Curr Microbiol (2008) 56:175–181 DOI 10.1007/s00284-007-9069-5
Antagonistic Activity of Potential Probiotic Lactobacilli Against the Ureolytic Pathogen Yersinia enterocolitica Paola Lavermicocca Æ Francesca Valerio Æ Stella L. Lonigro Æ Alfredo Di Leo Æ Angelo Visconti
Received: 12 June 2007 / Accepted: 4 October 2007 / Published online: 12 December 2007 Springer Science+Business Media, LLC 2007
Abstract Strains of Lactobacillus spp., isolated from sourdough and olive brines (seven strains), and three human isolates were screened for their antagonistic activity in coculture against the ureolytic pathogen Yersinia enterocolitica. A general reduction in the pathogen population was observed after 6 h when each Lactobacillus strain was cocultured with the pathogen at a ratio of 100:1 cfu ml-1, causing an almost complete inhibition of urease activity. Strains were also screened for their performances in in vitro tests such as adhesion ability to Caco2 cells, tolerance to low pH, bile salts, and simulated digestion, which enabled the differences between strains to be highlighted. Three strains, L. paracasei IMPC2.1 and L. plantarum ITM21B and ITM5BG, met the main criteria for selecting effective probiotics: the ability to inhibit the pathogen Y. enterocolitica and, consequently, its urease activity (ITM21B); survival of simulated digestion (ITM21B and IMPC2.1); strong adhesion ability to enterocytes and good survival at low pH and in the presence of bile salts (ITM5BG and IMPC2.1).
Introduction Lactobacilli and Bifidobacteria, having probiotic characteristics, play a key role in modulating gut microbiota and P. Lavermicocca F. Valerio (&) S. L. Lonigro A. Visconti Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126 Bari, Italy e-mail:
[email protected] A. Di Leo Section of Gastroenterology, DETO, University of Bari, Bari, Policlinico, Italy
maintaining host health [19]. Benefits deriving from a regular intake of probiotic strains are correlated with their ability to stimulate a protective immune response enhancing resistance to microbial pathogens and safeguarding microbial intestinal balance [6, 11]. In particular, increases in lactobacilli and bifidobacteria may result in acidification of the gut, in improvements to the nutritional status of gut epithelium, and in a decrease in intestinal permeability to toxic molecules [2]. Probiotics can improve gut health by inhibiting undesirable micro-organisms through competitive inhibition of pathogens for adhesion sites, competition for nutrients, and/or production of antimicrobial substances [4, 18, 20]. For some pathogens a possible mechanism of action comes through the activation of the enzyme urease, which catalyzes the hydrolysis of urea to yield toxic metabolites like ammonia and plays an essential role in diseases such as urolithiasis, peptic ulceration, hepatic encephalopathy, and yersiniosis [16]. Particularly, ammonia produced by urease activity represents a pathogenic factor for Yersinia enterocolitica, which is the major bacterial food-borne pathogen in the U.S. pork processing industry and which causes gastroenteritis [3]. Urease contributes to acid tolerance of the pathogen since ammonia derived from hydrolysis of urea causes a net increase in the environmental pH, allowing the pathogen to cross the stomach and to invade the intestinal mucosa [7, 16]. The interaction between Y. enterocolitica and Lactobacillus sakei has been investigated by developing a predictive model to describe the inhibition and/or inactivation of the pathogen [12, 23]. Urease activity and ammonia production also play a role in the survival and pathogenesis of Helicobacter pylori. Sgouras et al. [21] have recently studied the effect of the Lactobacillus casei strain Shirota on this highly ureolytic human pathogen: viability as well as urease activity was inhibited in the presence of the probiotic strain.
123
176
In our study, the ureolytic pathogen Y. enterocolitica was used in coculture experiments to select strains of lactobacilli which have fundamental probiotic characteristics and are capable of counteracting the pathogen’s growth and/or the corresponding ammonia production. Strains were characterized for several in vitro properties, such as their ability to survive simulated digestion and adhere to enterocytes, as requisites for their successful colonization and persistence in the gut. Materials and Methods Bacterial Strains The Lactobacillus strains listed in Table 1 were grown in still culture in de Man Rogosa Sharpe (MRS) medium (Difco Laboratories, Detroit, MI, USA) at 37C. Lactobacillus plantarum ITM21B (ITM: culture collection of Institute of Sciences of Food Production, CNR, Bari, Italy), Lactobacillus fermentum ITM18B, and Lactobacillus alimentarius ITM5Q were isolated from sourdough [5], while L. plantarum ITM5BG, ITM4TG, ITM2TP, and ITM4TP1 were isolated from olive brines [15]. Lactobacillus paracasei IMPC2.1 (IMPC: culture collection of Microbiology Institute of Catholic University of Piacenza, Italy), Lactobacillus rhamnosus GG ATCC53103 (ATCC: American Type Culture Collection, Rockville, MD, USA), and Lactobacillus rhamnosus ITMCAIII were human isolates. The ureasepositive human isolate Yersinia enterocolitica subsp. enterocolitica DSM4780 (DSM: Deutsche Sammlung von Mikroorganismen und Zellkulturen GMBH, Braunschweig, Germany) was grown in still culture in Brain Heart Infusion (BHI) medium (Difco) at 30C. All strains were stored at 80C in the appropriate substrate containing 20% (v/v) glycerol (Difco) and subcultured twice before use in experiments. Antagonistic Activity in Coculture Inhibition of Y. enterocolitica growth The interference of lactobacilli with the growth of Y. enterocolitica was evaluated by coculturing the pathogen with each lactic acid bacterial strain in the LAPTg medium (tryptone, 10 g L-1; yeast extract, 10 g L-1; peptone, 15 g L-1; glucose, 10 g L-1; Tween 80, 1 ml L-1) [17] containing 2 mM urea (U-LAPTg). All strains were subcultured (2%, v/v) in U-LAPTg medium and incubated for 24 h under the appropriate conditions. After incubation, the culture of the pathogenic strain (about 1 9 108 cfu ml-1) was diluted with the same medium in order to obtain about 2 9 106 cfu
123
P. Lavermicocca et al.: Inhibition of Y. enterocolitica
ml-1, and 5 ml of this suspension was mixed with 1 ml of an overnight culture of individual LAB strains previously diluted to obtain about 5 9 106 or 5 9 108 cfu ml-1. Cultures were incubated under still conditions at 37C for 24 h. At time intervals of 0, 6, and 24 h, aliquots were removed and serially diluted, and 100-ll aliquots were plated on BHI and MRS agar plates to determine the bacterial colony counts of Y. enterocolitica and lactobacilli, respectively. Plates were incubated under appropriate conditions. Y. enterocolitica colonies on BHI agar plates were clearly distinguished from lactobacilli on the basis of their morphology. After 24 h of incubation Y. enterocolitica formed whitish colonies 2– 3 mm in diameter with an irregular margin, while lactobacilli formed punctiform colonies with an entire margin. ULAPTg medium inoculated with Y. enterocolitica or lactobacilli alone under the coculture conditions were used as controls. The effect of lactic acid or low pH on pathogen growth and urease activity was evaluated. Six-hour-old cells of Y. enterocolitica (about 107 cfu ml-1) were suspended in U-LAPTg medium containing 20, 30, and 40 mM DL-lactic acid (85% syrup), obtaining final pH values 5.2, 4.2, and 4.0, respectively, or in medium acidified with HCl (3 N) to the same pH levels. Samples were incubated for 1 h at 37C. All assays were performed three times, with two replicates each. Lactic acid in cocultures was determined by a commercially available D-lactic/L-lactic acid assay kit (Roche Diagnostics, Basel, Switzerland).
Urease enzyme assay Urease activity was measured by evaluating the release of ammonia by the pathogen population deriving from coculture and from monocultures using the method described by Eaton et al. [8]. At each sampling time (0, 6, and 24 h) cells were collected by centrifugation (2900 g), washed in sodium phosphate buffer (10 mM, pH 7.0), resuspended in a reaction mixture (50 mM potassium phosphate buffer, pH 6.5, and 200 mM urea), and incubated for 2 h at 37C. Controls were Y. enterocolitica cells in buffer alone (no urea) or buffer containing urea. The reagents used were from a commercially available ammonia assay kit (Roche Diagnostics) except for the enzyme glutamate dehydrogenase (GIDH), which was from Roche and was used at a concentration of 50 U ml-1 (*10-fold higher than the activity recommended by the kit manufacturer). Ammonia determination was based on the following linked reactions. Urea ! ammonium þ CO2 urease
GIDH
Ammonium þ 2 oxoglutarate ! L glutamate þ NADþ
NADH
P. Lavermicocca et al.: Inhibition of Y. enterocolitica
177
Table 1 Growth of Yersinia enterocolitica in coculture with Lactobacillus strains and relevant ammonia production in the urease enzyme assay Strain
Bacterial count (log10 cfu ml-1 ± SD)a
Ammonia (mg L-1 ± SD)a
0h
0h
6h
24 h
6h
24 h 90.20 ± 0.91
Y. enterocolitica DSM4780 (Y.e.)
6.16 ± 0.09
7.80 ± 0.16
8.48 ± 0.16
4.6 ± 0.77
49.13 ± 8.17
Y.e. + L. plantarum ITM21B
6.27 ± 0.02
5.78 ± 0.20
\DLb
4.68 ± 0.75
2.74 ± 0.53
––
Y.e. + L. paracasei IMPC2.1
6.30 ± 0.02
6.37 ± 0.11
\DL
2.79 ± 0.71
4.73 ± 0.35
––
Y.e. + L. plantarum ITM5BG
6.27 ± 0.02
6.33 ± 0.04
\DL
2.45 ± 0.92
4.48 ± 0.19
––
Y.e. + L. rhamnosus GG ATCC53103
6.21 ± 0.01
6.40 ± 0.08
\DL
5.91 ± 0.66
3.85 ± 0.22
––
c
Y.e. + L. rhamnosus ITMCAIII
6.15 ± 0.08
6.54 ± 0.22
\DL
5.11 ± 0.26
4.20 ± 0.09
––
Y.e. + L. plantarum ITM4TP1 Y.e. + L. plantarum ITM2TP
5.93 ± 0.21 5.95 ± 0.31
6.26 ± 0.38 6.70 ± 0.68
\DL \DL
3.40 ± 0.03 4.50 ± 0.66
3.83 ± 0.42 3.58 ± 0.61
–– ––
Y.e. + L. plantarum ITM4TG
6.01 ± 0.25
6.55 ± 0.33
\DL
3.22 ± 1.50
4.03 ± 0.33
––
Y.e. + L. alimentarius ITM5Q
6.07 ± 0.22
6.67 ± 0.44
3.04 ± 0.62
5.70 ± 0.95
3.87 ± 0.92
4.20 ± 0.42
Y.e. + L. fermentum ITM18B
5.87 ± 0.29
6.00 ± 0.23
\DL
2.29 ± 1.26
3.37 ± 0.28
––
a
Data, expressed as means ± standard errors, are from three independent experiments with two replicates each (n = 6)
b
Detection limit: log10 2 cfu ml-1
c
Values not reported since bacterial counts were below the detection limit
The amount of ammonia produced by urease is proportional to the amount of NAD+ produced. The assay was modified such that GIDH was in excess and urease activity was the rate-limiting step. The urease activity was measured by a change in optical density at 340 nm. The results were reported as the average of three independent experiments with two replicates each. Low pH and Bile Tolerance Low pH and bile tolerance tests were performed in 96microwell plates, containing 190-ll aliquots of MRS, MRS (pH 2.5), or MRS containing 0.4% Oxgall bile salts.The wells were inoculated in quadruplicate with 10 ll of an overnight culture of each Lactobacillus strain grown in MRS. Media without inoculation were used as controls. At inoculation time and after 4 h of incubation at 37C, colony count was performed on MRS agar. Each experiment was performed three times.
Simulated Digestion Simulated digestion was tested as described by Fernande´z et al. [10]. Briefly, lactobacilli strains were inoculated at 2% (vol/vol) in 50 ml of LAPTg medium and incubated at 37C for 24 h. Cells were washed in sterile saline solution (0.9% NaCl), centrifuged, and suspended in 50 ml of artificial gastric juice with the following composition: NaCl, 125 mM; KCl, 7 mM; NaHCO3, 45 mM; and pepsin, 3 g L-1. The final pH was adjusted with HCl to pH 2 and with NaOH to pH 7. The resulting bacterial
suspensions were incubated under shaking (200 rpm) to simulate peristalsis. One hundred-microliter aliquots were taken for the enumeration of viable cells at 0 and 180 min. After 180 min incubation suspensions were centrifuged and cells were suspended in 50 ml artificial intestinal juice (0.1% pancreatin and 0.15% Oxgall bile salts in water, pH 8.0, with 5 M NaOH). The suspensions were incubated as above and samples for total viable counts were taken at 0 and 180 min. The results are reported as the average of three independent experiments with two replicates each.
Cell Lines The Caco-2 (HTL 97023) cell line was purchased from the Interlab Cell Line Collection (Genova, Italy) and cultured in 35-mm-diameter dishes in Dulbecco’s modified Eagle’s minimal essential medium (D-MEM) supplemented with 1% nonessential amino acid solution, 1 mM sodium pyruvate, 1% penicillin and streptomycin, and 10% heatinactivated fetal bovine serum, then incubated at 37C in 5% CO2. Reagents were all purchased from Gibco, Invitrogen (Grand Island, NY, USA). The culture medium was changed every 48 h. When the cultures reached confluence, they were maintained for 15 additional days and then the cells were used for the adhesion assay. The adhesion assays were carried out between the 46th and the 66th passage.
Adhesion Assay Caco-2 cells monolayers were washed twice with phosphate-buffered saline (PBS), 3 ml of nonsupplemented D-
123
178
Percentage inhibition of Y. enterocolitica in the coculture assay was calculated as follows: (1 – log10 cfu ml-1 treated cells/log10 cfu ml-1 control cells) 9 100. Percentage survival following simulated digestion and tolerance to low pH and bile salts were calculated using the following formula: (log10 cfu ml-1 treated cells/log10 cfu ml-1 control cells) 9 100. Data from all experiments were analyzed by one-way analysis of variance followed by the Dunnett, Fisher, or Tukey test. All statistical analyses were carried out with the STATISTICA 6.0 software (StatSoft software package, Tulsa, OK, USA).
Results and Discussion The ability of the lactobacilli strains to inhibit the in vitro growth of the ureolytic intestinal pathogen Y. enterocolitica was evaluated in coculture experiments. The effect of lactobacilli on the urease activity of the pathogen was monitored by evaluating the ability of the pathogen population deriving from the coculture to hydrolyze urea and release ammonia in the urease enzyme assay. In this assay, lactobacilli (monoculture) proved unable to hydrolyze urea (data not shown). In the growth medium, the pathogen (monoculture) multiplied during the first 6 h from log10 6.16 to log10 7.80 cfu ml-1 (pH 5.6) and then reached a value of log10 8.48 cfu ml-1 after 24 h (pH 5.0) (Table 1). Findings obtained after 6 h of incubation of Y. enterocolitica (about 106 cfu ml-1) with lactobacilli (about 106 cfu ml-1) led to an inhibition ranging from 0% to 4% of the pathogen population, while the ammonia production by surviving cells was reduced by 20%. Only after 24 h was the pathogen growth completely inhibited by almost all the
123
100
50
80
40
60
30
40
20
20
10
Lactic acid (mM)
Statistical Analysis
lactobacilli (data not shown). In order to observe an earlier effect on pathogen viability or modification in its urease activity, the lactobacilli concentration was increased by two logs (about 108 cfu ml-1). All lactobacilli had similar growth in coculture, with values ranging from log10 8.12 ± 0.04 cfu ml-1 to log10 8.65 ± 0.45 cfu ml-1 after 6 h and from log10 8.17 ± 0.09 cfu ml-1 to log10 9.43 ± 0.37 cfu ml-1 after 24 h. Initial pH values for cocultures were 5.25 ± 0.17, decreasing to 4.6 ± 0.09 after 6 h and to 4.13 ± 0.11 after 24 h. A reduced viability of Y. enterocolitica was observed after 6 h of incubation in coculture, with different percentages of inhibition (p \ 0.05) depending on strains: L. plantarum ITM21B showed the highest inhibitory ability (26%) by producing a two-log decrease in pathogen population with respect to the control. For the remaining strains, a one-log reduction in the Y. enterocolitica population was observed (from 13.6% to 23.2%) (Table 1). After 24 h of growth in coculture the population of the pathogen was reduced to values below the detection limit (log10 2 cfu ml-1) by all strains except for L. alimentarius ITM5Q, which caused a 64% growth reduction (Table 1). Even if Y. enterocolitica survived after 6 h with a considerable population of viable cells (about 106 cfu ml-1) in the coculture, the relevant ammonia production by the surviving population was reduced by at least 90.4% (Table 1, Fig. 1). In order to assess if lactic acid, the main metabolite produced by lactobacilli, was responsible for the reduced ability to hydrolyze urea by the surviving Y. enterocolitica population, the amount of lactic acid in coculture was measured (Fig. 1). Strains produced different lactic acid concentrations after 6 h but had a similar effect on the urease activity of the pathogen. A relationship between lactic acid production and inhibition of urease
ammonia reduction (%)
MEM was added to each dish, and cells were incubated for at least 30 min before the addition of bacteria. Cells of LAB strains grown for 24 h in MRS were washed in PBS and appropriately diluted with D-MEM to give a bacterial concentration of *108 cfu ml-1, and 120 ll of this suspension was added to each dish. After incubation for 1 h, all dishes were washed four times with PBS to remove nonadhering bacteria. Finally, cells were fixed with 3 ml of methanol for 5 min at room temperature. Cells were stained with 3 ml of Giemsa stain solution (1:20) for 30 min. Dishes were then washed with PBS until no color was observed, dried in air overnight, and examined microscopically (magnification, 9 100) under oil immersion. Adherent lactobacilli in 20 random microscopic fields were counted for each test. Each adhesion assay was performed in triplicate.
P. Lavermicocca et al.: Inhibition of Y. enterocolitica
0
0 21B
21
5BG
GG CAIII 4TP1 2TP 4TG
5Q
18B
Strains
Fig. 1 Inhibition of ammonia produced by Y. enterocolitica evaluated in the urease enzyme assay after 6 h of coculture (white bar). Lactic acid production in the coculture system after 6 h of incubation (black bar). Data, expressed as means ± standard errors, are from three independent experiments with two replicates each (n = 6)
P. Lavermicocca et al.: Inhibition of Y. enterocolitica
activity has been suggested in the Helicobacter pylori/ Lactobacillus casei strain Shirota coculture system [21]. Besides, Aiba et al. [1] established that 10 mM lactic acid produced by Lactobacillus salivarius exerts an inhibitory effect on the growth of H. pylori as well as on its urease activity. Moreover, other authors have demonstrated the role of lactic acid on the inhibition or inactivation of Y. enterocolitica in monoculture or in the presence of Lactobacillus sakei [12, 23]. In our study, when 6-h-old cells of Y. enterocolitica were incubated for 1 h in U-LAPTg medium containing 20, 30, and 40 mM lactic acid (about the same concentrations found in cocultures after 6 h), neither the growth of the pathogen nor its urease activity was affected (p [ 0.05; unreported results). When the pathogen was incubated for 1 h in the medium acidified with HCl (5.2, 4.2, and 4.0), a slight effect on urease activity was observed at pH 4.2 and 4.0 (from 44.8 ± 0.1 to 31.9 ± 1.1 and 32.6 ± 0 mg L-1, respectively; p \ 0.05). These results indicated that the surviving Y. enterocolitica population’s reduced ability to hydrolyze urea after 6 h of coculture was not related to the presence of lactic acid (Fig. 1). Only prolonged exposure (18 h) of the pathogen to at least 30 mM lactic acid or to pH 4.2 and 4.0 resulted in a complete inhibition of the bacterial growth, with a consequent reduction in ammonia production (unreported results). Therefore, it can be hypothesized that in our pathogen/lactobacilli system, Y. enterocolitica cells were exposed to a damage accumulation due to the gradual increase in toxic metabolites (including lactic acid) and pH lowering. Lactic acid bacterial strains were selected according to their ability to inhibit Y. enterocolitica’s growth and, consequently, the ammonia production, though several other criteria were taken into account for screening strains as potential probiotics. The first requirement for a probiotic bacterium is its ability to survive the harsh conditions of the stomach and to tolerate the presence of bile salts in the small intestine. Survival following 4 h of incubation at pH 2.5 was observed for 9 of the 10 strains tested at values ranging from 85.6% to 92.9% (Table 2). Strains had a relatively high tolerance to 0.4% of bile salts after 4 h of incubation, with survival percentages ranging from 86.5% to 99.8% (Table 2). In order to deeply discriminate strain behavior, the viability of bacterial cells during simulated gastric and intestinal digestion was assessed (Fig. 2). When samples were treated for 3 h with gastric juice at pH 7.0 and successively for 3 h with intestinal juice, the bacterial population declined slightly (p [ 0.05) (Fig. 2). After treatment with gastric juice at pH 2 and successive intestinal digestion, only L. plantarum ITM21B and L. paracasei IMPC2.1 populations survived (values above detection limit). The ability to survive passage through the acidic environment of the stomach and to reach the
179 Table 2 Adhesion properties and low pH and bile tolerances of Lactobacillus strains Strain
Adhesiona
Survivalb pH 2.5
0.4% ox-gall
ITM21B
22.97 ± 12.13
87.71 ± 1.04
98.27 ± 1.84
IMPC2.1
76.30 ± 38.50
89.51 ± 0.45
97.19 ± 2.27
ITM5BG GG
87.39 ± 21.79 38.22 ± 8.25
92.91 ± 8.98 89.20 ± 0.97
95.76 ± 3.27 97.94 ± 1.10
ITMCAIII
22.66 ± 17.04
88.92 ± 2.52
99.79 ± 2.42
ITM4TP1
36.01 ± 17.53
85.62 ± 3.08
98.10 ± 1.38
ITM2TP
45.24 ± 17.29
89.89 ± 3.11
99.86 ± 2.02
ITM4TG
15.16 ± 2.73
88.44 ± 3.21
97.88 ± 2.49
ITM5Q
51.97 ± 34.63
87.11 ± 0.84
95.92 ± 5.15
ITM18 B
27.29 ± 18.44
48.31 ± 10.21
86.47 ± 0.43
a
Average number of adhering lactobacilli in 20 microscopic fields ± standard error (n = 3)
b
Percentage surviving lactobacilli ± standard error after 4 h at pH 2.5 or in the presence of bile salts (n = 3)
intestine was an important discriminative parameter applicable to only four strains among those tested: viable populations of L. paracasei IMPC2.1 and L. plantarum strains ITM21B, ITM5BG, and ITM2TP were found after gastric digestion (Fig. 2). The adhesiveness of the strains to enterocytes seems to be related to their persistence in the gut [20]. In order to discriminate strains on the basis of their adhesion ability, the adhesion to enterocyte-like Caco-2 cells was used as a well-characterized cellular model (Table 2). Strains L. paracasei IMPC2.1 and L. plantarum ITM5BG could be considered strongly adhesive and significantly different (p \ 0.05) from the remaining strains. In fact, they behaved better in this assay than L. rhamnosus GG (p \ 0.05) (Table 2), which has strong adhesive properties to human intestinal epithelial cells [9]. All data (except those from the urease assay, which were not significantly different among strains; p [ 0.05) were considered for highlighting potential probiotic strains that could be applied to control the pathogen Y. enterocolitica. Three strains, L. paracasei IMPC2.1 and L. plantarum ITM21B and ITM5BG, meet the main criteria applied for selecting effective probiotics: the ability to inhibit the pathogen Y. enterocolitica and its relevant urease activity (ITM21B); survival of simulated digestion (ITM21B and IMPC2.1); adhesion to enterocytes and good survival at low pH and in the presence of bile salts (ITM5BG and IMPC2.1) (Tables 1 and 2, Fig. 2). These strains can therefore be selected for efficient application as probiotics in order to increase lactic acid populations in the gut, leading to an environmental acidification. Colonization may result in a competitive inhibition of the growth of
123
180
P. Lavermicocca et al.: Inhibition of Y. enterocolitica L. plantarum ITM21B
12
10
10
-1
12
8 6 4 detection limit
2
8 6 4
0
14
14
L. plantarum ITM5BG -1
8 6 4 detection limit
2
10
log10 CFU ml
-1
log10 CFU ml
10
8 6 4
0
L. rhamnosus ITMCAIII
12
10
10
-1
12
8 6 4 detection limit
2
L. plantarum ITM4TP1
14
log10 CFU ml
log10 CFU ml
-1
14
8 6 4 detection limit
2
0
0
14
L. plantarum ITM2TP
14
12
10
10
-1
12
log10 CFU ml
-1
detection limit
2
0
log10 CFU ml
L. rhamnosus GG
12
12
8 6 4 detection limit
2
L. plantarum ITM4TG
8 6 4 detection limit
2 0
0
14
14
L. alimentarius ITM5Q
12
10
10
-1
12
log10 CFU ml
-1
detection limit
2
0
log10 CFU ml
L. paracasei IMPC2.1
14
log10 CFU ml
log10 CFU ml
-1
14
8 6 4 detection limit
2 0
L. fermentum ITM18B
8 6 4 detection limit
2 0
0
180
0
Time (min)
180
0
180
0
180
Time (min)
Fig. 2 Survival of lactobacilli during sijmulated gastric (solid lines) and intestinal (dotted lines) digestion at pH 2 () and pH 7 (•). Data, expressed as means ± standard errors, are from three independent experiments with two replicates each (n = 6)
123
P. Lavermicocca et al.: Inhibition of Y. enterocolitica
urea-producing bacteria such as Y. enterocolitica and in a reduction in their ammonia production: a 1:100 ratio between pathogen and probiotic strain was necessary to obtain a significant decrease in viable cells of the pathogen after 6 h of coculture and an almost-complete inhibition of its ability to hydrolyze urea. Recently the L. paracasei IMPC2.1 and L. plantarum ITM21B strains have been incorporated into vegetable matrices (artichokes or table olives) and used in human feeding studies: strains successfully survived the harsh passage through the gastrointestinal tract and colonized, even transiently, the intestinal tract of volunteers [13, 14, 22]. Simulated gastric and intestinal digestion performed on L. paracasei IMPC2.1 and L. plantarum ITM21B populations anchored to artichokes or olives indicated that the presence of prebiotic substances and the physical structure of the vegetable could explain the improved survival of bacteria during the test [22]. These strains also possess technological features which are currently leading to their successful application in processing and storage of vegetable products for developing a new probiotic vegetable line (P. Lavermicocca et al., patent applications PCT Nos. WO 2006/037517 A1 and WO 2005/053430 A1). In conclusion, our selection indicated that Lactobacillus strains of human origin or isolated from foods show potential to develop efficacious probiotic foods, for prophylaxis or defined treatment targets. The use of these strains may moderate intestinal dysfunctions, affecting the likelihood of Y. enterocolitica survival by compromising urease functionality and cell viability. Acknowledgments This work was supported by Fondazione Cassa di Risparmio di Puglia (Bari, Italy) and by the Italian Ministry for University and Research (art. 12 D.M. 593/2000, D.D. 3300, 22 December 2005, tema 2) Project ‘‘Ortobiotici pugliesi’’ (D.M. 28830). The authors would like to thank Anthony Green for revising the English in the manuscript.
181
6.
7.
8.
9.
10.
11.
12.
13. 14.
15.
16. 17.
18. 19.
References 20. 1. Aiba Y, Suzuki N, Kabir AM, Takagi A, Koga Y (1998) Lactic acid-mediated suppression of Helicobacter pylori by the oral administration of Lactobacillus salivarius as a probiotic in a gnotobiotic murine model. Am J Gastroenterol 93:2097–2101 2. Bengmark S (1996) Econutrition and health maintenance–a new concept to prevent GI inflammation, ulceration and sepsis. Clin Nutr 15:1–10 3. Bhaduri S, Wesley IV, Bush EJ (2005) Prevalence of pathogenic Yersinia enterocolitica strains in pigs in the United States. Appl Environ Microbiol 71:7117–7121 4. Brassart D, Shiffrin EJ (1997) The use of probiotics to reinforce mucosal defence mechanism. Trends Food Sci Tech 8:321–326 5. Corsetti A, Lavermicocca P, Morea M, Baruzzi F, Tosti N, Gobbetti M (2001) Phenotypic and molecular identification and clustering of lactic acid bacteria and yeasts from wheat (species
21.
22.
23.
Triticum durum and Triticum aestivum) sourdoughs of southern Italy. Int J Food Microbiol 64:95–104 Cross ML (2002) Microbes versus microbes: immune signals generated by probiotic lactobacilli and their role in protection against microbial pathogens. FEMS Immunol Med Microbiol 34:245–253 De Koning-Ward TF, Robins-Browne RM (1995) Contribution of urease to acid tolerance in Yersinia enterocolitica. Infect Immun 63:3790–3795 Eaton KA, Brooks CL, Morgan CL, Kracowka S (1991) Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun 59:2470–2475 Elo S, Saxelin M, Salminen S (1991) Attachment of Lactobacillus casei K strain GG to human colon carcinoma cell line Caco-2: comparison with other dairy strains. Lett Appl Microbiol 13:154–156 Ferna´ndez MF, Boris S, Barbe`s C (2003) Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. J Appl Microbiol 94:449–455 Isolauri E, Su¨tas Y, Kankaanpa¨a¨ P, Arvilommi H, Salminen S (2001) Probiotics: effects on immunity. Am J Clin Nutr 73:444s– 450s Janssen M, Geeraerd AH, Logist F, De Visscher Y, Vereecken KM, Debevere J, Devlieghere F, Van Impe JF (2006) Modelling Yersinia enterocolitica inactivation in coculture experiments with Lactobacillus sakei as based on pH and lactic acid profiles. Int J Food Microbiol 111:59–72 Lavermicocca P (2006) Highlights on new food research. Dig Liver Dis 38(Suppl 2):S295–S299 Lavermicocca P, Valerio F, Lonigro SL, De Angelis M, Morelli L, Callegari ML, Rizzello CG, Visconti A (2005) Study of adhesion and survival of lactobacilli and bifidobacteria on table olives with the aim of formulating a new probiotic food. Appl Environ Microbiol 71:4233–4240 Lavermicocca P, Gobbetti M, Corsetti A, Caputo L (1998) Characterization of lactic acid bacteria isolated from olive phylloplane and table olive brines. Ital J Food Sci 10:27–39 Mobley HL, Island MD, Hausinger RP (1995) Molecular biology of microbial ureases. Microbiol Rev 59:451–480 Raibaud P, Galpin JV, Ducluzeau R, Mocquot F, Oliver G. (1973) Le genre Lactobacillus dans le tube digestif du rat. II. Caracte`res de souches he´te´rofermentaires isole´s de rats ‘‘holo’’ et ‘‘gnotoxe´niques. Ann Microbiol 124:223–235 Rolfe RD (2000) The role of probiotic cultures in the control of gastrointestinal health. J Nutr 130:396S–402S Saarela M, Lahteenmaki L, Crittenden R, Salminen S, MattilaSandholm T (2002) Gut bacteria and health foods—the European perspective. Int J Food Microbiol 78:99–117 Servin AL, Coconnier MH (2003) Adhesion of probiotic strains to the intestinal mucosa and interaction with pathogens. Best Pract Res Clin Gastroenterol 17:741–754 Sgouras D, Maragkoudakis P, Petraki K, Martinez-Gonzalez B, Eriotou E, Michopoulos S, Kalantzopoulos G, Tsakalidou E, Mentis A (2004) In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota. Appl Environ Microbiol 70:518–526 Valerio F, De Bellis P, Lonigro SL, Morelli L, Visconti A, Lavermicocca P (2006) In vitro and in vivo survival and transit tolerance of potentially probiotic strains carried by artichokes in the gastrointestinal tract. Appl Environ Microbiol 72:3042–3045 Vereecken KM, Devlieghere F, Bockstaele A., Debevere J, Van Impe JF (2003) A model for lactic acid-induced inhibition of Yersinia enterocolitica in mono- and coculture with Lactobacillus sakei. Food Microbiol 20:701–713
123
