Selection of lactic acid bacteria isolated from swine against pathogenic bacteria and porcine epidemic diarrhea (PED) virus

Abstract

Lactic acid bacteria (LAB) are commensal microbes in pig gastrointestinal tract and have been applied as probiotics due to their ability to benefit and improve pig health. The finding of suitable LAB strains to use as probiotics as substitution of antibiotic growth promoter in pig production in Thailand would lead to the increase of product quality and value. The objective of this research was to 1) isolate and pre-select acid- and bile- tolerant LAB from healthy antibiotic-free indigenous and commercial pigs in Thailand, characterize and identify the pre-selected acid and bile tolerant LAB by phenotypic characteristics (21 sugars), whole cell protein profile by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and complete 16S ribosomal RNA gene analysis, and investigate antimicrobial susceptibility following European Food Safety Authority (EFSA) recommendations; 2) study and observe their antiviral activity against porcine epidemic diarrhea virus (PEDV); 3) evaluate the probiotic properties and antibacterial activity against porcine enteric pathogenic bacteria included Enterohemorrhagic Escherichia coli (EHEC) Enterotoxigenic Escherichia coli (ETEC) Salmonella Typhimurium and Salmonella Choleraesuis. We were able to pre-select 34 acid and bile tolerant LAB strains (15 isolates from indigenous pigs and 19 isolates from commercial pigs) which were identified as Enterococcus faecium (11 isolates), Enterococcus hirae (9 isolates), Lactobacillus plantarum (4 isolates), Lactobacillus agilis (3 isolates), Pediococcus pentosaceus (6 isolates) and Pediococcus acidilactici (1 isolate). After, the results of their antimicrobial susceptibility profiles by disk diffusion showed seven isolates with susceptible to the broad spectrum antibiotics and antibiotics against Gram-positive bacteria. These seven isolates were selected for further confirmation of their antimicrobial susceptibility profiles by MICs following European Food Safety Authority (EFSA) microbiological cut-off values, and investigated for antiviral activity against PEDV. We found that five isolates included L. plantarum (22F, 25F, 31F), P. acidilactici (72N) and P. pentosaceus (77F) were acceptable as probiotic candidates followed the antimicrobial susceptibility recommendation by EFSA, and among the five isolates, both cell-free supernatant and live cells of L. plantarum (25F) possessed the best antiviral activity against PEDV by reducing cytopathic effects (CPE) from PEDV to <50% of high power field area. In addition, these five isolates were selected for in vitro evaluation of probiotic properties and antibacterial activity against EHEC, ETEC, Salmonella Typhimurium, Salmonella Choleraesuis and Streptococcus suis type II. The resuls revealed that L. plantarum 22F possessed better functional probiotic properties for most in vitro evaluations. When taken together all the experiments in this research, we could summarize that L. plantarum 22F and 25F were the most suitable for development as probiotic candidates for pig industry since L. plantarum 22F exhibited most of functional probiotic properties and live cells of L. plantarum 25F showed the best antibacterial and antiviral activity against pathogenic bacteria and PEDV