Identification of Novel Genes Mediating Survival of Salmonella on Low-Moisture Foods via Transposon Sequencing Analysis

Abstract

Salmonella enterica is the leading foodborne pathogen associated with outbreaks involving low-moisture foods (LMFs). However, the genes involved in Salmonella's longterm survival on LMFs remain poorly characterized. In this study, in-shell pistachios were inoculated with Tn5-based mutant libraries of S. Enteritidis P125109, S. Typhimurium 14028s, and S. Newport C4.2 at approximate 10 8 CFU/g and stored at 25 • C. Transposon sequencing analysis (Tn-seq) was then employed to determine the relative abundance of each Tn5 insertion site immediately after inoculation (T 0), after drying (T 1), and at 120 days (T 120). In S. Enteritidis, S. Typhimurium, and S. Newport mutant libraries, the relative abundance of 51, 80, and 101 Tn5 insertion sites, respectively, was significantly lower at T 1 compared to T 0 , while in libraries of S. Enteritidis and S. Typhimurium the relative abundance of 42 and 68 Tn5 insertion sites, respectively, was significantly lower at T 120 compared to T 1. Tn5 insertion sites with reduced relative abundance in this competition assay were localized in DNA repair, lipopolysaccharide biosynthesis and stringent response genes. Twelve genes among those under strong negative selection in the competition assay were selected for further study. Whole gene deletion mutants in ten of these genes, sspA, barA, uvrB, damX, rfbD, uvrY, lrhA, yifE, rbsR, and ompR, were impaired for individual survival on pistachios. The findings highlight the value of combined mutagenesis and sequencing to identify novel genes important for the survival of Salmonella in low-moisture foods.

Mechanisms of Salmonella Attachment and Survival on In-Shell Black Peppercorns, Almonds, and Hazelnuts

“…A number of previous studies suggested that the overall bacterial attachment and survival on various biotic and abiotic surfaces can be mediated by genes encoding transcription factors, regulators, virulence determinants, and cell surface components such as cellulose, flagella, and fimbriae (Barak et al, 2005;Marvasi et al, 2013;Salazar et al, 2013;Tan et al, 2016;de Moraes et al, 2017;Jayeola et al, 2020). We used a library of over 120,000 mutant strains, each with a random Tn5 transposon insertion, to identify the subset of mutants that is under selection during up to 14 days of ambient storage on almond shells.…”

“…A transposon insertion library in a fully sequenced S. enterica serovar Enteritidis PT4 strain was utilized, which had been constructed as previously described (de Moraes et al, 2017(de Moraes et al, , 2018Jayeola et al, 2020). Briefly, the EZ-Tn5 < T7/KAN-2 > promoter insertion kit (Epicentre Technologies Corporation, Madison, WI) was employed, and specific primers were used to add N 18 random barcodes to both sides of the EZ-Tn5 transposon for sequencing analysis (Supplementary Table 2).…”

“…To identify specific genes that were involved in S. enterica survival on almond shells, we adopted a published method (de Moraes et al, 2017;Jayeola et al, 2020), with minor modifications. Briefly, the entire Tn5 mutant library was surfaceinoculated on in-shell almonds using the inoculation protocol described above.…”

“…Enriched S. enterica cells were pelleted and plated onto LB agar containing kanamycin and incubated at 37 • C for 24 h. Genomic DNA was extracted using Generation Capture Columns from Qiagen (Hilden, Germany). The DNA was subsequently used as template for PCR using primers flanking the N 18 barcode, and the frequency of each barcode was determined by Illumina sequencing, as previously described (de Moraes et al, 2017(de Moraes et al, , 2018George et al, 2018;Jayeola et al, 2020).…”

“…As the plasmid has its own origin of replication, this observation was likely due to the copy number of the plasmid being higher relative to the copy number of the genome at later time points. Interestingly, a large number of Tn5 mutants in S. Enteritidis which were strongly selected on almonds were also found to be strongly selected in S. enterica on pistachios (Jayeola et al, 2020). Table 4 lists the S. enterica genes that were selected on both almonds and pistachios.…”