In vivo Antagonistic Effect of Lactobacillus helveticus CU 631 against Salmonella enteritidis KU101 infection

In vivo antagonistic effect of Lactobacillus helveticus CU 631 and Lactobacillus spp. against typical enteritis causing pathogen Salmonella enteritidis KU 101 have been determined, which showed an increase in survival rate and the decline in viable cell numbers of pathogen in liver and spleen at sacrifice. A signifcant difference in the antagonistic effect against KU 101 were observed, which was species and/or strain dependent of Lactobacillus (p<0.01), the survival rate of the mice in the Salmonella infection by feeding L. helveticus CU 631 has been shown to be 157%, whereas those of L. rhamnosus GG ATCC 53103, L. acidophilus ATCC 4356, L. johnsonii C-4 were 137%, 132%, 119% respectively on the basis of lactobacilli non-associated control KU101 fed mice to be 100%. Viable cells of S. enteritidis KU101 in the liver and in the spleen at sacrifice were decreased in Lactobacillus spp. fed group with no significant difference. The higher level of total secretory IgA concentration in the intestinal fluid of lactobacilli fed mice than control mice have been observed. In vitro antagonistic activity of Lactobacillus spp. against KU101 have been determined, a prominent antagonistic activity of CU 631 against KU 101 were demonstrated. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 3 : 430-434)


INTRODUCTION
Many strains of Salmonella enteritidis, E. coli and Campylobacter are reported to be agents of enteritis and food intoxication with principal symptoms of diarrhea.They inhabit in the gastrointestinal tract of domestic animals and are transmitted to animal and food products, hence they are recognized as an important hygienic indicator organism which causing acute enteritis and septicemia (Aserkoff et al., 1970;Bowmer, 1964).
Diarrheal disease has been one of the critical diseases and it can be fatal when it occurs in individuals with compromised immune system including infants and the elderly (Black, 1993).The control of these bacteria are of pivotal importance in preventing diarrheal diseases in humans and animals (Anon, 1990), The gastrointestinal tract contanis a complexes of microorganisms whose population's change are susceptible in response to various kinds of environmental factors such as antibiotics, medication, and diet.Enterobacterial pathogens may induce a disease when the host's normal flora has been disturbed, as diarrhea usually results from the disruption of the normal flora in the gastrointestinal tract (GI), the treatments that recovering the normal flora have been to be effective with the least side effects.In this respect, probiotics that have potentials to improve the microbial balance in the GI tract have long been used for the prophylactic and therapeutic agents against diarrheal diseases (Kateralis et al., 1995;Gonzales et al., 1994).The lactic acid-secreting bacteria such as lactobacilli and bifidobacteria work as probiotics (Gibson and Fuller, 2000).However not all lactic acid bacteria can be probiotic and their potencies can be different depending on strains, and good strains should be developed and characterized for a practical use.
In search for the probiotics exhibiting preventative effects against diarrheal diseases we evaluated 4 species of Lactobacillus with the Salmonella infection model in mice.Salmonella is the second most common cause of diarrheal diseases in the Unites States (Edwards, 1999).The antagonistic effect of Lactobacillus spp.against Salmonella enteritidis KU101 were determined.

Mice
Male specific pathogen free ICR mice were obtained from Daehan Biolink (Choongbook, Korea) and were acclimated for at least 1 wk before use.The mice were kept under the condition of 12 h light-dark cycle at a controlled temperature (22±2°C) and were supplied with water and feed ad libitum.

Bacterial strains and media
The strain and sources of bacteria used in this study were given in Table 1.Lactobacillus spp.were cultured in MRS broth (Difco, USA) at 37°C and maintained in 11% skim milk containing 0.75M adonitol at -70°C.Salmonella enteritidis was grown in Brain Heart Infusion (Difco, USA) and maintaned in BHI broth containing 30% glycerol at -70°C.

In vivo Antagonistic Effect of Lactobacillus helveticus CU 631 against
Salmonella enteritidis KU101 infection

In vivo antagonistic effect test against the Salmonella infection in mice
The lactobacilli were subcultured in 10 ml of MRS broth (Difco Laboratories) at 37°C overnight, the whole grown cells were transferred to 50 ml of same broth and were further incubated at 37°C, when the cell growth reached the log phase, the cells were harvested by centrifugation and were suspended in 10% skim milk with a desired suspension.The bacteria a dose of 2-4×10 9 cfu were administered orally to the mice for 8 consecutive days, the mice in the control group received skim milk only.One day after the last feeding was done, ICR mice (five per group) were orally challanged with 1×10 8 cfu of S. enteritidis, the dose of which is equivalent to LD 50 .And the percent survival (number of alive/total number of mice) was recorded every day for 21 days.

Viable cell count of S. enteritidis in liver and spleen
Viable cell count was conducted on the method described by Perdigon (1992).Liver and spleen tissue were mixed in 5 ml of 0.1% peptone and homogenized by homogenizer.Salmenella-Shigella agar (Difco, USA) was used and followed by the procedures of standard plate count (Marshall, 1993).Diluted samples were plated on SS agar and incubated for 24 h at 37°C and black colonies were counted

Salmonella enteritidis
The ultrasensitive assay of Lehrer et al. (1991) was used to determine the antimicrobial activities of Lactobacillus spp.MRS culture against Salmonella enteritidis KU101 which were grown overnight for 18 h at 37°C in 50 ml of tryptic soy broth (TSB), to obtain midlogarithmic phase KU101, 10 ml of tryptic soy broth was inoculated with 200 µl of cultured tryptic soy broth overnight culture and incubated additional 3 h at 37°C.The bacteria were pelleted by centrifugation at 5,500×g for 5 min at 4°C, washed once with PBS and resuspended in 10ml of cold PBS.The optical density (OD) of an aliquot was measured at 620nm and based on the relationship OD 620 0.2=5×10 7 CFU/ml, a volume containing 1-5×10 6 CFU was added to 10 ml of previously autoclaved, warm (42°C) 10 mM sodium phosphate buffer containing 3 mg of powdered TSB medium, 1%(w/v) of low-electroendosmosis-type agarose (Sigma no 6013, St Louis Mo), and a final concentration of 0.02% (v/v) Tween 20 (Sigma).After rapid dispersal with a laboratory vortex mixer, the agar was poured into petri dish to form a uniform layer approximately 1 mm deep.A 3 mm diameter gel punch was used to make nine evenly spaced wells per dish, Lactobacillus spp.samples (5 µl) were addd to each well.The plates were incubated for 3 h at 37°C and overlayed with 10 ml of sterile agar (TSB[6%, wt/vol], agarose [1% wt/vol]).After incubation for 18 to 24 h at at 37°C, the diameter of the inhibition area surrounding the wells was measured.The diameter of clearing was calculated by subtracting the diameter of central well (3 mm) and expressed in units (0.1 mm=1 U).

Determination of the level of sIgA from the intestinal contents
The intestinal contents from the ICR mice fed with Lactobacillus spp.were prepared; the inside of the small intestine was flushed with 1 ml of ice-cold PBS and centrifuged 16,000×g for 1 min and supernatant was taken in 1 ml PBS containing 0.1 mg/ml soy bean trypsin inhibitor, 50 mM EDTA (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Sigma), and the concentration of s Ig A from resulting intestinal contents were determined by the procedures described by Han et al. (1999).
50 µl of 0.1% bovine serum albumin (Sigma, USA) was added into the each wells of 96 well microplate (Falcon, USA), 50 ml of sample and 50 µl of intestinal content was added to the first well and mixed, the diluted samples were incubated 2 h at 37°C and maintained overnight at 4°C.And removed the samples from the plate and washed 3 times with cold PBS containing 0.05% Tween 20.
200 µl of bovine serum albumin was added to each well and incubated 1 h at 37°C and washed 3 times with cold PBS (pH 7.4) containing 0.05% Tween 20.50 µl of antimouse IgA (Sigma USA) conjugated with horse sera peroxidase which was diluted by 1:1,000 was added to each well and incubated 1 h at 37°C and washed 3 times with cold PBS (pH 7.4) containing 0.05% Tween 20. 100 µl of alkaline phosphatase substrate solution containing 1 mg/1 ml of ρ-nitrophenyl phosphate disodium (Sigma, USA) was added to each well and incubated 2 h at 37°C and determined the total IgA by measuring the optical density at 405 nm with ELIZA reader (Bio-rad Microplate reader Model 3550, USA).

Statistical analysis
Within the same treatment group, for the comparison of the inhibitory activity by lactobacilli, in vivo protection activities between the treatments values were compared using SAS Duncan's multiple-range test.

In vivo antagonistic effects of Lactobacillus spp against the Salmonella infection
To investigate if the CU 631, ATCC 4356, ATCC 53103 and C-4 monoassociated bacteria are able to protect the specific germfree mice against Salmonella enteritidis KU101 infection, and to screen the inhibitory strain that have preventive capacity against diarrheal disease, mice were infected with S. enteritidis KU101.The ICR mouse were fed with each Lactobacillus for 8 consecutive days, one day after last feeding S. enteritidis were challanged orally, when the survival rates were observed every day for 21 days.The mice started to become sick and the first death was observed on day 3 postinfection and a 32% mortality rate was observed by day 11 postinfection KU101 infected control mice, but not until 21 postinfection in the CU 631 monoassocated KU101 infected mice.As shown Figure 1(A), a highly delayed mortality was observed in the CU 631 monoassociated KU101 infected mice compared with the germ free KU101 infected mice (p<0.01)The most active in mortality delaying activity have been observed in L. helveticus CU 631, and followed by L. acidophilus ATCC 4356, L. rhamnosus ATCC 53103, and L. johnsonii C-4, with the Salmonella enteritidis KU 101 infection model in mice.The results indicate that the established lactobacilli species exert antimicrobial activity in vivo.
Figure 1(B) showed a strong effect of CU 631 on survival rate of mice (survival rate of 157%) comparing with the lactobacilli non-associated control mice infected with KU 101.Those of ATCC 4356, C-4 and ATCC 53103 monoassociated in the intestine of KU 101 infected mice revealed 137%, 132% and 119% respectively.In review report, Marie-Francoise et al. (1997)  adhering human Lactobacillus acidophilus strain LA1 demonstrated an antibacterial activity in conventional or germ free mouse models orally infected by Salmonella typhimurium.L. casei GG survives in the human gastrointestinal tract and causes clinically significant health benefits against human diarrhea (Isorauri et al., 1994).
In vivo antagonistic effect of CU 631 and lactobacilli have been shown in liver.(A) and in spleen(B) in Figure 2. he number of Salmonella enteritidis KU101 in the liver of the control mice was 3.2±0.08CFU log/organ but those numbers in CU631 fed mice were 2.3±0.07CFU log/organ with no significant difference.KU101 viable count in the spleen of the mice fed with Lactobacillus spp. on 21 days after the challenge was 2.6±0.10CFU log/organ, which could be considered as the result of the protective and antagonistic activity of lactobacilli in vivo.There was no statistically significant differences in viable number of KU 101 in the lactobacilli treated mice as shown in Figure 2(B).

In vitro inhibition of S. enteritidis by Lactobacillus spp
In vitro inhibition of S. enteritidis by Lactobacillus spp were given in Table 2. L. helveticus CU 631 showed most potent inhibition activity against Salmonella enteritidis, which agrees with those results of in vivo antagonistic effect test.Those results of in vitro and in vivo antagonistic effect test shows the fact that Lactobacillus helveticus CU 631 could be utilized as a probiotic starter strain.Lactobacilli are the most frequently used species in products for human consumption and can be found in infant foods, cultured milks and pharmaceutical preparations.L. rhamnosus GG survives in human gastrointestinal tract and causes clinically significant health benefits against human diarrhea (Isolauri et al., 1994;Kaila et al., 1992).In this research, a statistically significant inhibitory activity of L. helveticus CU 631 against S. enteritidis than that of L. rhamnosus GG has been shown in Table 2, in vitro studies have recently documented the antagonistic activity of 27 lactobacilli strains against enteropathogen Helicobacter pylori as a result of the competitive exclusion of adhesion of pathogenic bacteria to host cells CU 631 revealed most

Effect of Lactobacillus spp. feeding on total secretory IgA production
Previous studies have shown that some probiotics administered orally exhibit their beneficial effects by stimulating gut-associated lymphoid tissue (GALT) (Perdigon et al., 1992).As shown in Figure 3, while the concentration of IgA in the L. helveticus fed group was an average of 162 ng/ml, that of control group remained an average of 160 ng/ml, indicating that there is no significant change in the total IgA level by feeding L. helveticus cells.L. acidophilus ATCC 4356 fed group showed the highest IgA level in the intestinal fluid among the Lactobacillus spp.fed group.In pararell with the antimicrobial activity, lactobacilli are known to stimulate immunological defences against pathogens (Perdigon et al., 1992), it has also been demonstrated that when L. acidophilus was orally admistered to human in fermented milk, it was able to increase the blood phagocytic activity, as well as both the total immunoglobulin A levels in serum and the specific immunoglobulin A titers to S. typhimurium TY 21a in serum (Link-Amster et al., 1994), but our results indicated that the total Ig A level have been varied depending upon the lactobacilli strains as shown in Figure 3. Highly increased IgA level have been appeared in the B longum fed mice; .theconcentration of secretory IgA in intestinal content from B. longum treated group was 2.5 times higher than that of control group suggesting that the protective effects of B. longum may partly be attributed to the enhancement of local rather than systemic immunity (Han et al., 1999).

Figure 1 .
Figure 1.(A) Mortality due to S. enteritidis KU101 infection in germ free ICR mice associated with Lactobacillus spp.CU 631, ATCC 4356, ATCC 53103 and C-4 and not associated(control)mice.Mice were fed with Lactobacillus spp., following challenge with Salmonella enteritidis 1×10 8 cfu (B) Survival rate of mice fed with Lactobacillus spp.following challenge with Salmonella enteritidis after 21 days.Mean survival times (hours) of treated mice Survival rate (%) = Mean survival times (hours) of control mice × 100 * ,a-d Means in a row with no common superscript differ significantly (p<0.001).SEM (Standard error of mean):1.30.

Figure 2 .
Figure 2. The number of Salmonella enteritidis in the (A) liver and (B) spleen of the mice fed with Lactobacillus spp., 21 days after the challenge with Salmonella enteritidis

Table 1 .
Sources of strains of Lactobacillus spp., Salmonella enteritidis used in this investigation

Table 2 .
In vitro antagonistic effect of Lactobacillus spp.
a-dMeans in a row with no common superscript differ significantly (p<0.001).SEM: Standard error of mean.