Genetic determinants of Salmonella enterica critical for attachment and biofilm formation

Abstract

Salmonella is a bacterial pathogen frequently involved in human gastrointestinal infections including those associated with low-moisture foods such as dehydrated food powders/spices, vegetable seeds, and tree nuts. The survival/persistence of Salmonella on low moisture foods and in dry environments is enhanced by its ability in developing biofilms. This study was undertaken to identify the genetic determinants critical for Salmonella attachment and biofilm formation. E. coli SM10 lambda pir, with a kanamycin resistant marker on mini-Tn10 (mini-Tn10:lacZ:kan^r), an ampicillin resistant marker on the mini-Tn10-bearing suicidal plasmid pLBT and a streptomycin sensitive marker on the SM10 chromosome, was used as a donor (amp^r, kan^r, strep^s), and three Salmonella strains (amp^s, kan^s, strep^r) were used as recipients in a transposon mutagenesis study. The donor and each recipient were co-incubated overnight on tryptic soy agar at 37 °C, and mutant colonies (amp^s, kan^r, strep^r) were subsequently selected. A single-banded degenerate PCR product, amplified from each mutant genome using oligonucleotide primers derived from the end of min-Tn10 and restriction enzyme EcoR I- or Pst I-recognizing sequence, were analyzed using the Sanger sequencing technology. Acquired DNA sequences were compared to those deposited in the Genbank using BLAST search. Cells of Salmonella mutants accumulated either significantly more or less (P < 0.05) biofilms than their parent cells on polystyrene surface. Sequence analysis of degenerate PCR products revealed that the mini-Tn10 from pLBT had inserted into the cdg, trx, fadI or rxt on Salmonella chromosomes. Results of the research will likely help strategize future antimicrobial intervention for control of pathogen attachment and biofilm formation.

Keywords: Biofilm; Mini-Tn10; Mutagenesis; Salmonella.