Salmonella Transfer and Survival on Fresh-cut Fruits Yingshu He, - PowerPoint PPT Presentation

Salmonella Transfer and Survival on Fresh-cut Fruits Yingshu He, Ph.D. Research associate Illinois Institute of Technology Institute for Food Safety and Health Bedford Park, Illinois, United States � 1

Background Salmonella outbreaks associated with fresh produce � 2

Background Salmonella outbreaks associated with fresh produce Tomatoes Cantaloupes Cucumbers 81% Newport & Typhimurium (by cases) 32% (by cases) � 3

Background S. Newport and Typhimurium outbreaks associated with fresh fruits Tomatoes Cantaloupes Cucumbers 2006 S . Typhimurium outbreak 2012 S . Typhimurium & 2014 S .Newport outbreak 21 states, 183 cases, S . Newport outbreak 29 states, 275 cases, 22 hospitalized, 0 deaths 24 states, 261 cases, 48 hospitalized, 1 deaths 2002 S .Newport outbreak 94 hospitalized, 3 deaths 510 cases � 4

Hypothesis ๏ Main route of contamination: through fresh-cutting ๏ Inside fruits: adapt to the internal environment in fruits and survive for extended period of time (gene expression & metabolic pathways) � 5

Objectives ๏ Transfer of Salmonella Newport and Typhimurium strains on fresh fruit produces by cutting and their growth dynamics on fresh-cut fruits and in blended juice ๏ Transcriptomic responses of Salmonella Newport on fresh-cut tomatoes and cantaloupes � 6

Objective 1 - Method Processing by cutting -ve 2nd +ve 1st 3rd 4th � 7

Objective 1 - Method Processing by cutting -ve 2nd +ve 1st 3rd 4th � 7

Objective 1 - Method (Cont.) Store at : 4 ℃ , 90% ± 5% HM, Up to 7 days � 8

Objective 1 - Results (Transfer) Proportion of S . Newport population (%) in fruits after each cutting Cucumber (CFU/cm^2) A Tomato (CFU/cm^2) C Tomato (MPN/cm^2) E 19.55% 6.61% 3.14% 3.83% 9.03% 95.66% 4.34% 96.20% 3.80% 2.42% 71.42% 0.62% 0.39% 0.13% 0.04% 1st_spl 2nd_spl 3rd_spl 4th_spl 1st_spl 2nd_spl 3rd_spl 4th_spl 1st_spl 2nd_spl 3rd_spl 4th_spl B D F Apple (CFU/cm^2) Cantaloupe (CFU/cm^2) Cantaloupe (MPN/cm^2) 27.76% 1.26% 6.05% 3.57% 8.33% 95.87% 4.13% 96.32% 3.68% 1.46% 2.28% 0.95% 0.43% 0.14% 63.91% 1st_spl 2nd_spl 3rd_spl 4th_spl 1st_spl 2nd_spl 3rd_spl 4th_spl 1st_spl 2nd_spl 3rd_spl 4th_spl High inoculation High inoculation Low inoculation � 9

Objective 1 - Results (Transfer) Salmonella transfer rates (%) of cuttings vs 1st sample 7 -10 8 CFU/ml) — SN & ST on four fruits (10 Transfer Rate (vs 1st sample) 1st-2nd Cut 1st-3rd Cut 1st-4th Cut Apple Tomato Cucumber Cantaloupe * 70% Transfer rate (CFU/cm^2) * 60% * 50% 40% * 30% 20% 10% 0% SN- SN- ST- ST- SN- SN- ST- ST- SN- SN- ST- ST- SN- SN- ST- ST- Day1 Day2 Day1 Day2 Day1 Day2 Day1 Day2 Day1 Day2 Day1 Day2 Day1 Day2 Day1 Day2 1st-2nd Cut 25.49% 37.91% 38.61% 24.88% 35.34% 18.70% 24.67% 44.53% 6.26% 2.59% 4.61% 5.88% 5.08% 7.31% 5.55% 9.10% 1st-3rd Cut 9.20% 14.61% 12.22% 12.77% 13.78% 6.09% 5.00% 21.87% 0.94% 0.32% 1.44% 3.43% 0.69% 2.08% 0.75% 2.44% 1st-4th Cut 3.58% 7.34% 5.24% 4.40% 3.34% 1.89% 2.19% 4.34% 0.38% 0.16% 0.40% 1.96% 0.13% 0.52% 0.32% 0.51% * SN, Salmonella Newport. ST, Salmonella Typhimurium. ✦ Overall, transfer rate of 1st-2nd cut > rest cuttings ✦ Cucumber & apple > tomato & cantaloupe � 10

Objective 1 - Results (Transfer) S. Newport transfer rates (%) of cuttings vs 1st sample on tomatoes and cantaloupes (High vs Low) 10.00% * Tomato (High) 9.00% Tomato (Low) 8.00% Cantaloupe (High) Transfer rate (CFU/cm^2)* 7.00% * Cantaloupe (Low) 6.00% * 5.00% 4.00% 3.37% 3.00% 2.28% 2.02% 2.00% 1.65% 1.00% 0.69% 0.05% 0.00% 1st-2nd_spl 1st-3rd_spl 1st-4th_spl Sample • High: high inoculation level, 107-108 CFU/ml * Transfer rate was calculated based on CFU/cm^2 for high inoculation level and MPN/cm^2 for low • Low: low inoculation level, 103-104 MPN/ml inoculation level. � 11

Objective 1- Results (Survival) Survival of Salmonella on fresh-cut fruits and in blended juice at 4 o C (XLD) Mini Cucumber A Gala Apple B 9.00 9.00 8.50 8.50 Log10 CFU/ml (High) Log10 CFU/ml (High) 8.00 8.00 7.50 7.50 7.00 7.00 6.50 6.50 6.00 6.00 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Day Day Cum-Cube-SN Cum-Cube-ST Cum-Juice-SN Cum-Juice-ST Apl-Cube-SN Apl-Cube-ST Apl-Juice-SN Apl-Juice-ST Tomato C Cantaloupe D 9.00 9.00 8.50 8.50 Log10 CFU/ml (High) Log10 CFU/ml (High) 8.00 8.00 7.50 7.50 7.00 7.00 6.50 6.50 6.00 6.00 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Day Day Tom-Cube-SN Tom-Cube-ST Tom-Juice-SN Tom-Juice-ST Clp-Cube-SN Clp-Cube-ST Clp-Juice-SN Clp-Juice-ST * SN, Salmonella Newport. ST, Salmonella Typhimurium. � 12

Objective 2 - Method Store at : 4 ℃ , 90% ± 5% HM, For 1 hour RNA-Seq library preparation � 13

Objective 2 - Method (Cont.) RNA-Seq data analysis flow chart Sequencing cDNA library Fastq file FastQC e.g. HiSeq HiSat 2 Trimmomatic FastQC (.fasta -> .sam) (adaptor removal) Samtool Blast2Go Differential (.sam -> .bam) (edgeR) expression analysis Reference strain Annotation (.gff) KEGG GSEA � 14

Objective 2 - Results Gene differential expression analysis (volcano plots) Differential Expression (DE) Results 5000 4881 4500 4306 4000 No. of Genes 3500 Tom1h-Bpw1h Clp1h-Bpw1h 3000 2500 4881 4306 2000 1500 1000 500 325 358 159 160 166 198 0 Total DE Down-regulated � 15

Objective 2 - Results (Cont.) S .Newport carbohydrate utilization on tomato and cantaloupe slices Galactitol-1P Galactitol Cellodextrin Cellulose Tagatose-6P Cellobiose Galactose Sucrose Sorbitol-6P Glucose Fructose-6P Fructose Sorbitol Glucose-6P Maltose Glycolysis Cellobiose-6P Glycer- Cellobiose aldehyde-3P (extracellular) Maltose Glucose-6P (extracellular) Fructose-1,6diP Arbutin-6P Arbutin Salicin Fructose Fructose-1P Salicin-6P � 16

S .Newport carbohydrate utilization on tomato and cantaloupe slices Galactitol-1P Galactitol Cellulose Cellodextrin Cellobiose Tagatose-6P Galactose Sucrose Sorbitol-6P Glucose Fructose-6P Fructose Sorbitol Glucose-6P Maltose Glycolysis Cellobiose-6P Glycer- Cellobiose aldehyde-3P (extracellular) Glucose-6P Maltose (extracellular) Fructose-1,6 diP Arbutin-6P Arbutin Salicin Fructose Fructose-1P Salicin-6P � 17

S .Newport carbohydrate utilization on tomato and cantaloupe slices Galactitol-1P Galactitol Cellulose Cellodextrin Cellobiose Tagatose-6P Galactose Sucrose Sorbitol-6P Glucose Fructose-6P Fructose Sorbitol Glucose-6P Maltose Glycolysis Cellobiose-6P Glycer- Cellobiose aldehyde-3P (extracellular) Glucose-6P Maltose (extracellular) Fructose-1,6 diP Arbutin-6P Arbutin Salicin Fructose Salicin-6P Fructose-1P � 18

Objective 2 - Results (Cont.) Cantaloupe vs Tomato nutrition table* Nutrient (g /100g), selected Tomato Cantaloupe Carbohydrate 3.89 8.16 sugar, total 2.63 7.86 Sucrose —— 4.35 Glucose 1.25 1.54 Fructose 1.37 1.87 Maltose —— 0.04 Galactose —— 0.06 Starch —— 0.03 Amino Acids Tryptophan 0.006 0.002 0.021 Isoleucine 0.018 Leucine 0.025 0.029 Lysine 0.027 0.03 Methionine 0.006 0.012 Cystine 0.009 0.002 Phenylalanine 0.027 0.023 Tyrosine 0.014 0.014 Valine 0.018 0.033 Glutamic acid 0.431 0.209 * Source: USDA National Nutrient Database for Standard Reference Legacy April 2018 Software v.3.9.4 2018-05-17 � 19

Objective 2 - Results (Cont.) Nitrate and amino acid utilization of S . Newport on tomato slices N-Acetyl- N-Acetyl- N-Acetyl- glutamate Glutamate glutamate glutamyl-P semi-aldehyde Nitrate Glutamine (extracellular) N-Acetyl-ornithine Carbamoyl-P Ammonia (NH3) Nitrate Ornithine Citrulline Arginine Nitrite and proline metabolism Aspartate Arginine L- Citrate Asparagine Fumarate Arginosuccinate Cycle Up-regulated Not significantly regulated Involved in other pathways � 20

Objective 2 - Results (Cont.) Commonly up-regulated gene sets of S . Newport on tomato and cantaloupe slices Clp_NES_Sum Tom_NES_Sum Ribosomal subunit Cellular Component Extracellular region Structural molecule activity (ribosome) transaminase activity Molecular GO Category Function Binding (NADP/GTP/rRNA) Ribosom bogenesis Ion transport Carboxylic acid metabolism Biological Process amino acid metabolism Multi-organism process (Virulence) Carbohydrate derivative metabolism Carbon utilization 0.00 5.00 10.00 15.00 20.00 25.00 30.00 NES � 21

Summary • The transfer of S.enterica was most significant by the first two cuts of the un-inoculated fruit with a contaminated knife. • Salmonella can survive and adapt well on selected fresh-cut fruits and in blended juice under low temperature storage for at least 7 days. • Up-regulated gene sets involved in carbon utilization and amino acid metabolism may facilitate the adaptation and survival of S. Newport on fresh-cut tomatoes and cantaloupes. � 22