The effects of heat shock on the D70-values of Listeria monocytogenes on selected seafood matrices

MISSING IMAGE

Material Information

Title:
The effects of heat shock on the D70-values of Listeria monocytogenes on selected seafood matrices subtitle
Physical Description:
xix, 119 leaves : ill. ; 29 cm.
Language:
English
Creator:
Wood, Michael V
Publication Date:

Subjects

Subjects / Keywords:
Food Science and Human Nutrition thesis, Ph.D   ( lcsh )
Dissertations, Academic -- Food Science and Human Nutrition -- UF   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 2004.
Bibliography:
Includes bibliographical references.
Statement of Responsibility:
by Michael V. Wood.
General Note:
Printout.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 022483872
System ID:
AA00014268:00001


This item is only available as the following downloads:


Full Text












THE EFFECTS OF HEAT SHOCK ON THE D70-VALUES OF Listeria
monocytogenes ON SELECTED SEAFOOD MATRICES
















By

MICHAEL VINCENT WOOD


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004


































Copyright 2004

by

Michael Vincent Wood



































I dedicate this dissertation to my wife, Candice, my sons
Jordan and Kendall and my parents, Tommie and Joyce Wood.
Also, in memory of my uncle, Reverend Asa L. Dawson.















ACKNOWLEDGMENTS

I would like to extend thanks and gratitude to my

committee chairman and major advisor, Dr. Keith R.

Schneider. Without his guidance, this work would not have

been possible. Thanks are also due to my supervisory

committee members, Dr. Murat O. Balaban, Dr. Gary E.

Rodrick and Dr. Sally K. Williams, for all of their help

and guidance in completion of this research.

I would like to thank the University of Florida's

College of Agricultural and Life Sciences for its financial

support of the University Alumni Fellowship. I thank again

Dr. Keith R. Schneider for providing financial support in

the way of a L time assistantship.

The efforts of my coworkers, Raina Allen, Mark

Campbell, Kelly Felkey, Kristina Garner, Minh Lam, Ayana

McCoy, Doriliz Mestey, Donald Noel, Dirk Sampath and

Benjamin Warren, were invaluable in the completion of this

project. In conclusion, I would like to thank my parents,

Tommie and Joyce Wood, for always loving and supporting me

throughout my lifelong endeavors. I would like to thank my

grandparents, Velish and Dorothy McMullen, for teaching me








at an early age the importance of a proper education. I

would like to thank my brothers, Thomas and Christopher

Wood, for their constant love and support throughout

graduate school. I thank my other family members, too

numerous to mention, for love and support throughout

graduate school. I thank my other brothers, DaShaun Huston

and Waldo Nedd, for the support and inspiration to finish

this doctoral degree. I want to thank my children, Jordan

and Kendall, for inspiring and driving me to excel, so that

their futures may be bright. I thank my wife, Candice, for

carrying the load during the completion of my graduate

degrees. Candice has always given me the unconditional

love and support that I have needed in order to complete

graduate school, and I love her for that.
















TABLE OF CONTENTS
Page

ACKNOWLEDGMENTS ................. ...... ................... iv

LIST OF TABLES .............. ...... ....... .............. viii

LIST OF FIGURES ................. ......................... xiii

ABSTRACT ................................................xvii

CHAPTER

1 INTRODUCTION ................. ......... ............... 1

2 LITERATURE REVIEW ................. ... ................ 6

Listeria spp ............................................. 6
History .................. .............................. 8
Outbreaks ................. ............................. 9
Dairy Products ............. .. ....................... 9
Meat and Poultry ..................................... 11
Seafood ................... ........................ 12
Virulence Factors of Listeria spp. .................... 16
Species ................. ........... ............. 16
Genetic .................. ......................... 17
Clinical Manifestations ........................... 18
Mode of Infection ................................... 20
Regulation of L. monocytogenes ........................21
Listeriosis and Ready-To-Eat Foods .................... 23
Dairy Products .............. ....................... 23
Meat and Poultry ...................................... 24
Seafood ................... ........................ 25
Stress Proteins ....................................... 27
Decimal Reduction Times ...................................30

3 METHODS AND MATERIALS .................................... 32

Bacterial Culturing Protocol ...........................32
Nalidixic Acid Resistance ..............................32
Growth Curves .........................................34
Background Study ................ .................... 35



vi









Recovery Study ................ .......................... 36
Protein Electrophoresis .................. ............ 37
Western Blotting ........................ .. ............ 41
D7o-Value Determination for Heat Shocked Cultures ...... 46
D70-Value Reversion ........................................48
DTo-Values and Seafood................................. 49

4 RESULTS AND DISCUSSIONS ............................... 51

Growth Curves ................. .. ........ ............. 51
Background Microflora Study ............................58
Recovery Study ................... .................... 58
Protein Electrophoresis ............................... 60
Western Blot .......................................... 66
D70-value Determinations of Heat Shocked Cultures ......70
D7o-value Reversion Study .............................. 71
D7T-Values and Seafood.................................. 77

5 SUMMARY AND CONCLUSIONS ................... ............... 82

APPENDIX ................. ............... ................ 87

LIST OF REFERENCES ............... ....................... 111

BIOGRAPHICAL SKETCH .............. ....................... 119















LIST OF TABLES


Table page

1.Growth data of non-adapted Listeria cultures on MOX agar
at the end of stationary phase (12 hours).' ........ 53

2.Growth data of nalidixic acid adapted Listeria cultures
on MOX agar at the end of stationary phase (12
hours).* ............... ... .......... .............. 54

3.Growth data of non-adapted Listeria cultures on TSA at
the end of stationary phase (13 hours) ...........55

4.Growth data of nalidixic acid adapted Listeria cultures
on TSA at the end of stationary phase (12 hours). 56

5.Growth of nalidixic acid adapted Listeria cultures on
TSA/NX at the end of stationary phase (17 hours). 57

6.Total logio amounts recovered of Listeria-nx 1/2b cultures
on seafood matrices.* .............................. 58

7.Total logo amounts recovered of Listeria-nx 1/2a cultures
on seafood matrices.' ................ ............. 59

8.Total logo amounts recovered of Listeria-nx 4c cultures
on seafood matrices.* .............................. 59

9.Total logo amounts recovered of Listeria innocua-nx
cultures on seafood matrices.* .....................59

10.Proteins identified by GC-Mass Spectrometry. Organism
of origin is based on the number of matching amino
acids in the test sequence.* ....................... 65

11.D70-values of NHS and HS Listeria cultures. ........... 70

12.D70-values of NHS and HS Listeria cultures during
reversion study.* .................................. 73

13.D70-values of cooked shrimp inoculated with NHS and HS L.
monocytogenes.* ................................... 78









14.D70-values of crab meat inoculated with NHS and HS L.
monocytogenes. *................................ ... 78

15.D70-values of smoked salmon inoculated with NHS and HS L.
monocytogenes.. .................................... 78

A-1.Acquisition of nalidixic acid resistance by Listeria
cultures ........................ ................... 87

A-2.Raw Data of Growth of Listeria cultures on MOX agar.. 88

A-3.Raw Data of Growth of Listeria-nx cultures on MOX
agar. ........................... ................. 89

A-4.Raw Data of Growth of Listeria cultures on TSA ...... 89

A-5.Raw Data of Growth of Listeria-nx cultures on TSA. ... 90

A-6.Raw Data from the calculation of D70-values of non-heat
shocked Listeria monocytogenes 1/2a in TSB......... 93

A-7.Raw Data from the calculation of D70-values of heat
shocked Listeria monocytogenes 1/2a in TSB......... 93

A-8.Raw Data from the calculation of D70-values of non-heat
shocked Listeria monocytogenes 1/2b in TSB......... 94

A-9.Raw Data from the calculation of D70-values of heat
shocked Listeria monocytogenes 1/2b in TSB......... 94

A-10.Raw Data from the calculation of D70-values of non-heat
shocked Listeria monocytogenes 4c in TSB........... 95

A-ll.Raw Data from the calculation of D70-values of heat
shocked Listeria monocytogenes 4c in TSB........... 95

A-12.Raw Data from the calculation of D7o-values of non-heat
shocked Listeria innocua in TSB.................... 96

A-13.Raw Data from the calculation of D70-values of heat
shocked Listeria innocua in TSB. .................. 96

A-14.Raw Data from the calculation of D7o-values in the
reversion study of non-heat shocked Listeria
monocytogenes 1/2a in TSB .......................... 97

A-15.Raw Data from the calculation of D70-values in the
reversion study of non-heat shocked Listeria
monocytogenes 1/2b in TSB .......................... 97









A-16.Raw Data from the calculation of D70-values in the
reversion study of non-heat shocked Listeria
monocytogenes 4c in TSB............................ 98

A-17.Raw Data from the calculation of D70-values in the
reversion study of non-heat shocked Listeria innocua
in TSB ............................................. 98

A-18.Raw Data from the calculation of D70-values for day one
of the reversion study of heat shocked Listeria
monocytogenes 1/2a in TSB........................... 99

A-19.Raw Data from the calculation of D70-values for day one
of the reversion study of heat shocked Listeria
monocytogenes 1/2b in TSB........................... 99

A-20.Raw Data from the calculation of D70-values for day one
of the reversion study of heat shocked Listeria
monocytogenes 4c in TSB............................ 100

A-21.Raw Data from the calculation of D70-values for day one
of the reversion study of heat shocked Listeria
innocua in TSB.................................... 100

A-22.Raw Data from the calculation of D70-values for day two
of the reversion study of heat shocked Listeria
monocytogenes 1/2a in TSB ......................... 101

A-23.Raw Data from the calculation of D70-values for day two
of the reversion study of heat shocked Listeria
monocytogenes 1/2b in TSB ......................... 101

A-24.Raw Data from the calculation of D7o-values for day two
of the reversion study of heat shocked Listeria
monocytogenes 4c in TSB............................ 102

A-25.Raw Data from the calculation of D7o-values for day two
of the reversion study of heat shocked Listeria
innocua in TSB.................................... 102

A-26.Raw Data from the calculation of D70-values for day
three of the reversion study of heat shocked Listeria
monocytogenes 1/2a in TSB.......................... 103

A-27.Raw Data from the calculation of D70-values for day
three of the reversion study of heat shocked Listeria
monocytogenes 1/2b in TSB.......................... 103









A-28.Raw Data from the calculation of D70-values for day
three of the reversion study of heat shocked Listeria
monocytogenes 4c in TSB............................ 104

A-29.Raw Data from the calculation of D70-values for day
three of the reversion study of heat shocked Listeria
innocua in TSB.................................... 104

A-30.Raw Data from the calculation of D70-values of non-heat
shocked Listeria monocytogenes 1/2a on cooked
shrimp ............. ...... .... ..................... 105

A-31.Raw Data from the calculation of D70-values of heat
shocked Listeria monocytogenes 1/2a on cooked
shrimp....................... ...................... 105

A-32.Raw Data from the calculation of D70-values of non-heat
shocked Listeria monocytogenes 1/2b on cooked
shrimp. ........................... ............... 106

A-33.Raw Data from the calculation of D70-values of heat
shocked Listeria monocytogenes 1/2b on cooked
shrimp............................................ 106

A-34.Raw Data from the calculation of D70-values of non-heat
shocked Listeria monocytogenes 1/2a on crab
meat. ..................... ........ ............. 107

A-35.Raw Data from the calculation of D70-values of heat
shocked Listeria monocytogenes 1/2a on crab
meat. ............. ............ ................. 107

A-36.Raw Data from the calculation of D70-values of non-heat
shocked Listeria monocytogenes 1/2b on crab
meat. ........................................... 108

A-37.Raw Data from the calculation of D70-values of heat
shocked Listeria monocytogenes 1/2b on crab
meat. .......................................... 108

A-38.Raw Data from the calculation of D70-values of non-heat
shocked Listeria monocytogenes 1/2a on smoked
salmon ............................... ............... 109

A-39.Raw Data from the calculation of D70-values of heat
shocked Listeria monocytogenes 1/2a on smoked
salmon ............. ....................... ...... 109









A-40.Raw Data from the calculation of D7o-values of non-heat
shocked Listeria monocytogenes 1/2b on smoked
salmon ........................... ............... 110

A-41.Raw Data from the calculation of D"o-values of heat
shocked Listeria monocytogenes 1/2b on smoked
salmon. ................ ......... ........ ....... 110















LIST OF FIGURES


Figure page

1. Mode of intracellular spread for L. monocytogenes,
once engulfed in host cell.146 ...................... 21

2. Typical standard curve for the Bio-Rad Protein
Microassay, bovine serum albumin (BSA), bovine Gamma
globulin (IgG). OD595 corrected for blank. 1.25-25
ig/ml x 0.8 ml = 1-20pg of protein. 8 .............. 40

3. One hundred pl of 12-hour cultures of the respective
non-adapted Listeria strains were inoculated into
tryptic soy broth and incubated at 370C in a rotary
shaker for 12 hours. At each time point samples were
spread plated on MOX agar in triplicate. The mean
populations of the respective strains were combined
and plotted over time. No significant difference
between mean populations of strains were found
between strains at the end of stationary phase
(p < 0.05) ....................................... 53

4. One hundred pl of 12-hour cultures of the respective
adapted Listeria-nx strains were inoculated into
tryptic soy broth and incubated at 370C in a rotary
shaker for 12 hours. At each time point samples were
spread plated on MOX agar in triplicate. The mean
populations of the respective strains were combined
and plotted over time. No significant difference
between mean populations of strains were found
between strains at the end of stationary phase
(p < 0.05). ....................................... 54

5. One hundred pl of 13-hour cultures of the respective
non-adapted Listeria strains were inoculated into
tryptic soy broth and incubated at 370C in a rotary
shaker for 13 hours. At each time point samples were
spread plated on TSA agar in triplicate. The mean
populations of the respective strains were combined
and plotted over time. No significant difference
between mean populations of strains were found









between strains at the end of stationary phase
(p < 0.05) ....................................... 55

6. One hundred pl of 12-hour cultures of the respective
nalidixic acid adapted Listeria strains were
inoculated into tryptic soy broth and incubated at
370C in a rotary shaker for 12 hours. At each time
point samples were spread plated on TSA agar in
triplicate. The mean populations of the respective
strains were combined and plotted over time. No
significant difference between mean populations of
strains were found between strains at the end of
stationary phase (p < 0.05) ........................ 56

7. One hundred pi of 17-hour cultures of the respective
nalidixic acid adapted Listeria strains were
inoculated into tryptic soy broth and incubated at
370C in a rotary shaker for 17 hours. At each time
point samples were spread plated on TSA/NX agar in
triplicate. The mean populations of the respective
strains were combined and plotted over time. No
significant difference between mean populations of
strains were found between strains at the end of
stationary phase (p < 0.05)......................... 57

8. SDS-PAGE (10% gel) patterns of the whole protein
extract of (A) L. monocytogenes-nx 1/2a;(B) L.
monocytogenes-nx 4c; (C)L. monocytogenes-nx 1/2b; (D)
L. innocua-nx. Each culture was analyzed in
duplicate ......................................... 60

9. SDS-PAGE (10% gel) patterns of the whole protein
extract of (A) NHS Listeria monocytogenes-nx 1/2a;(B)
HS Listeria monocytogenes-nx 1/2a; (C) Protein
standard. Arrows denote changes in protein
patterns ........................................... 62

10. Densitometer software representation of protein
difference at the 57 kDa range between NHS L.
monocytogenes-nx 1/2a (A) and HS L. monocytogenes-nx
1/2a (B). The change in peak 1 as seen between graphs
A and B represents the induced increase in stress
protein due to heat stress. .......................... 63

11. SDS-PAGE (10% gel) patterns of the whole protein
extract of (A) Protein standard (B) HS Listeria
monocytogenes-nx 1/2b (C) NHS Listeria monocytogenes-









nx 1/2b. Arrows denote changes in protein
patterns. ......................... .................. 64

12. Densitometer software representation of protein
difference at the 85 kDa range between NHS L.
monocytogenes-nx 1/2b (A) and HS L. monocytogenes-nx
1/2b (B). Scan of protein difference at the 57 kDa
range between NHS L. monocytogenes-nx 1/2b (C) and HS
L. monocytogenes-nx 1/2b (D). The changes in peaks 1
as seen between graphs C and D represents the induced
increase in stress protein due to heat stress. ...... 66

13. Densitometer software representation of immunoblot
patterns of stressed L. monocytogenes-nx 1/2a and 1/2b
against GroEL antibodies. (A) Scan of immunoblot of
protein section of NHS L. monocytogenes-nx 1/2a
against GroEL antibodies. (B) Scan of immunoblot of
protein section of heat shock L. monocytogenes-nx 1/2a
against GroEL antibodies. (C) Scan of immunoblot of
protein section of NHS L. monocytogenes-nx 1/2b
against GroEL antibodies. (D) Scan of immunoblot of
protein section of heat shock L. monocytogenes-nx 1/2a
against GroEL antibodies. ............................ 68

14. Densitometer software representation of immunoblot
patterns of stressed L. monocytogenes-nx 4c and
innocua-nx against GroEL antibodies. (A) Scan of
immunoblot of protein section of NHS L. monocytogenes-
nx 4c against GroEL antibodies. (B) Scan of
immunoblot of protein section of heat shock L.
monocytogenes-nx 4c against GroEL antibodies. (C)
Scan of immunoblot of protein section of NHS L.
innocua-nx against GroEL antibodies. (D) Scan of
immunoblot of protein section of heat shock L.
innocua-nx against GroEL antibodies. ................ 69

15. SDS-PAGE (10% gel) patterns of the whole protein
extract of NHS and HS L. monocytogenes-nx 1/2a
cultures. Lanes represent sample day (A) NHS day 0;
(B) HS day 0; (C) HS day 1; (D) HS day 2; (E) Protein
standard. The arrow indicates the region of induced
protein production used for densitometer scans. .....74

16. Densitometer software representation of figure 18.
The protein difference in a scanned region of 50 to 75
kDa before and after heat shocking L. monocytogenes-nx
1/2a. (A) NHS L. monocytogenes-nx 1/2a. (B) HS L.
monocytogenes-nx 1/2a after day zero. (C) HS L.









monocytogenes-nx 1/2a after day one. (D) HS L.
monocytogenes-nx 1/2a after day two. ................ 75

17. SDS-PAGE (10% gel) patterns of the whole protein
extract of NHS and HS L. monocytogenes-nx 1/2b
cultures. Lanes represent sample day (A) NHS day 0;
(B) HS day 0; (C) HS day 1; (D) HS day 2; (E) Protein
standard. The arrow indicates the region of induced
protein production used for densitometer scans. .....76

18. Densitometer software representation of figure 20.
The protein difference in a scanned region of 50 to 75
kDa before and after heat shocking L. monocytogenes-nx
1/2b. (A) NHS L. monocytogenes-nx 1/2b. (B) HS L.
monocytogenes-nx 1/2b after day zero. (C) HS L.
monocytogenes-nx 1/2b after day one. (D) HS L.
monocytogenes-nx 1/2b after day two. ................ 77

A-1. Crab meat was inoculated with approximately 106 CFU of
each individual Listeria strain and air dried for 1
hour prior to treatment. Data represents triplicate
trials. ......................... .................. 91

A-2. Smoked salmon was inoculated with approximately 106 CFU
of each individual Listeria strain and air dried for 1
hour prior to treatment. Data represents triplicate
trials. ............................................ 92

A-3. Cooked shrimp was inoculated with approximately 106 CFU
of each individual Listeria strain and air dried for 1
hour prior to treatment. Data represents triplicate
trials ................. ............................. 92














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

THE EFFECTS OF HEAT SHOCK ON THE D70-VALUES OF Listeria
monocytogenes ON SELECTED SEAFOOD MATRICES

By

Michael V. Wood

May 2004

Chair: Keith R. Schneider
Major Department: Food Science & Human Nutrition



The objective of this research was to determine the

effects of heat shocking and subsequent protein production

on three strains of Listeria monocytogenes (1/2a, 1/2b,

4c), and Listeria innocua. The three strains of L.

monocytogenes utilized were chosen from three lineages; I

containing isolates pathogenic to humans, II including

isolates pathogenic to both humans and animals, and III

strains not linked to disease in humans.

Growth characteristics were examined on standard agar

media and on different seafood matrices. The stress

protein GroEL was also examined after heat shock

conditions. D70-values of heat shocked (HS) and non-heat

shocked (NHS) cultures were determined and compared.









Results showed D70-values of heat shocked L. monocytogenes

1/2a and 1/2b increased significantly (p < 0.05) from 1.04

and 0.94 to 2.07 and 2.24 minutes, respectively. D70-values

of L. monocytogenes 4c and L. innocua, both non-pathogenic,

did not show a similar increase.

Studies examining the presence of the stress protein

GroEL and other GroEL-like compounds, showed in all cases,

the NHS cultures had lower levels as compared to HS cells.

Densitometer readings of L. monocytogenes 1/2a showed an

approximate 2-fold increase in GroEL levels, presenting 687

ng and 351 ng for HS and NHS cultures, respectively. In

tests on HS L. monocytogenes 1/2b, GroEL levels were 913 ng

as compared to 209 ng for the NHS strain. Heat shocked L.

monocytogenes 4c presented total GroEL levels of 353 ng as

compared to 213 ng for the NHS cultures. L. innocua

expressed GroEL levels of 659 ng and 273 ng for HS and NHS

cultures, respectively.

The two L. monocytogenes cultures, 1/2a and 1/2b, had

significantly increased D7o-values, but reverted back to a

NHS state at the 24-hour sampling interval. The initial

D70-values for HS and NHS L. monocytogenes 1/2a were 2.38

and 1.25 minutes, respectively. The D70-values for Day-1 HS

were significantly higher (p < 0.05) when compared to NHS

culture on both days and to HS cells on days 1 and 2. The


xviii









same effect was seen for L. monocytogenes 1/2b with initial

D7T-values of 2.40 and 1.19 for heat and non-heat treated,

respectively.














CHAPTER 1
INTRODUCTION

Listeria monocytogenes is an intracellular pathogen

with ingestion of contaminated foodstuffs being the primary

vehicle of entry. Individuals with conditions resulting in

immunodeficiencies are more likely to contract listeriosis.

These include persons with Acquired Immune Deficiency

Syndrome (AIDS) and cirrhosis. Other high-risk groups who

are more likely to contract listeriosis are the elderly,

pregnant women and neonates. 49,59)

There is a 20%-25% mortality rate associated with

clinical manifestation of listeriosis. 38) These vary from

no visible symptoms, to flu-like manifestations, to severe

cases of meningitis and/or septicemia. Pregnant women in

their third trimester are particularly at risk for

contracting listeriosis. Pregnant women contracting

listeriosis will initially manifest flu-like symptoms.

These symptoms can evolve and cause miscarriages,

stillbirths, septicemia, or meningitis in the neonate.

Adults, children and newborns may suffer from complications

of the central nervous and pulmonary systems as well as








develop secondary illnesses such as pneumonia and

endocarditis. 32)

In a study conducted in 1997, 651 researchers

characterized Listeria strains utilizing polymerase chain

reaction-restriction fragment length polymorphism (PCR-

RFLP) of the virulence genes, hly, actA and inlA and

through the use of ribotyping. The use of virulence gene

alleles analysis and ribotyping separated L. monocytogenes

into three distinct lineages that have different pathogenic

potentials. Lineage I includes foodborne epidemic isolates

as well as isolates associated with sporadic outbreaks in

humans. Lineage II contains both human and animal isolates

associated with foodborne epidemics. Lineage III includes

those strains isolated only from animals.(651

Various strains of L. monocytogenes exhibit different

characteristics. Different strains of L. monocytogenes

have been shown to exhibit a higher decimal reduction time

(D-values) than others.(15s Differences in D-values can be

attributed to various factors. One factor that contributes

to pathogenicity is the presence or absence of heat shock

proteins. Many microorganisms produce heat shock proteins

as a means of protection from the debilitating effects of

heat. These heat shock proteins can work individually or

in conjunction with each other. Two specific heat shock









proteins, GroEL and GroES, can form a cage around a

thermally damaged protein. Within this cage, the damaged

protein is repaired. 31 Heat shock proteins are one of the

many stress-specific proteins produced by microorganisms.

Other examples of stress-induced proteins include cold,

osmotic and acid shock proteins.12'5'22,31)

Recently, there have been several recalls by

manufacturers due to food contaminated with L.

monocytogenes. The majority of these recalls have been

associated with processed meats such as hot dogs, cold cuts

and sausages.130 While there have been many listeriosis

outbreaks, only five recorded outbreaks have been

associated with seafood products. 34,37,40,47,62) Due to the

high mortality rate and the risk groups associated with L.

monocytogenes, the consequences of an outbreak could be

devastating.

In the 2003, United States Quantitative Assessment of

the Relative Risk to Public Health from Foodborne Listeria

monocytogenes Among Selected Categories of Ready-to-Eat

Foods, 300 seafood is placed into different risk categories.

While raw seafood is placed into the low-risk category,

smoked seafood and ready-to-eat crustaceans are placed into

the high-risk category. The risks associated with smoked

seafood involve the ability of the food matrix to support









the growth of L. monocytogenes during extended

refrigeration temperatures.(62) Other rationale for smoked

seafood being in the high-risk group stem from incidences

involving improper cooking times and temperatures used

during processing..34 For ready-to-eat crustaceans,

specifically cooked shrimp and canned crab-meat, the risks

are linked primarily to post-process contamination. 30)

L. monocytogenes is a problematic microorganism for

the ready-to-eat seafood industry. It has the ability to

survive and proliferate in a processing environment if

proper sanitation procedures are not maintained. 130

Improper cooking and/or other non-thermal processing

methods also contribute to Listeria contamination of

finished product.(0) The ability of L. monocytogenes to

produce stress-specific proteins also contributes to its

survival.(22) Studies have been conducted analyzing how

these proteins effect the D-values of certain strains of L.

monocytogenes in other types of foods.114'151 Few research

studies have examined the relationship among heat shock

proteins, D-values and ready-to-eat seafood.

The objectives of this study were to examine three

lineages of Listeria monocytogenes and one strain of

Listeria innocua to determine how the production of heat





5


shock proteins affected D-values in both growth media and

on inoculated ready-to-eat seafood.














CHAPTER 2
LITERATURE REVIEW

During the past two decades, listeriosis has emerged

as a major foodborne disease. With the emergence of more

ready-to-eat foods and their increased shelf-lives, the

prevention of contamination by Listeria monocytogenes has

become a necessity to the food and beverage industry. The

need for research on L. monocytogenes has increased with

the incidences of contamination. This chapter will examine

scientific literature that is pertinent to this study.

Listeria spp.

Listeria spp. are Gram-positive, non-spore forming,

non-capsulating motile rods, with motility most prominent

at 20C. Listeria are microaerophilic, thriving at reduced

oxygen levels. Listeria spp. are differentiated from other

Gram-positive bacteria by the production of hydrogen

sulfide and acid production from glucose.(60)

The pH can affect the growth of Listeria spp. with

optimal growth occurring between 4.3 to 9.5."1 At pH

values below 4.3, cells have the ability to survive but not

proliferate. Experimentally, the presence of up to 0.1%

acetic, citric or lactic acid in tryptose broth inhibits









the growth of L. monocytogenes. The antimicrobial

properties of these acids are related to their degree of

dissociation, with citric and lactic acid being less

detrimental than acetic acid at the same pH.(11

L. monocytogenes are halotolerant, able to grow in the

presence of 10 to 12% sodium chloride.1(4B The bacterium can

survive for 60 days at concentrations as high as 30% at

4.00C. However, survival is shortened to five days when

temperatures are raised to 370C.136)

Listeria spp. are able to proliferate at temperature

ranges from 0 to 450C. The average generation times for 39

L. monocytogenes strains tested were 43, 6.6 and 1.1 hours

at 4, 10 and 370C, respectively. It has been shown that

temperatures below 0C preserve or moderately inactivate the

organism. Survival and injury during frozen storage depend

on the substrate and the rate of freezing.161)

Investigators have cloned three genes (ItrA, ltrB and

ItrC) of L. monocytogenes that are essential for low

temperature growth.168) When a 1.2-kb internal fragment of

ItrB was used as a probe in Southern hybridizations of

HindIII-digested L. monocytogenes DNA, a 9.5-kb DNA

fragment that hybridized with the probe was found to be

unique to the epidemic-associated serotype 4b strains of L.

monocytogenes. (68)









While researching the survivability of L. monocytogenes

on glass beads and ceramic tile, investigators determined

survival was temperature dependent, with optimal growth

ranging from 300C to 370C. 64' Gray et al. (1966) determined

that the survival times for L. monocytogenes in straw, dry

fodder, soil and animal feces were six months, three

months, 295 days and two years, respectively. 33)

History

Listeria spp. are not a new type of bacterium. It was

first discussed in 1911 in veterinary outbreaks associated

with contaminated feed silage. 411 Researchers described the

bacterium in terms of large mononuclear leukocytes observed

in rabbits. This group of researchers named this new

organism Bacterium monocytogenes. 41) In 1927, another group

of researchers in South Africa discovered the microorganism

that caused Tiger River disease. They named this organism

Listerella hepatolytica. 441 Both research groups sent their

cultures to the National Type Collection at the Lister

Institute in London. These two cultures were found to be

the same organism. The two groups decided upon the name

Listerella monocytogenes. This name was rejected in 1939

and was changed in 1940 to what we now know as Listeria

monocytogenes.145' The hemolytic/pathogenic group remained

L. monocytogenes, while the nonpathogenic non-hemolytic L.









monocytogenes group became to be known as L. innocua from

the Latin word meaning harmless.160'

Outbreaks

There have been many listeriosis outbreaks worldwide.

The first documented epidemic of foodborne listeriosis was

in 1981 in Nova Scotia, Canada. 58) It was attributed to

contaminated coleslaw. Forty-one cases of listeriosis were

reported; seven adults and 34 cases of prenatal infections.

Investigators isolated L. monocytogenes from two unopened

cartons of coleslaw. The investigator found no Listeria

spp. in the manufacturing plant. The contaminated coleslaw

was traced to a field where the cabbage had been fertilized

by sheep dung. The investigators determined that two sheep

had contracted listeriosis and died recently on the farm. (58

Prenatal cases exhibited classical listeriosis symptoms

followed by spontaneous abortions, stillbirth, live

premature or term births of symptomatic infants. Only two

cases of nonsymptomatic non-infected infant births were

noted from mothers who had contracting listeriosis during

this outbreak. A 27% mortality rate was observed in all

live born infants with a Listeria infection.(58)

Dairy Products

Another epidemic occurred between June and August

1983.126) Epidemiological evidence indicated contamination








of whole and 2% milk, though no L. monocytogenes was found

in the tested product. The investigators determined that

12% of raw milk from a specific supplier's bunk tank

harbored the organism. A total of 49 cases of listeriosis

were reported with seven manifesting in neonates and

infants. The remaining 42 cases involved immunocompromised

persons. Fourteen fatalities were documented, yielding a

29% mortality rate.(26)

In 1996 in Illinois a listeriosis outbreak was

attributed to consumption of chocolate milk at a picnic. 19)

Forty-five people had symptoms for listeriosis, and stool

samples from 11 persons tested positive for L.

monocytogenes. The most common symptoms were diarrhea

(present in 79% of the cases) and fever (72%). Persons who

became ill had elevated levels of antibody to listeriolysin

0. Cultures isolated from stool specimens from infected

patients, the implicated chocolate milk, and a tank drain

at the dairy facility all contained L. monocytogenes

serotype 1/2b. These isolates were indistinguishable on

multilocus enzyme electrophoresis, ribotyping, and DNA

macrorestriction analysis. 19)

Another epidemic occurred in Los Angeles and Orange

Counties, California, in 1985. More than 142 cases were

traced to soft Mexican-style cheese. The investigators








determined two possible sources of contamination; raw whole

milk mixed with pasteurized milk and/or post-process

contamination. All cases were traced to a single

manufacturing plant in southern California. The outbreak

resulted in 42 deaths with 85% being neonates. Forty-two

neonates exhibited listeriosis symptoms within 24 hours of

birth.135

Meat and Poultry

Until recently, there had been only one confirmed

fatality from foodborne listeriosis involving meat

products. In 1988, an immunocompromised individual

consumed a turkey frank that had been cross contaminated

from a casing peeler.14)

An outbreak in 1998 had at least 50 cases of

listeriosis reported to the Centers for Disease Control and

Prevention (CDC) by 11 states.o30) Two pregnant women had

spontaneous abortions and six adults died. The CDC had

identified the cause of the illnesses as L. monocytogenes,

serotype 4b. The CDC, along with local and state health

departments, had also identified the means of transmission,

which included hot dogs and possibly deli meats. Both

types of meat were manufactured under many brand names, but

they were all manufactured in one Sara Lee manufacturing

facility.(24 Near the end of 1998, Sara Lee voluntarily








recalled various lots of hot dogs and deli meat. The CDC

isolated L. monocytogenes 4b as the causative agent, as

well as a different strain of L. monocytogenes from an

unopened package of hot dogs produced at the company's

processing facility. To date, Sara Lee has recalled over

six tons of hot dogs and deli meats. The mortality rate

for this outbreak was reported to be 20%, with seven deaths

being stillborn babies. Researchers have shown that this

outbreak was caused by dust in the light fixtures that was

contaminated with L. monocytogenes. These numbers make

this the largest listeriosis outbreak in history.16

Seafood

There have been several recorded outbreaks of

listeriosis associated with seafood products. In January,

1980, 22 cases of perinatal L. monocytogenes infections

occurred at three obstetric hospitals in Auckland, New

Zealand. Investigators looked for common foods among the

infected women and traced the cause of the epidemic to

contaminated shellfish and raw fish.137 Women presented in

term labor with signs of amnionitis (11 of 22 cases) and

associated fetal distress. A mild flu-like illness or

urinary tract symptoms were common in 18 of the 22 women.

Five fetal deaths occurred during this outbreak. Three

deaths occurred before 20 weeks of gestation. Most









liveborn affected babies had early respiratory symptoms (12

of 14 cases). Meningitis occurred in 4 of 14 infants, with

one child dying. Vaginal carriage of L. monocytogenes was

found in only 1 of 750 consecutive asymptomatic pregnant

women who were tested at the time of the epidemic. Rectal

carriage was found in 25 (3.3%) of the women tested.1371

An outbreak that occurred in Connecticut in 1990

involved L. monocytogenes. 47 The investigation was

initiated when two pregnant women presented with

listeriosis. Epidemiologists examining the outbreak

researched the incubation period, the possibility of mild

disease due to Listeria infection, and foods associated

with risk of disease. The only common exposure was food

consumed at a party. 47 Ten of 36 party-goers showed

typical symptoms, which included isolation of L.

monocytogenes from blood and/or stool, and/or two or more

of the following: fever, musculoskeletal symptoms, nausea,

vomiting, diarrhea. One of 25 stool cultures was

positively identified as L. monocytogenes. The two blood

isolates and one stool isolate were serotype 4b and

identical by enzyme typing. The incubation periods

recorded for the two pregnant women were 19 and 23 days.

Investigators focused on the consumption of large amounts

of shrimp, nonalcoholic beverages and cauliflower consumed









at this function. The consumption of shrimp was deemed the

significant risk factor for illness after investigating the

other foods. Conclusions from this investigation suggest a

milder illness may exist in healthy persons who consume

foods contaminated with L. monocytogenes due to the low

mortality associated with this outbreak. Additionally, the

report demonstrated a prolonged incubation period for this

disease.'47)

In Sweden in 1994, a listeriosis outbreak was

attributed to the consumption of gravad rainbow trout.1621

Gravad rainbow trout is made from raw fillets rubbed in

sugar, salt, pepper, and dill, then left to mature at

refrigeration temperatures for two days. It is then

packaged (sliced or whole) under vacuum and stored for 2-3

weeks at room temperature and under refrigeration

temperatures once opened. Food samples taken from

patients' refrigerators and from unopened packages were

contaminated with at least four different strains of L.

monocytogenes ranging in concentration from 100 to 2.5 x 106

CFU/g. Nine people were infected and two deaths

occurred. (62)

In 1999, a smaller outbreak of listeriosis occurred in

Finland. 40) Five people were infected by the consumption of

contaminated cold-smoked rainbow trout. None of these








patients died, but they all contracted febrile

gastroenteritis. Febrile gastroenteritis is characterized

by abdominal cramps, diarrhea, fever and vomiting. The

sample of suspected cold-smoked rainbow trout contained 2.0

x 105 CFU/g of L. monocytogenes. Improper storage

temperature of the fish at the retail outlet was

approximately 100C, rather than the range of 0 to 30C

recommended by the manufacturer. This high storage

temperature of the fish product could have allowed L.

monocytogenes to grow to such high levels. Stool samples

were taken from each patient the day after onset of the

gastroenteritis and showed Salmonella, Shigella,

Campylobacter and Yersinia, though no Listeria were

isolated. Stool samples taken one week later tested

positive for L. monocytogenes. Since no other enteric

pathogens were found in the second set of stool samples,

this strongly suggests that the outbreak was caused by L.

monocytogenes. (4 These results provide additional

supporting evidence for previous findings that L.

monocytogenes can cause febrile gastroenteritis in healthy

persons, if the food consumed is heavily contaminated. The

results also show that vacuum-packed, cold-smoked rainbow

trout can contain large numbers of L. monocytogenes and

must be considered a potential source of contamination.








Virulence Factors of Listeria spp.

Species

There are currently six species in the genus Listeria:

L. monocytogenes, L. innocua, L. seeligeri, L. welshimeri,

L. ivanovii and L. grayi. In a study conducted by Rocourt

et al. (1992), Listeria murrayi was deemed to be the same

organism as Listeria grayi. 50) This study evaluated the

genomic similarities between L. grayi and L. murrayi using

DNA/DNA hybridizations and multilocus enzyme

electrophoresis. The results of these examinations

indicated that they should be considered members of a

single species, Listeria grayi. 50) L. monocytogenes, L.

seeligeri and L. ivanovii are the only species that are

hemolytic. These three contain a virulence cluster and

have the ability to be pathogenic.59) This virulence

cluster consists of several virulence genes. Genes inlAB

and iap are associated with host entry. All other

identified virulence genes have been mapped to the same

chromosomal region around the hly gene. The hly gene

encodes for a hemolysin called listeriolysin O (LLO) that

is essential for lysis of the phagosomal membrane. This

gene is flanked by two operons for plcA-prfA and

lecithinase. The lecithinase operon contains at least two

genes involved in virulence: actA, required for actin








assembly and plcB, which encodes a lecithinase involved in

cell-to-cell spread. Upstream from hly, the plcA gene

encodes for phosphatidylinositol-specicfic phospholipase C,

which may contribute to the lysis of the phagosomal

membrane. The plcA gene is followed by the prfA gene,

which encodes a pleiotropic activator of plcA, hly, and the

lecithinase operon as well as inlAB.1481

L. monocytogenes has thirteen identified serovars;

1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and

7. Serovars 4b, 1/2a and 1/2b account for 95% of human

isolates. 48)

Genetic

A study by Weidmann et al. (1997) showed that there was

diversity within different strains of L. monocytogenes. 65)

L. monocytogenes strains were differentiated by ribotyping

to group strains by different levels of virulence.

Ribotyping is a molecular method for the characterization,

identification and typing of bacterial isolates.165s In

ribotyping, restriction enzymes are used to cut the genes

coding for 16s rRNA into pieces, followed by

electrophoresis which separates them by size using an

agarose gel."(12 Genetic probes then visualize locations of

different-size fragments of DNA in the gel, which appear as

bands. The banding pattern of DNA fragments corresponding








to the complimentary rRNA, is known as a ribotype. The

banding patterns are compared to a database of other L.

monocytogenes strains and grouped according to pattern

similarity. In experiments that characterized the

pathogenic potential of isolates that the researchers

collected from the smoked fish industry and ruminant

outbreaks, L. monocytogenes was separated into three

lineages."651 These lineages are as follows:

Lineage I
o Has the most potential for pathogenicity in
humans.
o Isolated from human sporadic isolates and
foodborne epidemic isolates.


Lineage II
o Has potential for human and animal pathogenicity.
o Isolated from human and animal sporadic isolates.


Lineage III
o Has the most potential for pathogenicity in
animals.
o Isolated from animal sporadic isolates and
epidemic isolates.

Clinical Manifestations

L. monocytogenes is an intracellular pathogen with

ingestion of contaminated foodstuffs being the primary

vehicle of entry.32) Individuals with predisposed

conditions resulting in immunodeficiencies are more likely

to contract listeriosis. These conditions include AIDS,

cirrhosis, hepatitis, malignancy, diabetes and ulcerative









colitis. A select demographic of people are more likely to

contract listeriosis as well. This demographic includes

persons over the age of 60, individuals on

immunosuppressing medication, pregnant women and

neonates.(32)

There is a 25 to 30% mortality rate associated with

clinical manifestation of listeriosis. 32) Infected

individuals can be asymptomatic or can present symptoms

ranging from gastroenteritis to severe cases of meningitis

and/or septicemia. Pregnant women who contract listeriosis

manifest flu-like symptoms that can result in miscarriage,

stillbirths, septicemia, or meningitis in the neonate.

Adults, children, and newborns may exhibit complications

involving the central nervous system, pulmonary system

(pneumonia) as well as endocarditis.1321 Incubation time is

approximately 12 hours after ingesting L. monocytogenes

contaminated foods. The first stages manifest as flu-like

symptoms with serious complications developing from one to

six weeks after infection.132) In non-pregnant adults, L.

monocytogenes has a particular tropism for the central

nervous system. Meningitis and meningoencephalitis

frequently occur in these cases of listeriosis. 32)









L. monocytogenes is a relatively rare infection and

until 1986 was not recorded by CDC. The reported cases of

listeriosis for 2000 were 2,078, with 390 fatalities. 16)

Researchers have reported that infection and fatality

rates varied greatly between populations when grouped by

age. Listeriosis associated with pregnant women occurred

at a rate of 12.4 cases per 100,000 births. An incidence

rate of 1 in 1,000,000 persons, with 11% mortality rate, in

persons from ages 1 to 40. The mortality rate associated

with listeriosis was estimated at 63% in persons over the

age of 60.(161

Mode of Infection

Macrophages from the host ingest the L. monocytogenes

and form a vacuole around the cell. The vacuole is lysed

by the protein LLO, allowing the bacterium to escape into

the cytoplasm. Once in the cytoplasm, L. monocytogenes

begin to replicate. Once intracellular replication begins,

L. monocytogenes induces the production of actin filaments

that form a cloud around the bacterial cell. The actin is

rearranged to a tail that consists of short filaments and

other actin binding proteins that stabilize this structure.

The formation of the actin tail at one end of the L.

monocytogenes cell produces a propulsive force that moves

it through the cytoplasm of the host cell. L.









monocytogenes cells that reach the surface of the infected

host cell induce the formation of pseudopod-like structures

at the opposite end of the actin tail filaments. The

neighboring cells take up the pseudopod structures within a

vacuole. Once this vacuole is lysed by LLO, the cycle of

intracellular spread renews itself.125 This infection cycle

is shown graphically in figure 1.146)





















http://www.med.ufl.edu/biochem/DLPURICH/Listeria.html

Figure 1. Mode of intracellular spread for L.
monocytogenes, once engulfed in host cell.146'


Regulation of L. monocytogenes

The infective dose for L. monocytogenes is not known.

Both infectious doses and onset time are likely to vary

with host susceptibility, number of organisms ingested,









food ingested, and/or the virulence of the particular

strain of L. monocytogenes. 251

The United States Food and Drug Administration's (FDA)

policy on L. monocytogenes is based on the information that

the organism can cause human illness, it can grow at

refrigeration temperatures and the infectious dose is

unknown but can be influenced by the food matrix.(30) Based

on this information, there is a concern about the presence

of L. monocytogenes in ready-to-eat seafood products

because of the potential for growth during cold storage.

FDA's current policy on L. monocytogenes in ready-to-eat

seafood products states that any detectable presence is

considered to be hazardous to health. The limit of

sensitivity for detection is currently 1 CFU/25 g. The FDA

requires recalls of any ready-to-eat food in which L.

monocytogenes is detected. A Class I recall, which

involves agents which can results in human death, is

initiated when there is a reasonable probability that the

use of, or exposure to, a violative product will cause

serious adverse health consequences or death. 171 In 2003,

there were seven recalls for ready-to-eat seafood products

due to Listeria monocytogenes contamination. 51-57)









Listeriosis and Ready-To-Eat Foods

Many ready-to-eat processed foods pose a high-risk of

spreading listeriosis to the susceptible populations.

These foods are usually refrigerated for extended periods

of time, offering a prime environment for the proliferation

of L. monocytogenes. Foods included in this category are

unpasteurized milk(26' and foods made from unpasteurized

milk, soft cheeses, (13 frankfurters, 281 delicatessen meats,

poultry products125) and some seafood. 39

Dairy Products

The outbreak caused by Mexican-style soft cheese was

likely caused by using unpasteurized milk for cheese

making.135) Studies show that L. monocytogenes will grow in

pasteurized milk with the numbers increasing 10-fold in

seven days at 40C. L. monocytogenes grows more rapidly in

pasteurized milk than in unpasteurized milk due to lack of

competing organisms. Therefore, milk that is contaminated

after pasteurization may attain very high numbers of L.

monocytogenes after one week. This was evident in the

Illinois chocolate milk listeriosis outbreak of 1996.(19'

Because of its resistance to temperature fluctuations,

ability to multiply at refrigeration temperatures and salt

tolerance, L. monocytogenes can survive the cheese

manufacturing and ripening process.143 Its growth in cheese









milk is retarded by lactic acid starter cultures. During

the manufacturing process, L. monocytogenes is primarily

found in the cheese curd, with only a small portion

appearing in the whey. The behavior of L. monocytogenes

cells in the curd is influenced by the type of cheese.

This is seen in the high growth rates found in feta cheese

and compared to significant inactivation in cottage cheese.

During ripening of the cheese, the numbers of L.

monocytogenes cells may gradually increase as in Camembert,

decrease gradually as in cheddar or Colby, or decrease

rapidly during early ripening and then stabilize as in bleu

cheese. 43

Meat and Poultry

L. monocytogenes attaches strongly to the surface of

raw meats and poultry and is difficult to remove or

inactivate. L. monocytogenes multiplies readily in meat

products, including vacuum-packaged beef. A study by

Farber et al. (1999) showed at pH values near 6.0, L.

monocytogenes increases ten-fold, conversely there was

little or no multiplication at a pH of approximately 5.0.

Ready-to-eat meat products that have received a heat

treatment followed by cooling in brine before packaging may

provide a particularly conducive environment for

proliferation of L. monocytogenes. The reduction in









competitive microflora and the high salt tolerance of the

organism contributed to its survival.1251

Seafood

In the United States Quantitative Assessment of the

Relative Risk to Public Health from Foodborne Listeria

monocytogenes Among Selected Categories of Ready-to-Eat

Foods 30) the seafood at risk were as follows:

Raw Seafood
Preserved Fish
Cooked Ready-To-Eat Crustaceans
Smoked Seafood


Seafood was further broken down based on risk potential

for L. monocytogenes contamination, ranging from high to

low. The group ratings were based on the amounts of L.

monocytogenes found commercially in these types of foods,

portion size and number of servings per year. Raw seafood

was placed in the low-risk category because it is assumed

that they will be cooked prior to serving, killing any L.

monocytogenes cells if prepared properly. Preserved fish

is also considered low-risk because the chosen preservation

method, either low acid or an anaerobic environment, has a

lethal effect on the L. monocytogenes. In addition, the

amount of preserved fish eaten in the US per serving/per

year is a low, further decreasing the number of projected

incidences.








In the US Risk Assessment (2003), smoked seafood and

ready-to-eat crustaceans were placed into the high-risk

category.(30) Smoked seafood was placed into the high-risk

category because of the amount of incidences of commercial

contamination by L. monocytogenes. Research has shown that

some cold-smoking processes do not completely kill L.

monocytogenes. 34) Ready-to-eat crustaceans fall into the

high-risk category because of the number of incidences

caused by commercial contamination with L. monocytogenes.

The presence of L. monocytogenes in cooked, ready-to-eat

foods is usually an indicator of post-process

contamination. 30)

In a study performed in 1990, Listeria spp. were

isolated in 81% of freshwater and 33% of seawater

samples.1'18 Of these combined water samples, 62% were

identified as L. monocytogenes. In this same study,

Listeria spp. were found in 30% of sediment samples, with

17% testing positive for L. monocytogenes.(18>

Numerous studies examining the presence of Listeria

spp. in seafood have been performed.(18'23'28'39) One study

showed 30% of fresh shrimp and crabmeat was positive for

Listeria, while 33% of frozen seafood, prawn meat and Black

pomphret, were Listeria positive. 23) Approximately 60% of

the frozen samples tested positive for Listeria spp. with









26% confirmed as L. monocytogenes. The frozen seafood

consisted of raw and cooked shrimp, lobster tail, cooked

crabmeat fish fillets and surimi products.1231 The presence

of Listeria spp. on the cooked products may be an

indication of post-process contamination. 30) In a study

Dillon et al. (1992) of smoked and fermented fish, 12.2% of

the samples were positive for L. monocytogenes.

Approximately 8.9% of hot-smoked fish, 13.6% of cold-smoked

fish and 25.8% of fermented fish were contaminated with L.

monocytogenes. Presence of L. monocytogenes on the hot-

smoked fish could be attributed to improper cooking times

and/or temperatures, or an indication of post-process

contamination. 23)

Stress Proteins

Stress adaptation occurs in numerous bacteria,

including L. monocytogenes. Resistance of L. monocytogenes

to heat or other lethal factors can be increased by heat

shock or adaptation to other stresses. Bacteria produce

proteins to protect themselves at times of stress.'63'

Extremes such as acidity, 11 cold, 15'68 heat, 22'42) salinity

and pressure 63) can induce the production of these stress

proteins. Some proteins are universal, being produced

during any type of stress. Other proteins are stress

specific, such as those for acid, cold, heat and salt









shock. Optimal heat shock temperatures for mesophilic

microorganisms are approximately 10-150C above the optimal

growth temperature for the specific microbe. L.

monocytogenes has optimal heat shock temperatures at this

range.'631 The magnitude of heat shock thermotolerance is

not only regulated by temperature, but also time. The time

of exposure to heat shock, heating matrix, the condition of

the L. monocytogenes culture, as well as the method used to

recover the cells, all have an effect on bacterial

thermotolerance. (67 Researchers have shown that heat

shocking L. monocytogenes at 420C for 60 minutes and then

subjecting the cells to 600C at various times resulted in a

D60-value increase of approximately two times versus the

non-heat shocked strain.114

In a study by Georgopoulus et al. (1993) it was shown

that L. monocytogenes exhibited a large increase in the

rate of synthesis of a particular set of proteins during

incubation at elevated temperatures. L. monocytogenes

produces proteins during heat shock conditions that have

molecular weights similar to the major heat shock proteins

(HSP) seen in E. coli; DnaK (69 KDa), GroEL (57 KDa), and

GrpE (22KDa) 31 This is expected due to the extreme

conservation of HSPs, not only throughout the bacterial

kingdom, but throughout evolution as a whole."2) These HSPs









are induced during elevated temperature incubation. 2'22'31'421

These proteins play a major role in acquired

thermotolerance.

Research has shown that the protective responses

induced by heat shocking are reversible.1451 Reversible

mechanisms, which may include post-translational

modifications such as phosphorylation/dephosphorylation is

responsible for the capture and release of damaged

polypeptides. Phosphorylation has been shown to induce

altered protein-binding properties in DnaK and GroEL upon

heat shock. This change in binding property is believed to

promote the repair of damaged polypeptides and can occur

independently of protein synthesis. Once repair is

complete, the polypeptide is release through the process of

dephosphorylation. (2

In the Gram-positive model, stress responses are

handled by four different regulatory mechanisms. Class I

heat shock genes encodes the regular chaperone-like

proteins, such as GroES, GroEL and DnaK. These proteins

are controlled by the HrcA repressor. 221 Class II genes

encode general stress proteins whose transcription is

dependent on the general stress transcription factor, known

as sigma factor B (0) .(22) Studies have shown that oB is

responsible for producing various cold shock proteins,15)









acid shocked proteins'631 and various HSPs.'66) Class III

heat shock genes encoding ClpP and two HSP100, Clp ATPases,

ClpC and ClpE, form part of the CtsR stress response

regulon. These genes are negatively controlled by CtsR,

the product of the first gene of the ClpC operon.(421 Class

IV includes stress response genes whose expression is

independent of HrcA, o" or CtsR and whose regulatory

mechanism has yet to be identified.31)

Decimal Reduction Times

Decimal reduction time (D-value) is the time required

to reduce a bacterial population by 90% or one log unit at

a given temperature. Food processors utilize D-values to

try to ensure the elimination of pathogens, including L.

monocytogenes from the food supply. 3)

The D-values for L. monocytogenes vary from strain to

strain and from study to study. D-values of L.

monocytogenes are dependent upon many different factors.

While some researchers have them as high as 3.25 hours1271 at

580C on hot smoked salmon and some as low as 1.5 seconds at

730C in milk. l15 Casadei et al. (1997) noted that different

strains of L. monocytogenes can have large variations in

thermal resistance and D-values. 115 In research done by

Bremer et al. (2003), D-values for L. monocytogenes varied






31


by seafood matrix, with smoked salmon having the highest

average Ds8-value of the matrices studied at 3.25 hours. (1o














CHAPTER 3
METHODS AND MATERIALS

Bacterial Culturing Protocol

Listeria cultures were grown in 10 ml of TSB/NX at 370C

overnight. A loopful of culture was placed into fresh

TSB/NX and incubated at 370C overnight. This process was

repeated for three consecutive days. On day four, cultures

were placed in phosphate buffered saline (PBS)(ICN

Biomedicals Inc., Aurora, OH) and centrifuged at 4000 g for

20 minutes.

Nalidixic Acid Resistance

For this experiment, four Listeria cultures were

introduced to increasing amounts of nalidixic acid in order

to develop a resistance to this antibiotic. This process

was performed to eliminate contamination from background

microflora. Nalidixic acid is an antibiotic that inhibits

DNA gyrase causing the bacteria to lose its ability to

divide. The cell fills with nuclear material and

eventually bursts. Gram-positive microbes have a greater

tolerance level than Gram-negative microbes and can

eventually become resistant to the effects of nalidixic

acid. 11) All experimental media was supplemented with 50









mg/1 of nalidixic acid, in order to screen out all

microflora with the exception of the adapted Listeria

cultures.

The cultures used for this experiment were Listeria

monocytogenes 1/2a, 1/2b, 4c and Listeria innocua. The L.

monocytogenes cultures were obtained from Dr. Martin

Weidman at Cornell University. The L. monocytogenes 1/2a,

1/2b and 4c cultures were originally obtained from smoked

fish, a human isolate and an animal isolate, respectively.

The Listeria innocua culture was obtained from Dr. Anita

Wright at the University of Florida.

One loopful of each culture was placed into 10 ml of

tryptic soy broth (TSB)(Difco, Becton Dickinson, Sparks,

MD) and incubated overnight at 370C. A stock solution of

nalidixic acid sodium salts (Sigma, St. Louis, MO) was

prepared at 50,000 mg/l by adding 12.5 g to 250 ml of

deionized water. Starting at 10 pL, nalidixic acid was

added to 10 ml tubes of TSB, yielding a final nalidixic

acid concentration of 50 mg/l. Tubes were then incubated

overnight at 370C. Survivors were then placed into TSB

tubes containing 100 mg/l of nalidixic acid and grown for

18-24 hours at 370C. The amount of nalidixic acid used in

the enrichment medium was increased 50 mg/l each day until

growth was observed at a final concentration of 350 mg/l.









Experiments were performed using a working solution of 50

mg/l of nalidixic acid. The 50 mg/1 concentration was

significantly less than the 350 mg/1 the cultures were

adapted too, but still high enough to screen out background

flora. This concentration allowed growth in a medium that

both selected against any background microflora and allowed

the adapted cultures to grow unencumbered.

Growth Curves

Growth curves were used to determine generation times

of target organisms on different growth media.

Additionally, populations were correlated to optical

density measurements at 600 nm. The growth phases of the

non-heat shocked cultures were compared against the

nalidixic acid adapted cultures. The results of this study

were used to determine the type of media to be used

throughout the experiment. The cultures that were used for

this project were the cultures adapted in the nalidixic

acid resistance study. These were designated L.

monocytogenes-nx 1/2a (nx denotes nalidixic acid

resistant), L. monocytogenes-nx 1/2b, L. monocytogenes-nx

4c and Listeria innocua-nx. The non-heat shocked types of

these four cultures were also used.

All of the cultures were grown for 18-24 hours in TSB

at 370C. One hundred p1 of each culture was placed into









500 ml of TSB; one ml of each culture was serially diluted

(1:10) in PBS and then spread-plated on modified oxford

agar (MOX)(Difco, Becton Dickinson, Sparks, MD), tryptic

soy agar (TSA)(Difco, Becton Dickinson, Sparks, MD) and TSA

supplemented with nalidixic acid (TSA/NX). An additional

one ml was placed into a glass cuvette for an optical

density measurement using a Spectronic 601

spectrophotometer (Milton Roy, Ivyland, PA) with the

absorbance read at 600 nm and recorded. Every hour the

same steps were repeated starting with one ml of each

culture being serially diluted (1:10) in PBS, until there

were three consecutive absorbance readings that did not

increase. Data was collected and analyzed.

Background Study

This study was performed to determine if there were

any bacteria native to the seafood matrices that had the

ability to grow on TSA/NX. Any background microflora could

potentially cause a problem in recovery studies by causing

apparent Listeria counts to be larger than they actually

were. Seafood matrices used in this experiment were

crabmeat (Callinectes sapidus), cooked shrimp (Penaeus

setiferus) and smoked salmon (Oncorhyncus gorbuscha) and

were obtained from Northwest Seafood, Gainesville, FL.

Crabmeat and salmon were processed prior to purchase.









Shrimp was boiled for five minutes in tap water and

refrigerated before each study. All seafood samples were

cut into 10 gram pieces and stomached in 90 ml of PBS in a

Mix 1 Laboratory blender (AES Laboratories, Paris, France).

Samples were serially diluted (1:10) and pour plated using

TSA/NX. Plates were incubated for 18-24 hours at 370C.

Data was collected and analyzed.

Recovery Study

This experiment was performed to determine the amount

of the Listeria spp. (L. monocytogenes-nx 1/2a, L.

monocytogenes-nx 1/2b, L. monocytogenes-nx 4c, L. innocua-

nx), which could be recovered from the three food matrices:

cooked shrimp, crab meat and smoked salmon.

Cultures were grown in TSB/NX overnight at 370C,

reculturing for three consecutive days before use.

Cultures were washed and centrifuged in PBS at 4000 g for

20 minutes. Shrimp was boiled for five minutes and chilled

before testing. Seafood samples were cut into 10 gram

pieces. One hundred pl(1.0 x 106 CFU/ml) of each culture

was placed onto each piece of seafood. Samples were

allowed to remain at room temperature for 60 minutes to

dry. Samples were stomached in 90 ml of PBS for 30

seconds. Samples were serially diluted (1:10) and pour









plated using TSA/NX. Plates were incubated for 18-24 hours

at 370C. Data was collected and analyzed.

Protein Electrophoresis

Protein electrophoresis was used to examine proteins

from the different strains of Listeria, both adapted and

non-adapted. Bacterial cells were lysed and the extracted

proteins were placed at one end of a polyacrylamide gel

(10% Criterion- Tris-HCl Precast Gels, Bio-Rad, Hercules,

CA). The gel was then subjected to a strong electrical

field inside of a vertical gel box (CriterionM cell, Bio-

Rad, Hercules, CA) at 100V for approximately two hours.

Proteins were pulled toward the positive pole of the

electric field, with the larger molecules traveling slower

through the gel matrix and small molecules traveling

quicker. This causes the proteins to become separated

according to their molecular weight.

When bacteria are stressed, different biochemical

mechanisms are initiated and uninitiated within the cell.

One result of stress-inducted mechanisms is the production

of stress proteins. Stress proteins can be general or they

can be stress specific, such as cold shock, heat shock or

acid shock proteins. For this experiment, the nalidixic

acid resistant Listeria samples (1/2a, 1/2b, 4c and

innocua) underwent a heat shocking protocol.(67) To heat









shock, the cultures were placed into a 500C water bath for

60 minutes. After 60 minutes, the heat shocked cultures

were given 15 to 30 minutes to recuperate.'67) The protein

profiles of the heat shocked Listeria samples were examined

against the protein profile of the non-heat shocked

cultures.

Two sets of the Listeria cultures were grown for three

consecutive days in TSB/NX at 370C. One set was non-heat

shocked, while the other underwent the heat shocking

protocol. On day three, cultures were removed and

centrifuged in PBS at 4000 g for 20 minutes. The culture

pellets were placed into fresh Universal Pre-Enrichment

Broth (UPB)(Difco, Becton Dickinson, Sparks, MD) for heat

shocking.

The non-heat shocked cultures were left at room

temperature. The cultures to be heat shocked underwent the

previously mentioned heat shocking protocol. All cultures

were centrifuged in PBS at 4000 g for 20 minutes.

The culture pellets were placed into 10 ml of CelLytic"

B Bacterial Cell Lysis Reagent (Sigma-Aldrich, St. Louis,

MO) along with 100 pl of a 10 mg/ml lysozyme solution, for

24 hours to ensure proper cell lysis and maximize protein

retrieval. Cultures were centrifuged at 4000 g for 20

minutes. The resulting supernatant contained the total









bacterial proteins. Seven hundred ul of the protein

supernatant solution was mixed with 285 pl of Laemmli

Sample Buffer (Bio-Rad, Hercules, CA) and 15 ul of P-

mercaptoethanol (Fisher Chemical, Fair Lawn, NJ). The P-

mercaptoethanol was added to break up insoluble inclusion

bodies which have the ability to attract proteins. The

sample was then heated at 900C for five minutes to aid in

cell lysis.

Prior, to placing the protein samples in the gel, the

protein amounts had to be made equal to one another. This

equilibration was carried out using the Bio-Rad Protein

Assay. This assay is based on the Bradford Assay and is a

procedure for determining the concentration of solubilized

protein."9) The lyophilized protein standard bovine gamma

globulin was rehydrated with 20 ml of sterile deionized

water. Eight hundred pl of each gamma globulin standard

and samples were pipeted into a clean, dry cuvette. This

assay was performed in triplicate. Two hundred ul of dye

reagent was added to each cuvette and mixed. Cuvettes were

incubated at room temperature for at least five minutes and

no more than 60 minutes. The cuvette was placed into a

Spectronic 601 spectrophotometer (Milton Roy, Ivyland, PA)

and the absorbance read at 595 nm. The linear range of the

assay for gamma globulin is 1.2 to 25pg/ml (figure 2).











0.6
Microassay procedure
0.5. (1-20 pg)

0.4- SA IgG

O0.3
0
0.2

0.1


2.5 5 7.5 10 12.5 15 17.5 20 22.5 25
Protein (.g,ml)

Figure 2. Typical standard curve for the Bio-Rad Protein
Microassay, bovine serum albumin (BSA), bovine
Gamma globulin (IgG). OD595 corrected for blank.
1.25-25 pg/ml x 0.8 ml = 1-20pg of protein.(8


Equal amounts of the total protein solution were placed

into a Criterion" pre-cast 10% polyacrylamide protein gel

(Bio-Rad, Hercules, CA). The gel was subject to 100V for

approximately two hours on a Criterion" electrophoresis

cell (Bio-Rad, Hercules, CA). The gel was washed three

times with sterile deionized water for a total of 15

minutes. GelCode" Blue staining reagent (Pierce Chemicals,

Rockford, IL) was added and placed on a Roto Mix Type 50800

turntable mixer (Thermolyne, Dubuque, IA) for 60 minutes.

Gel was washed for a total time of 60 minutes in cold,

sterile, deionized water. After washing, the gel was

examined using a ScanJet 5370C flatbed scanner (Hewlett-









Packard, Palo Alto, CA) and visualized using Corel

PhotoHousem imaging software (Corel Corporation, Ontario,

Canada). The protein amounts were calculated using UN-

SCAN-IT gel" densitometer software (Silk Software, Orem,

UT).

Western Blotting

The general stress protein, GroEL was identified using

a western blotting protocol. For this experiment, the

nalidixic acid resistant Listeria samples (1/2a, 1/2b, 4c

and innocua) underwent a heat shocking protocol.167 The

protein profiles of the heat shocked Listeria were examined

against the protein profile of the non-heat shocked

cultures. The western blotting protocol took place in

three steps: blotting, immuno-staining and color

development.

Two sets of the Listeria cultures were grown for three

consecutive days in TSB/NX at 37'C. One set was the non-

heat shocked, while the other underwent heat shocking. On

day three, cultures were removed and centrifuged in PBS at

4000 g for 20 minutes. The culture pellets were placed

into fresh UPB (Difco, Becton Dickinson, Sparks, MD) for

heat shocking. The non-heat shocked cultures were left at

room temperature, while the cultures to be heat shocked

underwent the aforementioned heat shocking protocol. All









cultures were centrifuged in PBS at 4000 g for 20 minutes.

The culture pellets were placed into CelLytic" B Bacterial

Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO) overnight

to ensure proper cell lysis and maximize protein retrieval.

Cultures were centrifuged at 4000 g for 10 minutes. The

resulting supernatant contained the total bacterial

proteins. Equal amounts of the total protein solution were

placed into a CriterionM pre-cast 10% polyacrylamide

protein gel (Bio-Rad, Hercules, CA). The gel was subjected

to 100V for approximately two hours on a Criterionm

electrophoresis cell (Bio-Rad, Hercules, CA).

The target protein was identified by using anti-GroEL

antibodies (Sigma-Aldrich, St. Louis, MO). Blotting was

performed using a Criterion" Blotter (Bio-Rad, Hercules,

CA), with the Amplified Alkaline Phosphatase Goat Anti-

Rabbit Immun-Blot Assay kit (Bio-Rad, Hercules, CA), used

for color visualization of the target proteins.

The blotting required the transfer of the proteins

from the polyacrylamide gel to a polyvinylidene difluoride

(PVDF) membrane. The membrane and filter papers were cut

to the dimensions of the gel. The gel was equilibrated in

Towbin transfer buffer (TTB) for 15 minutes. 7) The TTB

with 20% methanol was prepared with 100 ml of 10x

Tris/Glycine (TG)(Bio-Rad, Hercules, CA) buffer, 200 ml of









methanol (Sigma-Aldrich, St. Louis, MO) and 700 ml of

deionized water.

The blotter tank was filled with TTB with 10% methanol

to approximately half of the volume. TTB is composed of

100 ml of 10X TG buffer, 800 ml of sterile, deionized water

and 100 ml of methanol. The TTB was used to facilitate the

transfer of the proteins from the gel to the PVDF membrane.

A magnetic stir bar was placed in the bottom of the blotter

tank. Low temperatures are used to facilitate a better

transfer of proteins from the polyacrylamide gel to the

PVDF membrane; therefore an ice block was then placed in

the back of the cell.

The gel/membrane sandwich was prepared using

manufacturer's recommend procedure."7 The pre-equilibrated

gel was then placed on top of the filter paper. A roller

was used to remove any air bubbles that may have been

trapped underneath the gel. Once all of the components

were in place, the remaining TTB was added to the fill

level marked on the tank. The gel/membrane sandwich was

subjected to 100V for 30 minutes.

Before immuno-staining could take place, the stock

solutions were prepared according to manufacturer's

recommendations. To prepare the Tris-buffered saline (TBS;

Bio-Rad, Hercules, CA), 100 ml of 10x TBS was added to 900









ml of sterile, deionized water. The wash solution, TTBS,

was prepared by adding 450 1l of Tween-20 (Bio-Rad,

Hercules, CA) to 900 ml of lx TBS. The blocking solution

was prepared by adding 5.0 g of non-fat dry milk (Bio-Rad,

Hercules, CA) to 100 ml of TBS. The first antibody

solution was made by diluting the anti GroEL to its

appropriate titer (1:10,000) in 100 ml of TTBS. The second

antibody solution was prepared by adding 33 1p of

streptavidin (Bio-Rad, Hercules, CA) to 100 ml of the

biotinylated goat anti-rabbit serum (Bio-Rad, Hercules, CA)

to 100 ml of TTBS. To prepare the streptavidin-

biotinylated alkaline phosphatase complex (S-BAPC)(Bio-Rad,

Hercules, CA) 33 pL of streptavidin was added to 100 ml

TTBS. In addition, 33 1l of biotinylated-alkaline

phosphatase was added to the previously prepared

streptavidin solution. That complex was allowed to

incubate for no less than one and no more than three hours

at room temperature. The AP Color Development Buffer (Bio-

Rad, Hercules, CA) was made by adding sterile, deionized

water to the buffer at its appropriate titer (1:25).

The staining procedure began with the blocking step.

The PVDF membrane was immersed into the blocking solution

at a 450 angle. The solution was gently agitated on an









orbital shaking platform for one hour at room temperature.

The blocking solution was then decanted and TTBS added.

The solution was then washed for five to 10 minutes with

gentle agitation at room temperature. This washing step

was repeated once. The TTBS was decanted and the first

antibody solution was added to the membrane. The overnight

incubation was preformed. The first antibody solution was

then decanted and TTBS was added to the membrane to wash.

The wash lasted for five to 10 minutes with gentle

agitation at room temperature. This wash step was repeated

once more. The TTBS was decanted and the biotinylated goat

anti-rabbit solution (second antibody) was added to the

PVDF membrane. The membrane was allowed to sit in the

solution for no more than two hours with gentle agitation

to room temperature. While the PVDF membrane soaked in the

second antibody solution, the S-BAPC was prepared. The

complex was allowed to form for one hour, not exceeding

three hours, at room temperature. The second antibody

solution was then decanted and TTBS added. The solution

was then washed for five to ten minutes with gentle

agitation at room temperature. This washing step was

repeated once. The TTBS was decanted and the S-BAPC was

added to the PVDF membrane. The membrane was washed for

five to 10 minutes with gentle agitation at room









temperature. The S-BAPC was decanted and the PVDF membrane

was washed three times with TTBS.

The color development occurred in four steps.

Immediately before use, one ml of AP Color Development

Reagent A (Bio-Rad, Hercules, CA) and one ml of Color

Development Reagent B (Bio-Rad, Hercules, CA) was added to

100 ml of color development buffer (Bio-Rad, Hercules, CA)

at room temperature. The PVDF membrane was immersed in the

color development solution. Protein concentrations of 100

ng or higher became visible immediately as purple bands or

dots. Smaller amounts of proteins became visible within 30

minutes. Immersing the PVDF membrane in sterile, deionized

water for 10 minutes with gentle agitation at room

temperature stopped the color development. The water was

changed once at the five-minute mark of the total 10 minute

wash step. After washing, the PVDF membrane was examined

using a ScanJet 5370C flatbed scanner (Hewlett-Packard,

Palo Alto, CA) and visualized using Corel PhotoHousem

imaging software (Corel Corporation, Ottawa, Ontario,

Canada). Protein amounts were calculated using UN-SCAN-IT

gelM densitometer software (Silk Software, Orem, UT).

D7o-Value Determination for Heat Shocked Cultures

The D-value is defined as the amount of time at a set

temperature that is needed to reduce the viable bacterial









population by one logo unit. These experiments were

performed to determine the difference between the D70-values

of heat shocked and non-heat shocked Listeria cultures.

Two sets of the Listeria cultures were grown for three

consecutive days in TSB/NX at 370C. On day four, a total

of 72 tubes were inoculated. One half was non-heat

shocked, while the other half underwent heat shocking. On

day five, cultures were removed and centrifuged in PBS at

4000 g for 20 minutes. The culture pellets were placed

into fresh UPB to undergo the heat shocking protocol.>67

The non-heat shocked cultures were left at room

temperature.

All culture tubes were placed into a 700C Precision

Series 280 water bath (Winchester, VA). This temperature

was chosen based on the information in FDA's 1999 Food

Code, which states raw fish and foods containing raw fish

shall be cooked to heat all parts of the food to 630C or

above for 15 seconds. 29) A thermal inactivation temperature

of 700C (D70-value) was chosen as a point slightly higher

than the minimum heating temperature required. Tubes were

removed in sets of six (three heat shocked and three non-

heat shocked) every 30 seconds for five minutes and

immediately placed on ice to stop any further heat

inactivation. This was the thermal inactivation protocol









use for all subsequent experiments. Samples were serially

diluted (1:10) and pour plated on TSA. Plates were

incubated for approximately 18 to 24 hours at 370C. Data

was collected and the D70-values were analyzed.

D7o-Value Reversion

The purpose of this study was to determine when heat

shocked cultures reverted back to the non-heat shocked

state. This reversion was examined proteomically as well

as through the use of bacterial D7o-values.

Bacterial cultures first underwent the bacterial

culturing protocol. A total of 72 tubes were inoculated,

with one half being the non-heat shocked, while the other

half underwent the aforementioned heat shocking protocol.

Cultures were removed and centrifuged in PBS at 4000 g for

20 minutes. The culture pellets were placed into fresh UPB

for heat shocking. The non-heat shocked cultures were left

at room temperature, while cultures to be heat shocked

underwent the heat shocking protocol. A loopful of each

culture was placed into fresh TSB for use on the next day.

All culture tubes were placed into a Precision Series

280 700C water bath (Winchester, VA) and underwent the

thermal inactivation protocol. Samples were serially

diluted (1:10) and pour plated on TSA. Original samples

were kept and set aside for protein electrophoresis.









Plates were incubated for 18 to 24 hours at 370C. Data was

collected and the D?0-values were analyzed.

Cultures were reinoculated for three consecutive day

and both heat shocked and non-heat shocked proteins were

examined. The method used in the reversion study utilized

the same protocol that was previously described in the

protein electrophoresis study.

D7o-Values and Seafood

This experiment was performed to determine the

difference in D70-values of the heat shocked and the non-

heat shocked Listeria cultures when inoculated onto three

different types of seafood; cooked shrimp, crab meat and

smoked salmon. The seafood used for this experiment was

obtained from Northwest Seafood, Gainesville, FL. Shrimp

was boiled for five minutes. All seafood samples were cut

into 10 g pieces.

Bacterial cultures first underwent the bacterial

culturing protocol. Half of the cultures were heat shocked

according to the previous heat shock protocol, while the

remaining half were used as the non-heat shocked cultures.

Cultures were centrifuged at 4000 g for twenty minutes in

PBS.

One hundred pL of each culture was placed onto each 10 g

seafood sample. Samples were allowed to sit at room






50


temperature for 60 minutes to achieve bacterial attachment.

Seafood samples were placed into a Whirl-Pak bag (NASCO,

Fort Atkinson, WI) and underwent the thermal inactivation

protocol. To each sample, 90 ml of PBS was added to make a

1:10 dilution. Samples were then serially diluted in tubes

of PBS. From each dilution tube, one ml of sample was

pour-plated in TSA/NX. Plates were incubated at 370C for

approximately 18 to 24 hours. The plates were counted and

data was analyzed.














CHAPTER 4
RESULTS AND DISCUSSIONS

Growth Curves


Growth curves were performed for all of the Listeria

cultures, including nalidixic acid resistance strains

(Listeria-nx) on modified oxford media (MOX), tryptic soy

agar (TSA) and tryptic soy agar supplemented with 50 mg/l

(TSA/NX). Statistical analysis using the Least Square

Means (LSM) test was used to compare the growth of each

organism for each type media. Growth curves data is

presented in figures 3 through 7.

The MOX data showed a difference in growth patterns

when comparing non-adapted to nalidixic acid resistant

strains. An increase was seen in the lag phase associated

with the nalidixic acid resistant strains when compared to

the non-adapted cultures. This difference in population

was not significant (p < 0.05) after nine hours once the

non-adapted and nalidixic acid cultures reach stationary

phase.

MOX is a selective and differential agar for Listeria

species. The growth of the nalidixic acid resistant

strains on MOX showed no detrimental effects on the agar's






52


ability to differentiate Listeria spp. from background

flora. It was also concluded that there was no difference

between the non-adapted and nalidixic acid adapted strains

when grown on TSA, a general, non-selective media. When

comparing TSA versus TSA/NX, the data showed that there

were no significant differences between the growth patterns

seen for nalidixic acid adapted strains. No studies were

conducted on TSA/NX for non-adapted strains.

Statistical analysis showed no significant differences

in final bacterial titers when comparing media type to

counts achieved at the stationary for each organism (Tables

1 through 5).









Table 1. Growth data of non-adapted Listeria cultures on
MOX agar at the end of stationary phase (12
hours).

Time 1/2a 1/2b 4c innocua
(Hours) (Log1o CFU) (Logio CFU) (Log1o CFU) (Logio CFU)
12 10.08a 10.03a 10.05a 9.21a
'Values with different letters (a-b) in the same row differ
significantly (p < 0.05)




Growth of Listeria cultures on MOX


2 4 6 8 10 12
Time (Hours)

--X-1/2a --l1/2b --4c --innocua i


Figure 3. One hundred pl of 12-hour cultures of the
respective non-adapted Listeria strains were
inoculated into tryptic soy broth and incubated
at 370C in a rotary shaker for 12 hours. At
each time point samples were spread plated on
MOX agar in triplicate. The mean populations of
the respective strains were combined and plotted
over time. No significant difference between
mean populations of strains were found between
strains at the end of stationary phase (p <
0.05)









Table 2. Growth data of nalidixic acid adapted Listeria
cultures on MOX agar at the end of stationary
phase (12 hours).

Time 1/2a-nx 1/2b-nx 4c-nx innocua-nx
(Hours) (Log CFU) (Log CFU) (Log CFU) (Log CFU)
12 9.24a 9.18a 8.91a 9.61a

Values with different letters (a-b) in the same row differ
significantly (p < 0.05)


Growth Curves of Listeria-nx on MOX


10




6



0 2 4 6 8 10 12
Time (Hours)

[-K-1/2A ---1/2B --4C -A-innocua


Figure 4. One hundred pl of 12-hour cultures of the
respective adapted Listeria-nx strains were
inoculated into tryptic soy broth and incubated
at 370C in a rotary shaker for 12 hours. At
each time point samples were spread plated on
MOX agar in triplicate. The mean populations of
the respective strains were combined and plotted
over time. No significant difference between
mean populations of strains were found between
strains at the end of stationary phase (p <
0.05).









Table 3. Growth data of non-adapted Listeria cultures on
TSA at the end of stationary phase (13 hours).

Time 1/2a 1/2b 4c innocua
(Hours) (Log CFU) (Log CFU) (Log CFU) (Log CFU)
13 9.04a 9.03a 9.28a 9.24a
'Values with different letters (a-b) in the same row differ
significantly (p < 0.05)


Growth of Listeria cultures on TSA


10

8

6

4
0 2 4 6 8 10 12

Time (Hours)

---1/2A --1/2B --4C --innocua ]


Figure 5. One hundred pl of 13-hour cultures of the
respective non-adapted Listeria strains were
inoculated into tryptic soy broth and incubated
at 370C in a rotary shaker for 13 hours. At
each time point samples were spread plated on
TSA agar in triplicate. The mean populations of
the respective strains were combined and plotted
over time. No significant difference between
mean populations of strains were found between
strains at the end of stationary phase (p <
0.05).









Table 4. Growth data of nalidixic acid adapted Listeria
cultures on TSA at the end of stationary phase
(12 hours).

Time l/2a-nx 1/2b-nx 4c-nx innocua-nx
(Hours) (Log CFU) (Log CFU) (Log CFU) (Log CFU)
12 9.11a 9.07a 9.40a 9.11a
'Values with different letters (a-b) in the same row differ
significantly (p < 0.05)




Growth of Listeria-nx cultures on TSA

12

10

8

6 -- -----------

4
0 2 4 6 8 10 12
Time (Hours)

i_--l/2A --l1/2B -W-4C -A-innocua


Figure 6. One hundred pl of 12-hour cultures of the
respective nalidixic acid adapted Listeria
strains were inoculated into tryptic soy broth
and incubated at 370C in a rotary shaker for 12
hours. At each time point samples were spread
plated on TSA agar in triplicate. The mean
populations of the respective strains were
combined and plotted over time. No significant
difference between mean populations of strains
were found between strains at the end of
stationary phase (p < 0.05).









Table 5. Growth of nalidixic acid adapted Listeria
cultures on TSA/NX at the end of stationary
phase (17 hours).

Time 1/2a-nx 1/2b-nx 4c-nx innocua-nx
(Hours) (Log CFU) (Log CFU) (Log CFU) (Log CFU)
17 9.54a 9.08a 8.98a 9.11a
"Values with different letters (a-b) in the same row differ
significantly (p < 0.05)


Growth of Listeria-nx on TSA/NX


0 3 6 9 12 15 18
Time (Hours)

S-1-/2A -- 1/2B 2--4C -M--innocua

Figure 7. One hundred ul of 17-hour cultures of the
respective nalidixic acid adapted Listeria
strains were inoculated into tryptic soy broth
and incubated at 370C in a rotary shaker for 17
hours. At each time point samples were spread
plated on TSA/NX agar in triplicate. The mean
populations of the respective strains were
combined and plotted over time. No significant
difference between mean populations of strains
were found between strains at the end of
stationary phase (p < 0.05).


.~ --------^ --









Background Microflora Study

The results from this study showed that no organisms

native to cooked shrimp, crab meat or smoked salmon had the

ability to grow on the TSA/NX media. The use of TSA/NX was

beneficial for the final seafood study, in that no special

considerations were made when counting the number of

surviving colonies, since the naturally occurring

microflora present were excluded.

Recovery Study

The results from the recovery studies are shown in

tables 6 through 9. These studies show the amounts of the

individual Listeria-nx cultures that were recovered from

each of the seafood matrices. Statistical analysis using

the LSM assay showed no statistical difference between

inoculated and recovered levels of Listeria-nx from any of

the seafood matrices.

Table 6. Total logo amounts recovered of Listeria-nx
1/2b cultures on seafood matrices.*

Initial Amount Experimental Amount

Crab Meat 6.25a 6.16a
Shrimp 6.27a 6.25a
Smoked Salmon 6.27a 6.26a
Values with different letters (a-b) in the same row differ
significantly (p < 0.05)









Table 7. Total log1i amounts recovered of Listeria-nx
1/2a cultures on seafood matrices.*


Table 8. Total logio amounts recovered of Listeria-nx 4c
cultures on seafood matrices.*


significantly (p < 0.05)


Table 9. Total log10 amounts recovered of Listeria
innocua-nx cultures on seafood matrices.*









Protein Electrophoresis

Protein electrophoresis was performed to examine the

profiles of each individual Listeria-nx strain. Figure 8

shows the protein profile of the four Listeria-nx cultures.



1-- 00 kDa

-- 75 kDa



-- 50 kDa
A B C D

Figure 8. SDS-PAGE (10% gel) patterns of the whole protein
extract of (A) L. monocytogenes-nx 1/2a;(B) L.
monocytogenes-nx 4c; (C)L. monocytogenes-nx
1/2b; (D) L. innocua-nx. Each culture was
analyzed in duplicate.

Figure 8 showed differences between each of the

Listeria-nx strains. The most noticeable differences seen

were between the L. monocytogenes-nx strains and Listeria

innocua-nx. Additional protein bands were visible in all

the L. monocytogenes-nx strains as compared to the L.

innocua-nx culture. One notable difference was seen at the

50-60 kilo Daltons (kDa) range for each of Listeria-nx

cultures. When examining the lanes for all of the L.

monocytogenes-nx cultures, there was a recognizable double

band at the 50-60 kDa range, but only a single band at that

same region for the L. innocua-nx. Researchers have shown

that one of the proteins necessary for virulence,








listeriolysin-0 (LLO), is approximately 53 kDa.33" The

absence of this would make virulence impossible, thus it

follows that Listeria innocua, an avirulent species, would

lack this protein.

Protein electrophoresis was also used to examine the

differences between the non-heat shocked (NHS) Listeria-nx

cultures and their corresponding heat shocked (HS)

Listeria-nx counterparts. Figure 9 shows the difference in

protein production between HS L. monocytogenes-nx 1/2a and

NHS L. monocytogenes-nx 1/2a. There is one protein in the

HS cells that is noticeably reduced in the NHS cells

(denoted by the arrow). Using Un-Scan-ItT gel densitometer

software (Silk Scientific, Orem, UT) to scan the unknown

region between 50 and 75 kDa, it was calculated that the

protein concentration in the NHS cells was 337 ng, compared

to 1074 ng for HS cultures. It is hypothesized that the

heat shocking process caused this approximate three-fold

increase in the unknown protein.

This protein band fell between the 50 kDa and 75 kDa

markers, so its approximate mass was known. There are two

general stress proteins, GroEL and DnaK that fall between

these two masses. GroEL is approximately 57 kDa in mass,

while DnaK is approximately 69 kDa in mass. This is

consistent with published reports that note the








overproduction of these proteins in at times of

stress. (2,5,22, 31, 42,66)


A B C
Figure 9. SDS-PAGE (10% gel) patterns of the whole protein
extract of (A) NHS Listeria monocytogenes-nx
1/2a; (B) HS Listeria monocytogenes-nx 1/2a; (C)
Protein standard. Arrows denote changes in
protein patterns.





































Figure 10. Densitometer software representation of protein
difference at the 57 kDa range between NHS L.
monocytogenes-nx 1/2a (A) and HS L.
monocytogenes-nx 1/2a (B). The change in peak 1
as seen between graphs A and B represents the
induced increase in stress protein due to heat
stress.

Figure 11 represents the difference in protein

production of NHS L. monocytogenes-nx 1/2b and HS L.

monocytogenes-nx 1/2b. From the data seen in figure 11,

there were two proteins (denoted by arrows) induced in HS

L. monocytogenes-nx 1/2b. From the position and mass of

protein A (denoted by the dotted arrow), the molecule

weight fell between 75 and 100 kDa. There are no known

general stress proteins that fall in that range of








molecular masses. Protein B (denoted by the solid arrow)

was hypothesized to be GroEL, based on its molecular masses

between 50 and 75 kDa. As seen in the research by De

Angelis et al. (2003), GroEL is a protein that has the

ability to repair a wide variety of heat damaged

proteins.(20) Protein identification of this region showed

the protein to be GroEL along with approximately 50 other

proteins (Table 10). It is possible that some of the

additional proteins seen might be associated with the

GroEL/GroES protein repair complex.



















A B C
Figure 11. SDS-PAGE (10% gel) patterns of the whole protein
extract of (A) Protein standard (B) HS Listeria
monocytogenes-nx 1/2b (C) NHS Listeria
monocytogenes-nx 1/2b. Arrows denote changes in
protein patterns.











Table 10. Proteins identified by GC-Mass Spectrometry.
Organism of origin is based on the number of
matching amino acids in the test sequence.

Protein Organism

GroEL Bacillus species

Adhesin Bacillus species

NaDH2 Dehydrogenase Unknown

Anaerobic Dehydrogenase Bacillus species

ATPase Listeria species

General Protein Listeria species

DNA Binding Protein Unknown

Ribosomal Protein Bacillus species

Protein similarity searches were conducted using BLAST at the National
Center for Biotechnology Information nonredundant databases.

As seen in figure 12, densitometer software showed

that HS L. monocytogenes-nx 1/2b was produced approximately

488 ng of protein A. The NHS L. monocytogenes-nx 1/2b

produced approximately 178 ng. This was approximately half

the amount of the same protein when compared to the HS L.

monocytogenes-nx culture. This is similar to the results

seen the trials examining the NHS and HS L. monocytogenes-

nx 1/2a. Additionally, HS L. monocytogenes-nx 1/2b

produced approximately three times as much of protein B as








compared to NHS L. monocytogenes-nx 1/2b, 610 ng and 209

ng, respectively.


C U


Figure 12. Densitometer software representation of protein
difference at the 85 kDa range between NHS L.
monocytogenes-nx 1/2b (A) and HS L.
monocytogenes-nx 1/2b (B). Scan of protein
difference at the 57 kDa range between NHS L.
monocytogenes-nx 1/2b (C) and HS L.
monocytogenes-nx 1/2b (D). The changes in peaks
1 as seen between graphs C and D represents the
induced increase in stress protein due to heat
stress.

Western Blot

Western blots were performed on 10% SDS polyacrylamide

gels to determine if the proteins being induced were GroEL.

The proteins were transferred to a nitrocellulose membrane

where anti-GroEL antibodies were applied forming antibody-

antigen complexes. Once these complexes were formed, they

were visualized and presence of the target protein was

established. Using densitometer software, an approximate


A









amount of GroEL produced was determined for each strain.

Figures 13 and 14 show the densitometer scans of the

Western blots for L. monocytogenes-nx 1/2a, L.

monocytogenes-nx 1/2b, L. monocytogenes-nx 4c and L.

innocua-nx. These scans verify amounts of the stress

protein GroEL produced by each strain. Densitometer

readings seen in figures 13 and 14 confirm that more GroEL

was produced in the HS cells as compared to the NHS cells.

The increase in GroEL was seen in all four of the strains

tested. HS L. monocytogenes 1/2a cells had a GroEL

concentration of 687 ng as compared to NHS cells which

presented 351 ng. L. monocytogenes 1/2b HS cultures

produced 913 ng, while NHS cells had 209 ng. The HS L.

monocytogenes 4c cultures had 353 ng while the NHS cultures

had 213 ng. The HS L. innocua presented concentrations of

659 ng while the NHS strain produced 273 ng. This

experiment showed that GroEL was produced under both NHS

and HS conditions; however HS conditions produced more

GroEL than NHS conditions in each of the four strains.

















A :


B








D


Figure 13. Densitometer software representation of
immunoblot patterns of stressed L.
monocytogenes-nx 1/2a and 1/2b against GroEL
antibodies. (A) Scan of immunoblot of protein
section of NHS L. monocytogenes-nx 1/2a against
GroEL antibodies. (B) Scan of immunoblot of
protein section of heat shock L. monocytogenes-
nx 1/2a against GroEL antibodies. (C) Scan of
immunoblot of protein section of NHS L.
monocytogenes-nx 1/2b against GroEL antibodies.
(D) Scan of immunoblot of protein section of
heat shock L. monocytogenes-nx 1/2a against
GroEL antibodies.



















Figure 14. Densitometer software representation of
immunoblot patterns of stressed L.
monocytogenes-nx 4c and innocua-nx against GroEL
antibodies. (A) Scan of immunoblot of protein
section of NHS L. monocytogenes-nx 4c against
GroEL antibodies. (B) Scan of immunoblot of
protein section of heat shock L. monocytogenes-
nx 4c against GroEL antibodies. (C) Scan of
immunoblot of protein section of NHS L. innocua-
nx against GroEL antibodies. (D) Scan of
immunoblot of protein section of heat shock L.
innocua-nx against GroEL antibodies.


i .i









D7o-value Determinations of Heat Shocked Cultures

The D7o-values were calculated by using standard

regression analysis coupled with a standard ANOVA analysis

to determine statistical differences between cultures

(Table 11).

Table 11. D7o-values of NHS and HS Listeria cultures.

1/2a 1/2b 4c innocua

(minutes) (minutes) (minutes) (minutes)

NHS 1.04a 0.94a 0.95a 0.95a

HS 2.27b 2.02b 0.99a 0.95a

Values with different letters (a-b) in the same row differ
significantly (p < 0.05)

The data showed a statistical increase between the D7o-

values of the NHS as compared to the HS L. monocytogenes-nx

1/2a and 1/2b. The D70-values of HS L. monocytogenes-nx 4c

and L. innocua-nx were not significantly different. The

data showed that subjecting L. monocytogenes-nx 1/2a and

1/2b to heat shocking caused an increase in their D70-

values. L. monocytogenes-nx 4c and L. innocua-nx had D70-

values that did not increase when subjected to heat

shocking. Since no differences in D70-values were observed

for both L. monocytogenes-nx 4c and L. innocua-nx, no

additional D7o-value experiments were performed for these

cultures.








The D70-value data generated in this study showed that

pathogenic lineages I and II, as described by Wiedmann et

al. (1999), had significant increases, whereas non-

pathogenic lineage did not. Wiedmann et al. (1999)

separated L. monocytogenes cultures into three lineages

based on pathogenicity.165 The D70-values of the pathogenic

strains from lineages I (1/2b) and II (1/2a) increased when

heat shocked. Lineages III (4c) and L. innocua did not

have D70-value increases when subjected to thermal stress.

While showing an increase in GroEL production, there was no

increase in heat resistance as was noted for 1/2a and 1/2b.

This demonstrated that increased GroEL production seen

after thermal stress is not the only mechanism for enhanced

heat resistance.

D70-value Reversion Study

The D7o-values were calculated by using standard

regression analysis coupled with a standard ANOVA to

determine statistical differences between the D70-values

calculated for each day of the two day trial. After one

day HS Listeria-nx cultures reverted back to D7o-values

equivalent to those seen for NHS cultures (Table 12). This

return to the NHS state is also visualized through changes

in proteins seen on electrophoresis gels and amounts

calculated though densitometry (Figures 15 and 16).









From data collected through protein electrophoresis,

D7T-value determination and densitometry, it can be

concluded that protein production was induced by both L.

monocytogenes-nx 1/2a and 1/2b after being heat shocked.

Twenty four hours after reinoculating into fresh enrichment

both, proteins initially induced after the heat stressing,

returned to levels equivalent to those seen for NHS

cultures. Statistical analysis of the densitometer

readings of the proteins produced by the HS cultures on the

days one and two were shown to be equivalent to the amounts

produced by initial NHS cultures. Examining the

densitometer readings for day zero cultures, a large

increase was seen in total protein production at the 57 kDa

region in both HS L. monocytogenes-nx 1/2a and 1/2b. Total

protein levels increased in HS L. monocytogenes-nx 1/2a and

1/2b from 244 ng to 401 ng and from 250 ng to 580 ng,

respectively. For NHS L. monocytogenes-nx 1/2a, the day

one and day two concentrations at the 57 kDa region for

total protein production were both approximately 225 ng.

The amount of total protein production in this same region

seen in HS L. monocytogenes-nx 1/2a was approximately 400

ng. The amount of total protein recorded for L.

monocytogenes-nx 1/2b NHS and HS were approximately 260 ng

and 580 ng, respectively.









Results from this experiment showed the significance

of the heat-injury/repair mechanism of L. monocytogenes.

The findings suggest that heat shocked L. monocytogenes

cultures have the ability to recover from non-lethal heat

treatments after 24 hours once recultured into fresh

enrichment broth. This lessens the impact attributed to

sub-lethal injured pathogenic organisms described in the

scientific literature. DeAngelis et al. (2003) stated that

sub-lethally injured microorganisms have the ability to

survive better than non-injured cells due to the production

stress-induced proteins.120) However, if the stress induced

response only lasts 24 hours, the cross-protection

attributed to these injured microorganisms may not be a

factor in virulence.

Table 12. D70-values of NHS and HS Listeria cultures
during reversion study.*


(a-b) in the same row differ


vaui wiI UIIL ierent letters
significantly (p < 0.05)


Day 0 NHS Day 0 HS Day 1 HS Day 2 HS

(minutes) (minutes) (minutes) (minutes)

1/2a 1.25a 2.38b 1.41a 1.31a

1/2b 1.19a 2.40b 1.19a 1.16a

4c 1.09a 1.37a 0.95a 1.13a

innocua 1.13a 1.21a 0.84a 1.29a






74



















A B C D E
Figure 15. SDS-PAGE (10% gel) patterns of the whole protein
extract of NHS and HS L. monocytogenes-nx 1/2a
cultures. Lanes represent sample day (A) NHS
day 0; (B) HS day 0; (C) HS day 1; (D) HS day 2;
(E) Protein standard. The arrow indicates the
region of induced protein production used for
densitometer scans.


















A B










C "D


Figure 16. Densitometer software representation of figure
18. The protein difference in a scanned region
of 50 to 75 kDa before and after heat shocking
L. monocytogenes-nx 1/2a. (A) NHS L.
monocytogenes-nx 1/2a. (B) HS L. monocytogenes-
nx 1/2a after day zero. (C) HS L.
monocytogenes-nx 1/2a after day one. (D) HS L.
monocytogenes-nx 1/2a after day two.


























A B C D E
Figure 17. SDS-PAGE (10% gel) patterns of the whole protein
extract of NHS and HS L. monocytogenes-nx 1/2b
cultures. Lanes represent sample day (A) NHS
day 0; (B) HS day 0; (C) HS day 1; (D) HS day 2;
(E) Protein standard. The arrow indicates the
region of induced protein production used for
densitometer scans.



















A






i ,


C D

Figure 18. Densitometer software representation of figure
20. The protein difference in a scanned region
of 50 to 75 kDa before and after heat shocking
L. monocytogenes-nx 1/2b. (A) NHS L.
monocytogenes-nx 1/2b. (B) HS L. monocytogenes-
nx 1/2b after day zero. (C) HS L.
monocytogenes-nx 1/2b after day one. (D) HS L.
monocytogenes-nx 1/2b after day two.

D7o-Values and Seafood

The D70-values were calculated by using standard

regression analysis coupled with a standard ANOVA analysis

to determine statistical differences between the cultures

(Tables 13 through 15). D7o-values of the HS L.

monocytogenes-nx 1/2a and 1/2b inoculated on the seafood

matrices showed an approximate three-fold increase when


I I









compared to the corresponding NHS cultures (Tables 13

through 15).

Table 13. D70-values of cooked shrimp inoculated with NHS
and HS L. monocytogenes.*


'Values with different letters (a-b) in the same row differ
significantly (p < 0.05)

Table 14. D70-values of crab meat inoculated with NHS and
HS L. monocytogenes.*

Crab Meat (minutes)


NHS HS

1/2a 1.79a 4.46b


1/2b 1.42a 3.51b

4 .l in s e I


a ues w erenlt Letters a--D in thne same row difrrer
significantly (p < 0.05)

Table 15. D70-values of smoked salmon inoculated
and HS L. monocytogenes.*


with NHS


(a-b) in the same row differ


a ues w ereiletters
significantly (p < 0.05)


Smoked Salmon (minutes)


NHS HS

1/2a 1.67a 4.03b

1/2b 1.39a 3.21b

V 1 it7 df


'1









The differences between D7o-values for the NHS and HS

L. monocytogenes-nx 1/2a cultures on cooked shrimp were

1.39 and 3.67 minutes, respectively. For NHS and HS L.

monocytogenes-nx 1/2b on cooked shrimp, the D70-values were

1.35 and 3.47 minutes, respectively. On crab meat, D70-

values for the NHS and HS L. monocytogenes-nx 1/2a strains

were calculated as 1.79 and 4.46 minutes, respectively.

The D70-values for NHS and HS L. monocytogenes-nx 1/2b on

crab meat were 1.42 and 3.51 minutes, respectively. The

D70-value for NHS L. monocytogenes-nx 1/2a on smoked salmon

was 1.67 minutes. Smoked salmon inoculated with HS L.

monocytogenes-nx 1/2a had a D7o-value of 4.03 minutes

There was a significant increase in heat resistance

for the HS cultures (1/2a and 1/2b) on all three seafood

matrices. This increase was approximately double that seen

in D70-values trials of the Listeria-nx strains performed in

TSB. The D70-value of HS L. monocytogenes-nx 1/2a in TSB

was 2.27 minutes, while the average D70-value of the same

organism inoculated on the experimental seafood matrices

was 4.04 minutes. For the HS L. monocytogenes-nx 1/2b, the

D7o-value in TSB was 2.02 minutes, compared to 3.37 minutes

for the average D70-values of HS L. monocytogenes-nx 1/2b on

the tested seafood matrices.









From the increases seen in the D7o-values generated

from the Listeria cultures tested, all seafood matrices

imparted a protective effect against thermal inactivation.

HS Listeria cultures, when inoculated on a seafood matrix,

demonstrated their ability to increase D7o-values above that

seen in enrichment broth trials. When comparing D70-values

for each of the matrices, crab meat showed the most

protective effect, yielding an increase of 2.38 minutes.

The D7o-values for cooked shrimp and smoked salmon increased

2.20 and 2.11 minutes, respectively. There was no direct

correlation between the fat content and increased thermal

resistance for the matrices examined in this study. This

observation was supported in that the seafood that had the

highest fat content, smoked salmon at 8 g of fat per

serving, yielding the smallest increase in D70-value.

Research conducted Ben Embarek et al. (1993) also

noted an increase in D-values associated with Listeria

inoculated on seafood, although it was concluded that fat

content had a significant role in the increased

survivability. L. monocytogenes strains were inoculated on

several species of vacuum-packaged fish fillets and D6o-

values of the heated strains were compared with strains

inoculated in enrichment both. D60-values calculated were









found to be four times higher in the cultures inoculated on

the seafood matrices.6)

The protection effect seen in this study also concurs

with another thermal inactivation study conducted by

Casadei et al. (1998). In this experiment the Do6-values of

L. monocytogenes in TSB were compared to D60-values of L.

monocytogenes inoculated in cream. The researchers

observed a 23 to 47 seconds increase in the experimental

D6o-values in TSB and cream, respectively. 15

According to the current scientific literature, the

data presented in this research was within the recognized

range established for D70-values seen for Listeria spp.

Normalized D7o-values were calculated by using known D- and

Z-values published for L. monocytogenes from other relevant

research which utilized temperatures other than 700C.

Calculated D70-values from this study ranged from 0.008 to

34.4 minutes.127 This wide variation observed could be due

to differences in matrices, bacterial strain and/or growing

conditions utilized. The numbers found in this research

were consistent with normalized D70-values found in similar

studies.




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EE0Y7GDRO_P0J6JX INGEST_TIME 2013-09-28T00:32:05Z PACKAGE AA00014268_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES