Establishing a Method for Microbiological Evaluation of Fresh Produce at Risk for Salmonella Contamination Open Access

Pennington, Whitney Claire (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/d217qq21b?locale=en
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Abstract

Salmonellosis is one of the leading causes of gastroenteritis in the United States, with incidence of human cases highest in the southeastern states of Alabama, South Carolina, North Carolina, Florida, and Georgia. Of recent concern is the frequency and impact of foodborne outbreaks of Salmonella due to contamination of pre- and post-harvest produce, specifically leafy greens, tomatoes, and cantaloupe. While the source of contamination in these produce-related outbreaks is rarely specified, suspected causes include wildlife, proximity to agricultural farms whose wastes infiltrate water supply used for irrigation, and application of feces as a form of fertilizer. Here we will establish methods to evaluate the surface of fresh produce grown on farms using untreated surface water as source water for irrigation for the presence of Salmonella bacteria. We describe the standardization, optimization, and validation of a produce wash method that preserves and then resuscitates samples frozen at -80C. We confirm that freezing samples in a non-selective media supplemented with 15% glycerol for preservative followed by a 2-hour reincubation after being thawed is optimal to efficiently recover Salmonella from frozen samples. Moreover we show that this method is sufficient at recovering Salmonella from the surface of broccoli where the most probable number (MPN) Salmonella concentration of irrigation water is 0.26 MPN/100ml.

Table of Contents

Table of Contents

INTODUCTION............................................................................................................................................................ 1

METHODS.................................................................................................................................................................. 7

Phase 1..................................................................................................................................................................... 8

Phase 2.................................................................................................................................................................... 15

RESULTS................................................................................................................................................................... 20

Phase 1.................................................................................................................................................................... 20

Phase 2.................................................................................................................................................................... 21

DISCUSSION.............................................................................................................................................................. 23

Phase 1.................................................................................................................................................................... 23

Phase 2.................................................................................................................................................................... 27

Limitations................................................................................................................................................................ 30

Future Directions........................................................................................................................................................ 32

Conclusion................................................................................................................................................................. 33

FIGURES.................................................................................................................................................................... 35

REFERENCES............................................................................................................................................................... 41

Figures

Figure 1: Overview of experiments to determine the most efficient recovery of Salmonella from frozen water samples.................... 35

Figure 2: The standard Most Probable Number (MPN) protocol used by collaborators at the UGA-Tifton laboratory to determine the

concentration of Salmonella in irrigation ponds on local produce farms....................................................................................36

Figure 3: Schematic of protocol used in Emory laboratory to determine the optimal volume of glycerol and resuscitation time for

facilitation of Salmonella recovery from frozen water samples.......................................................................................... 37

Figure 4: Overview of a seeding experiment used to determine the limit of detection for a broccoli produce wash procedure............ 38

Tables

Table 1: Overview of the efficiency of recovery from each of the eight conditions tested in an experiment aimed at determining the

best procedure for recovering Salmonella from frozen water sample...................................................................................... 39

Table 2: The Salmonella recovery in MPN/100ml/630g broccoli washed during trial one a seeding experiment attempting to determine

the lower limit of detection of a broccoli produce wash method.............................................................................................40

Table 3: The Salmonella MPN/100ml of inoculum applied to broccoli and the corresponding MPN/100ml/630g broccoli recovered from

the produce wash of these applications ...........................................................................................................................40

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