The poultry industry is an important economic activity across the world. Serious economic losses resulted when poultry are exposed to stressful environmental conditions and diseases. Emergences of a large variety of pathogens and antibiotics resistance bacteria are the main limiting factors for productivity of the poultry industry . Inclusion of antibiotic growth promoters in chicken diets for disease management and growth promotion has been associated with the emergence of antibiotic resistance bacteria. As a result the global concern focuses on combating usage of antibiotics as growth promoter .
Chicken reared under tropical Africa conditions are considered to have a wide diversity of uncharacterized GIT microbiota which can be a good source of probiotics. Thus, isolation of endogenous probiotic microorganism is considered as a potential probiotic source to alleviate main problems related to chicken production .In the present study potential lactic acid bacteria strains were isolated from the GIT of RIR chicken purchased from local farmers in Ethiopia. According to the farmers these RIR were pure breed and were doing healthy.
From the gastrointestinal tract content of the five chicken the lactic acid bacteria population was isolated from gizzard, cecum, small intestine and crop. The highest number of lactic acid bacteria population was found in the crop region (8.1 log CFU g-1 ) and the smallest number was found in the gizzard (6.2 log CFU g-1).
Similar to the present study, Jannehet al., (2014) found from 7-9 log CFU g-1 lactic acid bacteria population in the crop, gizzard, small intestine and cecum regions of chicken. The variation in lactic acid bacteria population may caused by environment variation, producing facility, age of the chicken, feed and water . The presence of gastric juices, pepsin and hydrochloric acid in the gizzard reduces the pH, resulting in lower bacterial population number .
In previous studies, various species of probiotic bacteria such as L. reuteri[19, 20], L. salivarius[19, 21, 16,22], Enterococcus faecium and Enterococcus durans were isolated from chicken digestive tract. In the present study, four LAB isolates namely, IS3, IS4, IS6 and IS7 were identified based on sequence analysis of 16S rRNA gene. These isolates were identified as L. salivarius(IS3) and L. reuteri(IS4, IS6 and IS7). The sequence of these isolates is published in GenBank as Lactobacillus salivarius strain CEL1 (IS3), Lactobacillus reuteri strain CEC2 (IS4), Lactobacillus reuteri strain CEC3 (IS6) and Lactobacillus reuteri strain CEC4 (IS7).
In general, microorganisms with potential probiotic advantage share common characteristics . Lower pH tolerance, salt tolerance, bile acid resistance, utilization of different carbon sources (degradation of oligosaccharides), hemolytic properties, antibiotics sensitivity, antimicrobial activity and in vitro adherence properties are the major tests used to isolate probiotic microorganisms from different sources . For this reason, different tests were performed to check whether the selected strains display the listed probiotic properties. Conventionally, probiotic microorganisms should be; safe to the host, resistance to GIT in vivo conditions, adhere and colonize to intestinal tissue, survive and resist during processing and storage and should have antimicrobial activity .
The probiotics strains should have a property to grow and survive in the host digestive system as they exposed to a range of stressful conditions in the GIT which includes lower pH and high bile salt .The ability of probiotic strains to act as probiotics is also determined by the ability to survive in the low pH of the stomach and in the high concentration of bile salt of the gastrointestinal tract . In the present study, from 10 isolates 60% of the isolates were able to grow at pH1. On the other hand, bile salt tolerance assay showed that all isolates survived at 0.1% bile salt concentration and only 6 isolates showed resistant to 0.3% bile salt.
Similar results were reported by Ehrmann et al., (2002)  who found the strains of L. reuteri, L. salivarius and L. animalis that tolerate pH 2 and 1 for 4h and 1h, respectively. Janneh et al., (2014)  also found different L. salivarius strains that survived at pH 2, and different Lactobacillus strains including L. reuteri that exhibited 0.3% bile after 6hrs incubation were found . Similarly, Ehrmann et al., (2002)  reported L. reuteri that resist 2% ox gall. Bile released in the small intestine damages bacteria by destroying the bacterial cell membrane. Lactic acid bacteria had bile salt hydrolase enzyme (BSH), which hydrolyze bile salts and reduce their solubility .
The results of antibiotics susceptibility test showed that isolate IS1, IS2, IS5, IS7 and IS8 were resistance to all used antibiotics and IS10 was found to be sensitive to all the tested antibiotics. Apart from IS10, all isolates were resistance to ampicillin. IS3 showed intermediate resistance to ciprofloxacin and chloramphenicol. In addition IS9 showed intermediate resistance to ciprofloxacin. Jose et al., (2015)  also reported different Lactobacillus spp. pattern that showed resistance and sensitivity to different antibiotics including ciprofloxacin, erythromycin, ampicillin and chloramphenicol. Shakor et al., (2017)  reported that no influence of ampicillin on the growth of LAB population. Unlike the result of this study, Singh et al., (2014)  reported different strains of L. reuteri which were sensitive to erythromycin and chloramphenicol.
The resistance of the probiotics isolate to some antibiotics is considered as intrinsic property rather than transmissible, presenting no safety concerns in feed or food. Basically, intrinsic resistance to some antibiotics is regarded as an advantage of the probiotics . Probiotics strains may be exposed to antibiotics in the animal gastrointestinal tract when antibiotics is used as animal health therapeutics. As a result to be an effective probiotics, the probiotics strains should possess non-transferable resistance which aids them in vivo survival .
The present study showed that all the isolates were able to ferment galactose, glucose, fructose, mannitol, sorbitol and sucrose. Except IS2, IS5 and IS10, all isolates utilize maltose as a carbon source. According to Shokryazdan et al., (2014) , L.salivarius species were able to ferment galactose, glucose, fructose, mannitol, sorbitol and sucrose. On the other hand, according to Jannah et al., (2014) , L. salivarius species were unable to metabolize starch. Carbohydrates especially, oligosaccharide metabolism is essential for ecological fitness of lactobacilli. Lactobacillus species have different intracellular enzymes to metabolize various carbon sources. However most of lactobacilli species are not amylolytic . Besides lack of appropriate enzyme, absence of intracellular transport system to take up the carbohydrate may prevent the bacteria to metabolize that carbohydrate . Other studies also confirmed similar carbohydrate fermentation profiles of different lactobacilli species [21, 31]. Thus, it is difficult to accurately differentiate lactobacilli isolates through carbohydrate fermentation .
Antagonistic activity of probiotics microorganism against pathogens is regarded as a characteristic of probiotic to maintain the gut microflora balanced and to keep the gut rid of pathogens. Probiotics inhibits the growth of pathogenic bacteria through production of; nonspecific antimicrobial compounds such as short chain fatty acid, hydrogen peroxide and low-molecular-weight proteins .
The result of this study showed the antibacterial property of isolates against E.coli, S. aureus, S. typhimurium, S. intermedius andS. enteritidis. All the isolates exhibited variable range of inhibition against the growth of the selected pathogens. Maximum size of inhibition zone (17.83mm) was shown against S. typhimurium by IS6. IS2 showed the minimum size of zone of inhibition against all used pathogens except E. coli.
Different strains of lactic acid bacteria inhibit the growth of bacteria including E. coli, S. typhimurium, S. aureus, C. perfringens, Klebsiellaspp. and Proteus spp. by bonding to the specific receptors and causing cell damage . Jannehet al., (2014)  reported different L. salivarius strains that shown inhibition against E.coli andS. enteritidis. This antagonistic activity of probiotics microorganism against pathogens is a role of probiotic to maintain the gut microflora balanced and to keep the gut rid of pathogens.
As a safety requirement, probiotics should be harmless to the host[ 35] and one of the tests used to assess the safety of probiotics is haemolytic activity test . In this study none of the isolates showed any sign of haemolysis when grown on blood agar. Similar results were found by . This makes the isolates harmless to their host.
As shown from Figure 2, the isolates had different hydrophobicity result ranging from 26.4 to 79.3%. The ability to adhere to the intestinal mucosa is one of the more important selection criteria for probiotics because adhesion to the intestinal mucosa is considered to be a prerequisite for colonization . Cellular hydrophobicity indicates the adhesion ability of LABs to enterocytic cellular lines. High adhesive ability of probiotic bacteria has the greatest beneficial effects to colonize the host gut .
In this study, all isolates were able to survive from 25-500c. The optimum temperature for all isolates was 450c. This ability of isolates will enable them to survive under a wide range of temperature during processing, storage and transport . In the present study, the isolated lactic acid bacteria spp. showed a variable capacity to survive at different concentrations of NaCl. All the isolates were able to grow at 4% (0.69 mol/L) of NaCl, but none of the isolates could grow in 8% (1.36 mol/L) of NaCl. The most tolerant isolates to high NaCl concentrations could survive in the gastrointestinal tract of the animal. The ability to resist high salt concentrations is important for probiotic bacteria to maintain their osmotic balance in order to survive and grow in the gastrointestinal tract which has an osmolarity equivalent to 0.3mol/L .
According to Pundiret al., (2013)  LAB isolates were able to survive at temperature between 25 and 400C and 1.5-6% NaCl. According to Kobierecka et al., (2017)  different strains of L. salivarius showed variable resistance to 0.69 and 1mol/L NaCl and no L. reuteri strain showed survival on 0.69mol/L NaCl. To prevent excessive reduction of pH lactic acid bacteria pumps alkali outside and convert the free acid to its salt form. This causes elevation of the osmotic pressure on bacterial cells. For this reason, isolation of potential lactic acid bacteria strains especially for commercial production depends on high osmotolerance feature .