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

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

Jun 21, 2023 183 likes •436 views

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

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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

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Background

Salmonella outbreaks associated with fresh produce

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Background

Salmonella outbreaks associated with fresh produce

Newport & Typhimurium

32%

(by cases) Tomatoes Cantaloupes Cucumbers 81% (by cases)

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Background

Cucumbers 2014 S.Newport outbreak 29 states, 275 cases, 48 hospitalized, 1 deaths Tomatoes 2006 S. Typhimurium outbreak 21 states, 183 cases, 22 hospitalized, 0 deaths 2002 S.Newport outbreak 510 cases Cantaloupes 2012 S. Typhimurium &

  • S. Newport outbreak

24 states, 261 cases, 94 hospitalized, 3 deaths

  • S. Newport and Typhimurium outbreaks associated with fresh fruits

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๏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)

Hypothesis

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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

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Processing by cutting

+ve 1st

  • ve

2nd 3rd 4th

Objective 1 - Method

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Processing by cutting

+ve 1st

  • ve

2nd 3rd 4th

Objective 1 - Method

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Store at :

4℃, 90% ± 5% HM, Up to 7 days

Objective 1 - Method (Cont.)

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Objective 1 - Results (Transfer)

Proportion of S. Newport population (%) in fruits after each cutting

High inoculation High inoculation Low inoculation

95.66% 3.83% 0.39% 0.13% 4.34%

Tomato (CFU/cm^2)

1st_spl 2nd_spl 3rd_spl 4th_spl

96.20% 3.14% 0.62% 0.04% 3.80%

Tomato (MPN/cm^2)

1st_spl 2nd_spl 3rd_spl 4th_spl

95.87% 3.57% 0.43% 0.14% 4.13%

Cantaloupe (CFU/cm^2)

1st_spl 2nd_spl 3rd_spl 4th_spl

96.32% 1.26% 1.46% 0.95% 3.68%

Cantaloupe (MPN/cm^2)

1st_spl 2nd_spl 3rd_spl 4th_spl

71.42% 19.55% 6.61% 2.42% 9.03%

Cucumber (CFU/cm^2)

1st_spl 2nd_spl 3rd_spl 4th_spl

63.91% 27.76% 6.05% 2.28% 8.33%

Apple (CFU/cm^2)

1st_spl 2nd_spl 3rd_spl 4th_spl

A B C D E F

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Salmonella transfer rates (%) of cuttings vs 1st sample

— SN & ST on four fruits (10

7-10 8 CFU/ml)

Objective 1 - Results (Transfer)

✦Overall, transfer rate of 1st-2nd cut > rest cuttings ✦Cucumber & apple > tomato & cantaloupe

SN- Day1 SN- Day2 ST- Day1 ST- Day2 SN- Day1 SN- Day2 ST- Day1 ST- Day2 SN- Day1 SN- Day2 ST- Day1 ST- Day2 SN- Day1 SN- Day2 ST- Day1 ST- 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% 0% 10% 20% 30% 40% 50% 60% 70%

Transfer rate (CFU/cm^2)

Transfer Rate (vs 1st sample)

1st-2nd Cut 1st-3rd Cut 1st-4th Cut Apple Cucumber Tomato Cantaloupe

* SN, Salmonella Newport. ST, Salmonella Typhimurium.

* *

* *

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  • S. Newport transfer rates (%) of cuttings vs 1st sample
  • n tomatoes and cantaloupes (High vs Low)
  • High: high inoculation level, 107-108 CFU/ml
  • Low: low inoculation level, 103-104 MPN/ml

Objective 1 - Results (Transfer)

* Transfer rate was calculated based on CFU/cm^2 for high inoculation level and MPN/cm^2 for low inoculation level.

3.37% 0.69% 0.05% 2.28% 2.02% 1.65%

0.00% 1.00% 2.00% 3.00% 4.00% 5.00% 6.00% 7.00% 8.00% 9.00% 10.00% 1st-2nd_spl 1st-3rd_spl 1st-4th_spl

Transfer rate (CFU/cm^2)* Sample

Tomato (High) Tomato (Low) Cantaloupe (High) Cantaloupe (Low)

* * *

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Objective 1- Results (Survival)

Survival of Salmonella on fresh-cut fruits and in blended juice at 4oC (XLD)

6.00 6.50 7.00 7.50 8.00 8.50 9.00 1 2 3 4 5 6 7 8

Log10 CFU/ml (High) Day

Mini Cucumber

Cum-Cube-SN Cum-Cube-ST Cum-Juice-SN Cum-Juice-ST

6.00 6.50 7.00 7.50 8.00 8.50 9.00 1 2 3 4 5 6 7 8

Log10 CFU/ml (High) Day

Gala Apple

Apl-Cube-SN Apl-Cube-ST Apl-Juice-SN Apl-Juice-ST

6.00 6.50 7.00 7.50 8.00 8.50 9.00 1 2 3 4 5 6 7 8

Log10 CFU/ml (High) Day

Tomato

Tom-Cube-SN Tom-Cube-ST Tom-Juice-SN Tom-Juice-ST

6.00 6.50 7.00 7.50 8.00 8.50 9.00 1 2 3 4 5 6 7 8

Log10 CFU/ml (High) Day

Cantaloupe

Clp-Cube-SN Clp-Cube-ST Clp-Juice-SN Clp-Juice-ST

A B C D

* SN, Salmonella Newport. ST, Salmonella Typhimurium. 12

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Objective 2 - Method

RNA-Seq library preparation

Store at :

4℃, 90% ± 5% HM, For 1 hour

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RNA-Seq data analysis flow chart

cDNA library Sequencing e.g. HiSeq Fastq file FastQC Trimmomatic (adaptor removal) Differential expression analysis Blast2Go (edgeR) Reference strain Annotation (.gff) Samtool (.sam -> .bam) HiSat 2 (.fasta -> .sam) FastQC KEGG GSEA

Objective 2 - Method (Cont.)

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Gene differential expression analysis (volcano plots)

Objective 2 - Results

Differential Expression (DE) Results

  • No. of Genes

500 1000 1500 2000 2500 3000 3500 4000 4500 5000

Total DE Down-regulated

198 160 358 4306 4881 166 159 325 4306 4881

Tom1h-Bpw1h Clp1h-Bpw1h

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Maltose

(extracellular)

Maltose Fructose-6P Sucrose Cellulose Cellodextrin Glucose

Glycolysis

Galactose Fructose Fructose-1P Fructose-1,6diP Glycer- aldehyde-3P Sorbitol Cellobiose-6P Glucose-6P Galactitol Galactitol-1P Tagatose-6P Glucose-6P Salicin Arbutin Fructose Cellobiose

(extracellular)

Sorbitol-6P Arbutin-6P Salicin-6P Cellobiose

S.Newport carbohydrate utilization on tomato and cantaloupe slices

Objective 2 - Results (Cont.)

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Maltose (extracellular) Maltose Fructose-6P Sucrose Cellulose Cellodextrin Glucose

Glycolysis

Galactose Fructose Fructose-1P Fructose-1,6 diP Glycer- aldehyde-3P Sorbitol Cellobiose-6P Glucose-6P Galactitol Galactitol-1P Tagatose-6P Glucose-6P Salicin Arbutin Fructose Cellobiose (extracellular) Sorbitol-6P Arbutin-6P Salicin-6P Cellobiose

S.Newport carbohydrate utilization on tomato and cantaloupe slices

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Maltose (extracellular) Maltose Fructose-6P Sucrose Cellulose Cellodextrin Glucose

Glycolysis

Galactose Fructose Fructose-1P Fructose-1,6 diP Glycer- aldehyde-3P Sorbitol Cellobiose-6P Glucose-6P Galactitol Galactitol-1P Tagatose-6P Glucose-6P Salicin Arbutin Fructose Cellobiose (extracellular) Sorbitol-6P Arbutin-6P Salicin-6P Cellobiose

S.Newport carbohydrate utilization on tomato and cantaloupe slices

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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 Isoleucine 0.018 0.021 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

Cantaloupe vs Tomato nutrition table*

* Source: USDA National Nutrient Database for Standard Reference Legacy April 2018 Software v.3.9.4 2018-05-17

Objective 2 - Results (Cont.)

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Objective 2 - Results (Cont.)

Citrate Cycle Arginine and proline metabolism

N-Acetyl- glutamate semi-aldehyde Up-regulated Not significantly regulated Involved in other pathways Glutamate N-Acetyl- glutamate N-Acetyl- glutamyl-P N-Acetyl-ornithine Ornithine

Carbamoyl-P

Glutamine

Nitrate (extracellular) Nitrate Nitrite Ammonia (NH3) Citrulline

Asparagine Aspartate

L- Arginosuccinate Arginine Fumarate

Nitrate and amino acid utilization of S. Newport on tomato slices

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Commonly up-regulated gene sets of S. Newport

  • n tomato and cantaloupe slices

Objective 2 - Results (Cont.)

0.00 5.00 10.00 15.00 20.00 25.00 30.00 Carbon utilization Carbohydrate derivative metabolism Multi-organism process (Virulence) amino acid metabolism Carboxylic acid metabolism Ion transport Ribosom bogenesis Binding (NADP/GTP/rRNA) transaminase activity Structural molecule activity (ribosome) Extracellular region Ribosomal subunit

NES GO Category

Clp_NES_Sum Tom_NES_Sum Biological Process Molecular Function Cellular Component

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  • 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.

Summary

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This project is supported by the Agriculture and Food Research Initiative (AFRI) awards from the U. S. Department

  • f Agriculture (USDA) National Institute of Food and

Agriculture (NIFA).

Acknowledgments

  • Dr. Larry Beuchat (Co-PD)
  • Dr. Xiangyu Deng (Co-PD)

Yan Qi

  • Dr. Shaokang Zhang

Shaoting Li

  • Dr. Wei Zhang (PD)
  • Dr. Behzad Imanian

Ruin Chen Shimei Zhang Zihao Li Yuchen Tu Zhouyu Li Prinkesh Raka

  • Dr. Eric Brown
  • Dr. Mary Lou Tortorello
  • Dr. Joelle Salazar
  • Dr. Kristin Schill

Diane Stewart

  • Dr. Maria Brandl (Co-PD)

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