Details

Autor(en) / Beteiligte

• Jayeola, Victor Oladimeji

Titel

Identification of Molecular Mechanisms Mediating Long-Term Survival of Salmonella on Low-Moisture Foods

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2020

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Salmonella is a leading foodborne pathogen globally causing numerous cases of foodborne illnesses, hospitalizations and deaths annually. Low-moisture foods (LMFs) are important vehicles for Salmonella outbreaks due to the ability of this pathogen to survive on LMFs for long periods. However, the molecular mechanisms underlying the survival of Salmonella on LMFs are poorly characterized. The purpose of this dissertation was to identify molecular determinants required for the survival of Salmonella on LMFs.In the first study, the survival of Salmonella on a panel of dried fruit stored at 4 and 25 °C was assessed. Enumerations on growth media revealed rapid decline in the population of Salmonella on the dried fruit, with longer survival on dried apple stored at 4 °C compared to 25 °C. However, fluorescence microscopy and laser scanning confocal microscopy after staining with the LIVE-DEAD BacLight Bacterial Viability kit showed that a large proportion (56-85%) of Salmonella cells present on dried fruit were viable at the times when Salmonella could not be recovered on growth media. Findings from this study revealed that Salmonella can transition into the viable but non-culturable state on dried fruit. Therefore, microscopic and molecular methods should be used in addition to culture-based methods for efficient detection and enumeration of Salmonella contaminating dried fruit.In the second study, the global transcriptome of Salmonella on selected LMFs was analyzed. The LMFs were inoculated, dried to original water activity (Aw), and stored 25 °C in the dark. The total RNA of Salmonella on the LMFs after 3 hours of drying and at 1, 5 and 12 weeks was isolated, ribo-depleted, sequenced and analyzed. The transcriptional response of Salmonella on the LMFs was rapid as several genes were differentially expressed as soon as after 3 h of drying compared to the inoculum suspension. For all products, the transcriptome of Salmonella after drying at 3 h was noticeably different from transcriptome at 1, 5 and 12 weeks of storage, with no major changes in transcriptome thereafter. Several differentially-expressed genes were identified in all tested LMFs at all tested times, while some were upregulated only in specific LMF-time combinations. Some of the differentially expressed genes included those involved in fatty acid biosynthesis, osmoprotectant biosynthesis and transport, DNA repair and RNA degradation, biosynthesis of aromatic compounds and stringent response factors.Transposon sequencing analysis (Tn-Seq) was employed in the third study to determine the genetic determinants required for the survival of Salmonella on LMFs. Tn5-based mutant libraries of three different strains of Salmonella were negatively selected for survival on pistachios, followed by analysis of the distribution of distinct Tn5 insertion sites immediately after inoculation (T0), after drying (T1), and at 120 days (T120). Tn5 insertion sites with reduced relative abundance at T1 vs T0 or T120 vs T1 were localized in DNA repair, lipopolysaccharide biosynthesis and stringent response genes. Testing of 14 single-gene deletion mutants revealed that mutants in sspA, barA, uvrB, damX, rfbD, uvrY, lrhA, yifE, rbsR and ompR were impaired for survival on pistachios. The findings highlight the value of the combined mutagenesis and sequencing strategy to identify novel genes important for the survival of Salmonella in low-moisture foods.In the fourth study, we used a nitrocellulose-based screening protocol to identify Salmonella mutants with impaired desiccation tolerance. Multi-gene and single-gene deletion mutants of S. Typhimurium strain 14028s were screened for desiccation tolerance onnitrocellulose membrane, and mutations that resulted in impaired survival were identified. Several genes, including those involved in the biosynthesis of thin aggregative fimbriae (csgD and csgA), fimbriae genes (fimY and fimA), and recA were identified to be important for the desiccation tolerance of Salmonella. Survival assessment of mutants lacking these genes on in-shell pistachios revealed that while certain determinants such as thin aggregative fimbrae and recA were required for survival on model abiotic surfaces (NC) and LMFs (pistachios), others such as fimbriae that were important for survival on abiotic surfaces may not be required for survival on LMFs.The fifth study was performed to assess the capacity of a surrogate construct, S. Typhimurium MMH112 (harboring deletions in the major Salmonella pathogenicity islands SPI-1, SPI-2, SPI-3, SPI-4, and SPI-5) as an avirulent control in the Galleria mellonella infection model. Fourth-instar G. mellonella larvae were inoculated with bacterial suspensions of S. Typhimurium 14028s (WT) and S. Typhimurium MMH112 and monitored daily for survival. The mortality of inoculated G. mellonella larve was comparable between both strains, suggesting that S. Typhimurium MMH112 was virulent in G. mellonella and thus not suitable as an avirulent control in the infection model. Findings highlight the need for a Salmonella surrogate as an avirulent control in the G. mellonella infection model.In the final study, an ampicillin-based selection protocol was used to selectively enrich for Listeria monocytogenes mutants unable to grow on produce in order to identify genetic factors required for growth on produce. The protocol was validated by assessing the relative recovery of L. monocytogenes strain 2011L-2858 and its cold-sensitive mutant L1E4 (incapable of growth at 4 °C) following inoculation of cantaloupe rind with a 1:1 mixture of the strains and incubation at 4°C with or without ampicillin. Significant increase in the relative abundance of L1E4 compared to 2858 on cantaloupe rind in the presence of ampicillin proved the efficacy of this protocol to enrich for non-replicating cells. Application of this protocol to transposon mutant libraries from three L. monocytogenes strains yielded several mutants unable to grow on cantaloupe. Thus, ampicillin selection can facilitate discovery of genes essential for growth of L. monocytogenes on fresh produce.Findings from these studies showed diverse mechanisms employed by Salmonella to survive on LMFs. These mechanisms include the induction of the viable but non-culturable state, DNA recombination and repair, osmolarity regulation, lipopolysaccharide biogenesis, stringent response, stress-induced sigma factors, and fimbriae formation. Elucidation of the molecular basis of the survival of Salmonella on LMFs may aid the development of improved detection and inactivation strategies, thereby reducing the food safety burden associated with contamination of low-moisture foods.