Objective: The objective of this study was to characterize and assess lactic acid bacteria isolated from the gastrointestinal tract of broilers for their use in poultry farming as potential probiotic in Côte d'Ivoire. Materials and Methods: For this purpose, 90 colonies of lactic acid bacteria isolated from the crop and cecum of broilers were subjected to several probiotic tests: Thermoresistance, tolerance to acid pH and bile salts, self-aggregation and co-aggregation, hydrophobicity, antibacterial activity and sensitivity to antibiotics. Results: The results of this study showed that out of the 90 isolates 44 were resistant to pH 3 and 0.3% bile salt. Of the 44 isolates, 15 showed high probiotic potential. These isolates belong to the species Pediococcus acidilactici (4), Lactobacillus pentosus (4), Weissella confusa (2) Enterococcus faecium (2), Pediococcus pentosaceus (2) and Enterococcus faecalis (1). The heat map analysis also showed that the species with the best probiotic potential were, Lactobacillus pentosus JK (51 and 55) and Enterococcus faecium JK 96. Conclusion: The species Lactobacillus pentosus JK (51 and 55) and Enterococcus faecium JK 96 could be used for the production of probiotic feed for poultry farming in Côte d'Ivoire thus reducing the use of antibiotics.
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For more than fifty years, antibiotics have been added to the ration of factory-farmed animals not only to prevent certain infectious diseases but also to promote growth and improve feed efficiency. However, application of antibiotics growth promoters (AGPs) in poultry has been linked to the development and spread of resistant bacteria. The presence of antimicrobial residues in chicken products can affect human health. European countries banned their application in poultry feed in 20061. This situation has led researchers to develop alternatives to antibiotics such as organic acids, plant extracts, prebiotics and probiotics2,3. However, probiotics are the most widely used live microorganisms that confer a health benefit to the host (humans and animals) when administered in adequate amount4. They reduce enteric pathogens, improve the immune system or promote growth5. Probiotics can be isolated from the GIT of poultry that has abundant and dominant microbiota. Microbiota from the crop, gizzard, duodenum, jejunum, ileum, caecum and feces/excreta and colon have been studied. Many microbial species are used as probiotic agents. However, the most widely used belong to the group of lactic acid bacteria (LAB), mainly in the genera Lactobacillus, Bifidobacterium, Pediococcus, Streptococcus, Enterococcus and Lactococcus6,7. These microbial species can be used alone or in combination of two, three or more species. Many studies regarding various probiotics for poultry have been done in Europe but Africa particularly Côte d'Ivoire did not do such studies. Therefore, this study was designed to characterize and assess LAB strains isolated from broilers GIT with optimal probiotic properties for their use in poultry farming.
MATERIALS AND METHODS
Lactic acid bacteria and pathogenic strains: A total of 90 LAB isolated from gastrointestinal tract (crop and caecum) of broilers chickens were used in this study. The LAB cultures were prepared in the laboratory of biotechnology and food microbiology at Nangui Abrogoua University (Abidjan, Côte d’Ivoire). They were identified by MALDI-TOF MS method as Enterococcus faecium (3 isolates), Ent. faecalis (3 isolates), Pediococcus acidilactici (41 isolates), Pediococcus pentosaceus (19 isolates), Weissella confusa (3 isolates) and Lactobacillus pentosus (21 isolates). They were kept at -20°C in Man Rogosa and Sharp (MRS, Oxoid, France) broth with 40% glycerol.
Thermoresistance and NaCl tolerance assay: The LAB cultures were inoculated into MRS broth, then placed in a water bath at 63.5°C for 30 min. After sudden cooling, they were incubated at 30°C ±1°C for 48-72 hrs. Similarly, LAB cultures were inoculated into MRS broth containing increasing concentration of NaCl (2.0, 4.0 and 6.5%) and incubated at 37°C for 24 hrs. Cloudiness of the solution indicates positive result8.
Acid tolerance test: The 90 isolates were subjected for different pH tolerance (pH 2.0, 2.5 and 3) according to the method descried by Ramos et al.9 with slight modifications. Briefly, each isolate was grown on MRS broth for 24 hrs at 37°C. The cells were harvested by centrifugation at 5000 rpm for 10 min at 4°C and washed twice in sterile phosphate buffer saline (PBS, pH 7.0). Then, the washed cell density was adjusted to 0.2 optical density (OD) at 600 nm in PBS corresponding to approximately 108 cell mL1 and 1 mL was inoculated into 5 mL of PBS adjusted to pH 2.0, 2.5 and 3 with HCl. Cultures were incubated for 90 min at 37°C. Samples (0.1 mL) were obtained at time 0 and after 90 min and inoculated in MRS agar plates. Tolerance to different pH (pH 2.0, 2.5 and 3) was indicated by subsequent growth on MRS agar plates after 48 hrs of incubation at 37°C.
Bile salt tolerance test: The ability of the isolates to tolerate bile salts was determined according to the modified method described by Handa and Sharma10. The washed cells obtained above were inoculated into sterilized 10 mL of MRS broth containing 0.3, 1 and 2% (w/v) bile salt (Merck, Germany) respectively and incubated at 37°C for 72 hrs. The optical density (OD) at 620 nm was measured and compared to a bile salt-free MRS culture. The percent survival of cells was calculated using formula given below:
where, ΔOD 0%BS and ΔOD (0.3, 1, 2)%BS correspond to absorbance of cells cultivated in the presence of 0% and 0.3, 1 and 2% bile salt, respectively.
Cell surface hydrophobicity test: Bacterial cell surface hydrophobicity was assessed for the 15 acid tolerant isolates by measuring microbial adhesion to the non-polar solvent as described by Taheri et al.11 with slight modifications. Cells cultivated in MRS broth at 37°C for 24 hrs were washed twice in PBS and suspended in the same buffer. The optical density of the suspension was adjusted to 0.5 at 600 nm (A0). Then, 3 mL of cell suspension was mixed with 1 mL of toluene (VWR, France). The mixture was vortexed for 2 min and the phases were allowed to separate for 1 h at 37°C. The lower aqueous phase was carefully removed with a sterile Pasteur pipette and final optical density (A) was recorded at 600 nm to calculate cell hydrophobicity.
where, A0 and A measure cells optical density at the beginning and the end of the experiment, respectively.
Auto-aggregation and co-aggregation test: Auto-aggregation and co-aggregation assays were performed according to Kos et al.12 with slight modifications. The LAB and two pathogen strains (Salmonella enteritidis and Escherichia coli) were separately cultured at 37°C for 24 hrs in MRS broth and BHI broth. The pellet was washed twice in PBS and re-suspended in similar solution. The optical density of the suspension was adjusted to 0.3 at 600 nm. For auto-aggregation, the LAB suspension was vortexed and incubated at 37°C for 5 hrs without agitation. After 5 hrs, absorbance was determined at 600 nm and percentage of auto-aggregation was calculated using the following formula:
where, A0 and At measured at 600 nm, represent the absorbance of the mixture at 0 and 5 hrs, respectively.
For co-aggregation, equal volume of the LAB and pathogenic strain cultures (1:1 v/v) were mixed and incubated at 37°C for 5 hrs without agitation. Absorbance was determined at 600 nm and percentage of co-aggregation was calculated as followed:
Co-aggregation (%) = (((Ax+Ay)/2) - (Axy/(Ax+Ay))/2)X100
where, Ax, Ay and Axy represent the absorbance of individual pathogen, LAB and their mixture after incubation for 5 hrs, respectively.
Antimicrobial activity test: Six strains that are pathogenic to chickens were used as test pathogens to investigate the antagonistic activity of the LAB strains. They were Salmonella enteritidis ATCC 9186, Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25922, Staphylococcus gallinarum ATCC 35539, Pseudomonas aeruginosa ATCC 10145 and Bacillus cereus ATCC 10702. For detection of antimicrobial activity, the well diffusion assay described by Arici et al.13 was performed with some modifications. Briefly, the pathogenic strains were grown in BHI broth at 37°C for overnight. Simultaneously, the LAB strains were grown anaerobically overnight in MRS broth at 37°C. The cultures obtained were centrifuged and the supernatants were recovered and then filter-sterilized (0.45 mm, Millipore, BioRad, France). Aliquots of 60-80 μL of the sterile cell free supernatant were placed in 7 mm diameter wells on Muller-Hinton-agar plates previously seeded with the respective pathogenic strains. After 18 hrs of incubation at 37°C, the diameters of the zones of growth inhibition were measured.
Antibiotic sensitivity test: Antibiotic susceptibility testing of LAB was carried out according to the method described by Bauer et al.14. The antibiotic discs were chosen according to their importance in the different treatments in humans included Cephalothin (KF, 30 μg), Colistin (CST, 30 μg), Chloramphenicol (C, 30 μg), Oxacillin (Ox, 5 μg), Gentamycin (CN, 10 μg), Kanamycin (K, 30 μg), Imipenem (IPM, 10 μg), Amoxicillin (AML, 10 μg) and Erythromycin (E, 15 μg). The 100 μL of LAB strains were inoculated on MRS agar plate. The antibiotic discs were put on MRS agar surface and then incubated at 37°C for 24 hrs. The zones of inhibition around disc were measured. Inhibition diameters were measured and strains were classified as susceptible (S) or resistant (R) according to the recommendations of the Committee of Antibiogram of the French Society of Microbiology15.
Statistical analyses: All the experiments were performed in duplicate and repeated twice. The XLSTAT-2017 statistical software was used to calculate the mean and standard deviation as well as Principal Component Analysis (PCA) and heatmap.
Viability of lactic acid bacteria on inhibitory substances conditions: Of the 90 LAB probiotic strains isolated from the gastrointestinal tract of broilers chickens tested for acid and temperature tolerances, 15 strains were resistant to 63.5°C, 6.5% NaCl, pH 2 and 0.3% bile salt. They were: Weissella confusa (2), Pediococcus acidilactici (4), Pediococcus pentosaceus (2), Lactobacillus pentosus (4), Enterococcus faecalis (1), Enterococcus faecium (2) (Fig. 1).
Cell surface hydrophobicity of lactic acid bacteria strains isolated from the gastrointestinal tract of broilers chickens: All the 15 LAB strains showed high hydrophobicity ranged from 61-99.75%. Enterococcus faecium JK 92 was the most hydrophobic strains, followed by Pediococcus pentosaceus JK 86 (99.40%). Pediococcus acidilactici JK 37 was the least hydrophobic strains (61%) (Fig. 2). Statistical analyses showed 3 groups of lactic acid bacteria according to their hydrophobic properties. The first group of 10 LAB strains had values between 60 and 75%, the second of 1 LAB strain between 80 and 85% and the last group of 4 LAB strains between 95 and 100%.
Auto-aggregation properties of lactic acid bacteria strains isolated from the gastrointestinal tract of broilers chickens: All the 15 strains showed low auto-aggregation ability with values ranged from 0 to 20.22%. Enterococcus faecium JK 92 showed the most auto-aggregation. Pediococcus acidilactici JK 38 and Enterococcus faecalis JK 75 showed the least auto-aggregation (Fig. 3). Statistical analyses showed 3 groups of lactic acid bacteria according to their auto-aggregation properties. The first group of 9 LAB strains had values between 0 and 6%, the second of 4 LAB strains between 10 and 16% and the last group of 2 LABs strains between 18 and 21%.
Co-aggregation properties of lactic acid bacteria strains isolated from the gastrointestinal tract of broilers chickens: All the tested LAB strains showed co-aggregation ability with the pathogens Salmonella enteritidis and Escherichia coli. With Salmonella enteritidis, values were between 47.97% (Pediococcus pentosaceus JK 44) and 58.80% (Pediococcus pentosaceus JK 86) while with Escherichia coli, they varied from 49.21% (Lactobacillus pentosus JK 49) to 53.66% (Weissella confusa JK 25) (Fig. 4). Statistical analyses showed 2 groups of lactic acid bacteria according to their co-aggregation properties. The first group of 1 LAB strain had value between 30 and 35% for the co-aggregation with S. enteritidis and 35 and 40% for the co-aggregation with E. coli respectively. The second of 14 LAB strains had value between 45 and 60% for the co-aggregation with S. enteridis and 47 and 53% for the co-aggregation with E. coli, respectively.
Antimicrobial activity: The results for antimicrobial activity of the 15 LAB isolated from the gastrointestinal tract of broilers chickens against pathogenic bacteria is presented in Table 1. All 15 LAB strains showed antagonistic effects against Salmonella typhimurium, Pseudomonas aeruginosa and Bacillus cereus. The diameters of inhibition were ranged from 10-19 mm. The highest activity towards S. typhimurium was obtained by Lactobacillus pentosus JK 51 (18 mm). For Bacillus cereus, the highest activity was obtained by Lactobacillus pentosus JK 54 (18 mm) and Pediococcus pentosaceus JK 86 (18 mm). The highest activity towards Pseudomonas aeruginosa was obtained by Lactobacillus pentosus (JK 51 and 55) (19 mm). Salmonella enteritidis was inhibited by Enterococcus faecium JK 96 (19 mm). Escherichia coli was inhibited Lactobacillus pentosus (JK 51 and 55) (15 mm), Pediococcus acidilactici JK 38 (15 mm) and Weissella confusa JK 25 (12 mm). All 4 Lactobacillus pentosus strains and Enterococcus faecium JK 96 showed antagonistic activity towards all pathogens.