Isolation and identification of porcine-originated Escherichia coli Nissle 1917

Abstract:

[Objective] To verify that Escherichia coli Nissle 1917 also exists in swine as a natural isolate and could be isolated from swine faeces. The method for identifying the Escherichia coli Nissle 1917 using in situ hybridization was established. [Methods] All 135 pieces of fresh faeces from healthy weaned piglets were collected to prepare DNA template samples. And the DNA template samples were screened and tested by PCR with five pairs of specific primers targeting the chromosomal genes encoding the major ?mbrial subunit FimA (type 1 ?mbriae) and FocA (F1C ?mbriae), and the two cryptic plasmids pMUT1 and pMUT2, and mean while the human E. coli Nissle 1917 was chosen as the positive control. The 427 bp-long plasmid fragment named pMUT2(a) was purified from the gel and made into DNA probe using digoxigenin random primer labeling. [Results] The PCR result showed that the expected fragments of the five specific primers target were amplified from the two of 135 DNA template samples and the potential presence of Nissle 1917 in pigs was initially confirmed. Using the method of in situ hybridization with prepared pMUT2(a) probe, two positive strains were screened from the two positive faecal samples. Two positive clones were finally confirmed as positive strains of Nissle 1917 through further experiments of serological test, PCR and sequencing. [Conclusion] The isolation and identification of swine-originated probiotics Nissle 1917 laid a foundation for further study and application of excellent animal originated probiotic.

Key words:Escherichia coli Nissle 1917, Isolation and identification, Digoxigenin-labeled nucleic acid probe, Colony in situ hybridization