion ability of these two strains. Interestingly Inhibition of pathogens adhesion to epithelial cell HT29 the strain showing the lowest adhesion ability, L. fermentum 19, belongs to the same species that per- The inhibition of the adhesion of pathogens to the HT29 formed the higher adhesion (L. fermentum 88), indicat- epithelial cell line was found to be rather specific, depend- ing the strain specificity of this phenomenon, as it has ing on both the lactobacilli strain and the pathogen used,

Fig. 3 Adhesion (%) of potential probiotic Lactobacillus strains to HT29 cell line. The adhesion of each strains was compared with that of the reference strain L. rhamnosus GG by means of one-way ANOVA p <0.05,p <0.01,p<0.001 Ann Microbiol (2019) 69:61–72 69 which is in accordance with previous reports (Gueimonde may play a role on adhesion also favoring the attachment to et al. 2006). When compared to the positive control (path- other cells (Sommer et al. 1999). According to Fig. 4,itseems ogen alone) both selected strains (L. plantarum 9and that all lactobacilli would slightly increase the adhesion. These L. fermentum 88), as well as L. rhamnosus GG (included results obtained for the adhesion ability and the inhibition of for comparison purposes), reduced significantly (p <0.05) the adhesion of pathogens are in line with previous reports the adhesion of E. coli to the human intestinal cell line (Gueimonde et al. 2006; Collado et al. 2005, 2006, 2007), (Fig. 4). This result confirmed some reports indicating that indicating that the inhibition is not directly related to the ad- several probiotic agents are able to inhibit the cell associ- hesion ability of the strains. ation and adherence of pathogenic bacteria to intestinal epithelial cells. In fact, previous studies have shown that Hydrophobicity, aggregation, and coaggregation assays strains such as L. plantarum 299v and L. rhamnosus GG are able to induce the production of mucin by the epithelial To get further insight into the properties of these two cells inhibiting the adhesion of enteropathogenic E. coli Lactobacillus strains, their surface properties (hydrophobicity (Mack et al. 1999). Besides, some Lactobacillus strains and aggregation assay) were evaluated. These properties have share specific binding sites for carbohydrates with entero- been correlated to their potential adhesion ability to host tis- pathogenic bacteria, allowing their competition for the re- sues of the strains (Gómez Zavaglia et al. 2002). ceptor sites on host cells (Fujiwara et al. 2001;Nesseretal. L. plantarum 9 showed the highest hydrophobicity 23.9% 2000). Furthermore, it has been suggested that the (Table 3). These hydrophobicity percentages were lower than coaggregation of Lactobacillus strains with pathogens, that of the reference strain L. rhamnosus GG (64.14%). may constitute an important host defense mechanism Several authors have proposed that surface properties corre- (Reid et al. 1988). late with their adhesive capacity of the strains (Kos et al. 2003; Interestingly, L. fermentum 88 increased significantly Kotzamanidis and Kourelis 2010). In our study, L. plantarum (p < 0.05) the adhesion of the pathogen C. sakazaki (Fig. 4), 9andL. fermentum 88 revealed medium hydrophobic proper- while no effect on its adhesion to human intestinal cell line ties but a strong adhesion to HT29 cell lines (Fig. 3, Table 3), was observed for the other strains. Similarly to our observa- indicating that hydrophobicity was not directly related to the tions, increases in the adhesion of pathogen in the presence of adhesion to HT29 cells ability. In agreement with this obser- probiotic strains, including both bifidobacteria and vation, different authors have reported that, although hydro- lactobacilli, have been reported (Gueimonde et al. 2006; phobicity is a factor to consider, it does not directly correlate Arboleya et al. 2011). However, the biological significance with adhesion (Gómez Zavaglia et al. 2002; Savage 1992; of this phenomenon is unknown. It is possible that the patho- Gueimonde et al. 2005). gen adhered to the Lactobacillus strains and is no longer able Regarding autoaggregation activity, L. plantarum 9and to invade the epithelial cell. Therefore, it is a common trait of L. fermentum 88 presented higher values (70.74% and microbial pathogens to express adherence factors, permitting 64.89%, respectively) than the reference probiotic strain the bacteria to resist to the main lines of host internal defense L. rhamnosus GG (42.2%) (Table 3). The autoaggregation against them. In our specific case, according to Valcarce et al. of probiotic strains has also been correlated with adhesion (2002), C. sakazaki has a hydrophobic cell surface, which to intestinal epithelial cells (Jankovic 2012). These two

Fig. 4 Adhesion inhibition of pathogens to HT29 cell line by three Lactobacillus strains. The adhesion of Escherichia coli LMG 2092, Cronobacter ** sakazaki,andSalmonella ** typhimurium LMG 1586 in the ** * presence of each LAB was compared with the adhesion of the pathogen alone by means of one-way ANOVA. Differences were represented by asterisks (p <0.05,p <0.01,p <0.001) 70 Ann Microbiol (2019) 69:61–72

Table 3 Surface hydrophobicity, autoaggregation, and coaggregation properties of selected Lactobacillus isolates with indicator pathogens

Strains Hydrophobicity (%) Autoaggregation (%) % Coaggregation after 60 min with

S.typhimurium E.coli S. aureus IPT13 DH5 6538

L. plantarum 9 23.49 ± 0.02 70.74 ± 0.01 17.87 ± 0.015 13.65 ± 0.04 23.76 ± 0.055 L. fermentum 88 14.22 ± 0.08 64.89 ± 0.07 16.71 ± 0.03 25.51 ± 0.06 28.27 ± 0.06 L. rhamnosus GG 64.14 ± 0.08 42.2 ± 0.05 ND ND ND

Each value represents the mean value ±standard deviation (SD) from three trials. ND, not determinate strains were also able to coaggregate with the tested path- 2006;Makrasetal.2006). Xanthopoulos et al. (2000)reported ogens (Table 3). Previous studies have shown variable that L. paracasei and L. acidophillus isolated from infant feces coaggregation abilities of probiotics to different pathogens had weak antibacterial activity against E. coli, while our find- (Ramos et al. 2013; Saraniya and Jeevaratnam 2014), in- ings showed that strains belonging to L. rhamnosus had an dicating that the extent of coaggregation was strain-spe- interesting antibacterial activity against E. coli. Confirming to cific. These results suggested that L. plantarum 9and the study of Jacobsen et al. (1999), our findings showed that L. fermentum 88 can prevent adhesion of some pathogenic the tested Lactobacillus had a satisfactory abilities to inhibit bacteria by competition for binding sites on intestinal ep- various pathogenic bacteria. ithelial cells, and consequently these strains may be can- didates for reducing colonization by pathogens, therefore Antioxidant activity preventing infection (Gobin 2011). We have determined the antioxidant activity of intact cells and Antibacterial activity assay intracellular extracts of the two selected lactobacilli. DPPH scavenging activity ranged between 27 and 58%. The scav- The inhibitory activity of L. plantarum 9andL. fermentum 88 enging ability of the intact cells of the tested potential probi- strains against the human pathogens L. monocytogenes, otic strains was relatively medium compared to L. rhamnosus S. aureus and B. cereus,S. typhimurium, E. coli,and GG (91.7%) (Xanthopoulos et al. 2000). L. fermentum 88 P. aeruginosa was evaluated (Table 4). The results showed exhibited a higher ability to scavenge the radical DPPH that both strains revealed an inhibition against all the tested (58.98 ± 0.1%) than L. plantarum 9 (27% ± 0.05). Similar re- pathogenic bacteria. The detected antibacterial activity was sults were reported by Uugantsetseg and Batjargal (2014)that found against all the pathogens tested. Besides, L. plantarum showed inhibition rate in the range of 35.8 to L. fermentum 88 had a notable inhibitory activity towards S. 38%. This finding is also in agreement with the study of Mary typhimurium and L. monocytogenes while L. plantarum 9 et al. (2012)forLactobacillus isolated from African fermented showed higher efficacy than L. fermentum 88 for E. coli and foods. For the intracellular extracts, the maximum antioxidant P. aeruginosa. These findings of the antagonistic activity to- activity was again observed with L. fermentum 88 (43.87%), wards pathogenic bacteria lead to suggest the involvement of while L.plantarum 9 showed 30.32%. The intracellular cell- the organic acid, bacteriocins, or other compounds, like H2O2, free extracts from LAB had been found to have metal ion active in acidic conditions. In fact, organic acids have a strong chelating ability, reactive oxygen species scavenging ability, inhibiting power against Gram-negative bacteria and are con- and reduction activity (Lin and Yen 1999). These free radical sidered as essential antimicrobial compounds responsible for scavenging properties could help to create a low redox poten- the inhibitory activity of probiotics (De Keersmaecker et al. tial in the intestine after consumption of the strains.

Table 4 Antibacterial activity of selected Lactobacillus isolates against indicator pathogens

S. typhimurium E.coli S. aureus B. cereus L. monocytogenes P. aeruginosa IPT13 DH5 6538 49 CM15 ATCC 27853

L. plantarum 9 +(12 ± 0.04) ++ ++ + +(12 ± 0.04) ++(15 ± 0.01) (15 ± 0.03) (13 ± 0.015) (12 ± 0.05- 5) L. fermentum 88 ++(15 ± 0.03) +(12 ± 0.06) ++(13 ± 0.05) +(12 ± 0.05) ++(15 ± 0.015) +(12 ± 0.027)

Inhibition zone in mm; Each value represents the mean value ±standard deviation (SD) from three trials. (−), absence of inhibition zone; (+), d ≤ 12 weak inhibition activity; (++), medium activity with diameter 12 < d ≤ 15 Ann Microbiol (2019) 69:61–72 71

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