Isolation, characterization, and assessment of lactic acid bacteria toward their selection as poultry probiotics

Abstract

Background: Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, are now accepted as suitable alternatives to antibiotics in the control of animal infections and improving animal production. Lactic acid bacteria (LAB) with remarkable functional properties have been evaluated in different studies as possible probiotic candidates. The purpose of this study was to isolate, characterize and assess the potentials of LAB from poultry gastrointestinal tract as potential poultry probiotics.

Results: Potential LAB probiotics were isolated from broilers, characterized and evaluated for probiotic properties including antagonistic activity (against Escherichia coli, E. coli O157: H7, Enterococcus faecalis, Salmonella Typhimurium, S. Enteritidis and Listeria monocytogenes), survivability in simulated gastric juice, tolerance to phenol and bile salts, adhesion to ileum epithelial cells, auto and co-aggregation, hydrophobicity, α-glucosidase inhibitory activity, and antibiotic susceptibility tests. Most promising LAB strains with excellent probiotic potentials were identified by API 50 CHL and 16S rRNA sequencing as Lactobacillus reuteri I2, Pediococcus acidilactici I5, P. acidilactici I8, P. acidilactici c3, P. pentosaceus I13, and Enterococcus faecium c14. They inhibited all the pathogens tested with zones of inhibition ranging from 12.5 ± 0.71 to 20 ± 0 mm, and competitively excluded (P < 0.05) the pathogens examined while adhering to ileum epithelial cells with viable counts of 3.0 to 6.0 Log CFU/ml. The selected LAB strains also showed significant (P < 0.005) auto and co-aggregation abilities with α-glucosidase inhibitory activity ranging from 12.5 to 92.0%. The antibiotic susceptibility test showed 100.00% resistance of the LAB strains to oxacillin, with multiple antibiotic resistance indices above 0.5.

Conclusion: The selected LAB strains are ideal probiotic candidates which can be applied in the field for the improvement of poultry performance and control of pathogens in poultry, hence curtailing further transmission to humans.

Keywords: Antagonistic activity; Lactic acid bacteria; Poultry; Probiotics.

Fig. 1

Strain selection flow chart by phenotypic and genotypic methods. Strains without desired probiotics properties were excluded from subsequent examination. LAB: lactic acid bacteria

Fig. 2

Survival of lactic acid bacteria (LAB) strains from poultry crop in stimulated gastric juice pH 2.0. The data are the means of triplicate experiments, and error bars indicate SD. *values are significantly different (P < 0.05). A-H are LAB strains examined

Fig. 3

Adherence of poultry lactic acid bacteria (LAB) strains to poultry ileum epithelial cell. The data are the means of triplicate experiments, and error bars indicate standard deviations. *values are significantly different (P < 0.05)

Fig. 4

Tolerance to 0.10, 0.20, 0.30 and 0.40% phenol by lactic acid bacteria (LAB) strains from poultry. The data are the means of triplicate experiments, and error bars indicate standard deviations. *values are significantly different (P < 0.05)

Fig. 5

Competitive adherence lactic acid bacteria (LAB) strains and pathogens to poultry ileum epithelial cell. The data are the means of triplicate experiments, and error bars indicate standard deviations. *values are significantly different (P < 0.05). A-F are different LAB strains examined

Fig. 6

Adhesion of poultry lactic acid bacteria (LAB) strains to n- hexadecane. Results are expressed as mean ± SD of triplicate experiments. Values without * are not significantly different (P > 0.05)

Fig. 7

α-glucosidase inhibitory activity of lactic acid bacteria (LAB) strains from poultry. Data are expressed as mean expressed as mean ± SD from 3 independent experiments. *significant differences at P < 0.05

Fig. 8

Agarose gel electrophoresis of PCR products after amplification of 16S rRNA; lane M, 1 kb Marker (PROMEGA, USA), lane PC, Positive control (Lactobacillus casei ATCC 393), Lanes 1, 2, 3, 4, 5 and 6 are positive lactic acid bacteria (LAB) strains I2, I5, I8, I13, c3 and c14 at 1500 bp

Fig. 9

Phylogenic tree based on 16S rRNA gene sequences of potential lactic acid bacteria (LAB) strains in relation to the isolates and type strains. The isolates sequenced in the study are depicted in bold font mentioning the accession number and the source of the samples within bracket. Four strains are under the Pediococcus genus, one is Enterococcus faecium and another is Lactobacillus reuteri