Identification, Screening And ERIC-PCR Differentiation Of Lactobacilli Isolated From Weaning Piglet

231 suspected Lactobacillus strains were isolated from gastrointestinal tract of Yorkshire×Taihu weaning piglet. 26 bacteria strains (ZN8,SJBZ4,JJN1,SJNH7,SJBH4,ZB5,MNZ8,SNZ10,SJNZ4,WN8,WB5,HN1,JJB5,MBZ3,SBZ5,SBQ9,SNQ5,MNQ4,KBQ4,WN1,WB10,JJB3,ZB3,MBH5,KNZ4) were screened by acid resistance test from 75 bacteria strains.The 26 bacteria strains were identified as the members of the genus Lactobacillus by mobility test, nitrate reduction test, gelatin liquefaction test, indole test, hydrogen sulfide test and catalase test. 5 Lactobacillus species were identified by carbohydrate fermentation test: 7 Lactobacillus amylovorus strains, 3 Lactobacillus salivarius strains, 6 Lactobacillus reuteri strains, 9 Lactobacillus plantarum strains, 1 Lactobacillus acidophilus strains. Meanwhile, 3 Lactobacillus species were identified by 16S rRNA gene sequencing: 10 L. reuteri strains, 15 Lactobacillus johnsonii strains, 1 Weissella paremesenteroides strain.6 Lactobacillus strains, ZB3, WN8, WN1, SNQ5, SBZ5 and JJB3, which have strong inhibition on both Escherichia coli O₈ and Salmonella 339 were screened from the 26 bacteria strains by bacteriostatic test. 2 best Lactobacillus strains, SBZ5 and JJB3, were screened by bile tolerance test, heat-resistance test and growth test. Spontaneous rifampin-resistant (Rif¹) variants of the 2 Lactobacillus strains were isolated by spread plating 10⁹ CFU from an overnight culture onto MRS agar containing 10mg of rifampm/L and then were applied in weaning piglet feeding.Enterobacterial Repetitive Intergenic Consensus (ERIC), also named Intergenic Repetitive Unit (IRU), is a kind of intergenic repetitive sequence that exists predo minantly in Enterobacteria. ERIC sequences are dispersed throughout bacterial genomes and their copies or inter-ERIC distances are typical for given bacterial species and sometimes even for strains within a given species. So it can be used for classification, identification and differentiation of bacteria.The 20μL reaction mixture contained 10 mmol/L Tris-HCl, 50 mmol/L KC1, 3 mmol/L MgCl₂, 250μmol/L of each deoxynucleoside triphosphate, 1μmol/L of each primer (ERIC1R, 5'-ATGTAAGCTCCTGGGGATTCAC-3' and ERIC2, 5'-AAGTAAGTGACTG GGGTGAGCG-3'), 0.05 U of Long Taq DNA polymerase and 1μL of the respective template DNA. Samples were denatured for 5 min at 94℃and processed in the following manner: one cycle at 94℃for 3 min; followed by 5 cycles at 94℃for 1 min, at 32℃for 1 min, at 72℃for 2 min and 40 cycles at 94℃for 0.5 min, at 48℃for 0.5 min, at 72℃for 1 min and one cycle at 72℃for 7 min.ERIC-PCR was confirmed to be a powerful tool for a very rapid and highly reliable differentiation of Lactobacillus isolates at their strain level. ERIC-PCR for the 26 bacteria strains generated multiple DNA fragments ranging from 200 bp to 2000 bp with various intensities. The 26 isolates were grouped into 3 different groups with clearly distinguishable ERIC-PCR patterns, named as 26-A, 26-B, 26-C. The cluster 26-A included 7 L. reuteri strains(ZN8,SJBZ4,JJN1,SJNH7,SJBH4,ZB5,MNZ8), while 26-B and 26-C harbored 15 L. johnsonii strains (SNZ10,SJNZ4,WN8,WB5,HN1,JJB5,MBZ3,SBZ5,SBQ9,SNQ5,MNQ4,KBQ4,WN1,WB10,JJB3)and 3 L. reuteri strains (ZB3,MBH5,KNZ4) respectively. The genetic similarity value of group 26-A ranged between 52%-76%, while that of 26-B and 26-C ranged between 40%-92% and 37%-73% respectively. If 75% was set as the minimum genetic similarity value of identifying two strains as the same, 2 strains (ZN8 and SJBZ4) and 2 strains (SJNH7 and SJBH4) out of 26-A were the same strain, while 4 strains (WN8, WB5, HN1 and JJB5) and 3 stains (SBZ5, SBQ9 and SNQ5) out of 26-B were the same strain respectively. In addition, the 3 strains of 26-C were different from each other.