Correspondence: Zhen-quan Yang, College of Food Science and Engineering, Yangzhou University, 196 West Huayang Road, Yangzhou, Jiangsu 225009, China. Tel.: +86 514 89786037; fax: +86 514 87978128; e-mail: firstname.lastname@example.org
The aim of this study was to evaluate the probiotic effects of Lactobacillus strains against Vibrio parahaemolyticus causing gastroenteritis. Six-week-old ICR mice were pretreated with four Lactobacillus strains at three dosages, and then challenged with V. parahaemolyticus TGqx01 (serotype O3:K6). The results showed that V. parahaemolyticus TGqx01 caused severe intestinal fluid accumulation (FA) and villi damage in control mice which were pretreated with phosphate-buffered saline. In contrast, significant alleviation of FA was seen in mice pretreated by with a high dose of Lactobacillus strains (P <0.05, n =6) but not in mice that received low-dose pretreatments. Among middle-dose treatments, two highly adhesive strains, Lactobacillus rhamnosus H15 and Lactobacillus brevis Y29-4, significantly decreased intestinal FA and villi damage in treated mice (P <0.05). Two low-adhesive strains, Lactobacillus acidophilus Y14-3 and Lactobacillus fermentum F16-6, had no significant alleviating effects. At the same dosing levels, no significant differences in FA were observed in mice pretreated with strains with similar adhesive abilities but different antagonistic activities. Our findings suggest that Lactobacillus strains can alleviate V. parahaemolyticus-induced intestinal FA in mice, and the doses required for in vivo efficacy depend more on adhesive ability than on the antibacterial activity of strains.
Vibrio parahaemolyticus is a Gram-negative human pathogen present naturally in the marine environment and frequently isolated from a variety of seafood products (Su & Liu, 2007). Consumption of raw or undercooked seafood contaminated with V. parahaemolyticus can lead to acute gastroenteritis (McLaughlin et al., 2005). The enterotoxicity of V. parahaemolyticus is related to virulence properties including adhesiveness and other virulence factors such as exoenzymes and toxins (Takeda, 1988; Nishibuchi et al., 1992). When V. parahaemolyticus colonizes the small intestine, it produces virulence factors that lead to epithelial degeneration and exfoliation (Qadri et al., 2003), fluid accumulation (FA) and inflammation (Park et al., 2004; Hiyoshi et al., 2010), and effacement of the microvilli (Ritchie et al., 2012), in turn causing severe diarrhea and enteritis in patients and animals. Adhesion of V. parahaemolyticus to epithelial cells and mucus is a prerequisite for its colonization in the intestine (Yamamoto & Yokota, 1989; Chakrabarti et al., 1991). Factors such as pili, capsular polysaccharide, outer membrane proteins, and cell-associated hemoagglutinin play important roles in the adhesion of V. parahaemolyticus to its target cells (Nakasone & Iwanaga, 1990; Nagayama et al., 1994; Hsie et al., 2003). Adherence of clinical V. parahaemolyticus strains to erythrocytes, and Hep-2 and Caco-2 cell lines is generally inhibited by d-mannose (Falcioni et al., 2005), indicating that clinical V. parahaemolyticus strains can express mannose-specific adhesins (Msa) that mediate adherence to mannose-containing receptors on target cells.
Lactobacillus has shown great potential to control diseases caused by Vibrio anguillarum, Vibrio vulnificus, Vibrio alginolyticus and Vibrio harveyi in aquatic animals such as fish (Gildberg et al., 1997; Nikoskelainen et al., 2001) and shrimp (Villamil et al., 2003; Ajitha et al., 2004; Chiu et al., 2007; Kongnum & Hongpattarakere, 2012). The competition for adhesion sites and production of antibiotic substances are considered the main mechanisms for probiotic effects of Lactobacillus against pathogens (Kesarcodi-Watson et al., 2008). Msa have been found in Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus johnsonii and Lactobacillus paracasei (Ahrne et al., 1998; Neeser et al., 2000; Pretzer et al., 2005; Kang & Conway, 2006), suggesting that competition for mannose-containing receptors in vivo between V. parahaemolyticus and certain strains of lactobacillus. Moreover, the in vitro inhibitory activity of Lactobacillus strains against Vibrio species including V. parahaemolyticus has been reported in a previous study (Koga et al., 1998), and the results showed that V. parahaemolyticus was sensitive to a subset of the tested Lactobacillus strains. However, it is uncertain whether in vitro inhibitory activity can serve as a predictor for protective effects of Lactobacillus against V. parahaemolyticus in vivo.
The aim of this study was to evaluate protective effects of Lactobacillus against intestinal disease caused by V. parahaemolyticus. Twelve Lactobacillus strains were studied for their in vitro inhibitory activities against V. parahaemolyticus TGqx01 (clinical strain, serotype O3:K6) and their ability to adhere to intestine epithelial cells and mucus of mice. Four strains with significant differences in inhibitory and adhesive capabilities were assessed for their alleviating effects on V. parahaemolyticus TGqx01-induced intestinal FA and pathological changes in a mouse model.
Materials and methods
Bacterial strains and culture conditions
Twelve Lactobacillus strains that showed high acid tolerance in preliminary studies were used in this investigation (species and origins are shown in Table 1). The strains were grown anaerobically in de Man Rogosa and Sharpe broth (MRS) medium at 37 °C for 48 h. Vibrio parahaemolyticus strain TGqx01 (serotype O3:K6) was isolated from a clinical case in a food poison outbreak, and tested positive in both tdh and orf8 genes (Yang et al., 2008). The strain was cultured in Luria–Bertani (LB) medium supplemented with 2% NaCl (LBS) with shaking (150 r.p.m.) at 37 °C for 12 h. Viable cell concentration in cultures was determined by plate counting on MRS plates for lactobacillus strains or thiosulfate citrate bile salt sucrose plates for V. parahaemolyticus after serial dilution with sterile phosphate-buffered saline (PBS, pH 7.2). All cultures were centrifuged at 8000 g for 5 min at 4 °C and the live cells then collected and resuspended in an appropriate volume of sterile PBS to prepare suspensions of 109 colony-forming units (CFU) per mL.
Table 1. Antagonistic activity against Vibrio parahaemolyticus TGqx01 and adhesive ability of Lactobacillus strains to mouse epithelial cells and mucus
Values with no common superscript letters differ significantly (P <0.05).
Mean value ± standard deviation of the width of three inhibition halos around spots.
+ indicates the width of inhibition halo around wells is larger than 1.0 mm; – indicates no clear inhibition halo observed.
Mean value ± standard deviation of number of attached bacteria in three independent experiments.
Mean value ± standard deviation of OD570 nm readings from 10 replicate wells in two independent experiments.
5.3 ± 0.3a
32.1 ± 5.6a
0.30 ± 0.09a
4.8 ± 0.3b
2.7 ± 1.8e
0.05 ± 0.04c
4.7 ± 0.3b
8.9 ± 3.5d
0.05 ± 0.03c
4.3 ± 0.4b
27.0 ± 4.8b
0.21 ± 0.05a
4.3 ± 0.5b
19.5 ± 4.6c
0.12 ± 0.03b
4.3 ± 0.8b
10.8 ± 4.0d
0.11 ± 0.06c
4.0 ± 0.0c
37.4 ± 6.9a
0.23 ± 0.05a
4.0 ± 0.0c
32.0 ± 5.9a
0.16 ± 0.10b
2.5 ± 0.2d
40.1 ± 6.0a
0.34 ± 0.11a
2.5 ± 0.7d
5.7 ± 2.5d
0.09 ± 0.03c
2.3 ± 0.3d
31.1 ± 4.3a
0.20 ± 0.07b
2.1 ± 0.3d
21.7 ± 5.0b
0.16 ± 0.06b
Inhibition assays in vitro
The antibacterial activity of lactobacillus strains against V. parahaemolyticus TGqx01 in vitro was determined using the agar spot test and well-diffusion assay described by Schillinger & Lucke (1989). For the spot test, 5 μL of the lactobacillus suspensions (109 CFU mL−1) were spotted on the surface of MRS agar and anaerobically incubated at 37 °C for 48 h. The spots were then covered with LBS soft agar (0.5%) containing 107 CFU mL−1 of V. parahaemolyticus TGqx01 (as indicator). After incubation at 37 °C for 24 h, the widths (mm) of inhibition halos around spots were determined to estimate the inhibitory ability of each strain. For the well-diffusion assay, 500 μL of a 12-h culture of V. parahaemolyticus TGqx01 was spread onto LBS agar plates and air-dried at room temperature. Wells in the agar plates were made using sterile thin-walled steel tubing (7.5-mm-diameter). The wells were filled with 200-μL cell-free supernatants which were obtained by centrifuging the 48 h culture of each lactobacillus strain at 8000 g for 10 min and neutralizing to pH 6.5. Plates were then incubated at 37 °C for 16 h, and the diameters of clear inhibitory zones around wells were determined. Experiments were repeated three times and the results were expressed as mean value ± standard deviation.
Adhesion to mouse intestinal epithelial cell in vitro
The adhesiveness of lactobacillus strains to mouse intestinal epithelial cell was determined by a method modified from that described by Alwan et al. (1998). Briefly, epithelial cells were isolated from split small intestines of mice using a cell scraper and collected in 5 mL of PBS. Suspensions were then washed twice by centrifugation at 100 g for 10 min and adjusted to 1 × 106 cells mL−1 with PBS. A 1-mL aliquot of the lactobacillus suspension (109 CFU mL−1) was mixed with 1 mL of epithelial cells (106 cells mL−1) and incubated for 1.5 h at 37 °C. Epithelial cells with bound bacteria were centrifuged at 100 g for 10 min and washed twice with PBS, and 500 μL of the suspension was used to prepare microscope slides which were air-dried at room temperature and Gram-stained. The number of bacteria attaching to a single epithelial cell was counted at 400× magnification using a light microscope. Adhesiveness of each strain was expressed as the mean number of attached bacteria to 100 epithelial cells in 20 randomly selected fields.
Adhesion to mice intestinal mucus in vitro
Mucus from the small intestine of mice was prepared by a method modified from that described by Roos et al. (2000). Briefly, by gently scraping the inner surfaces of each mouse intestine (duodenum, jejunum and part of ileum segments) with a spatula, mucus was removed and collected in 250 μL of ice-cold PBS (pH 7.2). The resulting suspensions were mixed and centrifuged at 12 000 g for 10 min to remove cell debris and bacteria. The supernatants were collected, and the protein concentration was determined by measuring the absorbance at 280 nm using a UV-spectrophotometer. Adhesiveness was determined using a crystal violet method modified from that described by Vesterlund et al. (2005). Briefly, 150 μL of mucus extract (0.5 mg protein mL−1) was coated onto a 96-well microplate by incubation at 4 °C overnight. The wells were washed with PBS, and 100 μL of lactobacillus strain suspensions (109 CFU mL−1) and PBS (as control) were added and incubated at 20 °C for 2 h. Wells were then washed three times with PBS. The adherent bacteria were fixed at 60 °C for 20 min and stained with 100 μL of filtered 0.5% crystal violet solution for 45 min. Excess stain was removed by rinsing the wells five times with PBS. The crystal violet in adherent bacteria was released by adding 100 μL of citrate buffer (20 mM, pH 4.3) to wells and incubating for 45 min. The absorbance values at 570 nm were determined using a microplate reader (ELX800; Biotek Instruments Inc.). Five replicate wells were used in two independent experiments. The adhesive capability of strains to mucus was expressed as mean value of absorbance.
Animal administration and challenge with V. parahaemolyticus TGqx01
The specific pathogen-free mice were obtained from the laboratory animal center of Yangzhou University (Yangzhou, Jiangsu province, China). All animal experiments were performed with the approval of and in accordance with the institutional policies and the recommendations for care and use of laboratory animals. A total of 84 ICR mice (6 weeks old, 42 males and 42 females) were randomly divided into 14 groups, each group containing six mice (three males and three females). Mice in the 12 experimental groups received intragastric dose of high (5 × 108 CFU day−1), middle (5 × 107 CFU day−1) or low (5 × 106 CFU day−1) doses of Lactobacillus rhamnosus H15, Lactobacillus brevis Y29-4, Lactobacillus acidophilus Y14-3 or Lactobacillus fermentum F16-6, daily for seven consecutive days. The blank and control groups received PBS buffer alone. At 24 h after the last dose, the mice in the control and experimental groups were challenged intragastrically with 0.5 mL of V. parahaemolyticus TGqx01 suspension (109 CFU mL−1). Mice in the blank group received only 0.5 mL saline. All mice were sacrificed at 24 h post-challenge. Small intestines were removed for pathological and intestinal FA analysis.
Intestinal FA determination
Intestinal fluid secretion induced by V. parahaemolyticus TGqx01 was estimated by determining FA. Upper parts of the small intestine samples (with a length of 23–25 cm adjacent to the stomach) were removed from the experimental and control mice and put into Petri dishes before drying to constant weight in an air drying oven. Samples were electrically heated in an air drying oven at 70 °C for 6 h. The initial mass (M0, mg) and the final mass (M1, mg) were determined using an electronic analytical balance (XS205; Mettler Toledo Company, Changzhou, China). The degree of intestinal FA (mg cm−1) was estimated calculating (M0–M1)/length. The results are expressed as mean ± standard deviation of the six mice in each group.
To study possible histological changes induced by V. parahaemolyticus TGqx01, intestinal tissues were sampled from experimental, blank and control mice. Tissues were cut into small pieces, fixed in 10% neutral buffered formalin, dehydrated and embedded in paraffin wax. Serial sections (5 μm) were cut and stained with hematoxylin and eosin. Changes in small intestine histology were examined under a light microscopy and evaluated histologically by an experienced pathologist.
Statistical analysis was carried out by t-test statistical program using sigmaplot software (version 10; Systat Software Inc., Chicago, IL). All data were analyzed statistically using analysis of variance. Results were considered significant at P <0.05.
Results and discussion
Antagonistic and adhesive abilities of Lactobacillus strain in vitro
Antibacterial activity in vitro assays are often used as an important indicator to evaluate the potential of probiotic candidates (Spanggaard et al., 2001; Jensen et al., 2012). Our data showed that V. parahaemolyticus TGqx01 was inhibited by some strains of Lactobacillus in vitro (Table 1). This inhibition varied among the 12 tested strains in a strain-dependent manner. Higher levels of inhibition (≥ 4.5 mm) were observed in L. rhamnosus H15, L. acidophilus Y14-3 and L. plantarum FS-26, which were significantly higher than those observed in other strains such as L. brevis Y29-4, L. fermentum F16-6, and Lactobacillus casei YS42 and YL22 (≤ 2.5 mm). The well-diffusion assay showed that the neutralized supernatants from eight strains lost their antagonistic activity, whereas those from four strains with higher inhibitory levels in the agar spot test (L. rhamnosus H15, L. acidophilus Y14-3, L. plantarum FS-26 and F4-5) retained the antagonistic activity (inhibition halo width ≥ 1.0 mm) against V. parahaemolyticus TGqx01. These results suggested that the possible production of antibacterial compounds other than organic acids (e.g. bacteriocin or H2O2) may contribute to the higher inhibitory activity of Lactobacillus strains against V. parahaemolyticus (Koga et al., 1998; Kesarcodi-Watson et al., 2008; Kongnum & Hongpattarakere, 2012).
Competitive exclusion to reduce adhesiveness of pathogens in the intestinal tract is a common mechanism of lactobacillus probiotic effects against bacterial infection (Vine et al., 2004; Collado et al., 2007; Li et al., 2008). The 12 Lactobacillus strains had different levels of adhesion to both intestinal epithelial cells and mucus of mice (Table 1). The adhesive abilities of strains varied in a strain-dependent manner, as has been generally reported for probiotic bacteria (Lim & Im, 2008; Monteagudo-Mer et al., 2012). The L. rhamnosus H15 and two L. brevis strains Y29-4 and F4-3 exhibited higher adhesiveness to both intestinal epithelial cells (32.1, 40.1 and 37.4 bacteria cell−1, respectively) and mucus (OD570 nm values of 0.297, 0.336 and 0.229, respectively). Conversely, L. acidophilus Y14-3, L. fermentum F16-6 and L. plantarum FS-26 showed quite low adherence to mouse intestinal epithelial cells (2.7, 5.7 and 8.9 bacteria cell−1 respectively) and mucus (0.054, 0.089 and 0.046, respectively).
Adhesion of bacteria to cultured cells is generally used as an in vitro model for assessing the ability of bacteria to adhere to intestinal mucosa; however, this does not take into account possible adhesion to the mucus layer that covers epithelial cells in the intestine. In this study, 12 lactobacillus strains were tested for their adhesive abilities, using both the primary cultured epithelium and immobilized intestinal mucus of mouse as in vitro models. Interestingly, the data from these two in vitro models showed a strong correlation (R2 = 0.807). This suggests that factors important for Lactobacillus adhesion may be commonly presented on the epithelial cell surfaces and in mucus of the small intestine. Cell lines such as Caco-2 cells have been widely used to assess adhesion properties for Lactobacillus (Dunne et al., 2001); however, some Lactobacillus strains which adhere well to intestinal mucus showed quite poor adhesion to Caco-2 and HT-29 cell lines (Collado et al., 2007; Jensen et al., 2012). Therefore, the primary cultured epithelial cells may be more suitable than cell lines for assessing adherence of probiotic bacteria.
To understand the roles of antibacterial and adhesive properties of Lactobacillus strains in their probiotic effects in vivo, four strains (L. rhamnosus H15, L. brevis Y29-4, L. acidophilus Y14-3 and L. fermentum F16-6) which differ significantly in their antibacterial and adhesive abilities in vitro (P <0.001) were selected to study their protective effects against V. parahaemolyticus TGqx01 infection in a mouse model.
Effects of Lactobacillus strains on V. parahaemolyticus TGqx01-induced intestinal FA
The enterotoxicity of V. parahaemolyticus strains is often estimated by induced FA in rabbit (Nishibuchi et al., 1992; Raimondi et al., 1995) or rat (Baffone et al., 2005) ileal loop models. Data presented in Fig. 1 show that intragastric inoculation of mice with 5 × 108 CFU of V. parahaemolyticus TGqx01 resulted in severe FA in the small intestine. At 24 h post-challenge, the FA values of mice in the control group increased to 34.24 ±1.84 mg cm−1, which was significantly higher than that of unchallenged mice in the blank group (24.60 ±1.04 mg cm−1; P <0.001, n = 6). The duodenum, jejunum and part of the ileum of challenged mice exhibited different degrees of swelling and FA among differently treated groups. In general, mice pretreated with high doses of Lactobacillus had a less intensive distribution of swollen portions in the small intestines, and exhibited lower FA values than those of mice treated with low or no doses of Lactobacillus.
Experiments showed that administration of Lactobacillus strains before challenge alleviated V. parahaemolyticus-induced intestinal FA in mice; however, the degree of alleviation depended on the administered dose and strain. Compared with controls, the intestinal FA levels were significantly decreased in all mice treated with high doses (5 × 108 CFU day−1) but not in mice treated with low doses (5 × 106 CFU day−1), for all four of these strains, regardless of their in vitro antibacterial and adhesive properties. Among middle-dose treated mice (5 × 107 CFU day−1), L. rhamnosus H15 and L. brevis Y29-4 (both highly adhesive strains) alleviated FA significantly and reduced values to 29.72 ± 0.85 mg cm−1 (P <0.01 vs. controls) and 30.48 ± 1.55 mg cm−1 (P <0.05 vs. controls), respectively. Two low-adhesive strains, L. acidophilus Y14-3 and L. fermentum F16-6, did not significantly alleviate small intestinal FA at the dose of 5 × 107 CFU day−1. Comparing the same levels of dosage, no significant differences in FA reduction were observed (P >0.05) between treatments using strains with similar adhesive abilities and different antagonistic activities. Although the in vitro inhibitory activities against V. parahaemolyticus TGqx01 of L. rhamnosus H15 and L. acidophilus Y14-3 were greater than those of L. brevis Y29-4 and L. fermentum F16-6 (P <0.001), respectively, FA-alleviating effects between these strains did not significantly differ at any administered doses (P >0.46).
Effects of orally administrated viable probiotic are related to dosage (Reida et al., 2001; Li et al., 2011; Wen et al., 2012) and higher doses might be useful to prevent infections (Mane et al., 2011). Significant dose effects of all tested strains in alleviating FA were observed in this study. However, at the same dose levels, highly adhesive strains were more effective in alleviating V. parahaemolyticus-induced intestinal FA than were low-adhesive strains, regardless of their antibacterial properties. Inhibition of a pathogen by producing antagonistic compounds and by competition for adhesion sites have been widely suggested as modes of probiotic action for Lactobacillus (Kesarcodi-Watson et al., 2008). Although many in vitro studies have demonstrated antibacterial and adhesion properties of Lactobacillus spp., experimental validation is needed to confirm their relevance in anti-infection effects in vivo. Our results suggest that adhesive capability is a significant factor of the in vivo anti-infection effects of Lactobacillus strains against V. parahaemolyticus, and intestinal mucus attachment may be the prerequisite for probiotic action of Lactobacillus in vivo (Fuller, 1989; Valeur et al., 2004).
Intestinal histological changes following challenge with V. parahaemolyticus TGqx01
To determine the extent of intestinal tissue damage after challenge with V. parahaemolyticus TGqx01, tissue section and HE staining were performed (Fig. 2). In accordance with the FA examination, histological results showed that in the blank mice (PBS-treated, no challenge), intestinal villi were long, slim and regularly arranged, and had a normal appearance and distinct structure (Fig. 2a). In the control mice (PBS-treated, TGqx01 challenge), intestinal FA was the highest; correspondingly, the intestinal villi were seriously damaged and had lost their integrity (Fig. 2b). In contrast to mice in the control group, intestinal sections from mice fed with high and middle doses of Lactobacillus stains showed different degrees of alleviation of villi damage after challenge (Fig. 2–c–f). Among mice treated with middle doses of L. rhamnosus H15 and L. brevis Y29-4, intestinal villi were shortened, congested and irregularly arranged compared with the blank mice. Nevertheless, the intestinal villi in these two groups still retained their integrity and showed fewer lesions than did the control group, which suggests that these two strains protected the mice from V. parahaemolyticus infection. However, among mice treated with L. acidophilus Y14-3 and L. fermentum F16-6, the degree of alleviation was generally less significant than in those treated with L. rhamnosus H15 and L. brevis Y29-4.
Certain species or strains of Lactobacillus are generally regarded as safe and have been used as therapeutic or prophylactic supplements for humans and animals (Fuller, 1989; Mombelli & Gismondo, 2000; Ouwehand et al., 2002). Our results show that Lactobacillus can alleviate pathogenic V. parahaemolyticus-induced intestinal FA and villi damage in the mouse model, which suggests that some strains of Lactobacillus could be anticipated to become non-chemotherapeutic agents for controlling V. parahaemolyticus causing gastroenteritis.
This research was supported by the National Natural Scientific Foundation of China (Grant no. 30901047), Jiangsu Natural Scientific Foundation project (BK2009191) and grants (08KJD550003 and 2009KJA23001) from the Department of Education of Jiangsu Province. We thank Mrs. Haiyan Shen, Mr. Wei Chen and Mr. Hongliang Wang for their technical assistance in animal feeding and sample collection.
Conflict of interest
The authors have no conflict of interest to declare.