Detection of Filamentous
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Detection of Filamentous Salmonella on Low a w Food Items and Enumeration of Filamentous S almonella. Bingming Chen , Dr. Zachary Pratt, Dr. Charles Kaspar , Dr. Amy Wong* [email protected] or [email protected]

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Acknowledgements Mentors: Dr. Zachary Pratt, Dr. Amy Wong

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Acknowledgements mentors dr zachary pratt dr amy wong

Detection of Filamentous Salmonella on Low awFood Items and Enumeration of Filamentous Salmonella

Bingming Chen, Dr. Zachary Pratt, Dr. Charles Kaspar, Dr. Amy [email protected] or [email protected]

Department of Bacteriology & Food Research Institute, University of Wisconsin-Madison

Abstract

Filamentous Cells are found on low aw Food Items

Enumerating Filamentous Salmonella

Under certain stresses, Salmonella can form multi-chromosomal filaments (>10μm), which can septate when the cells encounter favorable conditions (Matticket al., 2000, AEM). Salmonellawith lower infectious doses are associated with outbreaks of foods that have low water activity (aw). One potential explanation for the low infectious dose is Salmonella may form multi-chromosomal filaments on these food items, which form only a single colony forming unit (CFU) in plate count assays. Filamentous Salmonella were detected on the surface of food items with low aw after their inoculation with Salmonella in stationary-phase. We developed a method to enumerate the viable units in a population of Stress Induced Filamentous Salmonella (SIFS). By incubating filamentous Salmonella cells in 1:5 diluted Luria-Bertani broth (0.2x LB) at room temperature for 8 hours, the percentage of filamentous cells in the population decreased from 70% to 18%. Concurrently, the concentration of CFU in culture increased 1-log, while the concentration of the non-filamentous Salmonella control sample remained unchanged over the same period of time. This method could be used to monitor food contamination and measure infectious doses on food surfaces that contain SIFS.

1. Pilot study

Control

Filaments

LB conc

LB conc

Peppercorn

20% peanut butter agar

50% peanut butter agar

Peanut Shell

Filamentous Salmonella can form on the surface of low-awfood items.

Filamentous cells have greater DNA/OD600 ratio than control cells

Introduction

  • Salmonella elongate during stress: low-aw, desiccation, UV radiation

  • Filamentous cell

    • Cell > 10μm

    • Multi-chromosomal

    • 1 CFU/filament

  • Filaments septatewhen favorable conditions encountered

  • Underestimate Salmonella viable unitswith traditional enumeration methods

  • 2. Enumerating Salmonella: 0.2x LB

    • Relative amount of DNA per OD of filamentous and control Salmonella strain 4539H and M-09.

    • Normalized the amount of DNA isolated in each sample to 1mL of culture at an OD600of 1.0.

    • Filamentous cells have higher DNA concentration

    CFU/mL

    OD600

    Methods

    Conclusion

    Future Studies

    1. Filamentous Cells on Food Items

    Inoculate food items with Salmonella during stationary-phase

    Peppercorn

    Peanut (Inner and outer shell, skin)

    Cheese (Swiss and Cheddar)

    Peanut butter (agar)

    Culture for 4 days at 30°C

    Harvest in PBS and look for filaments microscopically

    • Filamentous Salmonella can form on the surface of low aw food items

    • Incubating cultures of filaments in 0.2x LB at room temperature (22oC) for 8 hours was optimal for enumeration without cellular replication

    • Filamentous Salmonella have more DNA than non-filamentous cells with same biomass.

    • Further optimize enumeration of filaments

      • Higher temperature may shorten incubation time

      • Test with different strains of salmonellae

    • Apply enumeration method to inoculated food items

    2. Enumerating Filamentous Salmonella

    • Goal: allow filaments to septate without replication

    • Preparation of filamentous Salmonella:

    • - Control: Salmonella cultured on TSA plates

    • - Experiment: Salmonella cultured on TSA+7%NaCl (aw, 0.95)

    • - Incubate at 30oC for 4 days

    • Harvest cells and incubate in diluted LB broth at room temperature (22°C)

    • Measure optical density at 600nm (OD600), CFU/mL and percentage of filamentous cells periodically

    • Acknowledgements

    • Mentors: Dr. Zachary Pratt, Dr. Amy Wong

    • Wong and Kaspar Laboratories & Food Research Institute, UW-Madison

    • Undergraduate Symposium 2012 committee and CALS Undergraduate Symposium committee

    Enumeration is optimal after incubation in 0.2x LB at 22°C for 8 hours.


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