Residing in the human gastrointestinal tract is a large and complex microbial ecosystem that develops through infancy and childhood to form a diverse, but relatively stable community in adults (Vaughan et al., 2002).
These autochthonous bacteria interact with the diet and the host, contributing to protection against intestinal pathogens through colonization resistance and providing nutritional and colonic health benefits via their metabolic activities (Isolauri et al., 2002; Guarner and Malagelada, 2003).
It has become clear that these bacteria also interact with the hosts immune system and are essential for the maturation and homeostasis of a healthy immune system (Schiffrin and Blum, 2002).
Recognition of the importance of the intestinal microbiota to health has led to increasing interest in manipulating the composition and activity of the microbiota to improve both human and animal health.
Examples of bacteria demonstrated to have beneficial effects include Lactobacillus, Bifidobacterium, Escherichia coli Nissle 1917, Clostridium butyricium, Streptococcus salivarius thermophilus and a non pathogenic yeast Saccharomyces boulardii (Sartor, 2004; Donohue et al., 1998).
Mechanisms by which probiotics exert their therapeutic effects include (Guarner and Malagelada, 2003) modulation of barrier function (Sartor, 2004), mucosal trophic action (Guarner and Shaafsma, 1998),inhibition of pathogenic bacteria (Donohue et al., 1998), blockade of epithelial attachment and invasion by pathogenic bacteria (Sartor, 2004), modulation of intestinal cytokine production (Fioramonti et al., 2003), anti-inflammatory properties (Farina et al., 2001), enhancement of digestion and absorption of food (Donohue et al., 1998; Sartor, 2005).
Probiotics are defined as live microorganisms that when administered in adequate amounts confer a health benefit on the host (J-Boyle et al., 2006).
It is believed by many that the ideal probiotic should remain viable at the level of the intestine and should adhere to the intestinal epithelium to confer a significant health benefit. Some evidence supports the importance of viability in human studies, with viable bacteria having greater immunologic effects than nonviable bacteria and killed bacteria being associated with adverse effects in some instances (Aila et al., 1995; Irjavainen et al., 2003).
Some of the best characterized probiotics have also been shown to adhere strongly to intestinal epithelium in both in vitro and in vivo studies (Alander et al., 1999).
Probiotics must be resistant to gastric acid digestion and to bile salts to reach the intestinal intact and they should be nonpathogenic.
Most probiotics are strains of Bifidobacterium or Lactobacillus species. Some are derived from the intestinal microbiota of healthy humans and others are nonhuman strains used in the fermentation of dairy products. Species from other bacterial genera such as Streptococcus, Bacillus and Enterococcus have also been used as probiotics, but there are concerns surrounding the safety of such probiotics because these genera contain many pathogenic species, particularly Enterococcus (J-Boyle et al., 2006).
Nonbacterial microorganisms such as yeasts from the genus Saccharomyces have also been used as probiotics for many years.
The probiotic effects of this organism include the treatment of various types of diarrhea, alleviation of Crohns disease and balancing of intestinal micro flora through the growth modulation of bacteria present in the gastrointestinal tract (Ernet et al., 1994; Fuller, 1991; Kaila et al., 1992; Lidbeck et al. 1992).
Acute infectious gastroenteritis remains the most common cause of diarrhea world wide and is a leading cause of death in childhood. Despite improvements in public health and economic wealth, the incidence of intestinal infections remains high in the developed world and continues to be an important clinical problem with relevant morbidity (Casburn-Jones and Farthing, 2004).
The risk of diarrheal disease is increased in selected groups including young infants, immune deficient individuals (HIV, cancer, chemotherapy, malnutrition) and people with a high exposure to pathogens (informal settlements travellers, contaminated food and medications buffering gastric acid), etc. (Lilly and Stillwell, 1965). The use of probiotic microorganisms for the prevention or therapy of gastrointestinal disorders is the most usual application of probiotics because most health effects attributed to them are related directly or indirectly (i.e., mediated by the immune system) to the gastrointestinal tract (Vaughan et al., 2002).
In this study we identified Lactobacilli species by yogurt samples and investigated their antipathogenic activity.
MATERIALS AND METHODS
In this study 194 yogurt samples were collected from Iranian Ghashghaie and Bakhtiari tribes.
The samples had been cultured in MRS broth Medium and had been incubated in anaerobic condition with 5% Co2 and 37°C within 48 h. The grown samples were sub cultured in MRS agar for several times to get pure colony in anaerobic condition with 5% Co2 and 37°C for 48 h.
In each step Gram stain had been carried out for bacteria until getting pure colonies.
The pure colonies were cultured in TSI (triple-sugar-iron) agar Medium to study sugar fermentation and were cultured in SIM Medium for investigation of motility and production of H2S.
MRS broth Medium was prepared to culture bacteria without sugar. Then each of the carbohydrates was added into separated Medium in every tube to study fermentation. The bacteria were inoculated into these Mediums and were incubated in 37°C for 4 days. Lactobacillus casei was used as a positive control in all steps.
Two pathogens, E. coli O: 157 H: 7 and Salmonella typhi grew in Caso agar Medium and were incubated in 37°C for 24 hr to study antipathogenic activity.
A suspension was prepared in concentration of 0.5 McFarland by adding of 2-3 colonies from grown bacteria to 10 mL sterile distillated water. Then 2 mL of this suspension was added to semi solid Caso agar Medium.
Antibacterial activity was studied by spot test method, Lactobacillus strains were cultured in MRS agar in spot form at the middle of plate and were incubated in anaerobic condition with 5% co2 and 37°C within 48 h.
The pathogenic bacteria in semisolid Caso agar were distributed on the surface of grown Lactobacilli and were incubated in 37°C for 24 h. Then the inhibition effect was studied by assaying of each bacterial zone. The pathogenic bacterium without Lactobacillus was as a negative control and the Lactobacillus without pathogenic bacteria was as a positive control.
Lactobacilli strains were cultured in MRS broth Medium, centrifuged in 12000 rpm for 7 min to determine pathogenic bacteria death kinetic. Then a suspension was prepared in 0.5 McFarland concentrations from pathogenic bacteria that had grown in Caso agar. One ml of this suspension was added to Caso broth Medium in three separated parts. Lactobacilli species in concentration of 2, 5 and 10% were added to these suspensions at the same time. Then OD (optical density) of pathogenic bacteria was measured each h. There was one control with pathogen and without Lactobacilli.
102 species of Lactobacilli were isolated from 194 yogurt samples. All
of them were Lactobacillus in Gram Stain. The yeast samples were omitted
from this study. Lactobacilli species didnt have any motivation
in SIM Medium and production of H2S. All of Lactobacilli fermented
three types of sugar in TSI Medium.
There were used twelve types of sugar (Esculin, Maltose, Lactose, Melibiose, Raffinose, Salicin, Trehalose, Galactose, Mannitol, Cellobiose, Arabinose, and Mannose) to identify Lactobacilli strains. Some Lactobacillus strains such as L.casei, L.gasseri, L.acidophilus, L.salivarcius, L.delbrueckii and L.plantarum were identified.
Fifty nine Lactobacillus species had antagonistic effect against E.
coli and salmonella typhi morium. The mean diameter of zone for
E.coli was 40.4 mm and for salmonella was 26 mm. The highest
antagonistic effect was shown by L.casei. The inhibition activity of
Lactobacilli on E.coli is shown in Fig. 1. The
zone for E.coli was 30 mm up to 45 mm in diameter. The activity of Lactobacilli
by Bakhtiari tribe yogurts on E.coli is shown in Fig. 3.
||Zone of Lactobacillus for E. coli
||Zone of Lactobacillus for salmonella
||Zone of bakhtiari Lactobacilli on E.coli
The activity of Lactobacilli by Ghashghaie tribe yogurt against E.coli
is shown in Fig. 4.
The inhibition effect of Lactobacilli on Salmonella is shown
in Fig. 2. Inhibition zone for Salmonella was 18 mm
up to 32 mm in diameter. The activity of Lactobacilli by Bakhtiari tribe
yogurt against Salmonella is shown in Fig. 5.
||Zone of Ghashghaei lactobacilli on E. coli
||Zone of bakhtiari Lactobacilli on Salmonella
||Zone of ghashghaei Lactobacilli on salmonella
The activity of Lactobacilli by Ghashghaie tribe yogurt on Salmonella
is shown in Fig. 6.
The use of probiotic microorganisms for prevention or therapy of gastrointestinal disorders is an obvious measure and perhaps the most usual application of probiotics, because most health effects attributed to them are related directly or indirectly (i.e., mediated by the immune system) to the gastrointestinal tract. Many strains of probiotic microorganisms have shown inhibition of growth and metabolic activity as well as the adhesion of enteropathogenic bacteria (Salmonella, Shigella, enterotoxigenic E.coli and Vibrio cholerae) to intestinal cells to modulate (temporarily) the intestinal micro flora and to have immunostimulatory or -regulatory properties (Coconnier and et al., 1997; Gopal et al., 2001; De Vrese and Marteau, 2007).
Lactobacillus acidophilus culture has repeatedly demonstrated effectiveness at reducing E. coli O: 157 H: 7 in feedlot cattle up to 50% (LeJeune, 2007).
The antagonistic activity of Lactobacilli and Bifidobacteria against facultative anaerobic Gram-negative target bacteria; uropathogenic E. coli, S. enterica ssp. Enterica and Sh. sonnei was tested by co-cultivation experiments using micro aerobic or anaerobic conditions. E. coli was highly suppressed by L.rhamnosus GG and both of Bifidobacteria strains, but no significant activity was found against cystitic E.coli.
The effective probiotics against S.enterica ssp. enterica were L.paracasei 8700:2, L.plantarum 299v and L.fermentum ME-3 showing high activity in micro aerobic milieu. L.fermentum ME-3 and both Bifidobacteria expressed high activity against Sh.sonnei in anaerobic milieu. However, the highest antagonistic activity was expressed by L.rhamnosus GG, L.paracasei 8700:2 and L.plantarum 299v. The inhibitory activity of probiotic bacteria against strict anaerobic C.difficile strain was low (68 mm) expressed by both Bifidobacteria strains and L.paracasei 8700:2.
It was demonstrated the enhancement for eradication of Salmonella in chronic carriers due to administration of L.acidophilus. Some placebo-controlled double-blind studies have shown the positive effect of L.rhamnosus GG by reducing the incidence of travellers diarrhea (Hutt et al., 2006).
Perusal of the data pertaining to the antibacterial activity of L. acidophilus cultures against some common intestinal pathogenic organisms indicated that all the cultures of L. acidophilus were active against the tested intestinal pathogenic organisms. The zones of inhibition of pathogenic organisms tested were ranging from 10.5 -16.25 mm in diameter (Padmanabha et al., 2006).
But in this study the inhibition zone of E. coli O: 157 H: 7 was
30-45 mm in diameter and the inhibition zone of Salmonella typhi was
18-32 mm in diameter. In comparison with other studies, Lactobacilli
of Iranian tribes (Bakhtiari and Ghashghaie) had good antagonistic effect on
E.coli and Salmonella. These preventive activities are shown in
Fig. 1, 2, 3, 4,
5 and 6.
The effect of Lactobacillus casei (Yakult) on the growth of enterotoxigenic E. coli at different contact time was determined by Godiosa et al. (1993). At zero contact time, there was 32x103 colony forming units/mL (CFU/mL) with significant reduction to 9.7x 103 CFU/mL at 5 minutes contact time. At 5 minute intervals, there was progressive reduction in colony count to 0.8 x 103CFU/mL at 60 min contact time (Godiosa, Consignado, Adrian, Peña, Antoni and Jacalne, 1993).
In this study we assayed death kinetic of pathogens. 5% concentration of
Lactobacillus decreased OD of both E.coli and Salmonella
within 2 and 3 h after contact time. At zero time, OD of E.coli was 0.04
but it showed reduction to 0.03 after 3 h (Fig. 7, 8).
OD of Salmonella was 0.067 in zero time, but it showed reduction to
0.051 during 3 h after contact with Lactobacillus. In 10% concentration
of Lactobacillus, OD of E. coli decreased from 0.005 to 0 during
2 h after contact time and OD of Salmonella decreased from 0.011-0.005
3 h after contact time (Fig. 9, 10).
||Effect of 5 mL lactobacillus on OD of E.coli
||Effect of 5 mL lactobacillus on OD of Salmonella
||Effect of 10 mL Lactobacillus on OD of Salmonella
||Effect of 10 mL lactobacillus on OD of E. coli
The Lactobacillus species of Iranian tribes (Bakhtiari and ghashghaie) hadnt been studied. In this study we got significant results of antibacterial activity in comparison with others. In this study the inhibition zone for E.coli and salmonella were assayed and compared with the others.
These studies have been continued on probiotic bacteria. Specially probiotic of Iranian tribes should be studied for their antibacterial effect on another pathogen and they should be identified by molecular method. In conclusion, the activity of probiotics in prevention and treatment of infections by E.coli and salmonella are effective.
Financial support was provided by some Grants from Isfahan University of Medical Science (Dr Pishva), Medicine Faculty and Tehran University of Medical Science, Pharmacy Faculty (Dr Fazeli) .I thank Dr Havaee, Dr Pour Hossein, Mr. Jamalifar, Dr Shojaee, Dr Aghazadeh and Mr. Akbari.