Anaerobe 34 (2015) 169e173
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Screening of Propionibacterium spp. for potential probiotic properties Daniela Campaniello, Antonio Bevilacqua, Milena Sinigaglia, Clelia Altieri* Department of the Science of Agriculture, Food and Environment, University of Foggia, Via Napoli 25, 71122 Foggia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 November 2014 Received in revised form 10 June 2015 Accepted 11 June 2015 Available online 14 June 2015
The main topic of this paper is the evaluation of adhesion of propionibacteria to IPEC-J2 cells and the survival at pH 2.5 and with 0.3% bile salts added, bioactivity towards pathogens and antibiotic resistance of Propionibacterium freudenreichii subsp. shermanii, Propionibacterium jensenii, Propionibacterium acidipropionici and Propionibacterium thoenii. Adhesion to IPEC-J2 cell lines was ca. 25e35% and significantly increased with CaCl2. Moreover, propionibacteria showed a reduction of cell count of ca. 0.5% at pH 2.5 after 3 h, whereas cell count increased after 24 h with bile salts; finally, they significantly inhibited Escherichia coli O157:H7. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Propionibacteria Selection criteria Adhesion IPEC-J2 cells
1. Introduction Propionibacteria belong to the Actinobacteria class with a high G þ C content (64e68%); they are mesophilic, Gram-positive, catalase positive, non-motile pleomorphic rods, non-sporeforming, and anaerobic to aerotolerant bacteria. Some cells may be elongated, bifid or arranged in “Chinese characters” [1]. They grow at 15e40 C and at pH 5.1e8.5; the optimal temperature for growth is 30 C [1,2]. The current taxonomy describes 13 species that can be divided into two groups: “dairy or classical” and “cutaneous”; the classical propionibacteria (Propionibacterium acidipropionici, Propionibacterium cyclohexanicum, Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium microaerophilum, Propionibacterium thoenii) are generally isolated from milk and dairy environments, whereas the cutaneous propionibateria (Propionibacterium acidifaciens, Propionibacterium acnes, Propionibacterium australiense, Propionibacterium avidum, Propionibacterium granulosum, Propionibacterium humerusii, Propionibacterium propionicus) are from skin/intestine of human and animals. Propionibacteria metabolize different carbohydrates (glucose, galactose, lactose, fructose and others), alcohols (glycerol, erythritol and others) and organic acids (lactic and gluconic acids), and produce propionic and acetic acids and carbon dioxide as final products. In addition, bacteriocins and vitamins can be produced.
* Corresponding author. E-mail address: [email protected] (C. Altieri). http://dx.doi.org/10.1016/j.anaerobe.2015.06.003 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
Due to their antimicrobial activity, propionibacteria are used to enhance the technological properties of various food products, e.g. they are used to prolong the shelf life of bread, cakes, cheeses, fruits, vegetables and tobacco, as they suppress the growth of moulds and spoilage microorganisms [1]. Propionibacteria also stimulate the immune system and limit cancer progression although the mechanism involved is not defined. Cousin et al. [2] reported that dairy propionibacteria are able to prevent infections and allergies, promote immune system maturation, and reduce the risk of cancer because they bind carcinogenic compounds (mycotoxins, plants lectins and heavy metals). Cutaneous propionibacteria show a similar genetic and biochemical profile [3] and have been used as a pre-treatment in patients with colorectal carcinoma where they produced beneficial immunostimulation [2]. Propionibacteria persist transiently in the gut after ingestion for some weeks and exert positive effects on human health, because of their adaptability and high tolerance to digestive stress. Probiotic bacteria, delivered through food products, have to survive during the transit through the upper gastrointestinal tract, persist in the gut, and confer a health benefit [4,5]; on the other hand, the low pH of the stomach and secretion of bile salts into the small bowel could cause a bactericidal effect. Two desirable properties for probiotic bacteria are adhesion to intestinal mucosa and the antagonistic activity towards pathogens; EFSA (European Food Safety Agency) also requires the evaluation of antibiotic resistance as this trait could be a safety issue [6]. P. freudenreichii and P. acidipropionici,
170
D. Campaniello et al. / Anaerobe 34 (2015) 169e173
have been added to the QPS list (Qualified Presumption of Safety) [7]. Probiotics can be selected by using many criteria; the main aim of this paper was the evaluation of adhesion of propionibacteria to IPEC-J2 cells, used as a model of the ileal mucosa. Strains from a public collection (P. freudenreichii subsp. shermanii; ATCC 9614, P. jensenii ATCC 4870; P. acidipropionici ATCC 4875; P. thoenii ATCC 4874) were used as study subjects. As an additional aim, some other criteria were assessed (survival throughout the transit through the stomach and intestine, antagonistic activity towards some pathogens and resistance/susceptibility to antibiotics).
OptiPhase ‘Hisafe’ 2 (Fisher Chemicals, Louhghsborough Leisc., UK); radioactivity was measured through a scintillation counter (Wallac 1414 WinSpectral, Turku, Finland). Adhesion was expressed as the ratio of the radioactivity of lysed cells to the radioactivity of the labeled bacterial suspension. Lactobacillus reuteri 12002 [8] was used as a positive control. Each assay was performed in triplicate. A second assay was performed to analyze the effect of Ca2þ; the protocol was modified as follows: the experiment was performed by adding 950 mL of propionibacteria labeled with L-[methyl-3H] methionine and 50 mL of 200 mM of CaCl2 solution to wells containing IPEC-J2 monolayers, and the adhesion assay was performed as above.
2. Material and methods 2.3. Effect of acidic pH and bile salts 2.1. Microorganisms and growth conditions P. freudenreichii subsp. shermanii DSM 20270 (ATCC 9614; source: cheese), P. jensenii DSM 20279 (ATCC 4870; source: emmental cheese), P. acidipropionici DSM 20272 (ATCC 4875; source: emmental cheese), Propionibacterium thoeni DSM 20276 (ATCC 4874; source: emmental cheese) were used in this research. Strains were purchased from DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany) and grown in Propionibacterium medium (caseine peptone tryptic digest 10 g/L; yeast extract 5 g/L; Na-lactate 10 g/L; pH was adjusted to 7.0e7.02), with incubation at 30 C for 7 days, as suggested by the depositor. Cells were then harvested by centrifugation (5000 g for 10 min) and suspended in MRS broth (Oxoid, Hampshire, United Kingdom) containing 0.05% (w/v) L-cysteine (SigmaeAldrich, Milan) (cMRS) and incubated at 37 C for 5 days. Propionibacteria were stored at 18 C in cMRS containing 33% (v/v) sterile glycerol (J.T. Baker, Milan).
The effect of pH and bile salts was assessed on propionibacteria during stationary phase. cMRS acidified to pH 2.5 with HCl 5.0 mol/ L or containing 0.3% (w/v) bile salts (Oxoid) was inoculated to 7 log CFU/mL and incubated at 37 C for 3 or 24 h; viable counts were assessed by pour-plating on cMRS agar, with incubation at 37 C for 5 days under anaerobic conditions. cMRS broth (pH 6.0) inoculated with propionibacteria was used as a control. The experiments were performed on two independent batches; for each batch the analyses were performed twice. Data were modeled as viability loss (V.L.) (effect of pH) and growth index (DlogN) (effect of bile salts):
V:L: ¼ ð1 logNt=logN0 Þ*100 Dlog N ¼ log Nt log N0 where Nt is cell count after 3 or 24 h and N0 the initial cell count.
2.2. Adhesion assay
2.4. Bioactivity against foodborne pathogens
This assay was performed in Denmark (Department of Food Science, Faculty of Life Sciences, University of Copenhagen). IPEC-J2 cells were grown at 37 C in a 5% CO2, 95% air-humidified incubator in a medium containing a 1:1 mixture of Dulbecco's modified Eagle medium (DMEM; SigmaeAldrich) and F12 (SigmaeAldrich) supplemented with 100 mg/L streptomycin (Fluka Chemie GmbH, Steinheim, Switzerland), 100 mg/L penicillin (SigmaeAldrich), 2 mmol/L L-glutamine (SigmaeAldrich), 1 mmol/L pyruvate (SigmaeAldrich) and 10% fetal bovine serum (Cambrex Bio Science, Verviers, Belgium). Experiments were performed in DMEM medium containing F12 but without antibiotics. IPEC-J2 cells were seeded at a concentration of 5 105 cells per well (ca. 1 105 per cm2) in 12-well tissue culture plates (Corninc Inc, Corning, NY, USA) and grown to 100% confluence. Cell culture medium was changed every two days. cMRS broth (10 mL) was inoculated with propionibacteria and incubated at 37 C for 24 h; these cultures were used to inoculate 2 mL of cMRS broth containing 100 mL of metabolic radiolabelling 2 Mq/mL L-[methyl-3H] methionine (Amersham Biosciences, Uppsala, Sweden) and incubated at 37 C for 21 h. Bacteria were then centrifuged (6000 rpm for 2 min), washed three times with sterile PBS (9.0 g/L NaCl, 0.3 g Na2HPO4 *2H2O, pH 7) and suspended in DMEM containing F12 (7 log CFU/mL). The adhesion assay was performed by adding 1 mL of propionibacteria suspension separately to three wells containing an IPEC-J2 monolayer. After 1 h at 37 C, IPEC-J2 cells were washed three times with DMEM containing F12 and left overnight with 500 mL of NaOH/SDS solution (0.1 mmol/L NaOH, 1% w/v SDSAmersham Biosciences) to lyse cells. Cells were then treated with a buffer solution of Na2HPO4 *2H2O/citric (Na2HPO4 *2H2O 0.1 M; citric acid 0.1 M; pH 3) and added to 4.5 mL scintillation liquid
Listeria monocytogenes and Escherichia coli O157:H7, belonging to the Culture Collection of the Laboratory of Predictive Microbiology-Department of Science of Agriculture, Food and Environment (SAFE, University of Foggia, Italy) and isolated respectively from dairy and meat products, were used as test pathogens. Pathogens were grown in Nutrient Broth (Oxoid) (37 C for 24e48 h); aliquots of 100 mL of each pathogen (7 log CFU/mL) were plated on cMRS agar; thereafter, disks (9 mm) (Schleicher & Schuell Microscience, Dassel, Germany) were placed onto the surface of the agar and inoculated with 20 mL of the following suspensions: a) b) c) d)
propionibacteria cell cultures (pH 4.5); propionibacteria cell cultures, adjusted to pH 6.5; supernatant of propionibacteria; supernatant of propionibacteria adjusted to pH 6.5.
After the incubation (37 C for 48 h), a clear halo indicated the antimicrobial activity of propionibacteria against L. monocytogenes or E. coli O157:H7; the diameter of the inhibition halo was measured [8]. The experiments were performed on 3 different batches. 2.5. Susceptibility of Propionibacterium strains to antibiotics Susceptibility testing was performed as reported by NCCLS (National Committee for Clinical Laboratory Standards, 1993) [9]. Propionibacteria strains were grown in cMRS broth and inoculated onto the surface of cMRS agar. Antibiotic discs (Neo Sensitabs®, Taastrup, Denmark) (Table 1) were placed on inoculated plates and
D. Campaniello et al. / Anaerobe 34 (2015) 169e173
171
Table 1 Antibiotics used in this research. Antibiotics
Target
Group I: b-lactams Ampicillin Group II: no b-lactams Vancomycin (glycopeptide) Group III: protein synthesis inhibitors Erythromycin (macrolide saturated) Gentamycin (aminoglycoside) Streptomycin (aminoglycoside) Chloramphenicol Tetracyclines Group IV: other antibiotics Ciprofloxacin (quinolinone) Trimethoprim (sulphamide) a
Dosage (mg/disk)
Peptidoglycan
33
Peptidoglycan
70
50 S rRNA 30 S rRNA 30 S rRNA 50 S rRNA rRNA DNA gyrase DHFRa
78 40 100 60 80 10 52
DHFR ¼ dihydroflotareductase.
incubated at 37 C for 4 days, under anaerobic conditions. Susceptibility was assessed by measuring zone of inhibition. 2.6. Statistical analysis Data were analyzed through one-way analysis of variance (1way ANOVA), using Duncan's test as the post-hoc test, or through the t-test (paired comparison) (Statistica for Windows; Statsoft, Tulsa, Okhla.). 3. Results and discussion Selection of probiotics is a complex process and many traits should be assessed. In this study, we used a subset of selection criteria (survival at pH 2.5 and with 0.3% bile salts added, bioactivity towards pathogens and resistance to antibiotics) and focused on the use of a cell line derived from the normal mucosa of ileum to assess adhesion. 3.1. Adhesion assay Adhesion is generally evaluated through the use of model cell lines; Caco-2 cells (originally isolated from a human colon adenocarcinoma) [10] were used by several researchers [11,12]. Other authors used C2BBe1 [13], subcloned from Caco2 cells, or HT29 colonic adenocarcinomal human intestinal epithelial cell lines [11]. IPEC-J2 cells are nontumurogenic cells and possess traits of normal cells of ileum mucosa thus they represent an interesting alternative to Caco cell lines, generally used, for investigation of the adhesion ability of propionibacteria. Table 2 shows adhesion of propionibacteria and Lb. reuteri 12002 (positive control). Propionibacteria were able to adhere to IPEC-J2 cells, without significant differences amongst the strains. Adhesion ranged from 25.58% for P. acidipropionici to 35.09% for P. freudenreichii subsp. shermanii. Similar results were obtained for Lb. reuteri 12002 (33.50%).
Fig. 1. Viability loss of propionibacteria in acidified cMRS broth (pH 2.5) after 3 and 24 h. Mean values ± standard deviation. Values with different letters are significantly different (one-way ANOVA and Duncan's test) (P < 0.05).
Ouwehand et al. [14] studied adhesion of four P. freudenreichii strains to human intestinal mucus and observed that in vitro adhesion was moderate (0.2e6.5%). Collado et al. [15] tested various probiotic microorganisms (Lactobacillus rhamnosus GG, Lb. rhamnosus LC705, P. freudenreichii JS, Bifidobacterium breve 99) and observed that they were able to adhere to intestinal mucus and that P. freudenreichii JS showed the lowest adhesion percentage (0.9 ± 0.5%). When calcium was added, adhesion significantly increased to 47.95% for P. thoenii and 62.5% for P. jensenii; in particular, P. jensenii showed higher values than Lb. reuteri (62.5% vs 48.1%). Adhesion of P. freudenreichii subsp. shermanii was also studied by Lehto and Salminen [16]; they reported that this species showed adhesion percentages similar to L. rhamnosus GG, used as a positive control. Other researchers showed that propionibacteria adhered to intestinal cells both in vivo and in vitro [1,13,17] and that calcium et al. [18] reported that Propionibacteimproved adhesion. Zarate rium adhesion was calcium independent; nevertheless it could be enhanced when calcium was added. The effect of calcium probably relied upon the formation of ionic bonds between bacterial and epithelial cells. 3.2. Tolerance to pH 2.5 and bile salts The acidic environment of the stomach and the presence of bile in the small intestine represent two biological barriers that probiotics need to tolerate in order to exert their beneficial effects; thus it is necessary to study the survival of probiotic strains at pH 2.5 and with 0.3% bile salts to simulate the contact of microorganism with gastric juice and bile [19,20]. Fig. 1 shows the viability loss of propionibacteria at pH 2.5 after 3 and 24 h (this represented transit time through the stomach, and prolonged acid stress, respectively). After 3 h, the strains did not show any kind of viability loss (V.L.) thus confirming their significant resistance to acidic conditions. After 24 h P. freudenreichii
Table 2 Adhesion (%) of propionibacteria and Lactobacillus reuteri (positive control) on IPEC-J2 cell line. Mean values ± standard deviation. Control P. freudenreichii subsp. shermanii DSMZ 20270 P. acidipropionici DSMZ 20272 P. jensenii DSMZ 20279 P. thoenii DSMZ 20276 Lb. reuteri
35.09 25.58 29.87 25.65 33.50
± ± ± ± ±
CaCl2 200 mM 3.60aA 2.05aA 7.91aA 1.80aA 4.97aA
A,B Data in a column with different letters are significantly different (one-way ANOVA and Tukey's test) (P < 0.05). a,b Values in a line with different letters are significantly different (t-test, P < 0.05).
51.1 ± 1.38bA 48.10 ± 4.95bA 62.5 ± 5.67bB 47.95 ± 3.99bA 48.10 ± 3.06bA
172
D. Campaniello et al. / Anaerobe 34 (2015) 169e173
Recently Darilmaz and Beyatli [5] studied the inhibitory properties of propionibacteria against different pathogens and reported that E. coli ATCC 11229 and Shigella sonnei Mu:57 were generally inhibited. 3.4. Susceptibility of Propionibacterium to antibiotics
Fig. 2. Increase of cell count in cMRS broth þ0.3% of bile salts after 3 and 24 h. Mean values ± standard deviation. Values with different letters are significantly different (one-way ANOVA and Duncan's test) (P < 0.05).
subsp. shermanii, P. jensenii, P. thoenii showed a viability loss of 30%. Our results are not in agreement with other researchers; for example, Warminska-Radyko et al. [21] found different results: they studied 27 propionibacteria and found that at pH 2.5 tested strains generally survived for 1 h and only five strains survived for 2 h. Suomalainen et al. [22] also focused on this selection criterion and found that propionibacteria count was significantly reduced after 3 h at pH 2, but not at pH 4 or 3. Cousin et al. [23] found P. jensenii and P. acidipropionici (BIA64) did not survive to pH 2.5. Darilmaz and Beyatli [5] also observed a significant reduction in cell count at pH 2.0 and 3.0, for P. freudenreichii subsp. freudenreichii and P. jensenii. The different trend of tested strains from the results found in literature could be due to the strong strain-specificity to survive at acidic pH; this hypothesis was suggested by Boke et al. [24] for dairy propionibacteria. Concerning the effect of bile salts, propionibacteria experienced an increase in cell count ranging from 0.6 to 1.3 log CFU/mL after 24 h (Fig. 2), thus confirming the high tolerance of Propionibacterium spp. to intestinal conditions; Darilmaz and Beyatli [5] used a spectrophotometric approach to test the resistance of propionibacteria to oxgall (0.06, 0.15, and 0.3%) and found that they were highly susceptible to these compounds. This different trend could be the result of the medium used (MRS broth versus Sodium Lactate Broth), as it is well known that medium type can enhance the bioactivity of an antimicrobial or exert a protective effect.
3.3. Antimicrobial activity Propionibacteria inhibited E. coli O157:H7, but they did not affect the growth of L. monocytogenes; the inihibition zone ranged from 1.97 cm (P. freudenreichii) to 3.83 cm (P. thoenii). Neither cell cultures nor supernatant adjusted to pH 6.5 exerted a similar effect, thus suggesting that the antimicrobial activity could be attributed to low pH.
An important criterion to select a probiotic is the antibiotic resistance; the results for this assay are reported in Table 3 and expressed as resistant (), moderately susceptible (þ), susceptible (þþ) and very susceptible (þþþ). Propionibacteria strains tested were resistant to vancomycin and ciprofloxacin and sensitive to ampicillin (P. thoenii, P. freudenreichii and P. acidipropionici) and chloramphenicol (P. freudenreichii and P. acidipropionici), with inhibition zones ranging from 29.5 mm to 35.5 mm (data not shown). Tested strains were susceptible to erythromycin, tetracycline and trimethoprim (inhibition zone of 21e30 mm); however, the effect of tetracycline was reversible. Finally propionibacteria were moderately susceptible to gentamycin and streptomycin (inhibition zone of 10e20 mm). Concerning the antibiotic resistance of propionibacteria, some authors reported a moderate/strong sensitivity to vancomycin, tetracycline, chloramphenicol, ampicillin, erythromycin and bacitracin using different strains than were assayed in the study reported here [22], thus our results were quite different and suggested the strong strain-dependence of this trait; in addition, the different protocol used could strongly affect the output: in fact, the authors in the reference [22] used a micro-dilution approach, whilst we performed the experiment through a disk diffusion assay. Spreading of antibiotic resistance is an increasing threat and pathogens can acquire this resistance by conjugation [25,26]. The genes encoding antibiotic resistance can be placed on the chromosome or on plasmids; it is strongly recommend to avoid the use of starter or probiotic microorganisms carrying these genes on plasmids, as they can transfer genes to pathogens into the gut [26]. However, some authors reported that the genes of antibiotic resistance of propionibacteria are carried out on the chromosome, thus the resistance to antibiotics is not a problem for these microorganisms [27]. 4. Conclusion Probiotic characterization of propionibacteria (PAB) has been carried out extensively in the past. However, the most important drawback of other studies is the use of cell lines from non-normal mucosa (e.g. Caco-2 from colon cancer or similar lines). IPEC-J2 are from the normal ileum of pig, thus this cell line retains all the traits and properties of the intestinal mucosa. In addition, we assessed some other criteria considered important for assessment of potential probiotic strains, such as viability at low pH and in the presence of bile salts, both under normal conditions (3 h) and under prolonged stress conditions (24 h). The tested strains were able to adhere to mammalian epithelial cells, survive in acidic environments and in presence of bile salts, and exert a moderate antimicrobial activity towards E. coli O157:H7.
Table 3 Antibiotic-resistance. Resistant (), moderately susceptible (þ; Inhibition zone: 10e20 mm), susceptible (þþ; inhibition zone: 21e30 mm) and very susceptible (þþþ; inhibition zone > 31 mm). Strains
Ampi
Vanc
Eryt
Gent
Streptom
Cloramfen
Tetr
Ciprof
Trim
P. P. P. P.
þþ þþþ þþþ þþþ
e e e e
þþ þþ þþ þþ
þ þ þ þ
þ þ þ þ
þþ þþ þþþ þþþ
þþ þþ þþ þþ
e e e e
þþ þþ þþ þþ
jensenii thoenii freudenreichii acidipropionici
D. Campaniello et al. / Anaerobe 34 (2015) 169e173
References , Dairy propionibacteria: less conventional probiotics to improve the [1] G. Zarate human and animal health, in: E.C. Rigobelo (Ed.), Probiotic in Animals, InTech Publisher, Rijeka, Croatia, 2012, pp. 153e202. , G. Jan, Dairy propionibacteria as human [2] F.J. Cousin, D.D.G. Mater, B. Foligne probiotics: a review of recent evidence, Dairy Sci. Technol. 91 (2011) 1e26. rez, B. Mayo, Identification, typing and characterization of [3] S. Delgado, A. Sua Propionibacterium strains from healthy mucosa of the human stomach, Int. J. Food Microbiol. 149 (2011) 65e72. [4] FAO/WHO: Guidelines for the evaluation of probiotics in food. Food and Agriculture Organization in the United Nations and World Health Organization Working Group Report. Available from: http://www.fao.org/es/ESN/food/ foodandfood. [5] D.O. Darilmaz, Y. Beyatli, Acid-bile, antibiotic resistance and inhibitory properties of propionibacteria isolated from Turkish traditional home-made cheeses, Anaerobe 18 (2012) 122e127. [6] A. Bevilacqua, D. Campaniello, B. Speranza, M. Sinigaglia, M.R. Corbo, Selection of promising strains of probiotics for foods: proposal for a possible flow-chart with a special focus on the dark side of probiotics, in: A. Roma (Ed.), Probiotics in Health and Disease: New Research, Nova Publishers, New York, 2014, pp. 1e22. [7] A. Thierry, S.M. Deutsch, H. Falentin, M. Dalmasso, F.J. Cousin, G. Jan, New insights into physiology and metabolism of Propionibacterium freudenreichii, Int. J. Food Microbiol. 149 (2011) 19e27. [8] A. Bevilacqua, C. Altieri, M.R. Corbo, M. Sinigaglia, L. Ouoba II, Characterization of lactic acid bacteria isolated from Italian Bella di Cerignola table olives: selection of potential multifunctional starter cultures, J. Food Sci. 75 (2010) M536eM544. [9] NCCLS (National Committee for Clinical Laboratory Standards), 24, Performance Standards for Antimicrobial Disc Susceptibility Test: Tentative Standards, vol. 13, National Committee for Clinical Laboratory Standards, Villanova, PA, 1993. NCCLS Documents M2eA5. [10] M. Pinto, S. Robine-Leon, M.D. Appay, M. Kedinger, N. Triadou, E. Dussaulx, B. Lacroix, P. Simon-Assmann, K. Haffen, A. Fogh, A. Zweibaum, Enterocytelike differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture, Biol. Cell 47 (1983) 323e330. [11] R.K. Duary, Y.S. Rajput, V.K. Batish, S. Grover, Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells, Indian J. Med. Res. (2011) 664e671. [12] G. Chauviere, M.H. Coconnier, S. Kerneis, J. Fourniat, A.L. Servin, Adhesion of Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells, J. Gen. Microbiol. 138 (1992) 1689e1696. [13] Y. Huang, M.C. Adams, An in vitro model for investigating intestinal adhesion of potential dairy propionibacteria probiotic strains using cell line C2BBe1, Lett. Appl. Microbiol. 36 (2003) 213e216.
173
€lkko €, S. Salminen, In vitro adhesion of [14] A.C. Ouwehand, T. Suomalainen, S. To propionic acid bacteria to human intestinal mucus, Lait 82 (2002) 123e130. [15] M.C. Collado, L. Jalonen, J. Meriluoto, S. Salminen, Protection mechanism of probiotic combination against human pathogens: in vitro adhesion to human intestinal mucus, Asia Pac. J. Clin. Nutr. 15 (2006) 570e575. [16] E. Lehto, S. Salminen, Adhesion of two Lactobacillus strains, one Lactococcus and one Propionibacterium strain to cultured human intestinal Caco-2 cell line, Biosci. Microflora 16 (1997) 13e17. [17] Y. Huang, L. Kotula, M.C. Adams, The in vivo assessment of safety and gastrointestinal survival of an orally administered novel probiotic, Propionibacterium jensenii 702, in a male Wistar rat model, Food Chem. Toxicol. 41 (2003) 1781e1787. , V. Morata De Ambrosini, A. Perez Chaia, S. Gonza lez, Some factors [18] G. Zarate affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells, Can. J. Microbiol. 48 (2002) 449e457. [19] A. Cebeci, C. Gürakan, Properties of potential probiotic Lactobacillus plantarum strains, Food Microbiol. 20 (2003) 511e518. [20] C.N. Jacobsen, V. Rosenfeldt Nielsen, A.E. Hayford, P.L. Mǿller, K.F. Michaelsen, € m, M. Tvede, M. Jakobsen, Screening of probiotic A. Pǽrregaard, B. Sandstro properties of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of colonization ability of five selected strains in humans, Appl. Environ. Microbiol. 65 (1999) 4949e4956. [21] I. Warminska-Radyko, L. Laniewska-Moroz, A. Babuchowski, Possibilities for stimulation of Bifidobacterium growth by Propionibacteria, Lait 82 (2002) 113e121. €, S. Tynkkynen, In vitro and in vivo [22] T. Suomalainen, P. Sigvart-Mattila, J. M€ atto gastrointestinal survival, antibiotic susceptibility and genetic identification of Propionibacterium freudenreichii ssp. shermanii JS, Int. Dairy J. 18 (2008) 271e278. [23] F.J. Cousin, S. Louesdon, M.B. Maillard, S. Parayre, H. Falentin, S.M. Deutsch, G. Boudry, G. Jan, The first dairy product exclusively fermented by Propionibacterium freudenreichii: a new vector to study probiotic potentialities in vivo, Food Microbiol. 32 (2012) 135e146. [24] H. Boke, B. Aslim, G. Alp, The role of resistance to bile salts and acid tolerance of exopolysaccharides (Epps) produced by yogurt starter bacteria, Arch. Biol. Sci. 62 (2010) 323e328. [25] M. Jahan, G.G. Zhanel, R. Sparling, R.A. Holley, Horizontal transfer of antibiotic resistance from Enterococcus faecium of fermented meat origin to clinical isolates of E. faecium and Enterococcus faecalis, Int. J. Food Microbiol. 199 (2015) 78e85. [26] J.R. Huddleston, Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes, Infect. Drug Resist 7 (2014) 167e176. [27] L. Meile, G. Le Blay, A. Thierry, Safety assessment of dairy microorganisms: Propionibacterium and Bifidobacterium, Int. J. Food Microbiol. 126 (2008) 316e320.
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Screening of Propionibacterium spp. for potential probiotic properties Daniela Campaniello, Antonio Bevilacqua, Milena Sinigaglia, Clelia Altieri* Department of the Science of Agriculture, Food and Environment, University of Foggia, Via Napoli 25, 71122 Foggia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 November 2014 Received in revised form 10 June 2015 Accepted 11 June 2015 Available online 14 June 2015
The main topic of this paper is the evaluation of adhesion of propionibacteria to IPEC-J2 cells and the survival at pH 2.5 and with 0.3% bile salts added, bioactivity towards pathogens and antibiotic resistance of Propionibacterium freudenreichii subsp. shermanii, Propionibacterium jensenii, Propionibacterium acidipropionici and Propionibacterium thoenii. Adhesion to IPEC-J2 cell lines was ca. 25e35% and significantly increased with CaCl2. Moreover, propionibacteria showed a reduction of cell count of ca. 0.5% at pH 2.5 after 3 h, whereas cell count increased after 24 h with bile salts; finally, they significantly inhibited Escherichia coli O157:H7. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Propionibacteria Selection criteria Adhesion IPEC-J2 cells
1. Introduction Propionibacteria belong to the Actinobacteria class with a high G þ C content (64e68%); they are mesophilic, Gram-positive, catalase positive, non-motile pleomorphic rods, non-sporeforming, and anaerobic to aerotolerant bacteria. Some cells may be elongated, bifid or arranged in “Chinese characters” [1]. They grow at 15e40 C and at pH 5.1e8.5; the optimal temperature for growth is 30 C [1,2]. The current taxonomy describes 13 species that can be divided into two groups: “dairy or classical” and “cutaneous”; the classical propionibacteria (Propionibacterium acidipropionici, Propionibacterium cyclohexanicum, Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium microaerophilum, Propionibacterium thoenii) are generally isolated from milk and dairy environments, whereas the cutaneous propionibateria (Propionibacterium acidifaciens, Propionibacterium acnes, Propionibacterium australiense, Propionibacterium avidum, Propionibacterium granulosum, Propionibacterium humerusii, Propionibacterium propionicus) are from skin/intestine of human and animals. Propionibacteria metabolize different carbohydrates (glucose, galactose, lactose, fructose and others), alcohols (glycerol, erythritol and others) and organic acids (lactic and gluconic acids), and produce propionic and acetic acids and carbon dioxide as final products. In addition, bacteriocins and vitamins can be produced.
* Corresponding author. E-mail address: [email protected] (C. Altieri). http://dx.doi.org/10.1016/j.anaerobe.2015.06.003 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
Due to their antimicrobial activity, propionibacteria are used to enhance the technological properties of various food products, e.g. they are used to prolong the shelf life of bread, cakes, cheeses, fruits, vegetables and tobacco, as they suppress the growth of moulds and spoilage microorganisms [1]. Propionibacteria also stimulate the immune system and limit cancer progression although the mechanism involved is not defined. Cousin et al. [2] reported that dairy propionibacteria are able to prevent infections and allergies, promote immune system maturation, and reduce the risk of cancer because they bind carcinogenic compounds (mycotoxins, plants lectins and heavy metals). Cutaneous propionibacteria show a similar genetic and biochemical profile [3] and have been used as a pre-treatment in patients with colorectal carcinoma where they produced beneficial immunostimulation [2]. Propionibacteria persist transiently in the gut after ingestion for some weeks and exert positive effects on human health, because of their adaptability and high tolerance to digestive stress. Probiotic bacteria, delivered through food products, have to survive during the transit through the upper gastrointestinal tract, persist in the gut, and confer a health benefit [4,5]; on the other hand, the low pH of the stomach and secretion of bile salts into the small bowel could cause a bactericidal effect. Two desirable properties for probiotic bacteria are adhesion to intestinal mucosa and the antagonistic activity towards pathogens; EFSA (European Food Safety Agency) also requires the evaluation of antibiotic resistance as this trait could be a safety issue [6]. P. freudenreichii and P. acidipropionici,
170
D. Campaniello et al. / Anaerobe 34 (2015) 169e173
have been added to the QPS list (Qualified Presumption of Safety) [7]. Probiotics can be selected by using many criteria; the main aim of this paper was the evaluation of adhesion of propionibacteria to IPEC-J2 cells, used as a model of the ileal mucosa. Strains from a public collection (P. freudenreichii subsp. shermanii; ATCC 9614, P. jensenii ATCC 4870; P. acidipropionici ATCC 4875; P. thoenii ATCC 4874) were used as study subjects. As an additional aim, some other criteria were assessed (survival throughout the transit through the stomach and intestine, antagonistic activity towards some pathogens and resistance/susceptibility to antibiotics).
OptiPhase ‘Hisafe’ 2 (Fisher Chemicals, Louhghsborough Leisc., UK); radioactivity was measured through a scintillation counter (Wallac 1414 WinSpectral, Turku, Finland). Adhesion was expressed as the ratio of the radioactivity of lysed cells to the radioactivity of the labeled bacterial suspension. Lactobacillus reuteri 12002 [8] was used as a positive control. Each assay was performed in triplicate. A second assay was performed to analyze the effect of Ca2þ; the protocol was modified as follows: the experiment was performed by adding 950 mL of propionibacteria labeled with L-[methyl-3H] methionine and 50 mL of 200 mM of CaCl2 solution to wells containing IPEC-J2 monolayers, and the adhesion assay was performed as above.
2. Material and methods 2.3. Effect of acidic pH and bile salts 2.1. Microorganisms and growth conditions P. freudenreichii subsp. shermanii DSM 20270 (ATCC 9614; source: cheese), P. jensenii DSM 20279 (ATCC 4870; source: emmental cheese), P. acidipropionici DSM 20272 (ATCC 4875; source: emmental cheese), Propionibacterium thoeni DSM 20276 (ATCC 4874; source: emmental cheese) were used in this research. Strains were purchased from DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany) and grown in Propionibacterium medium (caseine peptone tryptic digest 10 g/L; yeast extract 5 g/L; Na-lactate 10 g/L; pH was adjusted to 7.0e7.02), with incubation at 30 C for 7 days, as suggested by the depositor. Cells were then harvested by centrifugation (5000 g for 10 min) and suspended in MRS broth (Oxoid, Hampshire, United Kingdom) containing 0.05% (w/v) L-cysteine (SigmaeAldrich, Milan) (cMRS) and incubated at 37 C for 5 days. Propionibacteria were stored at 18 C in cMRS containing 33% (v/v) sterile glycerol (J.T. Baker, Milan).
The effect of pH and bile salts was assessed on propionibacteria during stationary phase. cMRS acidified to pH 2.5 with HCl 5.0 mol/ L or containing 0.3% (w/v) bile salts (Oxoid) was inoculated to 7 log CFU/mL and incubated at 37 C for 3 or 24 h; viable counts were assessed by pour-plating on cMRS agar, with incubation at 37 C for 5 days under anaerobic conditions. cMRS broth (pH 6.0) inoculated with propionibacteria was used as a control. The experiments were performed on two independent batches; for each batch the analyses were performed twice. Data were modeled as viability loss (V.L.) (effect of pH) and growth index (DlogN) (effect of bile salts):
V:L: ¼ ð1 logNt=logN0 Þ*100 Dlog N ¼ log Nt log N0 where Nt is cell count after 3 or 24 h and N0 the initial cell count.
2.2. Adhesion assay
2.4. Bioactivity against foodborne pathogens
This assay was performed in Denmark (Department of Food Science, Faculty of Life Sciences, University of Copenhagen). IPEC-J2 cells were grown at 37 C in a 5% CO2, 95% air-humidified incubator in a medium containing a 1:1 mixture of Dulbecco's modified Eagle medium (DMEM; SigmaeAldrich) and F12 (SigmaeAldrich) supplemented with 100 mg/L streptomycin (Fluka Chemie GmbH, Steinheim, Switzerland), 100 mg/L penicillin (SigmaeAldrich), 2 mmol/L L-glutamine (SigmaeAldrich), 1 mmol/L pyruvate (SigmaeAldrich) and 10% fetal bovine serum (Cambrex Bio Science, Verviers, Belgium). Experiments were performed in DMEM medium containing F12 but without antibiotics. IPEC-J2 cells were seeded at a concentration of 5 105 cells per well (ca. 1 105 per cm2) in 12-well tissue culture plates (Corninc Inc, Corning, NY, USA) and grown to 100% confluence. Cell culture medium was changed every two days. cMRS broth (10 mL) was inoculated with propionibacteria and incubated at 37 C for 24 h; these cultures were used to inoculate 2 mL of cMRS broth containing 100 mL of metabolic radiolabelling 2 Mq/mL L-[methyl-3H] methionine (Amersham Biosciences, Uppsala, Sweden) and incubated at 37 C for 21 h. Bacteria were then centrifuged (6000 rpm for 2 min), washed three times with sterile PBS (9.0 g/L NaCl, 0.3 g Na2HPO4 *2H2O, pH 7) and suspended in DMEM containing F12 (7 log CFU/mL). The adhesion assay was performed by adding 1 mL of propionibacteria suspension separately to three wells containing an IPEC-J2 monolayer. After 1 h at 37 C, IPEC-J2 cells were washed three times with DMEM containing F12 and left overnight with 500 mL of NaOH/SDS solution (0.1 mmol/L NaOH, 1% w/v SDSAmersham Biosciences) to lyse cells. Cells were then treated with a buffer solution of Na2HPO4 *2H2O/citric (Na2HPO4 *2H2O 0.1 M; citric acid 0.1 M; pH 3) and added to 4.5 mL scintillation liquid
Listeria monocytogenes and Escherichia coli O157:H7, belonging to the Culture Collection of the Laboratory of Predictive Microbiology-Department of Science of Agriculture, Food and Environment (SAFE, University of Foggia, Italy) and isolated respectively from dairy and meat products, were used as test pathogens. Pathogens were grown in Nutrient Broth (Oxoid) (37 C for 24e48 h); aliquots of 100 mL of each pathogen (7 log CFU/mL) were plated on cMRS agar; thereafter, disks (9 mm) (Schleicher & Schuell Microscience, Dassel, Germany) were placed onto the surface of the agar and inoculated with 20 mL of the following suspensions: a) b) c) d)
propionibacteria cell cultures (pH 4.5); propionibacteria cell cultures, adjusted to pH 6.5; supernatant of propionibacteria; supernatant of propionibacteria adjusted to pH 6.5.
After the incubation (37 C for 48 h), a clear halo indicated the antimicrobial activity of propionibacteria against L. monocytogenes or E. coli O157:H7; the diameter of the inhibition halo was measured [8]. The experiments were performed on 3 different batches. 2.5. Susceptibility of Propionibacterium strains to antibiotics Susceptibility testing was performed as reported by NCCLS (National Committee for Clinical Laboratory Standards, 1993) [9]. Propionibacteria strains were grown in cMRS broth and inoculated onto the surface of cMRS agar. Antibiotic discs (Neo Sensitabs®, Taastrup, Denmark) (Table 1) were placed on inoculated plates and
D. Campaniello et al. / Anaerobe 34 (2015) 169e173
171
Table 1 Antibiotics used in this research. Antibiotics
Target
Group I: b-lactams Ampicillin Group II: no b-lactams Vancomycin (glycopeptide) Group III: protein synthesis inhibitors Erythromycin (macrolide saturated) Gentamycin (aminoglycoside) Streptomycin (aminoglycoside) Chloramphenicol Tetracyclines Group IV: other antibiotics Ciprofloxacin (quinolinone) Trimethoprim (sulphamide) a
Dosage (mg/disk)
Peptidoglycan
33
Peptidoglycan
70
50 S rRNA 30 S rRNA 30 S rRNA 50 S rRNA rRNA DNA gyrase DHFRa
78 40 100 60 80 10 52
DHFR ¼ dihydroflotareductase.
incubated at 37 C for 4 days, under anaerobic conditions. Susceptibility was assessed by measuring zone of inhibition. 2.6. Statistical analysis Data were analyzed through one-way analysis of variance (1way ANOVA), using Duncan's test as the post-hoc test, or through the t-test (paired comparison) (Statistica for Windows; Statsoft, Tulsa, Okhla.). 3. Results and discussion Selection of probiotics is a complex process and many traits should be assessed. In this study, we used a subset of selection criteria (survival at pH 2.5 and with 0.3% bile salts added, bioactivity towards pathogens and resistance to antibiotics) and focused on the use of a cell line derived from the normal mucosa of ileum to assess adhesion. 3.1. Adhesion assay Adhesion is generally evaluated through the use of model cell lines; Caco-2 cells (originally isolated from a human colon adenocarcinoma) [10] were used by several researchers [11,12]. Other authors used C2BBe1 [13], subcloned from Caco2 cells, or HT29 colonic adenocarcinomal human intestinal epithelial cell lines [11]. IPEC-J2 cells are nontumurogenic cells and possess traits of normal cells of ileum mucosa thus they represent an interesting alternative to Caco cell lines, generally used, for investigation of the adhesion ability of propionibacteria. Table 2 shows adhesion of propionibacteria and Lb. reuteri 12002 (positive control). Propionibacteria were able to adhere to IPEC-J2 cells, without significant differences amongst the strains. Adhesion ranged from 25.58% for P. acidipropionici to 35.09% for P. freudenreichii subsp. shermanii. Similar results were obtained for Lb. reuteri 12002 (33.50%).
Fig. 1. Viability loss of propionibacteria in acidified cMRS broth (pH 2.5) after 3 and 24 h. Mean values ± standard deviation. Values with different letters are significantly different (one-way ANOVA and Duncan's test) (P < 0.05).
Ouwehand et al. [14] studied adhesion of four P. freudenreichii strains to human intestinal mucus and observed that in vitro adhesion was moderate (0.2e6.5%). Collado et al. [15] tested various probiotic microorganisms (Lactobacillus rhamnosus GG, Lb. rhamnosus LC705, P. freudenreichii JS, Bifidobacterium breve 99) and observed that they were able to adhere to intestinal mucus and that P. freudenreichii JS showed the lowest adhesion percentage (0.9 ± 0.5%). When calcium was added, adhesion significantly increased to 47.95% for P. thoenii and 62.5% for P. jensenii; in particular, P. jensenii showed higher values than Lb. reuteri (62.5% vs 48.1%). Adhesion of P. freudenreichii subsp. shermanii was also studied by Lehto and Salminen [16]; they reported that this species showed adhesion percentages similar to L. rhamnosus GG, used as a positive control. Other researchers showed that propionibacteria adhered to intestinal cells both in vivo and in vitro [1,13,17] and that calcium et al. [18] reported that Propionibacteimproved adhesion. Zarate rium adhesion was calcium independent; nevertheless it could be enhanced when calcium was added. The effect of calcium probably relied upon the formation of ionic bonds between bacterial and epithelial cells. 3.2. Tolerance to pH 2.5 and bile salts The acidic environment of the stomach and the presence of bile in the small intestine represent two biological barriers that probiotics need to tolerate in order to exert their beneficial effects; thus it is necessary to study the survival of probiotic strains at pH 2.5 and with 0.3% bile salts to simulate the contact of microorganism with gastric juice and bile [19,20]. Fig. 1 shows the viability loss of propionibacteria at pH 2.5 after 3 and 24 h (this represented transit time through the stomach, and prolonged acid stress, respectively). After 3 h, the strains did not show any kind of viability loss (V.L.) thus confirming their significant resistance to acidic conditions. After 24 h P. freudenreichii
Table 2 Adhesion (%) of propionibacteria and Lactobacillus reuteri (positive control) on IPEC-J2 cell line. Mean values ± standard deviation. Control P. freudenreichii subsp. shermanii DSMZ 20270 P. acidipropionici DSMZ 20272 P. jensenii DSMZ 20279 P. thoenii DSMZ 20276 Lb. reuteri
35.09 25.58 29.87 25.65 33.50
± ± ± ± ±
CaCl2 200 mM 3.60aA 2.05aA 7.91aA 1.80aA 4.97aA
A,B Data in a column with different letters are significantly different (one-way ANOVA and Tukey's test) (P < 0.05). a,b Values in a line with different letters are significantly different (t-test, P < 0.05).
51.1 ± 1.38bA 48.10 ± 4.95bA 62.5 ± 5.67bB 47.95 ± 3.99bA 48.10 ± 3.06bA
172
D. Campaniello et al. / Anaerobe 34 (2015) 169e173
Recently Darilmaz and Beyatli [5] studied the inhibitory properties of propionibacteria against different pathogens and reported that E. coli ATCC 11229 and Shigella sonnei Mu:57 were generally inhibited. 3.4. Susceptibility of Propionibacterium to antibiotics
Fig. 2. Increase of cell count in cMRS broth þ0.3% of bile salts after 3 and 24 h. Mean values ± standard deviation. Values with different letters are significantly different (one-way ANOVA and Duncan's test) (P < 0.05).
subsp. shermanii, P. jensenii, P. thoenii showed a viability loss of 30%. Our results are not in agreement with other researchers; for example, Warminska-Radyko et al. [21] found different results: they studied 27 propionibacteria and found that at pH 2.5 tested strains generally survived for 1 h and only five strains survived for 2 h. Suomalainen et al. [22] also focused on this selection criterion and found that propionibacteria count was significantly reduced after 3 h at pH 2, but not at pH 4 or 3. Cousin et al. [23] found P. jensenii and P. acidipropionici (BIA64) did not survive to pH 2.5. Darilmaz and Beyatli [5] also observed a significant reduction in cell count at pH 2.0 and 3.0, for P. freudenreichii subsp. freudenreichii and P. jensenii. The different trend of tested strains from the results found in literature could be due to the strong strain-specificity to survive at acidic pH; this hypothesis was suggested by Boke et al. [24] for dairy propionibacteria. Concerning the effect of bile salts, propionibacteria experienced an increase in cell count ranging from 0.6 to 1.3 log CFU/mL after 24 h (Fig. 2), thus confirming the high tolerance of Propionibacterium spp. to intestinal conditions; Darilmaz and Beyatli [5] used a spectrophotometric approach to test the resistance of propionibacteria to oxgall (0.06, 0.15, and 0.3%) and found that they were highly susceptible to these compounds. This different trend could be the result of the medium used (MRS broth versus Sodium Lactate Broth), as it is well known that medium type can enhance the bioactivity of an antimicrobial or exert a protective effect.
3.3. Antimicrobial activity Propionibacteria inhibited E. coli O157:H7, but they did not affect the growth of L. monocytogenes; the inihibition zone ranged from 1.97 cm (P. freudenreichii) to 3.83 cm (P. thoenii). Neither cell cultures nor supernatant adjusted to pH 6.5 exerted a similar effect, thus suggesting that the antimicrobial activity could be attributed to low pH.
An important criterion to select a probiotic is the antibiotic resistance; the results for this assay are reported in Table 3 and expressed as resistant (), moderately susceptible (þ), susceptible (þþ) and very susceptible (þþþ). Propionibacteria strains tested were resistant to vancomycin and ciprofloxacin and sensitive to ampicillin (P. thoenii, P. freudenreichii and P. acidipropionici) and chloramphenicol (P. freudenreichii and P. acidipropionici), with inhibition zones ranging from 29.5 mm to 35.5 mm (data not shown). Tested strains were susceptible to erythromycin, tetracycline and trimethoprim (inhibition zone of 21e30 mm); however, the effect of tetracycline was reversible. Finally propionibacteria were moderately susceptible to gentamycin and streptomycin (inhibition zone of 10e20 mm). Concerning the antibiotic resistance of propionibacteria, some authors reported a moderate/strong sensitivity to vancomycin, tetracycline, chloramphenicol, ampicillin, erythromycin and bacitracin using different strains than were assayed in the study reported here [22], thus our results were quite different and suggested the strong strain-dependence of this trait; in addition, the different protocol used could strongly affect the output: in fact, the authors in the reference [22] used a micro-dilution approach, whilst we performed the experiment through a disk diffusion assay. Spreading of antibiotic resistance is an increasing threat and pathogens can acquire this resistance by conjugation [25,26]. The genes encoding antibiotic resistance can be placed on the chromosome or on plasmids; it is strongly recommend to avoid the use of starter or probiotic microorganisms carrying these genes on plasmids, as they can transfer genes to pathogens into the gut [26]. However, some authors reported that the genes of antibiotic resistance of propionibacteria are carried out on the chromosome, thus the resistance to antibiotics is not a problem for these microorganisms [27]. 4. Conclusion Probiotic characterization of propionibacteria (PAB) has been carried out extensively in the past. However, the most important drawback of other studies is the use of cell lines from non-normal mucosa (e.g. Caco-2 from colon cancer or similar lines). IPEC-J2 are from the normal ileum of pig, thus this cell line retains all the traits and properties of the intestinal mucosa. In addition, we assessed some other criteria considered important for assessment of potential probiotic strains, such as viability at low pH and in the presence of bile salts, both under normal conditions (3 h) and under prolonged stress conditions (24 h). The tested strains were able to adhere to mammalian epithelial cells, survive in acidic environments and in presence of bile salts, and exert a moderate antimicrobial activity towards E. coli O157:H7.
Table 3 Antibiotic-resistance. Resistant (), moderately susceptible (þ; Inhibition zone: 10e20 mm), susceptible (þþ; inhibition zone: 21e30 mm) and very susceptible (þþþ; inhibition zone > 31 mm). Strains
Ampi
Vanc
Eryt
Gent
Streptom
Cloramfen
Tetr
Ciprof
Trim
P. P. P. P.
þþ þþþ þþþ þþþ
e e e e
þþ þþ þþ þþ
þ þ þ þ
þ þ þ þ
þþ þþ þþþ þþþ
þþ þþ þþ þþ
e e e e
þþ þþ þþ þþ
jensenii thoenii freudenreichii acidipropionici
D. Campaniello et al. / Anaerobe 34 (2015) 169e173
References , Dairy propionibacteria: less conventional probiotics to improve the [1] G. Zarate human and animal health, in: E.C. Rigobelo (Ed.), Probiotic in Animals, InTech Publisher, Rijeka, Croatia, 2012, pp. 153e202. , G. Jan, Dairy propionibacteria as human [2] F.J. Cousin, D.D.G. Mater, B. Foligne probiotics: a review of recent evidence, Dairy Sci. Technol. 91 (2011) 1e26. rez, B. Mayo, Identification, typing and characterization of [3] S. Delgado, A. Sua Propionibacterium strains from healthy mucosa of the human stomach, Int. J. Food Microbiol. 149 (2011) 65e72. [4] FAO/WHO: Guidelines for the evaluation of probiotics in food. Food and Agriculture Organization in the United Nations and World Health Organization Working Group Report. Available from: http://www.fao.org/es/ESN/food/ foodandfood. [5] D.O. Darilmaz, Y. Beyatli, Acid-bile, antibiotic resistance and inhibitory properties of propionibacteria isolated from Turkish traditional home-made cheeses, Anaerobe 18 (2012) 122e127. [6] A. Bevilacqua, D. Campaniello, B. Speranza, M. Sinigaglia, M.R. Corbo, Selection of promising strains of probiotics for foods: proposal for a possible flow-chart with a special focus on the dark side of probiotics, in: A. Roma (Ed.), Probiotics in Health and Disease: New Research, Nova Publishers, New York, 2014, pp. 1e22. [7] A. Thierry, S.M. Deutsch, H. Falentin, M. Dalmasso, F.J. Cousin, G. Jan, New insights into physiology and metabolism of Propionibacterium freudenreichii, Int. J. Food Microbiol. 149 (2011) 19e27. [8] A. Bevilacqua, C. Altieri, M.R. Corbo, M. Sinigaglia, L. Ouoba II, Characterization of lactic acid bacteria isolated from Italian Bella di Cerignola table olives: selection of potential multifunctional starter cultures, J. Food Sci. 75 (2010) M536eM544. [9] NCCLS (National Committee for Clinical Laboratory Standards), 24, Performance Standards for Antimicrobial Disc Susceptibility Test: Tentative Standards, vol. 13, National Committee for Clinical Laboratory Standards, Villanova, PA, 1993. NCCLS Documents M2eA5. [10] M. Pinto, S. Robine-Leon, M.D. Appay, M. Kedinger, N. Triadou, E. Dussaulx, B. Lacroix, P. Simon-Assmann, K. Haffen, A. Fogh, A. Zweibaum, Enterocytelike differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture, Biol. Cell 47 (1983) 323e330. [11] R.K. Duary, Y.S. Rajput, V.K. Batish, S. Grover, Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells, Indian J. Med. Res. (2011) 664e671. [12] G. Chauviere, M.H. Coconnier, S. Kerneis, J. Fourniat, A.L. Servin, Adhesion of Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells, J. Gen. Microbiol. 138 (1992) 1689e1696. [13] Y. Huang, M.C. Adams, An in vitro model for investigating intestinal adhesion of potential dairy propionibacteria probiotic strains using cell line C2BBe1, Lett. Appl. Microbiol. 36 (2003) 213e216.
173
€lkko €, S. Salminen, In vitro adhesion of [14] A.C. Ouwehand, T. Suomalainen, S. To propionic acid bacteria to human intestinal mucus, Lait 82 (2002) 123e130. [15] M.C. Collado, L. Jalonen, J. Meriluoto, S. Salminen, Protection mechanism of probiotic combination against human pathogens: in vitro adhesion to human intestinal mucus, Asia Pac. J. Clin. Nutr. 15 (2006) 570e575. [16] E. Lehto, S. Salminen, Adhesion of two Lactobacillus strains, one Lactococcus and one Propionibacterium strain to cultured human intestinal Caco-2 cell line, Biosci. Microflora 16 (1997) 13e17. [17] Y. Huang, L. Kotula, M.C. Adams, The in vivo assessment of safety and gastrointestinal survival of an orally administered novel probiotic, Propionibacterium jensenii 702, in a male Wistar rat model, Food Chem. Toxicol. 41 (2003) 1781e1787. , V. Morata De Ambrosini, A. Perez Chaia, S. Gonza lez, Some factors [18] G. Zarate affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells, Can. J. Microbiol. 48 (2002) 449e457. [19] A. Cebeci, C. Gürakan, Properties of potential probiotic Lactobacillus plantarum strains, Food Microbiol. 20 (2003) 511e518. [20] C.N. Jacobsen, V. Rosenfeldt Nielsen, A.E. Hayford, P.L. Mǿller, K.F. Michaelsen, € m, M. Tvede, M. Jakobsen, Screening of probiotic A. Pǽrregaard, B. Sandstro properties of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of colonization ability of five selected strains in humans, Appl. Environ. Microbiol. 65 (1999) 4949e4956. [21] I. Warminska-Radyko, L. Laniewska-Moroz, A. Babuchowski, Possibilities for stimulation of Bifidobacterium growth by Propionibacteria, Lait 82 (2002) 113e121. €, S. Tynkkynen, In vitro and in vivo [22] T. Suomalainen, P. Sigvart-Mattila, J. M€ atto gastrointestinal survival, antibiotic susceptibility and genetic identification of Propionibacterium freudenreichii ssp. shermanii JS, Int. Dairy J. 18 (2008) 271e278. [23] F.J. Cousin, S. Louesdon, M.B. Maillard, S. Parayre, H. Falentin, S.M. Deutsch, G. Boudry, G. Jan, The first dairy product exclusively fermented by Propionibacterium freudenreichii: a new vector to study probiotic potentialities in vivo, Food Microbiol. 32 (2012) 135e146. [24] H. Boke, B. Aslim, G. Alp, The role of resistance to bile salts and acid tolerance of exopolysaccharides (Epps) produced by yogurt starter bacteria, Arch. Biol. Sci. 62 (2010) 323e328. [25] M. Jahan, G.G. Zhanel, R. Sparling, R.A. Holley, Horizontal transfer of antibiotic resistance from Enterococcus faecium of fermented meat origin to clinical isolates of E. faecium and Enterococcus faecalis, Int. J. Food Microbiol. 199 (2015) 78e85. [26] J.R. Huddleston, Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes, Infect. Drug Resist 7 (2014) 167e176. [27] L. Meile, G. Le Blay, A. Thierry, Safety assessment of dairy microorganisms: Propionibacterium and Bifidobacterium, Int. J. Food Microbiol. 126 (2008) 316e320.
