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2-3-2014 Investigating the Potential of Plant-derived Antimicrobials and Probiotic Bacteria for Controlling Listeria monocytogenes Abhinav Upadhyay University of Connecticut - Storrs, [email protected]

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Recommended Citation Upadhyay, Abhinav, "Investigating the Potential of Plant-derived Antimicrobials and Probiotic Bacteria for Controlling Listeria monocytogenes" (2014). Doctoral Dissertations. 326. https://opencommons.uconn.edu/dissertations/326 Investigating the Potential of Plant-derived Antimicrobials and Probiotic Bacteria

for Controlling Listeria monocytogenes

Abhinav Upadhyay, PhD

University of Connecticut, 2014

Listeria monocytogenes has emerged as one of the major foodborne pathogens resulting

in multiple foodborne outbreaks, characterized by high hospitalizations and case fatality rates.

The ubiquitous distribution of L. monocytogenes in the environment along with its ability to form

biofilms results in frequent contamination of food processing facilities and food products. The

drug of choice for treating listeriosis has been antibiotics, however, there have been reports of

development of antibiotic resistance in L. monocytogenes , and multidrug resistant strains have

been isolated from cattle carcasses and meat. The increasing antibiotic resistance in L.

monocytogenes and growing concerns over the use of synthetic chemicals in the food industry

have generated an interest in exploring the potential of various natural approaches as an

alternative strategy to prevent food contamination and control listeriosis in humans . In this

dissertation, the efficacy of six plant-derived antimicrobials (PDAs), namely trans-

cinnamaldehyde, carvacrol, thymol, eugenol, β-resorcylic acid and caprylic acid, and five

probiotic bacteria, namely Bifidobacterium bifidum (NRRL-B41410), Lactobacillus reuteri (B-

14172), L. fermentum (B-1840), L. plantarum (B-4496), and Lactococcus lactis subspecies lactis

(B-633) was investigated for controlling L. monocytogenes . Specifically, the efficacy of aforementioned PDAs in reducing L. monocytogenes biofilms on abiotic surfaces, and pathogen populations on popular high-risk foods such as frankfurter and cantaloupe was investigated. In Abhinav Upadhyay- University of Connecticut, 2014

addition, the efficacy of PDAs and probiotic bacteria in attenuating L. monocytogenes virulence was studied in vitro and in an invertebrate model, Galleria mellonella. Low concentrations of trans-cinnamaldehyde, carvacrol, thymol, and eugenol significantly reduced L. monocytogenes biofilm formation, while concentrations greater than the minimum bactericidal concentrations of

PDAs rapidly inactivated pre-formed biofilms. Trans-cinnamaldehyde and β-resorcylic acid also significantly reduced L. monocytogenes counts on frankfurters and cantaloupes. In addition, trans-cinnamaldehyde, carvacrol, thymol, and eugenol either alone or in combination with probiotic bacteria reduced L. monocytogenes motility, adhesion, invasion of intestinal epithelial cells, production of virulence factor listeriolysin O, and the expression of virulence genes critical for motility, adhesion-invasion, intracellular survival, and cell-to-cell spread in the host.

Moreover, the PDA-probiotic treatments significantly enhanced the survival of G. mellonella infected with lethal doses of L. monocytogenes. The above results underscore the potential use of aforementioned PDAs and probiotics in reducing L. monocytogenes on foods, and controlling listeriosis in humans.

2

Investigating the Potential of Plant-derived Antimicrobials and Probiotic Bacteria

for Controlling Listeria monocytogenes

Abhinav Upadhyay

B.V.Sc & A.H., Rajiv Gandhi Institute of Veterinary Education and Research, 2007

M.V.Sc., Rajasthan University of Veterinary and Animal Sciences, 2009

A Dissertation

Submitted in Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy

at the

University of Connecticut

2014

ii APPROVAL PAGE

Doctor of Philosophy Dissertation

Investigating the Potential of Plant-derived Antimicrobials and Probiotic Bacteria

for Controlling Listeria monocytogenes

Presented by

Abhinav Upadhyay, B.V.Sc. & A.H., M.V.Sc.

Major Advisor……………………………………………………………….

Dr. Kumar Venkitanarayanan

Associate Advisor……………………………………………………………

Dr. Cameron Faustman

Associate Advisor…………………………………………………………….

Dr. Kristen Govoni

Associate Advisor…………………………………………………………….

Dr. Paulo Verardi

University of Connecticut

2014

i ACKNOWLEDGEMENTS

I would like to express my deepest admiration and gratitude to my major advisor Dr.

Kumar Venkitanarayanan, for his guidance, support and motivation from the very early stage of my PhD program at University of Connecticut. I would never find words to thank him for his dedication to scientific research that made my job a lot easier. His perspicacious remarks, capacity to combine critique with empathy, penchant for punctuality and involvement has nourished my intellectual maturity that I will benefit from, for a long time to come.

I would like to extend my gratitude to Dr. Cameron Faustman, Dr. Kristen Govoni and Dr.

Paulo Verardi for their teachings, constructive suggestions, and encouragement during my research.

I also want to take this opportunity to thank my advisors from veterinary school, Dr. S.

Ram Kumar and Dr. A. K. Kataria who were instrumental in motivating and helping me to pursue doctoral program at University of Connecticut.

I would like to thank my senior colleagues at the Food microbiology laboratory, Dr. Anup

Kollanoor-Johny, Dr. Mary Anne Roshni Amalaradjou, and Dr. Sangeetha Ananda Bhaskaran for their help. Heartfelt thanks are also due to my lab mates Indu, Shan, Deepti, Hsin-Bai, Varun,

Meera, Genevieve, and Samantha for their support at all times. Many thanks go in particular to my Husky friends, Uday, Nidhish, Ansin, Apoorba, Ananya, Bushra, and the Bajrangiis

(Robbins, Sharad, Vineet, Saurabh, Gaurav and Ujjwal) for being my surrogate family here. I also thank my friends Meenakshi, Sid, Amit, Vishesh, Shruti, and Khushboo for their encouragement and belief in my dreams.

ii I would also like to extend my heartfelt appreciation to the faculty and staff of the

Department of Animal Science for their assistance and co-operation with various administrative works.

My parents deserve special mention for their unflinching support, prayers and sacrifice.

My father, Dr. Amlendu Kant Upadhyay, who made me realize and enjoy intellectual pursuit and my mother, Mrs. Anubha Upadhyay, the one who sincerely raised me with her caring and gentle love. I am also thankful to my relatives and my cousins Sadam, Zulu and Barbie for their affection.

Finally, I would like to thank everybody who was important to the successful completion of the thesis, as well as express my apology that I could not mention personally one by one.

iii TABLE OF CONTENTS

Approval page…………………………………………………………………………………….i

Acknowledgements……………………………………………………………………………….ii

Table of contents………………………………………………………………………………….iv

List of figures……………………………………………………………………………………..x

List of tables…………………………………………………………………………………….xiv

List of abbreviations…………………………………………………………………………….xvi

Chapter I: Introduction…………………………………………………………………………1

Chapter II: Review of Literature……………………………………………………………….6

1. Taxonomy of Listeria …………………………………………………………………………...8

2. Morphology, biochemical and physiological characteristics of L. monocytogenes……………. .9

3. Epidemiology of Listeria monocytogenes...... 10

3.1 Listeria in the environment...... 11

3.2 Listeria in food animals and products…...... 12

3.3 Listeria biofilms……...... 12

4. Mechanisms of survival...... 13

5. Listeria Genomics...... 14

6. Pathogenesis of Listeria infection in humans...... 15

6.1 Pathophysiology...... 15

6.2 Molecular determinants involved in host colonization and intracellular survival...... 17

6.3 Molecular determinants responsible for virulence regulation…...... 18

7. Antimicrobial resistance in Listeria monocytogenes…...... 20

8. Plant derived antimicrobials and probiotics…...... 21

iv 8.1 Control of L. monocytogenes in food processing environment using PDAs and probiotic

bacteria...... 23

8.2 Control of L. monocytogenes in foods using PDAs and probiotic bacteria...... 26

8.3 Control of L. monocytogenes virulence using PDAs and probiotic bacteria...... 32

References...... 37

Chapter III: Antibiofilm effect of plant-derived antimicrobials

on Listeria monocytogenes...... 67

Abstract…………………………………………………………………………………………..68

1. Introduction…………………………………………………………………………………....69

2. Materials and Methods……………………….……….……….……….……….………...... 72

2.1 Bacterial strains and culture conditions...... 72

2.2 Plant-derived antimicrobials and determination of SIC and MBC ...... 73

2.3 Biofilm inhibition and inactivation assay on polystyrene microtiter plates ………...... 73

2.3.1 Organic matter contamination on polystyrene plates ...... 74

2.4 Preparation of stainless steel coupons ...... 75

2.5 Biofilm inhibition and inactivation assay on stainless steel coupons...... 75

2.5.1 Organic matter contamination on stainless steel coupons ...... 76

2.6 Quantification of exopolysaccharide (EPS) production by ruthenium red staining...... 76

2.7 RNA isolation and real-time quantitative PCR ...... 77

2.8 Confocal microscopy ...... 78

2.9 Statistical analysis ...... 79

3. Results...... 79

3.1 Determination of SIC and MBC of plant-derived antimicrobials ...... 79

v 3.2 Effect of SICs of PDAs on L. monocytogenes biofilm formation on polystyrene microtiter

plates and stainless steel coupons ...... 79

3.3 Effect of PDAs in inactivating pre-formed L. monocytogenes biofilm on polystyrene

microtiter plates and stainless steel coupons ...... 80

3.4 Efficacy of PDAs in inactivating LM biofilms in presence of organic matter...... 81

3.5 Effect of PDAs on EPS production ...... 81

3.6 Confocal microscopy ...... 81

3.7 Effect of PDAs on expression of genes critical for biofilm formation in L. monocytogenes

4. Discussion...... 82

References...... 87

Chapter IV: Inactivation of Listeria monocytogenes on frankfurters by

plant-derived antimicrobials alone or in combination with hydrogen peroxide...108

Abstract...... 109

1. Introduction...... 111

2. Materials and Methods...... 113

2.1. Bacterial strains and culture conditions ...... 113

2.2. Frankfurters ...... 113

2.3. Inoculation and treatments ...... 114

2.4. Enumeration of L. monocytogenes on frankfurters ...... 115

2.5. pH determination ...... 115

2.6. Statistical analysis ...... 115

3. Results and Discussion...... 116

vi References...... 120

Chapter V: Efficacy of plant-derived antimicrobials combined with hydrogen peroxide as

antimicrobial wash and coating treatment for reducing

Listeria monocytogenes on cantaloupes...... 127

Abstract...... 128

1. Introduction…………………………………………………………………………………..129

2. Materials and Methods……………………………..……………………………...... 131

2.1. Bacterial strains, growth conditions and inoculum preparation ……………...... 131

2.2. Preparation and inoculation of cantaloupe rind plugs ……………………………...... 132

2.3. PDA wash treatment of cantaloupe rind plugs ……………………………...... 133

2.4. L. monocytogenes transfer from cantaloupe surface to inside while cutting...... 134

2.5. Preparation of PDA coating solution and application ……………………………...... 135

2.6. Statistical analysis ……………………………..……………………………...... 136

3. Results……………………………..……………………………..…………………...... 136

4. Discussion……………………………..………………………………….……….………....138

References……………………………..……………………………..…………………………143

Chapter VI: Effect of plant-derived antimicrobials and probiotic bacteria on

Listeria monocytogenes virulence in vitro and in invertebrate model, Galleria mellonella.. 156

Abstract……………………………..……………………………..……………………………157

1. Introduction…………………………………………………………………………………..158

2. Materials and Methods……………………………..……………………………..…….……160

vii 2.1 Bacterial strains and growth conditions ……….……….……….……...... ….……….160

2.2 Plant-derived antimicrobials, probiotics and SIC determination ………...... ….…….161

2.3 Motility assay ……….……….……….……….……….……….……...... ….……….162

2.4 Cell Culture ……….……….……….……….……….……….……...... ….……….162

2.4.1 Adhesion and invasion assay ……….……….……….……….………...... ……….162

2.5 E-Cadherin binding assay ……….……….……….……….……….………...... ……….163

2.6 Hemolysis assay ……….……….……….……….……….……….……….…...... …….164

2.7 RNA isolation and real-time quantitative PCR ……….……….……….………...... 165

2.8 In vivo studies using Galleria mellonella larvae ……….……….……….………...... 166

2.8.1 Galleria mellonella injection and determination of L. monocytogenes

infectious dose...... 166

2.8.2 Galleria mellonella survival assay ...... 167

2.8.3 Galleria mellonella peptide assay ……….……….……….……….……….……….....167

2.8.4 Statistical analysis ……….……….……….……….……….……….……….………...168

3. Results

3.1 Determination of SICs of plant compounds and probiotic culture supernatant ………...168

3.2 Effect of SICs of PDAs with or without probiotic supernatant on L. monocytogenes

motility ……….……….……….……….……….……….……….……….……….………....169

3.3 Effect of SICs of PDAs either alone or in combination with probiotic bacteria on L.

monocytogenes adhesion to and invasion of Caco-2 cells. ……….……….……….……….169

3.4 Effect of SICs of PDAs either alone or in combination with probiotic bacteria on L.

monocytogenes binding to human epithelial E-Cadherin. ……….……….……….………..170

viii 3.5 Effect of SICs of PDAs either alone or in combination with probiotic culture supernatant

on L. monocytogenes hemolysin production. ……….……….……….………...... 170

3.6 Effect of SICs of PDAs and probiotic supernatants on expression of L. monocytogenes

virulence genes and G. mellonella antimicrobial peptide genes. ……….……….……….....171

3.7 Effect of PDAs and probiotic culture supernatant on G. mellonella survival...... 171

4. Discussion...... 172

References...... 214

Chapter VIII: Summary ...... 223

ix List of Figures

Title Page

Chapter III

FIG. 1 Effect of MIC of trans-cinnamaldehyde (TC; 5.0, 10.0 mM), carvacrol (CR; 101

5.0, 10.0 mM), thymol (TY; 3.3, 5.0 mM) and eugenol (EG; 18.5, 25.0 mM)

on Listeria monocytogenes Scott A biofilm on microtiter plates at 37°C (A),

25°C (B), and 4°C (C).

FIG. 2 Effect of MIC of trans-cinnamaldehyde (TC; 5.0, 10.0 mM), carvacrol (CR; 103

5.0, 10.0 mM), thymol (TY; 3.3, 5.0 mM) and eugenol (EG; 18.5, 25.0 mM)

on Listeria monocytogenes Scott A biofilm on stainless steel coupons at 37°C

(A), 25°C (B), and 4°C (C).

FIG. 3 Scanning confocal micrographs of Listeria monocytogenes Scott A biofilm 105

before PDA treatment (A) and after treatment with MICs of (B) trans-

cinnamaldehyde (TC; 5.0 mM), (C) carvacrol (CR; 5.0 mM), (D) thymol

(TY; 3.3 mM) and (E) eugenol (EG; 18.5 mM).

FIG. 4 Effect of SICs of trans-cinnamaldehyde (TC; 0.05, 0.75 mM), carvacrol (CR; 106

0.50, 0.65 mM), thymol (TY; 0.33, 0.50 mM) and eugenol (EG; 1.8, 2.5 mM)

on exopolysaccharide production in Listeria monocytogenes Scott A biofilm

on microtiter plates at 37°C (A), 25°C (B), and 4°C (C).

x Chapter IV

FIG. 1 Inactivation of Listeria monocytogenes on frankfurters by dip treatment with 124

β-resorcylic acid (BR; 1.5%), carvacrol (CR, 0.75%), or trans-

cinnamaldehyde (TC; 0.75%) alone or in combination with hydrogen

peroxide (HP, 0.1%) at 55°C for 60 s.

FIG. 2 Inactivation of Listeria monocytogenes on frankfurters by dip treatment with 125

β-resorcylic acid (BR; 1.5%), carvacrol (CR, 0.75%), or trans-

cinnamaldehyde (TC; 0.75%) alone or in combination with hydrogen

peroxide (HP, 0.1%) at 65°C for 30 s.

Chapter V

FIG. 1 Inactivation of L. monocytogenes on cantaloupe surface by wash treatment 149

with caprylic acid (CA), carvacrol (CR), thymol (TH), β-resorcylic acid (BR)

alone or in combination with hydrogen peroxide (HP) at (A) 25°C for 3, 6, and

10 min (B) 55°C for 1, 3, and 5 min (C) 65°C for 1, 3, and 5 min.

Chapter VI

FIG. 1 Effect of SICs of (A) trans-cinnamaldehyde (TC), (B) carvacrol (CR), (C) 184

thymol (TY) and (D) eugenol (EG) either alone or in combination with

probiotic bacterial supernatant (6.25, 12.5%) on Listeria monocytogenes

(Scott A) motility.

xi FIG. 2 Effect of SICs of (A) trans-cinnamaldehyde (TC), (B) carvacrol (CR), (C) 188

thymol (TY) and (D) eugenol (EG) either alone or in combination with

probiotic bacteria on Listeria monocytogenes (Scott A) adhesion to Caco-2.

FIG. 3 Effect of SICs of (A) trans-cinnamaldehyde (TC), (B) carvacrol (CR), (C) 192

thymol (TY) and (D) eugenol (EG) either alone or in combination with

probiotic bacteria on Listeria monocytogenes (Scott A) invasion of Caco-2.

FIG. 4 Effect of Listeria. monocytogenes (Scott A) concentration on E-Cadherin 196

binding.

FIG.5 Effect of SICs of (A) trans-cinnamaldehyde (TC), (B) carvacrol (CR) (C) 197

thymol (TY) and (D) eugenol (EG) either alone or in combination with

probiotic bacteria on E-cadherin binding of Listeria monocytogenes (Scott A).

FIG. 6 Effect of SICs of (A) plant-derived antimicrobials, (B) probiotic supernatant or 201

(C) combination on hemolysis of 3% sheep RBC by Listeria monocytogenes

(Scott A).

FIG. 7A Dose-dependent survival of Galleria mellonella larvae inoculated with 206

Listeria monocytogenes (Scott A).

xii FIG. 7B Effect of SICs of plant-derived antimicrobials (PDAs) on survival of Galleria 207

mellonella larvae inoculated with Listeria monocytogenes (Scott A).

FIG. 7C Effect of SICs of probiotic culture supernatant on survival of Galleria 208

mellonella larvae inoculated with Listeria monocytogenes (Scott A).

FIG. 7D Effect of SICs of plant-derived antimicrobials (PDAs) either alone or in 209

combination with Bifidobacterium bifidum culture supernatant on survival of

Galleria mellonella larvae inoculated with Listeria monocytogenes (Scott A).

FIG. 8 Relative fold change in the expression level of antimicrobial peptide genes 210

(gallerimycin, lysozyme) of uninoculated Galleria mellonella in response to

SICs of (A) TC, CR, TY, and EG (B) probiotic cultures supernatant (12.5%) or

(C) their combinations.

FIG. 9 Relative fold change in the expression level of antimicrobial peptide genes 212

(gallerimycin, lysozyme) of Galleria mellonella inoculated with

Listeria monocytogenes in response to SICs of (A) TC, CR, TY, and EG (B)

probiotic cultures supernatant (12.5%) or (C) their combinations.

xiii

List of Tables

Chapter III

TABLE 1. List of primers used in the study 94

TABLE 2. Effect of sub-inhibitory concentrations of trans-cinnamaldehyde (TC), 96

carvacrol (CR), thymol (TY) and eugenol (EG) on Listeria monocytogenes

biofilm formation on microtiter plates at 37°C (A), 25°C (B), and 4°C (C).

The treatments included control, TC (0.50 mM, 0.75 mM), CR (0.50 mM,

0.65 mM), TY (0.33 mM , 0.50 mM), EG (1.8 mM, 2.5 mM).

TABLE 3. Effect of sub-inhibitory concentrations of trans-cinnamaldehyde (TC), 98

carvacrol (CR), thymol (TY) and eugenol (EG) on Listeria monocytogenes

biofilm formation on stainless steel coupons at 37°C (A), 25°C (B), and

4°C (C). The treatments included control, TC (0.5 mM, 0.75 mM), CR (0.5

mM, 0.65 mM), TY (0.33 mM, 0.50 mM), EG (1.8 mM, 2.5 mM).

TABLE 4. Relative fold change in the expression level of Listeria monocytogenes 100

biofilm associated genes in response to SICs of TC, CR, TY, and EG.

xiv Chapter IV

TABLE 1. Effect of PDA and HP dip treatments on pH of frankfurters. 126

Chapter V

TABLE 1. PDA wash and coating treatments 152

TABLE 2. Effect of PDA-HP wash treatments at (A) 55°C for 5 min and (B) 65°C for 153

5 min on transfer of Listeria monocytogenes from cantaloupe surface to

inside during cutting. Treatments included caprylic acid (CA), carvacrol

(CR), thymol (TY), β-resorcylic acid (BR) in combination with hydrogen

peroxide (HP).

TABLE 3A. Effect of chitosan based coating of cantaloupe surface with caprylic acid 154

(CA), carvacrol (CR), thymol (TY), β-resorcylic acid (BR) alone or in

combination with hydrogen peroxide (HP) on Listeria monocytogenes.

TABLE 3B. Survival of Listeria monocytogenes on cantaloupe surface pre-coated with 155

caprylic acid (CA), carvacrol (CR), thymol (TY), β-resorcylic acid (BR)

alone or in combination with hydrogen peroxide (HP).

CHAPTER VI

TABLE 1. List of primers used in the study 179

xv TABLE 2. Relative fold change in the expression level of L. monocytogenes virulence 181

genes in response to SICs of (A) TC, CR, TY, and EG (B) probiotic

cultures supernatant (12.5%) or (C) their combinations.

xvi List of Abbreviations

BR Beta-Resorcylic acid

CA Caprylic acid

CDC Centers for Disease Control and Prevention

CR Carvacrol

DMSO Dimethyl sulfoxide

EG Eugenol

EPS Extracellular polymeric substance

FAO Food and Agriculture Organization

FDA Food and Drug Administration

GRAS Generally recognized as safe

HACCP Hazard analysis and critical control points

HP Hydrogen peroxide

LM Listeria monocytogenes

MBC Minimum bactericidal concentration

MRS DeMan-Rogosa-Sharpe media

PDA Plant-derived antimicrobial

RT-qPCR Real-time quantitative polymerase chain reaction

RTE Ready to eat

SAS Statistical analysis software

SIC Sub-inhibitory concentration

xvii TC Trans-cinnamaldehyde

TSA Tryptic soy agar

TSB Tryptic soy broth

TY Thymol

USDA United States Department of Agriculture

USDA FSIS United States Department of Agriculture Food Safety and Inspection Service

WHO World Health Organization

xviii

Chapter I

Introduction

1 Despite significant advancements in food safety, foodborne illnesses remain a major

challenge in the United States. Annually, an estimated 48 million foodborne illnesses, 128,000

hospitalizations and 3,000 deaths are caused by foodborne pathogens in the United States

(Scallan et al., 2011), which amount to estimated healthcare costs of $77.7 billion (Scharff,

2012). Listeria monocytogenes has emerged as one of the major foodborne pathogens resulting in nearly 19% of all deaths due to foodborne infections in the US (Scallan et al., 2011; WHO,

2012). Recognized as a foodborne pathogen in the 1980s (Schlech et al., 1983), this bacterium has been implicated in more than 24 multistate foodborne outbreaks with high hospitalization rates and case fatalities in the past two decades (Cartwright, 2013).

Listeriosis occurs by the ingestion of contaminated foods, especially ready-to-eat (RTE) meat products (USFSIS, 2004), soft cheese, unpasteurized milk (Latorre et al., 2011) and fresh produce such as cantaloupes (Scallan et al., 2011). The most susceptible population includes the elderly, infants, immuno-compromised patients and pregnant women. The major symptoms of listeriosis include febrile gastroenteritis, flu-like manifestations followed by septicemia and meningitis (Begley et al., 2003). In pregnant women, infection may result in placentitis leading to abortion, stillbirth or premature termination of pregnancy (Rocourt and bille, 1997). Unlike most foodborne infections, listeriosis is characterized by a high case fatality rate of nearly 20 to

30% in susceptible populations (Schuchat et al., 1991).

Due to the ubiquitous distribution of L. monocytogenes in the environment (Beuchat,

1996; Steele and Odumeru, 2004) along with its ability to tolerate desiccation, fluctuating temperatures, low pH and high osmalarity, L. monocytogenes is a frequent contaminant of food

processing and packaging facilities (Chaturongakul et al., 2008; Donnelly, 2001). Moreover, L.

monocytogenes’ presence in soil, decaying vegetation, and irrigation water increases the risk of

2 potential contamination of fresh produce during the pre-harvest stage in fields (Hedberg et al.,

1994; Tauxe, 1997). In food processing facilities, L. monocytogenes is able to attach and form biofilms on a variety of surfaces, including polystyrene, stainless steel, polymers, plastic, teflon and rubber (Borucki et al., 2003; Chavant et al., 2002; Moretro and Langsrud, 2004). Once L. monocytogenes forms biofilms, it can survive for extended periods of time (Fatemi and Frank,

1999). The persistence of L. monocytogenes in food processing environments constitutes a significant food safety hazard, since biofilms protect the underlying bacteria from sanitizers

(Borucki et al., 2003; Folsom and Frank, 2007), and serve as a continuous source for contamination of food products.

Once inside the host, L. monocytogenes adheres to and invades the intestinal epithelium, followed by systemic spread to other organs via lymphatics and blood (Parida et al., 1998). An array of cell surface proteins and virulence factors critical for attachment to and invasion of intestinal epithelium, subsequent escape into cytoplasm, intracellular proliferation, and cell-to- cell spread have been characterized in L. monocytogenes (Vazquez-Boland et al., 2001).

The drug of choice for the treatment of listeriosis has been antibiotics such as β-lactam and cephalosporins. However, there have been reports of development of antibiotic resistance in

L. monocytogenes (Krawczk-Balska et al., 2012; Cekmez et al., 2012), and multidrug resistant strains have been isolated from cattle carcasses (Wieczorek, et al., 2012) and meat (Pesavento et al., 2010). This increasing antibiotic resistance in L. monocytogenes and growing concerns over the use of synthetic chemicals in the food industry has ignited an interest in exploring the potential of various natural approaches as an alternative strategy to control L. monocytogenes on foods and combat foodborne listeriosis in humans.

3 Since ancient times, plant extracts have been used as food preservatives, flavor enhancers and dietary supplements to prevent food spoilage, and maintain human health. In addition, fermented foods containing beneficial microbiota have been a part of traditional diets for their health benefits. The antimicrobial activity of several plant-derived compounds has been documented (Burt, 2004; Holley and Patel, 2005; Nychas and Skandamis, 2003; Osbourn, 1996;

Hoet et al., 2004; Tagboto and Townson, 2001; Ginsburg et al., 2011; Antony and Singh, 2011;

Nash et al., 2011; Shahid et al., 2009; Newman and Cragg, 2012), and an array of active components has been identified (Dixon, 2001). A majority of these compounds are secondary metabolites, and are produced as a result of reciprocal interactions between plants, microbes and animals (Reichling, 2010). Some of the plant-derived compounds that have been reported to possess significant antimicrobial properties include trans-cinnamaldehyde (TC), eugenol (EG), thymol (TY), carvacrol (CR), β-resorcylic acid (BR) and caprylic acid (CA). All these compounds have been shown to exert antimicrobial effects against Gram-positive and Gram- negative bacteria (Cosentino et al., 1999; Dorman and Deans, 2000; Mitsch et al., 2004; Si et al.,

2006).

The Food and Agriculture Organization (FAO) and World Health Organization (WHO) define probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host.” Probiotic bacteria exert multiple benefits to the host, including protection against enteric pathogens (Candela et al., 2008; Fukuda et al., 2011), facilitating digestion and assimilation of nutrients (Sonnenburg et al. 2005; Yatsunenko et al. 2012) and potentiating host immune function (Olszak, T. et al. 2012). Although several species have been identified, the majority of probiotic bacteria supplemented in human diets belong to Lactobacilli and Bifidobacteria (Fuller, 1989). Interestingly, recent studies have shown that plant-derived

4 antimicrobials (PDAs) that are highly bactericidal towards enteric pathogens exert low

antimicrobial effect against commensal gut microbiota (Hawrelak et al., 2009; Pasqua et al.,

2005). Since PDAs and probiotics exert their antimicrobial effects by different mechanisms

(Shipradeep et al., 2012), a combinatorial approach using both could be more effective in

controlling pathogens as compared to their usage separately. However, research investigating

their synergistic interactions on bacterial virulence is scanty. Thus it was hypothesized that plant-

derived compounds TC, CR, TY, EG, CA, and BR exert significant antimicrobial/antibiofilm

effects against L. monocytogenes on foods and abiotic surfaces. Moreover, the PDAs alone or in combination with probiotic bacteria reduce L. monocytogenes virulence.

The overall objective of this dissertation proposal was to investigate the antimicrobial

potential of PDAs and selected probiotic bacteria for controlling L. monocytogenes . The specific

objectives include:

1. To investigate the efficacy of TC, CR, TY and EG in controlling L. monocytogenes biofilm

on abiotic surfaces.

2. To investigate the efficacy of TC, CR, TY, EG, BR and CA as an antimicrobial dip for

reducing L. monocytogenes on skinless frankfurter.

3. To investigate the efficacy of TC, CR, TY, EG, BR and CA as an antimicrobial wash and

coating for reducing L. monocytogenes on cantaloupes.

4. To investigate the efficacy of TC, CR, TY and EG alone or in combination with probiotic

bacteria, Bifidobacterium and Lactobacillus in reducing L. monocytogenes virulence in vitro

and in the invertebrate model, Galleria mellonella.

5

Chapter II

Review of Literature

6 Listeria monocytogenes, formerly Bacterium monocytogenes, is a Gram-positive, motile, facultative anaerobic, non-spore forming, rod-shaped bacterium belonging to the Firmicutes phylum. The bacterium was first described in 1926 by E. G. D. Murray in Cambridge, United

Kingdom, as a cause of epizootic infection in laboratory rabbits and guinea pigs (Murray et al.,

1926). In the following year, J. H. H. Pirie isolated the organism from wild gerbils in South

Africa (Pirie et al., 1927), and gave the bacterium its present name in 1940 (Gray and Killinger,

1966). Nyfeldt reported the first confirmed case of human listeriosis in 1929, and since then, sporadic cases of human illnesses have been recorded, often as a result of zoonotic transmission of the bacterium from infected animals (Cain and McCann, 1986). However, during the early to mid-1980s, a significant rise in the number of foodborne human listeriosis cases in North

America and Europe was observed. Multiple listeriosis outbreaks were linked to the consumption of contaminated coleslaw (Schlech et al., 1983), milk (Fleming et al., 1985), and soft cheese

(Bille and Glauser, 1988; Linnan et al., 1988). These outbreaks, characterized by severe septicemia, meningitis, meningoencephalitis, abortions, and high case-fatality rates of 20 to 40%

(Farber and Peterkin, 1991) demonstrated L. monocytogenes as a major foodborne pathogen with a significant impact on public health and the food industry.

Analysis of L. monocytogenes revealed that the organism possesses a majority of characteristics required for being a successful foodborne pathogen, including the ability to survive in food processing environments and a variety of food products, tolerance to a wide variety of stresses commonly encountered during food preservation, and ability to cause a potentially fatal disease in humans. Listeriosis was added to the list of nationally notifiable diseases in 2001 (CDC, 2013a). Despite a low incidence rate of infection (0.25 per 100,000),

7 recent estimates rank listeriosis as the second most common cause of death from food-borne bacterial infections in the United States (CDC, 2011, 2013a).

1. Taxonomy of Listeria:

The genus Listeria contains eight species, namely L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L seeligeri, L. grayi, and the newly discovered L. marthii (Graves et al.,

2010) and L. rocourtiae (Leclercq et al., 2009). L. monocytogenes is pathogenic to humans and animals (Robinson et al, 2000), whereas L. ivanovii is primarily an animal pathogen, affecting large and small ruminants (Cossart, 2011). The other species of Listeria are nonpathogenic free- living saprophytes, and no human illnesses have been documented due to these species.

L. monocytogenes strains are differentiated based on serological reactions of somatic (O) and flagellar (H) antigens into 14 serotypes, comprising of 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab,

4b, 4c, 4d, 4e, 7 and nt (Liu et al., 2006; Gianfranceschi et al., 2009). Strains of L. monocytogenes within serovars 1/2a, 1/2b, 1/2c and 4b are responsible for greater than 98% of human infections (Borucki and Call, 2003; Liu et al., 2006). Based on pulse-field gel electrophoresis and multilocus enzyme electrophoresis, two lineages of L. monocytogenes were initially identified, with a third lineage subsequently recognized based on virulence gene variation, ribotyping, and DNA arrays (Piffaretti, et al., 1989; Rasmussen et al., 1991; Graves et al., 1994; Brosch et al., 1994; Wiedmann et al., 1997; Doumith et al., 2004). Lineage I includes serotypes 1/2b, 3b, 4b, 4d, 4e and 7, lineage II contains serotypes 1/2a, 1/2c, 3a, and 3c, whereas lineage III comprises of serotype 4a and 4c. The different lineages are further classified into clonal complexes using multilocus sequence typing. The precise delineation of lineages and clonal complexes is necessary to characterize the association between genetic diversity,

8 pathogenic potential and virulence in this organism, which could be useful for the development of effective measures to control foodborne listeriosis.

2. Morphology, biochemical and physiological characteristics of L. monocytogenes

All members of Listeria form short rods, 0.4 to 0.5 by 1 to 2 m, with blunt ends. The cell membrane of L. monocytogenes is 90 Å thick, and is composed of proteins (55 to 60%), lipids

(30 to 35%) and carbohydrates (1.3 to 2.3%). Lipids primarily include phospholipids (80 to 85%) such as phosphatidylglycerol and phosphoglycolipids (Bierne and Cossart, 2007; Verheul et al.,

1997). Greater than 90% of the total fatty acid content in L. monocytogenes membrane is branched chain fatty acids (Annous et al., 1997). The composition of fatty acids changes with temperature variation in the external environment. For example, cold stress induces an increase in the anteiso-C15:0 content to maintain optimal membrane fluidity, facilitating survival at refrigeration temperatures (Annous et al., 1997; Edgcomb et al., 2000; Nichols et al., 2002;

Schlech et al., 1983).

The cell wall of Listeria primarily contains peptidoglycan (35% dry weight of cell wall), teichoic acids (60% dry weight of cell wall), and lipoteichoic acids. The peptidoglycan layer contains cross-linkage with mesodiaminopimelic acid (Ghosh and Murray, 1967). The teichoic acid component contains N-acetylmuramic acid, N-acetylglucosamine, rhamnose, ribitol and phosphorus (Ullmann and Cameron, 1969; Hether and Jackson, 1983). Ribitol, rhamnose and lipoteichoic acid (O-factor) together with flagellar antigen (H-factor) form the basis for the classification of various serotypes described in Listeria (Fiedler, 1988; Seeliger and Hohne,

1979).

All species of Listeria are motile with five to six peritrichous flagella. Extensive research in the past few decades has revealed that flagella contribute to virulence in many foodborne

9 pathogens, either as the effectors of motility, colonization and invasion in intestine, or as

mediators of toxin secretion inside the host (O'Neil and Marquis, 2006). In addition, recent

evidence suggests that flagella participate in processes such as biofilm formation and modulation

of the host immune response (Duan et al., 2013). In L. monocytogenes, flagella aid biofilm

formation on surfaces in the external environment (Lemon et al., 2007) and facilitate intestinal

epithelial cell invasion in the host (Bigot et al., 2005; Dons et al., 2004). The transition from

ambient to human body temperature (37ºC) is sensed by means of a protein thermosensor,

GmaR, that transforms the changes in the environment into signals that affect flagellar function,

motility and pathogenesis inside the host (Kamp and Higgins, 2011).

Listeria spp. are relatively nutritionally undemanding. The metabolism is aerobic and facultatively anaerobic. L. monocytogenes can grow in a wide range of food products at temperatures ranging from 0 and 45ºC. The growth at low temperature is relatively slow, with a maximum doubling time of 1 to 2 days at 4ºC in dairy (Ryser and Marth, 2007) or meat products

(Hudson and Mott, 1993).

3. Epidemiology of Listeria monocytogenes

Epidemiological surveillance of foodborne pathogens plays a crucial role in implementing effective control measures and preventing foodborne illnesses. In the last three decades, the changing food production and distribution systems, globalization of the food market, and changing food habits such as increased consumption of refrigerated or ready-to-eat, minimally processed products, have increased the risk of foodborne infections, especially listeriosis. The widespread distribution of L. monocytogenes in diverse ecological niches such as water, soil, and vegetation, coupled with its ability to survive in food processing facilities leads to reoccurring food contamination and human infections. This is mediated by an extensive interacting network

10 of stress tolerance mechanisms that facilitate rapid adaptations to changing environments (Van

der Veen et al., 2007) such as variations in pH (5 to 9), temperature (1 to 44ºC) and salinity (up

to 12%) that are commonly encountered during food processing.

3.1 Listeria in the environment

Listeria spp. are commonly found in farm effluents, agro-industrial waste, sewage, and

river water, however, they are rarely isolated from unpolluted sea, springs or groundwater (Huss

et al., 2000; Schaffter and Parriaux, 2002; Hansen et al., 2006). Surface water has been reported

to possess a wide prevalence (6 to 60%) of L. monocytogenes (Wilkes, 2011). Colburn and

coworkers (1990) analyzed 37 fresh or low salinity estaurine samples from the California coast

and isolated 30 (81%) Listeria spp. and 19 (51%) L. monocytogenes strains. MacGowan and co- workers (1994) reported that approximately 93% (108/115) of sewage water samples were positive for Listeria spp, of which 60% (61/108) were L. monocytogenes. Nightingale and coworkers (2004) conducted a case-controlled study to understand the transmission and ecology of Listeria monocytogenes in a farm environment. The overall prevalence of L. monocytogenes

was approximately 24% on cattle farms and 32% on small ruminant farms with 30 and 45% soil

samples testing positive for the pathogen. MacGowan and coworkers (1994) obtained similar

results, where Listeria were present in 20 (14.7%) out of 130 soil samples from the United

Kingdom. In a recent cross-sectional study, L. monocytogenes was detected in 17.5% of fields

and 30% of irrigation and non-irrigation water samples (n=74) procured from 263 fields of 21

produce farms in New York State (Strawn et al., 2013). These reports suggest that the incidence

of Listeria spp. in water and soil potentially increases with an increase in animal and human

activity.

11 3.2 Listeria in food animals and products

A wide range of food types has been implicated in causing listeriosis, including seafood, meat, dairy and vegetable products. A majority of listeriosis outbreaks has been primarily due to the consumption of dairy and ready-to-eat (RTE) meat products such as frankfurters contaminated with L. monocytogenes (CDC 1999; CDC 2000; CDC 2002; Mead et al., 2006).

However, fresh produce has been increasingly associated with L. monocytogenes outbreaks in the last decade (Beuchat, 1996; CDC 2012a; FDA 2010). In 2011, a nation-wide foodborne outbreak, affecting 146 people from 28 states with 30 deaths and 1 miscarriage, was linked to the consumption of cantaloupes contaminated with L. monocytogenes (CDC, 2012b). In investigations following the outbreak, numerous deficiencies in fruit packing and processing, and plant sanitation were observed (FDA, 2011). In addition, livestock farms have been hypothesized as natural reservoirs of L. monocytogenes (Wesley, 1999) resulting from the contamination of feed or silage (Smith and Sherman, 1994), thereby leading to clinical infection or a latent carrier state in food animals, including cattle, sheep, goats, pigs and poultry (Wesley,

1999; Meng and Doyle, 1997; Pell, 1997). In food processing plants, L. monocytogenes present in raw material such as chicken, pork, beef or milk is effectively inactivated by the time- temperature treatment combinations approved for processed foods (Nightingale et al., 2004).

However, post-processing contamination of food products with L. monocytogenes can occur from the processing plant environment (Gravani, 1999).

3.3 Listeria biofilms

The environmental persistence of L. monocytogenes is attributed in part, to its ability to form biofilms. Biofilms are microbial communities adhered to biotic or abiotic surfaces enclosed in a self-produced extracellular polymeric matrix (Silva and Martinis, 2012). L. monocytogenes

12 is able to form biofilms of moderate thickness with average pathogen density of 10 4 to 10 7

CFU/cm 2 (Gram et al., 2007) on a variety of surfaces, including polystyrene, polypropylene,

glass, and stainless steel (Silva et al., 2008). In food-processing environments, L.

monocytogenes biofilms may represent a persistent source of food contamination (Chmielewski

and Frank, 2003; Brooks and Flint, 2008; Shi and Zhu, 2009) since they protect the pathogen

from adverse environmental factors such as desiccation, salinity, disinfectants, toxic metals,

acids, and antimicrobials (Hall-Stoodley et al., 2004; Carpentier and Cerf, 2011; Silva and

Martinis, 2012).

The major factors that modulate biofilm formation in L. monocytogenes include contact

surface properties (electrostatic charge and hydrophobicity), presence of flagella, serotype

diversity, extracellular polymeric substance (EPS) composition and environmental factors such

as pH and temperature (Borucki et al. 2003; Mai and Conner 2007; Palmer et al. 2007; Adrião et

al. 2008; Di Bonaventura et al. 2008; Harmsen et al. 2010). The EPS mediates bacterial adhesion

to surfaces, and provides protection by forming a tridimensional polymeric network around the

cells. In addition, it also modulates bacterial physiology inside the biofilm by facilitating enzyme

acquisition systems necessary for metabolism (Flemming and Winggender, 2010) and quorum

sensing. Recent research has revealed that horizontal gene transfer and biofilm formation are

interconnected, with biofilms being potential hot spots for genetic exchange between bacteria of

the same or different species (Madsen et al., 2012).

4. Mechanisms of survival

Being a robust foodborne pathogen, L. monocytogenes has the capability to tolerate a

variety of stresses encountered in the external environment or gastro-intestinal tract of humans.

13 Inside the stomach, L. monocytogenes , under the control of stress-responsive alternative sigma factor ( σB), synthesizes an array of compounds such as glutamate decarboxylase (GAD) and antiporter proteins BetL, Gbu, OpuC to tolerate extracellular acids. Specifically, GAD converts glutamate gamma aminobutyrate (GABA) via decarboxylation and transports it out of the cell.

This process utilizes cytoplasmic protons, thereby increasing cytoplasmic pH and facilitating survival in acidic environments (Sleator et al., 2005).

From the stomach, L. monocytogenes reaches the small intestine along with food

contents, where bile salts generate a high salinity environment (0.3 M NaCl). In response, L.

monocytogenes produces transporters BetL, Gbu, and OpuC for uptake of osmolytes such as

glycine, betaine and carnitine that prevent water loss from the bacterial cytoplasm. It also

secretes proteins Bsh (bile salt hydrolase) and BtlB (Bile tolerance locus B with bile acid

dehydrotase activity) to decrease the potency of bile salts. In addition, L. monocytogenes utilizes

the bile exclusion system (BilE) to remove excess bile salts from bacterial cytoplasm (Sleator et

al., 2005).

5. Listeria Genomics

The first genome sequence of L. monocytogenes (strain EGD-e, serovar 1/2a) and closely

related L. innocua was published in 2001 (Glaser et al., 2001). The sequence revealed a large

number of genes encoding surface proteins, sugar acquisition system, and transcriptional

regulators to facilitate survival in a variety of ecological niches. However, approximately 15% of

L. monocytogenes genes were found to be absent in the non-pathogenic L. innocua, particularly a

10-kb virulence locus LIPI-1, which encodes factors critical for intracellular survival in the host.

14

6. Pathogenesis of Listeria infection in humans

Listeria monocytogenes causes a severe, invasive, multi-systemic infection in high-risk

individuals that include the elderly, infants, pregnant women and immune-compromised patients.

In non-pregnant susceptible adults (50 to 70%), listeriosis is primarily associated with central

nervous system infection manifested as meningitis, meningoencephalitis, and brain abscess.

Bacteremia is another important manifestation of L. monocytogenes infection in adults that leads to septicemia, endocarditis, pneumonia, osteomyelitis and pyogranulomatous dermatitis (Slutsker and Schuchat, 1999; Gallaguer and Watakunakorn, 1998; Doganay, 2003). Fetomaternal or neonatal listeriosis results from maternal infection leading to colonization of the placenta, placentitis, fetal infection, stillbirth or abortion. However, in healthy humans, L. monocytogenes causes mild gastro-enteritis with flu like manifestations, without a concomitant invasive infection

(Cossart and Archambaud, 2009; Ooi and Lorber, 2005). Listeriosis has one of the highest hospitalization (90%) and case fatality rates (20 to 40%) of all food-borne infections (Vazquez-

Boland et al., 2001; Schuchat et al., 1991; Adak et al., 2002). Being a multi-host pathogen, L.

monocytogenes also infects a wide variety of animal species, including mammals and birds.

6.1 Pathophysiology

Since approximately 99% of L. monocytogenes infections occur via contaminated foods

(Swaminathan and Gerner-Smidt, 2007), the gastrointestinal tract is the major portal of entry of

L. monocytogenes into the host (Vazquez-Boland et al., 2001; Farber and Peterkin, 1991). L.

monocytogenes induces its own internalization without significantly affecting the host cellular

architecture and crosses the intestinal epithelial barrier, either via direct invasion of enterocytes

lining the absorptive epithelium or through phagocytosis by the M cells of Peyer’s patches

15 (Marco et al., 1997; Corr et al., 2006). The M cell translocation is non-specific, whereas the

enterocyte invasion mechanism involves specific ligand-receptor interactions. Apart from the

internalization step, the intracellular life cycle of the bacterium in phagocytes or nonphagocytic

mammalian cells is essentially identical. After crossing the intestinal epithelial barrier, L. monocytogenes localizes within macrophages in inflammatory foci present in the lamina propria, followed by their replication and dissemination to the mesenteric lymph nodes. Subsequently, through a lympho-hematogenous route, the pathogen reaches the spleen and liver, which are the primary target organs. Hepatocytes are highly permissive to receptor-mediated internalization and intracellular replication of L. monocytogenes , and are the primary site of colonization after

intestinal translocation. The infected cells become surrounded by neutrophils and macrophages,

leading to the development of pyogranulomatous lesions in the liver (Conlan and North, 1991;

Portnoy et al., 1992; Dramsi et al., 1995; Gregory and Liu, 2000). In mice, L. monocytogenes is

known to induce apoptosis of the splenic cells, leading to depletion of both CD4+ and CD8+

lymphocytes, thereby transiently attenuating early innate immune response and facilitating

bacterial colonization (Marco et al., 1991; Carrero et al., 2004a,b). However, it is unclear if a

similar process occurs in humans. In a healthy host, L. monocytogenes infectious foci present in

the primary target organs are efficiently contained by cytotoxic (CD8+) T lymphocytes, leading

to complete resolution of the pyogranulomas by 6 to 7 days after infection (Pamer, 2004).

However, in an immune-compromised host, the primary infectious foci are inefficiently

controlled, resulting in the dissemination of the pathogen to various organs in the body via

infected phagocytes (Berche, 1995; Blanot et al., 1997; Bakardjiev et al., 2006, Drevets, 1999;

Drevets et al., 1995).

16 The prognosis and outcome of listeriosis in humans depends on three major variables, namely dose, strain characteristics, and immune status of the host. In immune-competent individuals, ingestion of low to moderate doses of L. monocytogenes (<10 5 colony forming units,

CFUs) rarely cause the disease, however, higher doses (>10 6) may lead to acute gastroenteritis within 24 h of consumption of contaminated food.

6.2 Molecular determinants involved in host colonization and intracellular survival

Adhesion to, and invasion of, intestinal epithelial cells by L. monocytogenes is mediated by two bacterial surface proteins of the Internalin family, namely Internalin A (InlA) and Internalin B

(InlB) that bind to E-Cadherin and Met epithelial cell receptors, respectively (Hamon et al.,

2006; Pizarro-Cerda and Cossart, 2006; Bonazzi et al., 2009). Once inside the host cell, L. monocytogenes escapes from the phagocytic vacuole by means of the pore-forming toxin listeriolysin O (LLO). Pore formation starts by oligomerization of cholesterol-associated monomers that insert in the membrane lipid bilayer (Schnupf and Portnoy, 2007). The LLO- dependent perforation results in changes in vacuolar pH and calcium concentrations leading to membrane disruption. In addition, phospholipase A (PlcA) and phospholipase B (PlcB) facilitate the action of LLO, and are critical for the lysis of the secondary vacuole in the host cell

(Vazquez-Boland et al., 1992). Although the contribution of host factors is less well-defined, reports suggest that host response is crucial for LLO activation and release of the pathogen from phagosomes. For example, Singh and co-workers (2008) found that gamma-interferon-inducible lysosomal thiol oxidoreductase (GILT) reduces protein disulfide bonds in LLO, thereby leading to its activation in the phagosome by the thiol reductase mechanism. In addition, the phagosomal escape of L. monocytogenes was significantly delayed in GILT-negative macrophages. In another study, Radtke and co-workers, (2011) revealed that cystic fibrosis trans-membrane

17 conductance regulator (CFTR) protein transiently increases phagosomal chloride concentration

after infection, potentiating LLO pore formation, vacuole lysis, and bacterial escape into the

cytoplasm.

The listerial actin assembly-inducing protein (ActA) is another important virulence factor

found on bacterial cell surface that is critical for cell-to-cell spread of the pathogen. In the host

cell, ActA recruits the Arp2/3 complex leading to actin polymerization, formation of actin tail,

intracellular movement and dissemination of the pathogen intracellularly (Van Troys et al.,

2008). In addition to facilitating cell-to-cell spread of L. monocytogenes , ActA prevents autophagy of the pathogen by preventing sequestration of the bacterium by ubiquitinated protein that leads to autophagosome formation (Yoshikawa et al., 2009; Birmingham et al., 2007).

6.3 Molecular determinants responsible for virulence regulation

For facultative pathogens such as Listeria monocytogenes with two physiologically distinct lifestyles: saprophytic, primarily in decaying vegetation and soil; and parasitic in the host, it is critical to rapidly adapt to changing external conditions in order to ensure transmissibility to a host and perpetuation in the environment. The L. monocytogenes EGDe genome encodes an unusually high (7.3%, ~ 209 regulatory genes) number of transcriptional regulators to facilitate rapid transition between saprophytic/pathogenic lifestyles (Glaser et al., 2001). The major regulators include positive regulatory factor A (PrfA), the transcriptional regulators MogR and

DegU involved in the regulation of flagellar motility (Grundling et al., 2004; Knudsen et al.,

2004; Williams et al., 2005), the response regulators Agr involved in quorum sensing (Autret et al., 2003), Clp stress protein regulator CtsR (Nair et al., 2000), PerR and FurR that regulate iron uptake and storage (Rea et al., 2004), CodY proteins critical for adaptations to post-exponential

18 phase growth (Bennett et al., 2007), VirR controlling surface components’ modifications

(Mandin et al., 2005) and a number of two-component regulatory systems such as LisrR/K, Ces

R/K and CheA/Y responsible for stress tolerance (Cotter et al., 1999; Kallipolities and Ingmer,

2001) and chemotaxis (Dons et al., 2004), respectively.

The positive regulatory factor A (PrfA) is a 237-residue, 27kDa protein of the Crp/Fnr

family of bacterial transcription factors (Kreft and Boland, 2001; Scortti et al., 2007) that

coordinates the expression of virulence genes critical for host colonization, invasion, phagosomal

escape, cytosolic replication, and cell-to-cell spread (Vazquez-Boland et al., 2001; Portnoy et al.,

2002; Dussurget et al., 2004; Hamon et al., 2006). PrfA is weakly active in environmental

habitat, however during intracellular infection it is strongly induced (>300-fold), which is

mediated by a conformational shift in the transcription factor caused by the binding of a low-

molecular-weight cofactor to the N-terminal β–roll domain (Scortti et al., 2007). As of now, only

10 ( inlA, inlB, inlC, plcA, plcB, hly, actA, mpl, hpt, orfX ) of the 2853 coding sequences of the L. monocytogenes EGDe genome (Glaser et al., 2001) have been unambiguously identified as PrfA- dependent, by transcriptional profiling (Milohanic et al., 2003; Marr et al., 2006) and mechanistic studies (Ripio et al., 1997; Chico- Calero et al., 2002; Mengaud et al., 1991;

Lingnau et al., 1995; Engelbrecht et al., 1996). These genes are strongly induced during intracellular infection (Sheehan et al., 1995, Braun et al., 1997). Moreover, 145 additional coding sequences of L. monocytogenes EGDe genome exhibit some degree of PrfA mediated differential expression on transcriptomic profiling (Milohanic et al., 2003; Marr et al., 2006), however this regulation is potentially indirect since only a few of the differentially regulated genes are preceded by putative PrfA transcriptional boxes.

19 7. Antimicrobial resistance in Listeria monocytogenes

In recent years, antibiotic resistance has emerged as a significant concern in Listeria monocytogenes . The ubiquitous nature of this organism facilitates its frequent encounter with antibiotics, both from human (treatment) and non-human usage (growth promoters in food animals) of drugs. The currently employed therapy for the treatment of human listeriosis includes a combination of ampicillin and aminoglycoside such as gentamicin (Dutta et al., 2009; Poros-

Gluchowska and Markiewicz, 2003; Temple and Nahata, 2000; Swaminathan and Gerner-Smidt,

2007) in conjunction with supportive therapy. However, reports of reduced susceptibility and development of resistance in L. monocytogenes to these antibiotics are on the rise (Cekmez et al.,

2012; Krawczk-Balska et al., 2012; Li et al., 2007; Walsh et al., 2001). Multidrug resistant strains of L. monocytogenes have been isolated with increasing frequency in farm animals

(Srinivasan et al., 2005), cattle carcasses (Wieczorek, et al., 2012), meat (Pesavento et al., 2010), food products (Poyart-Salmeron et al., 1990; Rota et al., 1996; Walsh et al., 2001; Antunes et al.,

2002), and from sporadic human cases (Tsakris, et al., 1997; Safdar and Armstrong, 2003).

Antibiotic resistance in L. monocytogenes is primarily mediated by three mobile genetic

elements: self-transferable plasmids, mobilizable plasmids, and conjugative transposons

(Charpentier et al., 1995; Lungu et al., 2011). In addition, efflux pump-mediated antibiotic

tolerance has been reported in Listeria (Godreuil et al., 2003).

Given the increasing number of reports of antibiotic resistance in L. monocytogenes and

growing concerns over the use of synthetic chemicals in the food industry, there is renewed

interest in exploring the potential of various natural approaches as an alternative strategy to

prevent food contamination by L. monocytogenes and combat foodborne listeriosis in humans.

20 8. Plant-derived antimicrobials and probiotics

Since ancient times, plant extracts have been used as food preservatives, flavor enhancers

and dietary supplements to prevent food spoilage, and maintain human health. In addition,

fermented foods containing beneficial microbiota have been a part of traditional diets for their

health benefits. The antimicrobial properties of several plant-derived antimicrobials (PDAs) have

been documented (Ahmad and Beg, 2001; Bhatt and Negi, 2012; Kubo et al., 1993; Negi et al.,

1999, 2003a, 2003b, 2005, 2010; Silva et al., 1996; Zeng et al., 2012). A great majority of these

compounds are secondary metabolites, and are produced as a result of reciprocal interactions

between plants, microbes, and animals (Reichling, 2010). These secondary metabolites could be

species or genera specific in their action, and do not primarily contribute to major metabolic

processes in plants, but potentiate their ability to survive local environments (Harborne, 1993)

and defend plants against microorganisms such as bacteria, fungi and viruses (Kennedy and

Wightman, 2011).

The major benefit of using PDAs as food preservatives is that they do not exert deleterious

effects usually associated with synthetic chemicals (VanWyk and Gericke, 2000). Moreover,

there is evidence that the antimicrobial action of PDAs does not induce resistance in pathogens

(Ali et al., 2005; Ohno et al., 2003). The major groups of PDAs include polyphenols, flavonoids,

alkaloids, lectins, and tannins (Cowan, 1999; Geissman, et al., 1963). Some of the PDAs that

possess significant antimicrobial activity include trans -cinnamaldehyde, eugenol, thymol, carvacrol, β-resorcyclic acid, and caprylic acid. All the aforementioned compounds have been shown to exert significant antimicrobial effects against both Gram-positive and Gram-negative bacteria, primarily by damaging the cell wall and compromising membrane integrity, thereby leading to leakage of cellular contents and cell death (Burt, 2004). In addition, a majority of

21 PDAs are generally recognized as safe (GRAS) compounds with low mammalian cytotoxicity

and quick biodegradability in soil and water (Isman, 2000), thus making them safe and

environment-friendly antimicrobials.

The choice of PDAs for application in different foods to enhance food safety depends upon a

variety of factors. The antimicrobial efficacy of PDAs is determined by physicochemical

properties such as pH, pKa, hydrophobicity, solubility in aqueous solutions, and stability (Negi,

2012; Stratford and Eklund, 2003). The presence of fat (Cava-Roda et al., 2010), sugars

(Gutierrez et al., 2008a), and proteins (Cerrutti and Alzamora, 1996; Hyldgaard et al., 2012;

Kyung, 2012) modulate the antimicrobial efficacy of essential oils in foods. In addition, extrinsic factors such as temperature, water activity and atmospheric composition exert a significant impact on the antimicrobial properties of phytochemicals (Gould, 1989). Thus, besides the antimicrobial efficacy, the choice of phytochemicals depends upon the target food product

(Owen and Palombo, 2007).

The Food and Agriculture Organization (FAO) and World Health Organization (WHO) define probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Probiotic bacteria exert multiple benefits to the host, such as nutrient digestion and assimilation (Sonnenburg et al. 2005; Yatsunenko et al. 2012), potentiating host immune function (Olszak et al. 2012), and protection against enteric pathogens (Candela et al.,

2008; Fukuda et al., 2011). Although several species of bacteria have been identified, the majority of probiotic bacteria supplemented in human diets belong to Lactobacillus and

Bifidobacteria . The major scientific studies that investigated efficacy of natural compounds and probiotic bacteria to control L. monocytogenes in food processing environments, high-risk foods and virulence are summarized below.

22 8.1 Control of L. monocytogenes in food processing environment using PDAs and probiotic

bacteria

The widespread presence of L. monocytogenes in the environment potentially leads to

reoccurring introduction of the pathogen in food processing areas (Tompkin, 2002).

Implementation of good manufacturing practices, and hazard analysis critical control points are

important for controlling the persistence of L. monocytogenes in food processing facilities

(Tompkin et al., 1999). In this regard, both preventing the formation of L. monocytogenes biofilms as well as eradication of mature biofilms are critical, since biofilms contribute to the environmental persistence of L. monocytogenes . Chemical disinfectants commonly used for cleaning and sanitation include surfactants and alkali/acid chemicals such as quaternary ammonium compounds, hypochlorite (Krysinski et al., 1992), chlorine, peracetic acid, and peroctanoic acid (Fatemi and Frank, 1999). In addition, hydrogen peroxide and ozone treatments are employed to control L. monocytogenes biofilms in processing plants (Robbins et al., 2005).

These treatments disintegrate food residues by decreasing surface tension, emulsifying fats, and denaturing proteins (Hayes and Forsyth, 1998; Maukonen et al., 2003; Mosteller and Bishop,

1993; Simoes et al., 2010); however, they are not very effective in completely inactivating L. monocytogenes biofilms, especially in the presence of organic matter and low temperatures (Heir et al., 2004; Holah et al., 2002; Pan et al., 2006; Romanova et al., 2002). Moreover, hardness of water and chemical inhibitors negatively affect the disinfection efficiency (Bremer et al., 2002;

Cloete et al., 1998; Kuda et al., 2008; Simoes et al., 2010). Consequently, the efficacy of alternate approaches such as plant-derived antimicrobials; competitive exclusion bacteria and/or their antimicrobial metabolites have been investigated for controlling L. monocytogenes in food- processing environments.

23 In a recent study, Desai and co-workers (2012) found that thyme oil, oregano oil and

carvacrol at 0.1 to 0.5% concentrations were effective in inactivating L. monocytogenes mature biofilms developed from 21 L. monocytogenes strains representing 13 serotypes. Plant-derived antimicrobials have also been used with surfactants to improve the disinfection efficacy. The use of PDAs in combination with surfactants reduces the low water solubility of essential oils, thereby increasing the effective compound concentration and antimicrobial activity. Perez-

Conesa and coworkers investigated the efficacy of micellar encapsulated eugenol and carvacrol in reducing L. monocytogenes colony biofilms (Perez-Conesa et al., 2006) and L. monocytogenes

biofilms on steel chips (Perez-Conesa et al., 2011). These researchers encapsulated the essential

oils in micellar nonionic surfactants, and observed that the two antimicrobials were very

effective in inactivating L. monocytogenes biofilms, and decreased viable bacterial counts by 3.5 to 4.08 log CFU/cm 2 within 20 min of exposure.

The aforementioned studies primarily focused on inactivating pre-formed mature biofilms,

however, with increasing advancement in the understanding of molecular processes involved in

biofilm formation, new methodologies are targeted at interrupting critical cell-to-cell signaling

for enhancing biofilm control. These include bacterial quorum sensing antagonists (Dunstall et

al., 2005; Rasmussen et al., 2005a), two-component signal transduction inhibitors (Worthington

et al., 2012) and antibiofilm chemical modulators (Worthington et al., 2012) such as eukaryotic

protein kinase inhibitors (Wenderska et al., 2011). The selection of such compounds is based on

their ability to interact with a wide range of molecular targets critical for biofilm formation in

bacteria. Plant compounds have been reported to inhibit quorum sensing in human pathogens

(Adonizio et al., 2006; Vattem et al., 2007). Several quorum-sensing inhibitory phytochemicals,

including polyphenols have been effective in reducing biofilm formation in pathogenic bacteria

24 (Cragg et al., 1997; Huber et al., 2003) such as Burkholderia cenocepacia (Riedel et al., 2006),

Cronobacter sakazakii (Amalaradjou and Venkitanarayanan, 2011) and Pseudomonas aeruginosa (Sarabhai et al., 2013).

Physical interactions and chemical cross talk between bacteria of different species with the production of antagonistic metabolites is known to modulate bacterial colonization and biofilm formation in a niche (Carpentier and Chassaing, 2004; Kives et al., 2005; Rossland et al., 2005;

Simoes et al., 2010; Tait and Sutherland, 1998; Valle et al., 2006). Previously, Zhao and co- workers (2004) found that microorganisms obtained from floor drains of food processing facilities with no history of detectable L. monocytogenes exhibited significant antilisterial activity, and were able to inhibit biofilm formation by L. monocytogenes. The inhibitory bacteria isolated were identified as Enterococcus durans , Lactococcus lactis subsp. lactis and L. plantarum . Similarly, in a study by Leriche and Carpentier (2000), Staphylococcus sciuri biofilms were found to limit the adhesion and growth of L. monocytogenes on stainless steel surfaces. Although the exact mechanism of antibiofilm action induced by probiotics remains unclear, it appears to be through competition, exclusion and displacement from adhesion surfaces, and nutrient scarcity (Woo and Ahn, 2013). The ability of probiotic bacteria to synthesize and secrete anti-adhesive chemicals has been observed by several researchers (Desai and Banat, 1997; Nitschke and Costa, 2007; Rodrigues et al., 2004; van Hamme et al., 2006).

This includes antagonism against major foodborne pathogens such as Staphylococcus aureus

(Rodrigues et al., 2004), Salmonella (Mireles et al., 2001) and L. monocytogenes (Garcia-

Almendarez et al., 2008; Schobitz et al., 2014). In addition, bacterial metabolites such as nisin and reuterin have been investigated for their antibiofilm potential against various microorganisms, including L. monocytogenes (Bower et al., 1995; Chi-Zhang et al., 2004;

25 Dufour et al., 2004; Mahdavi et al., 2009). Although the use of PDAs and probiotics as bio- disinfectants is promising, further studies are needed to investigate the safety, feasibility and effectiveness of such approaches in commercial settings, specifically relating to their effects on the shelf life, sensory attributes and consumer acceptance of the final food products (Mosteller and Bishop, 1993; Wirtanen et al., 2000).

8.2 Control of L. monocytogenes in foods using PDAs and probiotic bacteria

Approximately 99% of all L. monocytogenes infections occur via contaminated food

(Swaminathan and Gerner-Smidt, 2007). Due to the high case fatality rates (20 to 30%) associated with listeriosis, the United States Department of Agriculture Food Safety and

Inspection Service (USDA-FSIS) has adopted a zero tolerance policy for the presence of L. monocytogenes on RTE foods (Gerba et al., 1996; U.S Food Safety and Inspection Service,

1989), which implies that L. monocytogenes detection ( ≥ 1 log CFU/25 g of sample) on RTE foods initiates a recall of the product leading to significant economic losses. Additionally, the

USDA-FSIS alternatives 1 and 2 of the L. monocytogenes regulations mandate that all RTE meat-processing plants apply post-processing treatments to food products, which may include the use of antimicrobials to inactivate or suppress L. monocytogenes growth (Anonymous, 2003 and

U.S. Food Safety and Inspection Service, 2004). Traditionally, various standard food preservation techniques, including refrigeration, desiccation, pasteurization, and synthetic antimicrobials for the control of Listeria in foods have been investigated with varying degrees of success (Davidson et al., 2001). Primarily, they are targeted at enhancing food safety and shelf- life without compromising the sensory and nutritional attributes of food products (Campos et al.,

2011; Gandhi and Chikindas, 2007). However, in light of the recent reports on foodborne

26 listeriosis outbreaks (CDC, 2011; CDC, 2012a,b; CDC, 2013), the contamination of food

products with L. monocytogenes still remains a significant challenge that needs to be adequately addressed. Modern methods of food preservation such as activated antimicrobial films, irradiation, and modified atmosphere packaging have limited application owing to their deleterious effects on the organoleptic properties and consumer acceptability of foods (Negi,

2012). With increasing consumer demands for minimally processed, microbiologically safe, and organic foods, there is significant focus on the use of natural preservatives to enhance the shelf- life and microbiological safety of food products.

Plant-derived antimicrobials are viable candidates to improve the safety and shelf-life of food products. In addition to the significant antimicrobial effects exerted by these compounds, the major benefits of using PDAs in foods include reduced application of potentially harmful synthetic chemical preservatives, increase in flavor, nutritional, and medicinal value of foods.

The majority of PDAs are GRAS-status compounds, and are approved for addition in foods by the US FDA.

8.2.2.1 Essential oils

Essential oils are volatile, aromatic, oils extracted from various plant parts, such as flowers, bark, leaves and fruits (Deans and Ritchie, 1987; Sanchez et al., 2010) by distillation (Negi, 2012) or supercritical fluid extraction (Bakkali et al., 2008; Pereira and Meireles, 2010). Individual active components present in essential oils are also synthesized chemically. Essential oils contain a variety of compounds such as terpenes, phenols, aldehydes or esters, which are classified as

GRAS substances. The antimicrobial action of essential oils or their active components involves multiple mechanisms with several cellular targets (Burt, 2004). Thus, the development of bacterial resistance against these natural interventions is a formidable challenge for pathogens.

27 Essential oils from cinnamon, clove, oregano, thyme, nutmeg, bay, and coriander exhibit a high

degree of antimicrobial activity against L. monocytogenes (Barbosa et al., 2009; Djenane et al.,

2011; Firouzi et al., 2007; Gutierrez et al., 2008a,b; Singh et al., 2003, Smith-palmer et al., 1998;

Zhang et al., 2009). Clove and cinnamon extracts have also been found to exert significant antilisterial effect in other food systems such as chicken meat (Hoque et al., 2008) and cheese

(Vrinda Menon and Garg, 2001; Smith-Palmer et al., 2001). In addition to their antimicrobial properties, essential oils such as wintergreen or clove oil containing high concentrations of phenols have been found to exhibit significant anti-inflammatory, antioxidant (Bhat et al., 2011;

Hame et al., 2012) and cardioprotective benefits to the host (Smith, 2012).

8.2.2.2 Plant extracts

Plant extracts have potential application in foods as preservatives and flavor enhancers. The antibacterial activity of plant extracts is primarily due to bacterial membrane disruption and cell content leakage (Otake et al., 1991). The extracts of cinnamon bark and fruit have been reported to possess antimicrobial activity (Agnihotri and Vaidya, 1996). Yuste and Fung (2002) reported a significant reduction in the number of L. monocytogenes in apple juice supplemented with 0.1-

0.3% cinnamon. Similarly, plant extracts have reduced L. monocytogenes in meat products (Hao et al., 1998; Ward et al., 1998). Water-soluble extracts of arrowroot tea extract (Kim and Fung,

2004) at 6% (w/w) concentration were found to significantly reduce L. monocytogenes in ground beef samples, however, the extracts of green and jasmine tea exerted no antimicrobial effect

(Kim et al., 2004). A combination of oregano and cranberry extracts significantly reduced L.

monocytogenes at pH 6.0, however the antilisterial effect was lesser at neutral pH. Likewise,

Ruiz and coworkers (2009) observed that rosemary extract was more effective in reducing L.

monocytogenes counts in turkey and ham meat when used in combination with nisin. The

28 antimicrobial efficacy of plant extracts is generally lesser than the individual antimicrobial

components, probably due to the use of crude extracts and the presence of compounds such as

sugars that protect the bacteria (Kapoor et al., 2007; Parvathy et al., 2009).

8.2.2.3 Organic acids

Acetic, lactic, and citric acids have been commonly used for food preservation. The

antimicrobial effect of organic acids is exerted primarily through pH-induced stress, acidification

of cytoplasm, reduced enzymatic activity, metabolism and cell injury (Samelis and Sofos, 2003).

In addition, extrinsic stress factors such as heat and low pH enhance the antimicrobial efficacy of

organic acids. Organic acids are widely applied in preventing microbial contamination and

extending the shelf-life of ready-to-eat meat products (Campos et al., 2011; Georgaras et al.,

2006; Murphy et al., 2006). Currently, sodium or potassium lactate (2%) in combination with

0.05 to 0.15% sodium diacetate is commonly used in the food industry as a preservative in meat

due to a strong synergistic antimicrobial effect (Abou-Zeid et al., 2007) and minimal effect on

organoleptic quality of the food (Abou-Zeid et al., 2007; Porto-Feit et al., 2011).

An increasing body of evidence has demonstrated the potential of application of

antimicrobials on food surfaces to inactivate foodborne pathogens (Garcia et al., 2007; Mattson

et al., 2011), since bacteria are primarily present at the product surface in cases of post-

processing contamination. For example, a dip treatment of frankfurters inoculated with L. monocytogenes in a solution containing 2% acetic acid, 1% lactic acid, or 0.1% benzoic acid followed by steam treatment for 1.5 s inhibited growth of the pathogen for 14 weeks at 7°C

(Murphy et al., 2006). Other combinations of organic acids that exhibit significant antilisterial activity on foods include acetic acid and monocaprylin on frankfurters (Garcia et al., 2007), propionate with lactate on pork (Porto-Feit et al., 2011), and lactate in combination with

29 diacetate on sausage (Georgaras et al., 2006) and ham (Stopforth et al., 2010). Moreover, since

the amount of antimicrobial necessary for surface application is less, the deleterious effect on

organoleptic properties of foods are minimal. A newer approach to increase the antimicrobial

efficacy of organic acids is using them as constituents of edible films and gels. The incorporation

of antimicrobials in edible coatings increases the contact time of the antimicrobial with meat

surface (Siracusa et al., 1992), thereby potentially increasing their efficacy. Lactic and acetic

acids incorporated in calcium alginate gels were found to be more effective in inactivating L. monocytogenes than acid treatment alone (Siracusa et al., 1992).

Probiotic bacteria such as lactic acid bacteria and their antimicrobial metabolites are a viable and safe (GRAS-status) approach to inhibit spoilage and pathogenic microorganisms in foods (Holzapfel et al., 1995; Ross et al., 2002). The use of probiotic bacteria as biocides in foods depends upon the viability and stability of probiotics and their beneficial properties during the product’s shelf life (Mattila-Sandholm et al., 2002). The various metabolites produced by these bacteria such as bacteriocins, diacetyl, hydrogen peroxide and organic acids are used for preserving food products without significantly affecting the organoleptic characteristics of foods

(Galvez et al., 2007).

8.2.2.4 Bacteriocins

Bacteriocins are ribosomally synthesized, antimicrobial peptides mostly produced by food- grade microbes (Balciunas et al., 2013; Galvez et al., 2007). They are promising candidates as bioprotective agents by virtue of their safety, nontoxic nature, and acid heat tolerance, coupled with a wide antimicrobial spectrum against spoilage and foodborne pathogens. Most bacteriocins exert their antimicrobial effects by forming a pore in the cell membrane, thereby disrupting the membrane potentially leading to cell death. The bacteriogenic strains can either be used as starter

30 cultures or as protective co-culture with a starter culture in fermented or non-fermented foods

(Galvez et al., 2007). Due to this reason, bacteriocins are being used in several applications, including biopreservation and shelf-life extension of food products (Balciunas et al., 2013).

Nisin, a class I bacteriocin, first discovered in 1928 as a metabolite of Lactococcus lactis

(Roger, 1928), is a USFDA-approved bio-preservative. The efficacy of nisin in inactivating L. monocytogenes has been demonstrated in many foods, especially cheese (Davies et al., 1997), sausages (Hampikyan and Ugur, 2007), and pork (Kouakou et al., 2008). The form of nisin commonly available commercially is Nisaplin (Danisco), which consists of 2.5% nisin in sodium chloride and non-fat dried milk (McAuliffe and Jordan, 2012). In addition, the class IIa group that includes pediocin-like peptides, in particular Pediocin PA-1, has received substantial attention due to their significant antimicrobial efficacy (Rodriguez et al., 2002) specifically against L. monocytogenes (Katla et al., 2001, 2002; Naghmouchi et al., 2006). The stability of pediocin PA-1 in foods such as cheese, frankfurters, and sausage has also been demonstrated

(Nieto-Lozano et al., 2010). Other bacteriocins with significant efficacy against L. monocytogenes in foods include divergicin M35 (Tahiri et al., 2004, 2009), enterocin AS-48

(Ananou et al., 2005, Molinos et al., 2008), lacticin 3147 (Morgan et al., 1999, 2001) and piscicosin CS526 (Azuma et al., 2007). These bacteriocins have been used successfully in various food systems to reduce L. monocytogenes contamination. For example, enterocin AS-48 reduced L. monocytogenes counts in sausage (Ananou et al., 2005), soy-based desserts (Martinez et al., 2009) and processed vegetables (Molinos et al., 2005).

Recent studies have investigated the efficacy of bacteriocins in combination with emerging technologies such as antimicrobial packaging for increasing shelf-life and food safety (Blanco et al., 2008, 2012; Ercolini et al., 2006; Mauriello et al., 2004). Alginate films containing enterocin

31 at 2000 activity units/cm 2 was effective in controlling L. monocytogenes in vacuum-packaged, cooked ham during refrigerated storage (Marcos et al., 2007). Similarly, the combination of chitosan and divergicin M35 (class IIa bacteriocins) exhibited an additive effect against L. monocytogenes (Benabbou et al., 2009). The use of antimicrobial film is especially applicable for solid food products such as frankfurters, where the surface contaminants come in close contact with the antimicrobial films. Foods can either be supplemented with bacteriocins produced ex situ, or by inoculation of bacteriocin-producing cultures (Stiles, 1996). However, stability of the strain, ability to produce sufficient concentrations of bacteriocins, economic feasibility, and deleterious effects of bacteriocins on food properties are some of the challenges in the application of bacteriocins in foods (Fallico et al., 2010). In addition, various extrinsic factors such as interaction with food chemicals and components, inactivation, or precipitation of proteins in the food matrix need to be considered while selecting bacteriocins for commercial application in foods. Due to these reasons, there are strict regulatory requirements for the use of bacteriocins in foods.

8.3 Control of L. monocytogenes virulence using PDAs and probiotic bacteria

The human intestinal tract is colonized by large and complex communities of bacterial

species that include as many as 10 12 cells per 1g of fecal mass in an average human being

(Hattori and Taylor, 2008; Savage et al., 1977). The gut microbiota interacts with the host intestinal tissue to perform various biological processes (Dethlefsen et al., 2007), including nutrition, immune-homeostasis and defense against intestinal pathogens (Sekirov et al., 2010).

With advances in high throughput sequencing, metagenomics, and development of gnotobiotic animals, the ability to explore the variations in gut microbiota composition and their effect on human health has markedly advanced (Gordon et al., 2011; Khor et al., 2011). Increasing gut

32 microbial diversity and abundance has been associated with improved gut health and reduced

susceptibility to foodborne infections (Dominguez-Bello and Blaser, 2008). In addition, changes

in dietary components have been associated with fluctuations in the composition of gut microbial

population and diversity (Ley et al., 2006; Duncan et al., 2007), which in turn modulate host

metabolic functions (Brown et al., 2012) and susceptibility to gastro-intestinal bacterial

infections (Ghosh et al., 2011).

Recent research has revealed that low concentrations of plant essential oils can reduce

bacterial pathogenicity by modulating gene transcription (Goh et al., 2002; Tsui et al., 2004) and

virulence protein production in many foodborne pathogens (Azizkhani et al., 2013; de Souza et

al., 2010; De Wit et al., 1979; Gonzalez-Fandos et al., 1994; Li et al., 2011; Parsaeimehr et al.,

2010; Qiu et al., 2010a,b, 2011a,b, 2012), including L. monocytogenes (Smith-Palmer et al.,

2002). Carvacrol and thymol have also been found to exert a similar effect on virulence attributes of other foodborne pathogens, including Salmonella Enteritidis (Upadhyaya et al., 2013) and

Staphylococcus aureus (Qiu et al., 2010; Smith-Palmer et al., 2004). Several researchers have

investigated the potential of plant-derived compounds as quorum sensing inhibitors (Koh et al.,

2013; Persson et al., 2005; Rasmussen et al., 2005a,b), since quorum sensing partially regulates

the expression of virulence determinants in pathogenic bacteria. This strategy is unlikely to

contribute to the development of multidrug resistant pathogens due to the absence of selection

pressure on bacteria (Koh et al., 2013). Nakayama and coworkers (2009) reported that ambuic

acid has the potential to inhibit quorum sensing in L. innocua by inhibiting signal peptide

biosynthesis.

Bifidobacterium and Lactobacillus , two genera of the human gut microflora, are among the

well-characterized candidates proposed to possess potential probiotic effects (Guerin-Danan et

33 al., 1998). Toure and coworkers (2003) screened and characterized isolates of Bifidobacteria

with significant antilisterial potency from newborn infants. Moroni and coworkers (2006) found

that bacteriocin-producing bifidobacterial isolates ( Bifidobacterium thermacidophilum , B. thermophilum ) from humans (Toure et al., 2003) reduced adhesion and invasion of L. monocytogenes on human intestinal cells (Caco2, HT-29) in vitro. Visualization of adhesion sites by fluorescent in situ hybridization revealed the adherence of probiotic bacteria and L. monocytogenes in close proximity. Similarly, Corrs and co-workers (2007) observed a reduction

in the adhesion and invasion efficacy of L. monocytogenes in the presence of Lactobacillus and

Bifidobacterium . In addition, these authors demonstrated that interactions between probiotic

bacteria and epithelial cells significantly affect the host immunological response. Similar

probiotic-mediated modulation of host intestinal cell immune response to prevent

enterohaemorrhagic E. coli O157:H7 infection has been observed by Jandu and co-workers

(2009). These authors demonstrated that the presence of probiotic L. helveticus R0052 reduced

pathogen-mediated disruption of IFN-gamma-STAT-1 signaling.

The antilisterial efficacy of probiotic bacteria has been recently demonstrated in gnotobiotic

mice (Bambirra et al., 2007; dos Santos et al., 2011). Archambaus and others (2012) studied the

effect of two Lactobacillus species, L. paracasei CNCM I-3689 and L. casei BL23 on L.

monocytogenes and orally acquired listeria infection in gnotobiotic mouse. The probiotic bacteria

decreased L. monocytogenes counts in intestinal tissue and reduced systemic spread of the

pathogen to various organs, including spleen. Moreover, whole genome intestinal

transcriptomics analysis revealed a modulation in host gene expression, primarily IFN-stimulated

genes and remodulation of the L. monocytogenes transcriptome.

34 Another approach for using probiotic bacteria as a prophylactic or therapeutic strategy to control listeriosis in humans is based on molecular mimicry of host receptor or targeted clearance of the pathogen. Previously, E. coli strain expressing glycosyltransferase genes and producing a chimeric LPS ending in a mimic of ganglioside GM1 exhibited cholera toxin binding property against Vibrio cholerae (Focareta et al., 2006). In another study, L. paracasei expression of

Listeria adhesion protein was tested for its efficacy to prevent L. monocytogenes adhesion, invasion and translocation in a Caco-2 cell culture system (Koo et al., 2012). These investigators observed that the recombinant probiotic was more effective than the normal probiotic strains in reducing L. monocytogenes infection in vitro. Recent studies have shown that PDAs that are highly bactericidal towards enteric pathogens exert low antimicrobial effects against commensal gut microbiota (Hawrelak et al., 2009; Di Pasqua et al., 2005). Since PDAs and probiotics exert their antimicrobial effects by different mechanisms (Shipradeep et al., 2012), a combinatorial approach using PDAs and probiotics could be more effective in controlling L. monocytogenes , however, studies eliciting their synergistic interactions are lacking.

In summary, L. monocytogenes is a significant foodborne bacterial pathogen causing life- threatening infections in susceptible populations. The widespread presence of L. monocytogenes in the environment along with its ability to form biofilms results in its frequent persistence in food processing and packaging facilities, thereby contaminating a variety of foods, especially ready-to-eat (RTE) meat products. In addition, fresh produce is increasingly recognized as a vehicle of foodborne pathogens, including L. monocytogenes. In the host, L. monocytogenes infections can result in high hospitalizations and case-fatality rates. Although antibiotics are the drugs of choice for treating listeriosis, emerging antibiotic resistance in L. monocytogenes underscores the need for alternative approaches for controlling this pathogen in humans.

35 Hypothesis:

Based on published literature and preliminary research, it was hypothesized that plant-derived

compounds TC, CR, TY, EG, CA, and BR exert significant antimicrobial/antibiofilm effects

against L. monocytogenes on foods and abiotic surfaces. Moreover, the PDAs alone or in combination with probiotic bacteria reduce virulence in L. monocytogenes.

The specific objectives of this dissertation were:

1. To investigate the efficacy of TC, CR, TY and EG in controlling L. monocytogenes

biofilm on abiotic surfaces.

2. To investigate the efficacy of TC, CR, TY, EG, BR and CA as an antimicrobial dip for

reducing L. monocytogenes on skinless frankfurter.

3. To investigate the efficacy of TC, CR, TY, EG, BR and CA as an antimicrobial wash and

coating for reducing L. monocytogenes on cantaloupe.

4. To investigate the efficacy of TC, CR, TY and EG alone or in combination with probiotic

bacteria, Bifidobacterium and Lactobacillus in reducing L. monocytogenes virulence in

vitro and in the invertebrate model, Galleria mellonella.

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65

Chapter III

Antibiofilm effect of plant-derived antimicrobials on Listeria monocytogenes

Published in Food Microbiology, 2013, 36: 79-89

66 Abstract

The present study investigated the efficacy of sub-inhibitory concentrations (SICs,

concentrations not inhibiting bacterial growth) and bactericidal concentrations (MBCs) of four,

generally recognized as safe (GRAS), plant-derived antimicrobials (PDAs) in inhibiting Listeria monocytogenes biofilm formation and inactivating mature L. monocytogenes biofilms, at 37, 25 and 4°C on polystyrene plates and stainless-steel coupons. In addition, the effect of SICs of

PDAs on the expression of L. monocytogenes genes critical for biofilm synthesis was determined by real-time quantitative PCR. The PDAs and their SICs used for inhibition of biofilm were TC at 0.0075% (0.50mM), and 0.01% (0.75 mM) concentration, CR at 0.0075% (0.50 mM), and

0.01% (0.65 mM) concentration, TY at 0.005% (0.33 mM) and 0.0075% (0.50 mM) concentration, and EG at 0.03% (1.8 mM), and 0.04% (2.5 mM) level, whereas the PDA concentrations used for inactivating mature biofilms were 0.1% (5.0 mM) and 0.2% (10.0 mM) for TC, CR; 0.1 (3.3 mM) and 0.2% (5.0 mM) for TY, and 0.3% (18.5 mM) and 0.4% (25.0 mM) for EG. All PDAs inhibited biofilm synthesis and inactivated fully formed L. monocytogenes biofilms on both matrices at three temperatures tested (P < 0.05). Real- time quantitative PCR data revealed that all PDAs down-regulated critical L. monocytogenes biofilm-associated genes

(P < 0.05). Results suggest that TC, CR, TY, and EG could potentially be used to control L. monocytogenes biofilms in food processing environments, although further studies under commercial settings are necessary.

67 1. Introduction

Listeria monocytogenes is a major foodborne pathogen responsible for
19% of all deaths resulting from foodborne illnesses in the United States (Scallan et al., 2011). Due to its ubiquitous distribution in the environment, coupled with the ability to tolerate a variety of harsh conditions such as desiccation, low temperature, low pH and high osmolarity, L. monocytogenes is a frequent contaminant of food processing and packaging environment (Chaturongakul et al.,

2008; Donnelly, 2001; Farber and Peterkin, 1991; Jay, 2000; Seeliger and Jones, 1986). In food processing facilities, L. monocytogenes is able to attach and form biofilms on a variety of food

processing equipment surfaces, including polystyrene, stainless steel, polymers, plastic, teflon

and rubber (Blackman and Frank, 1996; Borucki et al., 2003; Chavant et al., 2002; Lunden et al.,

2002; Meyer, 2003; Moretro and Langsrud, 2004). Once L. monocytogenes establishes biofilm, it can survive in food processing environments for extended periods of time (Fatemi and Frank,

1999; Frank and Koffi, 1990). L. monocytogenes cells in biofilms are surrounded by a self- generated matrix of hydrated extracellular polymeric substances (EPS) that constitute their immediate environment. The EPS accounts for a majority of the dry mass of the biofilm, and mainly consist of polysaccharides, proteins, nucleic acids and lipids (Flemming and Wingender,

2010). Because of the presence of EPS, cells in biofilms are more resistant to desiccation and disinfectants than planktonic cells (Chmielewski and Frank, 2003; Flemming and Wingender,

2010; Keskinen et al., 2008).

The persistence of L. monocytogenes in food processing facilities constitutes a significant

food safety hazard, since biofilms protect the underlying bacteria from action of antimicrobials

and sanitizers (Borucki et al., 2003; Folsom and Frank, 2007), and serve as a continuous source

for contamination of food products. There are several reports of listeriosis outbreaks due to

68 consumption of food products contaminated with persistent L. monocytogenes strains present on

food processing surfaces (Gottlieb et al., 2006; Kathariou, 2002; Krysinski et al., 1992; Kumar

and Anand, 1998; Pan et al., 2006; Tompkin, 2002). Therefore, controlling L. monocytogenes

biofilms in food processing facilities is critical for improving food safety and reducing listeriosis

in humans.

Most bacterial biofilms develop in a five-step process comprising of initial attachment,

irreversible attachment, microcolony formation, biofilm maturation, and cell dispersion (Davey

and O'Toole, 2000; Mclandsborough et al., 2006; Stoodley et al., 2002). Flagella-mediated initial

cell attachment has been reported to be crucial for biofilm formation in L. monocytogenes

(Lemon et al., 2007; O'Neil and Marquis, 2006; Todhanakasem and Young, 2008;

Vatanyoopaisarn et al., 2000). The major genes involved in L. monocytogenes attachment

include those associated with flagellar synthesis and motility ( flaA, fliP, fliG, flgE, motA, motB, and mogR ) (Chang et al., 2012; Grundling et al., 2004). Once the bacterial cells are attached, further maturation of biofilm occurs by means of complex cellular mechanisms involving quorum sensing. Two component systems have been found to be involved in the regulation of quorum sensing and biofilm formation in bacteria (Gueriri et al., 2008). A pleiotropic response regulator, DegU has been shown to coordinate biofilm formation in Bacillus subtilis and L. monocytogenes (Kobayashi, 2007; Murray et al., 2009). Similarly, the Agr-dependent quorum sensing system also plays a critical role in L. monocytogenes biofilm development (Rieu et al.,

2007). Biofilm formation was significantly decreased in an agrD mutant of L. monocytogenes

(Riedel et al., 2009). In addition, DnaK, a class I heat-shock response protein involved in stress

resistance in L. monocytogenes , has been shown to contribute to biofilm formation and tolerance

to disinfectants (Van der Veen and Abee, 2010). Moreover, the key transcriptional activator of

69 virulence genes, PrfA, plays a major role in biofilm formation in L. monocytogenes , and mutants lacking prfA were found to be defective in biofilm synthesis (Lemon et al., 2010).

Routine plant hygiene and sanitation are critical for controlling the persistence of L.

monocytogenes in food processing facilities (Tompkin et al., 1999). Although a variety of FDA-

approved disinfectants, including quaternary ammonium compounds and hypochlorite, have

been evaluated for plant sanitation (Krysinski et al., 1992), they were found to be not very

effective in controlling L. monocytogenes biofilms, especially in the presence of organic matter

and low temperatures (Pan et al., 2006; Heir et al., 2004; Holah et al., 2002; Romanova et al.,

2002). Moreover, the presence of chemical residues and potential formation of organochlorine

compounds from chlorine is of concern due to associated health risks (Donato and Zani, 2010;

Parish et al., 2003). Thus, there is an increasing interest in identifying safe and effective

antimicrobials for controlling L. monocytogenes biofilms in food processing plants.

Since ancient times, herbs and spices have been used to preserve foods as well as enhance

food flavor. The antimicrobial activity of several plant-derived compounds has been previously

demonstrated (Burt, 2004; Holley and Patel, 2005), and various active components of these oils

have been identified. Trans-cinnamaldehyde (TC) is an aldehyde present in extract of cinnamon

barks ( Cinnamomum zeylandicum ). Carvacrol (CR) and thymol (TY) are antimicrobial

compounds in oregano oil obtained from Origanum glandulosum . Similarly, eugenol (EG) is an

active ingredient in the oil of clove ( Eugenia caryophyllus ). All the aforementioned plant derived

antimicrobials (PDAs) are classified as generally recognized as safe by the U.S. Food and Drug

Administration (Adams et al., 2004, 2005; Kabara, 1991; Knowles et al., 2005).

70 This study was undertaken to investigate the efficacy of TC, CR, TY and EG in inhibiting

biofilm formation and inactivating mature biofilms of L. monocytogenes at 37, 25, and 4°C on two different matrices, namely polystyrene and stainless steel. Moreover, the effect of PDAs on exopolysaccharide synthesis and biofilm architecture was investigated. In addition, the effect of

PDAs on L. monocytogenes genes critical for biofilm formation was determined. For determining

ability of PDAs in preventing biofilm synthesis from planktonic cells, reducing

exopolysaccharide production, and down-regulating biofilm-associated genes, sub-inhibitory

concentrations (SICs, concentrations below MIC that do not inhibit bacterial growth) of TC, CR,

TY, and EG were used, whereas the inactivation of pre-formed biofilms of L. monocytogenes was investigated using minimum bactericidal concentration (MBC) and a concentration above the MBC of the PDAs.

2. Materials and Methods

2.1. Bacterial strains and culture conditions

All bacteriological media were purchased from Difco (Becton Dickinson, Sparks, MD). Three strains of L. monocytogenes , including ATCC 19115, Scott A and Presque-598 were used in this study. In order to study the growth pattern of L. monocytogenes , each strain was cultured

separately in 10 mL of sterile tryptic soy broth containing 0.6% yeast extract (TSBYE) in 30 ml

screw-cap tubes and incubated at 37°C for 18 h. Following incubation, the cultures were

sedimented by centrifugation (3600 × g for 15 min) at 4°C. The pellet was washed twice,

resuspended in 10 mL of sterile phosphate buffered saline (PBS, pH 7.0) and serial ten-fold

dilutions were cultured on duplicate tryptic soy agar (TSA) and oxford agar plates, followed by

incubation at 37°C for 24 h.

71 2.2. Plant-derived antimicrobials and determination of SIC and MBC

The SIC and MBC of each PDA were determined as previously described (Johny et al., 2010).

Sterile 24-well polystyrene tissue culture plates (Costar, Corning Incorporated, Corning, NY)

containing TSBYE (1 mL/well) were inoculated separately with
6.0 log CFU of L. monocytogenes , followed by the addition of 1 to 10 l of TC, CR, TY or EG (Sigma–Aldrich)

with an increment of 0.5 l. The plates were incubated at 37°C for 24 h, and bacterial growth

was determined by culturing on duplicate TSA and oxford agar plates. The two highest

concentrations of each PDA below its MIC that did not inhibit bacterial growth after 24 h of

incubation as compared to control samples were selected as its SICs for this study, whereas the

lowest concentration of PDAs that reduced L. monocytogenes population in TSBYE by
5.0 log

CFU/ml after incubation at 37°C for 24 h was taken as the MBC. Duplicate samples were

included and the experiment was repeated three times.

2.3. Biofilm inhibition and inactivation assay on polystyrene microtiter plates

The effect of TC, CR, TY and EG in inhibiting L. monocytogenes biofilm formation on

microtiter plates was determined according to a previously published method (Ayebah et al.,

2006). L. monocytogenes strains, grown separately in TSBYE at 37°C for 24 h were centrifuged

(3600 × g, 15 min, 4°C), washed in PBS, appropriately diluted and resuspended in TSBYE. Two

hundred microliters of the washed culture (
6.0 log CFU) were used to inoculated sterile 96-well

polystyrene tissue culture plates (Costar), followed by the addition of SICs of TC, CR, TY or

EG. The biofilms of L. monocytogenes were developed at three temperatures (37, 25 and 4°C).

For developing biofilms at 37 and 25°C, the tissue culture plates were incubated for a period of

96 h, whereas for developing biofilms at 4°C the plates were incubated for 7 days. The L.

monocytogenes population associated with the biofilm was enumerated at 24 h interval. The

72 wells were gently rinsed with PBS three times and the number of planktonic population

recovered in the PBS was enumerated. Subsequently, the wells were scraped with sterile pipet tip

for 5 min/well and the detached cells from the biofilm were plated on duplicate TSA and Oxford

agar plates (Amalaradjou et al., 2010; Ayebah et al., 2006). The plates were incubated at 37°C

for 48 h before counting bacterial colonies. The inactivation of pre-formed L. monocytogenes biofilms by TC, CR, TY, and EG was determined by a microtiter assay, as described by

Djordjevic et al. (2002). Sterile 96-well polystyrene tissue culture plates were inoculated with

200 l of the bacterial inoculum (
6.0 log CFU) and incubated at 37, or 25°C for 96 h, or at 4°C for 7 days. After biofilm formation, the effect of respective MBC and a concentration greater than the MBC of TC, CR, TY, and EG were tested with a contact time of 0, 15, 30, 60, 90, and

120 min. The surviving L. monocytogenes population in the biofilm at each sampling time was

determined as described above, however no washing step was followed here.

2.3.1. Organic matter contamination on polystyrene plates

The efficacy of TC, CR, TY and EG in inactivating biofilms of L. monocytogenes in the presence of dried skim milk was determined using method of Fatemi and Frank (1999). Sterile rehydrated dried skim milk (100 g milk solids/liter of TSBYE) (SACO Foods, Inc, Middleton, WI) was used to simulate organic matter contamination in food processing plants. To determine the biofilm inactivation efficacy of TC, CR, TY, and EG in the presence of organic matter, sterile 96-well polystyrene tissue culture plates were inoculated with 200 l of the bacterial inoculum (
6.0 log

CFU) suspended in TSBYE containing 10% dried skim milk and incubated at 37, or 25°C for 96

h, or at 4°C for 7 days. After biofilm formation, TC, CR, TY and EG were added, and the

surviving L. monocytogenes population in the biofilm was determined as described above.

Duplicate samples were used for each treatment and the assay was repeated three times.

73 2.4. Preparation of stainless steel coupons

Stainless steel coupons (type 304; diameter 1 cm) with no. 4 finish were prepared, as described

by Djordjevic et al. (2002). The coupons were rinsed in acetone for 10 min followed by washing

in distilled water. Coupons were cleaned with 100% ethanol, and rinsed in distilled water.

Finally, the coupons were sterilized in an autoclave for 15 min at 121°C.

2.5. Biofilm inhibition and inactivation assay on stainless steel coupons.

The inhibition of L. monocytogenes biofilm formation and inactivation of mature biofilm by the

PDAs on stainless steel coupons was determined as previously described (Kim et al., 2008). L.

monocytogenes strains grown to stationary phase in TSBYE at 37°C for 24 h were centrifuged

(4000 × g, 15 min, 4°C), and cells were resuspended in PBS (pH 7.4). Suspensions (25 ml) of

each L. monocytogenes strain (
6.0 log CFU) were deposited in separate sterile 50-ml test tubes containing a sterile stainless steel coupon. After the coupons were incubated at 4°C for 24 h to facilitate bacterial attachment, they were removed from the test tubes with a sterile forceps, immersed in 400 ml of sterile distilled water (22°C) for 15 s, and rinsed in 200 ml of sterile distilled water with gentle agitation for 5 s.

For the biofilm inhibition assay, the rinsed stainless steel coupons were deposited separately in

10 ml of TSBYE (day 0 or 0 h) containing the respective SICs of TC, CR, TY or EG and then were incubated at 37 or 25°C for 96 h or 4°C for 7 days. The numbers of L. monocytogenes in biofilms formed on coupons were determined on day 0 and at every 24 h incubation interval (for

96 h at 37 and 25°C and for 7 days at 4°C).

To investigate the bactericidal effect of plant compounds on fully formed, mature L. monocytogenes biofilms, following bacterial attachment, the coupons were deposited separately in 10 ml of TSBYE, and incubated for 96 h at 37 or 25°C or 7 days at 4°C to facilitate biofilm

74 formation. The coupons were rinsed in 25 ml of sterile distilled water with gentle agitation for 5

s, transferred to a sterile tube containing 10 ml of TSBYE with or without the respective MBC

and a concentration greater than the MBC of each PDA, and then incubated at 37, 25 or 4°C for

0, 15, 30, 60, 90 and 120 min. At each sampling point, coupons were removed from the medium

and rinsed in 25 ml of sterile water (22°C) with agitation for 15 s. After another rinse in sterile

distilled water (25 ml) for 5 s, the coupons were placed in 50 mL centrifuge tubes containing 3 g

of glass beads (Fisher Scientific, diameter 2 mm) and 10 mL of sterile PBS and vortexed for 1

min. The PBS suspension was serially diluted and surface plated (0.1 ml, in duplicate) on TSA

and Oxford agar plates, and incubated at 37°C for 24 h. When L. monocytogenes was not

detected by direct plating, the PBS samples were tested for surviving bacteria by enriching 1 ml

in 20 ml of TSBYE at 37°C for 24 h, followed by streaking on Oxford agar, and incubation at

37°C for 24 h.

2.5.1. Organic matter contamination on stainless steel coupons

The efficacy of PDAs in inactivating L. monocytogenes biofilms in the presence of organic matter on stainless steel coupons was determined as above (Fatemi and Frank, 1999).

2.6. Quantification of exopolysaccharide (EPS) production by ruthenium red staining

The effect of TC, CR, TY and EG in reducing exopolysaccharide matrix production in L. monocytogenes biofilm was determined by ruthenium red staining assay, as described previously

(Borucki et al., 2003; Zameer et al., 2010). L. monocytogenes strains cultured separately in

TSBYE at 37°C for 24 h were centrifuged (3600 × g, 15 min, 4°C), washed in PBS,

appropriately diluted and suspended in TSBYE. Sterile 96-well polystyrene plates were

inoculated with 200 l of the culture suspension (
6.0 log CFU), followed by the addition of

SICs of the PDAs. The tissue culture plates were incubated at 37, or 25°C for 96 h or at 4°C for 7

75 days for biofilm development. At the end of respective incubation periods, the developed

biofilms were washed with PBS three times, followed by addition of 200 l of aqueous 0.01%

ruthenium red (Sigma Aldrich) to each well. Wells without L. monocytogenes biofilms were also added with 200 l of ruthenium red stain to act as blanks. The plates were incubated for 60 min

at room temperature after which the liquid containing the residual stain was transferred to a fresh

microtiter plate, and the optical density (450 nm) was measured using synergy HT multi-mode

microplate reader (BioTek, Winooski, Vt). The amount of dye bound to the biofilm was

determined by the formula OD BF = OD B − OD S where OD B refers to optical density (450 nm) measured for blanks and OD S refers to the optical density (450 nm) obtained from residual stain collected from sample wells respectively (Zameer et al., 2010).

2.7. RNA isolation and real-time quantitative PCR

The effect of TC, CR, TY, and EG on the expression of L. monocytogenes genes critical for biofilm synthesis ( flaA, fliP, fliG, flgE, motA, motB, prfA, degU, mogR, dnaK, agrA, agrB, agrC )

was investigated using RT-qPCR (Ollinger et al., 2009). Each L. monocytogenes strain was

grown separately with or without the respective SICs of PDAs at 37°C in TSBYE to mid-log

phase (6 h) in sterile polystyrene plates, and total RNA was extracted using RNeasy RNA

isolation kit (Qiagen, Valencia, CA). Complementary DNA (cDNA) synthesis was performed

using the Superscript ІІ Reverse transcriptase kit (Invitrogen). The cDNA synthesized was used

as the template for RT-qPCR. The amplification product was detected using SYBR Green

reagents. The primers for each gene (Table 1) were designed from published GenBank L.

monocytogenes sequences using Primer Express® software (Applied Biosystems, Foster city,

CA). Relative gene expression was determined using the comparative critical threshold (Ct)

value method using a 7500 Step one Real Time PCR system (Applied Biosystems, Carlsbad,

76 CA) and expressed as fold change in expression relative to control (0 mM PDAs). Data were

normalized to the endogenous control (16S rRNA) and the level of candidate gene expression

between control samples ( L. monocytogenes not exposed to SIC of PDAs) and treated samples

(L. monocytogenes cultured in presence of SIC of PDAs) was compared to study the effect of

TC, CR, TY, and EG on the expression of each biofilm associated gene.

2.8. Confocal microscopy

To visualize the three-dimensional structure of L. monocytogenes biofilm and study the effect of

PDAs in inactivating L. monocytogenes biofilm, in situ confocal laser scanning microscopy was performed using a Leica true confocal scanner SP2 microscope with a water immersion lens.

Biofilms were formed at 37°C in TSBYE using a Lab-Tech four-chamber no. 1 borosilicate glass coverslip system (Lab-tek, Nalge Nunc International, Rochester, NY). Confocal microscopy was performed according to a previously published method (Chae and Schraft, 2000). The mature (96 h) L. monocytogenes biofilms formed on coverslips were exposed to PDA treatments, followed by gentle washing with PBS three times. The live and dead bacteria in the biofilm were imaged after staining with 0.0025 mM SYTO (Molecular Probes, Oregon) and 0.005 mM propidium iodide (PI) (Molecular probes) for 20 min. Biofilms of L. monocytogenes , not exposed to PDA treatments (controls) were also visualized to determine the normal architecture of the biofilm.

Fluorochromes were excited using a krypton–argon mixed-gas laser with a PMT2 filter. For each treatment, four randomly selected fields were analyzed. Optical sections of approximately 0.2

m in height were collected starting from the bottom and moving upward through the entire biofilm.

77 2.9. Statistical analysis

A completely randomized design was used for the study. For each treatment and control, the data from independent replicate trials were pooled and analyzed using the PROC MIXED subroutine of the Statistical Analysis Software (SAS ver. 9.2). The least significant difference test was used to determine significant differences (P < 0.05) due to treatments on bacterial counts. Data comparisons for the gene expression study were made by using Student's t test. Differences were considered significant when the P value was ≤ 0.05.

3. Results

Since the antibiofilm effect of TC, CR, TY, and EG was not significantly different among the three L. monocytogenes strains studied (P > 0.05), only the results obtained with ATCC-Scott A strain are reported in the manuscript.

3.1. Determination of SIC and MBC of plant-derived antimicrobials

The MBCs of TC, CR, TY, and EG on L. monocytogenes were 0.1% (5.0 mM), 0.1% (3.3 mM),

0.1% (3.3 mM), and 0.3% (18.5 mM), respectively. The two highest SICs of the PDAs that did not reduce L. monocytogenes growth as compared to control were 0.0075% (0.50 mM) and

0.01% (0.75 mM) for TC, 0.0075% (0.50 mM) and 0.01% (0.65 mM) for CR, 0.005% (0.33 mM) and 0.0075% (0.50 mM) for TY, and 0.03% (1.8 mM), 0.04% (2.5 mM) for EG. The average number of L. monocytogenes planktonic cell population recovered after 24 h of incubation was approximately 8.5 log CFU/ml in both control and PDA treatment wells.

3.2. Effect of SICs of PDAs on L. monocytogenes biofilm formation on polystyrene microtiter plates and stainless steel coupons

All four PDAs decreased (P < 0.05) L. monocytogenes biofilm formation on microtiter plates and stainless steel coupons at 37, 25, and 4°C (Table 2 and 3). On polystyrene plates, the SICs of TC,

78 CR, TY, and EG decreased the bacterial counts in the biofilm by
2.5–3.5 log CFU/ml after 96 h of incubation at 37 and 25°C, and 7 days of incubation at 4°C (Table 2A–C). However, the control wells yielded a biofilm-associated bacterial count of
7.5 log CFU/ml at the end of 96 h at 37 and 25°C, and
6.5 log CFU/ml after 7 days at 4°C (Table 2A–C). A similar inhibitory effect of PDAs on biofilm formation was observed on stainless steel coupons incubated at 37, 25, and 4°C (Table 3A–C). Moreover, the inhibitory effect of TC, CR, TY and EG on L. monocytogenes biofilm synthesis was similar on polystyrene and stainless steel surfaces (P >

0.05).

3.3. Effect of PDAs in inactivating pre-formed L. monocytogenes biofilm on polystyrene microtiter plates and stainless steel coupons

The efficacy of TC, CR, TY, and EG in inactivating pre-formed biofilms of L. monocytogenes on microtiter plates and stainless steel coupons at 37, 25, and 4°C is depicted in Figs. 1 and 2. The average L. monocytogenes population recovered from the biofilms on polystyrene plates after 96 h of incubation at 37 and 25°C was
7.5 log CFU/ml (Fig. 1A–B), whereas the counts at 4°C were
6.5 log CFU/ml (Fig. 1C). On microtiter plates incubated at 37°C, both concentrations of

EG completely inactivated the biofilm (enrichment negative) at 30 min of incubation, whereas

CR and TY decreased the biofilm-associated bacteria to undetectable levels (enrichment negative) at 60 min of treatment time (Fig. 1A). However, TC reduced L. monocytogenes in the biofilms by
4.5–5.0 log CFU/ml at 120 min of incubation at 37°C (Fig. 1A). Similar results were observed at 25°C, where all PDAs except TC completely inactivated L. monocytogenes biofilm after 60 min of incubation, while TC resulted in about 4.5 log CFU/ml reduction in biofilm counts compared to control samples (Fig. 1B). However at 4°C, all four PDAs were found to be more effective, resulting in similar magnitudes of bacterial reduction as early as 15

79 min (Fig. 1C). In case of stainless steel coupons, a similar trend in the inactivation of biofilms was observed for TC, which reduced L. monocytogenes in the biofilms by
4.5 log CFU/ml at

120 min of incubation at all the three temperature tested (Fig. 2A–C). However, CR, TY, and EG were found to be more effective, reducing biofilm-associated L. monocytogenes counts to undetectable levels (enrichment negative) by 15 min of treatment at the three incubation temperatures (Fig. 2A–C).

3.4. Efficacy of PDAs in inactivating L. monocytogenes biofilms in presence of organic matter

No significant difference (P > 0.05) was found in the antibiofilm effect of PDAs in the presence of organic matter on both polystyrene plates and stainless steel coupons (data not shown).

3.5. Effect of PDAs on EPS production

EPS production in L. monocytogenes biofilm (controls) was significantly higher at 37°C than at

25 and 4°C ( P < 0.05) (Fig. 4A–C). All PDAs reduced EPS production in L. monocytogenes biofilm on polystyrene plates after 96 h of incubation at 37, 25°C, and 7 days of incubation at

4°C in comparison to control ( P < 0.05). However, the magnitude of reduction in EPS production brought about by PDAs was greater at 37 and 25°C than at 4°C.

3.6. Confocal microscopy

The effect of PDAs on L. monocytogenes biofilm structure as revealed by confocal laser scan microscopy is depicted in Fig. 3A–E. In control L. monocytogenes biofilm (not exposed to PDA treatment), a dense uniform layer of live cells (stained green by SYTO dye) on the coverslip surface were observed (Fig. 3A), while the biofilm exposed to PDA treatments revealed presence of dead cells (stained red by propidium iodide dye) and patchy breaks in the biofilms with disrupted organization (Fig. 3B–E).

3.7. Effect of PDAs on expression of genes critical for biofilm formation in L. monocytogenes

80 Real time quantitative PCR results revealed that the SIC of TC (0.75 mM), CR (0.65 mM), TY

(0.50 mM) and EG (2.5 mM) substantially down-regulated (P < 0.05) the expression of the

majority of genes involved in biofilm formation in L. monocytogenes (Table 4).

4. Discussion

For controlling the persistence of L. monocytogenes in food processing facilities, both preventing the establishment of Listeria biofilm as well as eradication of mature biofilms in the food-processing environment are critical. Therefore, this study investigated the efficacy of PDAs for inhibiting biofilm formation from planktonic cells, and inactivating mature and fully formed

L. monocytogenes biofilms. This was based on the hypothesis that SICs of PDAs prevent biofilm production from planktonic cells by down-regulating critical genes, whereas bactericidal concentrations eradicate pre-formed biofilms by directly killing L. monocytogenes in biofilms.

The results revealed that the PDAs at bactericidal concentrations were considerably effective in killing mature L. monocytogenes biofilms and at SICs exerted an inhibitory effect on biofilm synthesis.

Previously, Conesa and coworkers investigated the effect of eugenol and carvacrol in controlling colony biofilms of L. monocytogenes developed on agar plates (Perez-Conesa et al.,

2006), or biofilms developed on steel chips (Perez-Conesa et al., 2011); however, these authors did not investigate the effect of plant compounds on biofilms developed at various temperatures encountered in the food processing environment or on plastic surfaces. In another study by Desai et al. (2012), the efficacy of several plant essential oils in inactivating L. monocytogenes biofilms developed at 22–24°C (room temperature) was investigated. In their study, L. monocytogenes biofilms were exposed to essential oils for durations ranging from 4 to 24 h, whereas we investigated the antibiofilm effect of the active ingredients of essential oils. In addition, these

81 studies did not investigate the effect of sub-inhibitory concentrations of essential oils on biofilm

architecture or the genes responsible for biofilm formation. In the present study, we determined

the inhibitory effect of SICs of PDAs on L. monocytogenes biofilm synthesis from planktonic

cells, and their effect on Listeria genes critical for biofilm production. Additionally, the present

study determined the efficacy of PDAs in inactivating L. monocytogenes biofilms at room and

refrigeration temperatures, besides 37°C, the optimal temperature for L. monocytogenes growth.

Moreover, since food processing plant sanitation for controlling bacterial biofilm involves clean in place (CIP) regimes with treatment times ranging from 10 to 60 min (Bremer et al., 2006;

Romney, 1990), the biofilm inactivation efficacy of the PDAs was investigated with contact times ranging from 15 to 120 min in our study as against 4 and 24 h used by Desai et al. (2012).

All three strains of L. monocytogenes used in the study were able to attach and form biofilm on both microtiter plates and stainless steel coupons at 37, 25, and 4°C, indicating their ability to adapt to different surfaces and environmental temperatures. However, the biofilm production was less at 4°C compared to 37 or 25°C (P < 0.05). For example, at 37 and 25°C, L. monocytogenes counts recovered from the biofilm on microtiter plates and stainless steel coupons were
7.5 log CFU/ml at 96 h, whereas, the counts even after 7 days of incubation at 4

°C were consistently lesser by at least 1.0 log CFU/ml (P < 0.05) (Table 1). This could be attributed to the decreased growth rate and reduced cell surface hydrophobicity of L. monocytogenes at 4°C. Similar results were obtained in EPS quantification assay, where a greater amount of EPS was produced by L. monocytogenes at 37 and 25°C than at 4°C (Fig. 4A–

C). Previously, Bonaventura et al. (2008), using a crystal violet based assay, also found that L. monocytogenes strains produced significantly less biofilm on polystyrene and steel at 4°C compared to that at 37 or 25°C. These authors found that cell surface hydrophobicity of L.

82 monocytogenes critical for biofilm synthesis reduced with decrease in temperature, and was

significantly lower at 4°C compared to that at 37 or 25°C.

Although TC, CR, TY and EG were effective in inhibiting (P < 0.05) L. monocytogenes

biofilm production on polystyrene and steel surfaces as compared to control, all PDAs at their

SICs, were not different from one another in their ability to decrease biofilm associated counts of

the pathogen on both matrices (P > 0.05) (Table 2). However, when tested at higher

concentrations for killing pre-formed biofilms, CR, TY and EG were more effective (P < 0.05)

than TC on both matrices at the three tested temperatures. For example, on microtiter plates at 37

and 25°C, all PDAs except TC decreased biofilm-associated L. monocytogenes counts to

undetectable levels (enrichment negative) at 60 min of exposure, whereas greater than 2.0 log

CFU/ml of the pathogen survived in TC-treated wells at the end of 120 min (Fig. 1A–B). These

findings were further confirmed by confocal microscopy, where Listeria biofilms exposed to CR,

TY, EG treatments revealed the presence of mostly dead cells in comparison to TC-treated

biofilms, which had both live and dead cells (Fig. 3B–E). Likewise, on stainless steel coupons,

both tested concentrations of CR, TY and EG completely inactivated L. monocytogenes biofilm

(enrichment negative) as rapidly as 15 min; however, countable populations of the pathogen were recovered from TC-treated biofilms even at 120 min of incubation at the three temperatures

(Fig. 2A–C). In general, there was a trend for increased antibiofilm effect of the compounds with increase in their concentrations. This was more marked for TC, especially in the biofilm inactivation experiments, where a concentration-based reduction in L. monocytogenes counts was observed on both matrices (Fig. 1 and Fig. 2A–C).

Although there was no difference in the inhibitory effect of four plant compounds on biofilm formation on polystyrene plates at the three tested temperatures (P > 0.05) (Fig. 1A–C),

83 the PDAs were somewhat more effective (P < 0.05) in inactivating L. monocytogenes

populations present in the biofilm on steel coupons at 4°C than the other two temperatures (Fig.

2A–C), Similarly, in the inactivation experiments on polystyrene plates, TC, CR, TY and EG

were more effective in killing L. monocytogenes at 4°C than at 37 or 25°C. Carvacrol, TY and

EG reduced L. monocytogenes to undetectable levels at 15 min of exposure at 4°C (Fig. 1C),

whereas complete inactivation of the pathogen was observed only at 60 min at 37 or 25°C (Fig.

1A–B). This enhanced antibiofilm effect of PDAs could be attributed to the reduced cell surface

hydrophobicity of the bacterium at 4°C, which potentially resulted in a weaker biofilm that is

more sensitive to the plant compounds.

Plant derived antimicrobials, including antibiotics, at their sub-inhibitory concentrations

are reported to modulate bacterial gene transcription (Amalaradjou and Venkitanarayanan, 2011;

Goh et al., 2002; Kollanoor-Johny et al., 2012; Tsui et al., 2004; Yim et al., 2011). Since SICs of

the four PDAs were used in biofilm inhibition experiments, the reduction observed in L.

monocytogenes biofilm formation in treated wells was not due to the killing of bacteria, but

could be attributed to their potential effect in modulating the expression of biofilm-associated

genes. Therefore, we used RT-qPCR to determine the effect of the SIC of TC, CR, TY, and EG

on the transcription of known genes critical for various steps involved in biofilm formation in L.

monocytogenes . Real time-qPCR results revealed that the four PDAs significantly down-

regulated the expression of many L. monocytogenes genes critical for biofilm, including those involved in motility, initial attachment on surface, and quorum sensing. However, the magnitude down-regulation of the various genes differed with each PDA. For example, CR and TY were more effective in down-regulating genes critical for initial attachment ( flaA, fliP, fliG, flgE, motA, motB ), quorum sensing ( agrA, agrB, agrC ), stress response ( dnaK ) and transcriptional

84 regulation ( prfA, degU, mogR ) as compared to TC and EG (Table 4). Likewise, EG was more effective than TC in down-regulating genes contributing to initial attachment ( flaA, fliP, flgE , motA ), stress response contributing to biofilm formation ( dnaK ), and transcriptional activation

(prfA ). However, quorum sensing genes were equally affected by both TC and EG. These findings suggest that the four plant compounds may act through different mechanisms.

In conclusion, TC, CR, TY and EG were effective in inhibiting biofilm formation and inactivating mature biofilms of L. monocytogenes on microtiter plates and stainless steel coupons at 37, 25, and 4°C. Since L. monocytogenes has the ability to persist on food processing surfaces and form biofilm at a wide range of temperatures, these compounds (TC, CR, TY, EG) could potentially be used as effective and environmentally friendly antimicrobials to control L. monocytogenes biofilm, thereby reducing food contamination and subsequent infections in humans. Future investigations validating the antibiofilm effect of the PDAs under commercial settings are warranted before recommending their usage.

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92 Table 1. List of primers used in the study

Gene with accession number Primer Sequence (5'- 3')

16S-rRNA* Forward 5'-AGCTTGCTCTTCCAAAGT- 3' (Somer and Kashi, 2003; Reverse 5'-AAGCAGTTACTCTTATCCT- 3' Burkholder et al. 2009) flaA Forward 5'-GACTTGTTACAAACAGCGGATTCA- 3' (NC_003210.1) Reverse 5'-ATTGACGCATACGTTGCAAGAT- 3' fliP Forward 5'-TTGGCCGGGTGTGAATGT- 3' (NC_003210.1) Reverse 5'-CCATTTACACCAAGCGAATCC- 3' fliG Forward 5'-AATCGCGACCGAAGTGGTT- 3' (NC_003210.1) Reverse 5'-CTCGTGCAAGGCGTTGTTT- 3' flgE Forward 5'-CAGCAGGTTCCCCGACTTC- 3' (NC_003210.1) Reverse 5'-CGGCCTTGTAGTGCTGCAT- 3' motA Forward 5'-CAACGCTCGGTGTACTTGGA- 3' (NC_003210.1) Reverse 5'-TTTCGCCCATCGCATGA- 3' motB Forward 5'-TGCAAAAAAATTCGAACAAATGG- 3' (NC_003210.1) Reverse 5'-CTGCCGCGCCTTCCT- 3' prfA Forward 5'-TGAGCAAGAATCTTACGCACTTTT- 3' ( NC_012488.1) Reverse 5'-GCTAGGCTGTATGAAACTTGTTTTTG- 3' degU Forward 5'-ACGCATAGAGAGTGCGAGGTATT- 3' (NC_003210.1) Reverse 5'-CCCAATTCCGCGGTTACTT- 3' mogR Forward 5'-AACTGCCGAAGAAATCTACCATTT- 3'

(NC_003210.1) Reverse 5'-CGATTCCACCGTGTTCTTCA- 3' dnaK Forward 5'-CAACACCTTCTGTAGTTGGTTTCAA- 3'

(NC_003210.1) Reverse 5'-TGCAGCACGTTTCGCTACTT- 3'

93 agrA Forward 5'-GCAGCCGGACATGAATGG- 3'

(NC_002973.6) Reverse 5'-AACCACGCGGATCAAACTTC- 3' agrB Forward 5'-TCGCTCCGGCAGACACA- 3'

(NC_003210.1) Reverse 5'-TTTTTAGTGTTTTCCGGTGTTCTTC- 3' agrC Forward 5'-TATTTTGCTAGATAATGCGGTTGAA- 3'

(NC_003210.1) Reverse 5'-CGCGATTCGAATAACTGGATTT- 3'

* Endogenous control

94 Table 2. Effect of sub-inhibitory concentrations of trans -cinnamaldehyde (TC), carvacrol (CR), thymol (TY) and eugenol (EG) on Listeria monocytogenes biofilm formation on microtiter plates at 37°C (A), 25°C (B), and 4°C (C). The Treatments included Control, TC (0.50 mM, 0.75 mM),

CR (0.50 mM, 0.65 mM), TY (0.33 mM , 0.50 mM), EG (1.8 mM, 2.5 mM).

(A) L. monocytogenes counts in biofilm (Log CFU/ml) at respective time of incubations (h)

Treatments 0 h 24 h 48 h 72 h 96 h Control 6.23±0.50 7.26±0.24 7.25±0.08 7.37±0.09 7.63±0.21 TC 0.50 mM 6.21±0.45 6.25±0.14 5.92±0.05 6.10±0.15 5.31±0.33 TC 0.75 mM 6.02±0.12 6.07±0.33 5.88±0.11 5.65±0.31 4.66±0.28 CR 0.50 mM 6.52±0.21 6.35±0.26 5.97±0.13 6.12±0.23 5.30±0.19 CR 0.65 mM 6.14±0.11 6.17±0.17 5.80±0.24 5.81±0.06 4.72±0.51 TY 0.33 mM 6.42±0.24 6.46±0.31 5.99±0.17 6.27±0.41 5.22±0.11 TY 0.50 mM 6.01±0.34 6.14±0.22 5.48±0.09 5.66±0.22 4.76±0.24 EG 1.80 mM 6.20±0.41 6.13±0.12 5.86±0.22 5.90±0.29 5.15±0.09 EG 2.50 mM 6.21±0.21 5.74±0.09 5.53±0.31 5.58±0.34 4.76±0.11

(B) L. monocytogenes counts in biofilm (Log CFU/ml) at respective time of incubations (h)

Treatments 0 h 24 h 48 h 72 h 96 h Control 6.02±0.10 7.03±0.07 7.41±0.11 7.3±0.45 7.57±0.61 TC 0.50 mM 6.10±0.21 5.98±0.09 5.75±0.14 5.91±0.32 5.21±0.53 TC 0.75 mM 6.32±0.08 5.93±0.11 5.28±0.21 5.72±0.26 4.48±0.24 CR 0.50 mM 6.21±0.12 6.34±0.23 5.97±0.31 6.01±0.11 4.87±0.31 CR 0.65 mM 6.07±0.23 6.13±0.16 5.80±0.24 5.73±0.09 4.73±0.09 TY 0.33 mM 6.23±0.07 6.46±0.27 5.99±0.09 6.06±0.41 4.85±0.36 TY 0.50 mM 6.53±0.14 6.14±0.22 5.48±0.08 5.82±0.36 4.80±0.44 EG 1.80 mM 6.23±0.11 6.13±0.30 5.86±0.10 5.89±0.28 4.85±0.08 EG 2.50 mM 6.14±0.24 5.40±0.07 5.31±0.07 5.44±0.18 4.81±0.22

95 (C) L. monocytogenes counts in biofilm (Log CFU/ml) at respective time of incubations (Days)

0 h Day 1 Day 3 Day 5 Day 7 Control 6.0±0.22 5.10±0.21 5.33±0.13 5.84±0.14 6.42±0.08 TC 0.50 mM 6.1±0.44 4.0±0.13 3.81±0.15 3.41±0.13 3.43±0.06 TC 0.75 mM 6.3±0.12 3.91±0.17 3.52±0.25 3.32±0.27 3.14±0.07 CR 0.50 mM 6.2±0.24 3.51±0.24 4.21±0.18 4.33±0.17 3.81±0.17 CR 0.65 mM 6.0±0.21 3.52±0.04 4.03±0.16 3.61±0.18 3.23±0.14 TY 0.33 mM 6.2±0.11 4.22±0.08 4.11±0.07 3.40±0.16 3.24±0.20 TY 0.50 mM 6.5±0.32 4.01±0.06 3.95±0.05 3.54±0.21 3.12±0.21 EG 1.80 mM 6.2±0.08 4.31±0.11 4.13±0.32 3.52±0.25 3.52±0.05 EG 2.50 mM 6.1±0.14 4.25±0.23 3.74±0.33 3.21±0.24 3.01±0.04

96 Table 3. Effect of sub-inhibitory concentrations of trans -cinnamaldehyde (TC), carvacrol (CR), thymol (TY) and eugenol (EG) on Listeria monocytogenes biofilm formation on stainless steel coupons at 37°C (A), 25°C (B), and 4°C (C). The Treatments included Control, TC (0.5 mM,

0.75 mM), CR (0.5 mM, 0.65 mM), TY (0.33 mM, 0.50 mM), EG (1.8 mM, 2.5 mM).

(A) L. monocytogenes counts in biofilm (Log CFU/ml) at respective time of incubations (h)

0 h 24 h 48 h 72 h 96 h Control 6.20±0.11 7.30±0.31 7.59±0.41 7.87±0.13 7.83±0.11 TC 0.50 mM 6.35±0.21 6.25±0.40 5.92±0.23 5.10±0.20 4.81±0.23 TC 0.75 mM 6.40±0.23 6.07±0.32 5.84±0.11 5.25±0.04 4.16±0.09 CR 0.50 mM 6.25±0.09 6.35±0.07 5.97±0.24 5.62±0.08 5.30±0.08 CR 0.65 mM 6.42±0.17 6.17±0.14 5.80±0.39 5.11±0.11 4.72±0.07 TY 0.33 mM 6.50±0.14 6.46±0.13 5.99±0.08 5.57±0.24 5.22±0.32 TY 0.50 mM 6.30±0.09 6.14±0.11 5.48±0.09 5.36±0.31 4.16±0.09 EG 1.80 mM 6.29±0.13 6.13±0.24 5.80±0.11 5.50±0.36 5.15±0.12 EG 2.50 mM 6.40±0.23 5.70±0.32 5.50±0.21 5.20±0.10 4.35±0.16

(B) L. monocytogenes counts in biofilm (Log CFU/ml) at respective time of incubations (h)

Treatments 0 h 24 h 48 h 72 h 96 h Control 6.40±0.20 7.03±0.14 7.21±0.33 7.37±0.41 7.67±0.20 TC 0.50 mM 6.35±0.12 5.58±0.09 5.25±0.21 5.16±0.32 4.91±0.10 TC 0.75 mM 6.40±0.30 5.30±0.39 5.08±0.14 4.72±0.25 4.6±0.05 CR 0.50 mM 6.25±0.44 6.34±0.21 5.97±0.09 5.51±0.11 4.5±0.09 CR 0.65 mM 6.42±0.25 6.13±0.30 5.50±0.15 5.33±0.24 4.30±0.41 TY 0.33 mM 6.50±0.19 6.46±0.37 5.99±0.21 5.67±0.35 4.81±0.30 TY 0.50 mM 6.30±0.24 6.14±0.24 5.48±0.17 5.23±0.10 4.89±0.25 EG 1.80 mM 6.29±0.21 6.13±0.14 5.86±0.07 5.89±0.21 4.85±0.21 EG 2.50 mM 6.40±0.35 5.74±0.36 5.53±0.21 5.74±0.42 4.81±0.08

97

(C) L. monocytogenes counts in biofilm (Log CFU/ml) at respective time of incubations (Days)

0 h Day 1 Day 3 Day 5 Day 7 Control 6.50±0.33 5.94±0.35 5.91±0.04 6.18±0.14 6.41±0.14 TC 0.50 mM 6.64±0.12 5.27±0.28 3.92±0.11 3.85±0.20 3.25±0.09 TC 0.75 mM 6.40±0.25 4.92±0.14 3.54±0.09 3.29±0.21 3.10±0.04 CR 0.50 mM 6.59±0.17 4.91±0.17 3.29±0.17 3.28±0.31 3.15±0.06 CR 0.65 mM 6.29±0.32 4.54±0.11 3.05±0.32 3.05±0.33 3.01±0.14 TY 0.33 mM 6.56±0.08 4.83±0.31 4.12±0.28 3.62±0.05 3.28±0.10 TY 0.50 mM 6.33±0.09 4.51±0.25 3.71±0.09 3.28±0.07 3.12±0.14 EG 1.80 mM 6.92±0.05 4.95±0.07 4.12±0.07 3.54±0.04 3.34±0.20 EG 2.50 mM 6.42±0.14 4.53±0.06 3.90±0.05 3.46±0.09 3.11±0.17

98 Table 4. Relative fold change in the expression level of Listeria monocytogenes biofilm associated genes in response to SICs of TC, CR, TY, and EG.

Relative fold change*

Gene Gene product or TC CR TY EG Function (0.75 mM) (0.65 mM) (0.50 mM) (2.5 mM) flaA Structural flagella protein -1.71±0.5 -16.74±2.5 -25.45±3.3 -12.93±1.1 fliP Flagellar biosynthesis -0.08±0.03 -7.76±1.0 -18.37±1.6 -1.09±0.2 protein fliG Flagellar motor switch -1.52±0.1 -4.67±0.9 -7.78±0.5 -1.46±0.5 protein flgE Flagellar hook protein -0.06±0.02 -7.20±0.5 -16.62±1.3 -0.99±0.05 motA Flagellar motor protein -1.44±0.1 -14.90±1.2 -18.20±0.9 -12.80±0.6 motB Flagellar motor protein -0.04±0.01 -5.54±0.2 -13.57±0.5 -0.76±0.01 prfA Transcriptional regulator -1.09±0.9 -20.83±2.2 -19.20±2.2 -8.40±0.8 degU Transcriptional regulator/ -8.55±0.3 -40.31±7.4 -47.99±8.1 -7.36±1.1 quorum sensing mogR Transcriptional regulator for -10.21±0.6 -67.98±6.3 -50.19±7.2 -8.58±1.2 motility dnaK Molecular chaperon involve -15.88±1.5 -93.47±14.1 -81.14±9.1 -17.44±5.1 in biofilm formation agrA Quorum sensing -6.49±1.1 -42.69±8.8 -40.27±4.4 -6.48±1.1 agrB Quorum sensing -6.58±2.1 -45.77±12.1 -27.86±5.0 -6.24±2.1 agrC Quorum sensing -8.49±1.5 -70.54±13.3 -45.24±6.3 -5.85±0.8

*Fold change in expression of biofilm associated genes relative to control (0 mM PDA). All genes except fliP, flgE, motB, (TC) and flgE, motB (EG) were significantly down-regulated in comparison to control ( P<0.05).

99 Fig.1.

Effect of MIC of trans -cinnamaldehyde (TC; 5.0, 10.0 mM), carvacrol (CR; 5.0, 10.0 mM), thymol (TY; 3.3, 5.0 mM) and eugenol (EG; 18.5, 25.0 mM) on Listeria monocytogenes Scott A biofilm on mic rotiter plates at 37°C (A), 25°C (B), and 4 °C (C). Error bars represent SEM

(n = 6). Sterile 96 well plates were inoculated with 200 l of L. monocytogenes cell suspension

(
6 log CFU) and incubated for 96 h (37, 25°C) or 7 days (4°C) respectively. After biofilm formation, the effect of various PDAs was tested at exposure times of 0, 15, 3 0, 60, 90, and

120 min at 37, 25 or 4°C and L. monocytogenes populations were enumerated.

Fig.1A.

100 Fig.1B.

Fig.1C.

101 Fig. 2.

Effect of MIC of trans-cinnamaldehyde (TC; 5.0, 10.0 mM), carvacrol (CR; 5.0, 10.0 mM), thymol (TY; 3.3, 5.0 mM) and eugenol (EG; 18.5, 25.0 mM) on Listeria monocytogenes Scott A biofilm on stainless steel coupons at 37°C (A), 25°C (B), and 4°C (C). Error bars represent SEM

(n = 6). Sterile stainless steel coupon was inoculated with LM (
6.0 log CFU) and incubated at

4°C for 24 h. Following bacterial attachment, the coupons were incubated for 96 h at 37 or 25 °C or 7 days at 4 °C to facilitate biofilm forma tion. After biofilm formation, the effect of various

PDAs was tested at exposure times of 0, 15, 30, 60 , 90 and 120 min at 37, 25 or 4 °C and

L. monocytogenes populations were enumerated

Fig.2A.

102 Fig.2B.

Fig. 2C.

103 Fig.3. Scanning confocal micrographs of Listeria monocytogenes Scott A biofi lm before PDA treatment (A) and after treatment with MICs of (B) trans -cinnamaldehyde (TC; 5.0 mM), (C) carvacrol (CR; 5.0 mM), (D) thymol (TY; 3.3 mM) and (E) eugenol (EG; 18.5 mM). Biofilms were formed at 37°C for 96 h on borosilicate glass cove rslip. The mature (96 h) L. monocytogenes biofi lms were exposed to PDAs, followed by gentle washing with PBS. The li ve and dead bacteria in the biofi lm were imaged after staining with 0.002 5 mM SYTO and 0.005 mM PI for 20 min. Biofi lms of L. monocytogenes , not exposed to PDA treatments (controls) were also visualized to determine the normal architectur e of the biofi lm. Fluorochromes were excited using a kryptonearg on mixed -gas laser with a PMT2 fi lter in a Leica true confocal scanner SP2 microscope.

A B C

D E

104

Fig.4. Effect of SICs of trans-cinnamaldehyde (TC; 0.05, 0.75 mM), carvacrol (CR; 0.50, 0.65 mM), thymol (TY; 0.33, 0.50 mM) and eugenol (EG; 1.8, 2.5 mM) on exopolysaccharide production in Listeria monocytogenes Scott A bio film on microtiter plates at 37° C (A), 25°C (B), and 4 °C (C). Error bars represent SEM (n = 6). Bars with different letters represent a significant difference between treatments (P < 0.05). Sterile 96 -wel l plates were inoculated with L. monocytogenes suspension (
6.0 log CFU), with or wi thout the presence of SICs of PDAs and incubated at 37, or 25°C for 96 h or at 4 °C for 7 days. The developed biofilms were stained with

0.01% ruthenium red for 60 min at 25 °C. The amount of dye bound to the biofilm was determined by the formula OD BF = OD B − OD S where OD B is the optical density (450 nm) measured for blanks while OD S is the optical density obtained from residual stain respectively.

Fig.4A.

105 Fig.4B.

Fig. 4C.

106

Chapter IV

Inactivation of Listeria monocytogenes on frankfurters by plant-derived antimicrobials

alone or in combination with hydrogen peroxide

Published in International Journal of Food Microbiology 2013, 163:114-118

107 Abstract

Listeria monocytogenes is a significant foodborne pathogen associated with outbreaks

involving contaminated ready-to-eat (RTE) products, including frankfurters. The USDA-FSIS

has established a zero tolerance policy for L. monocytogenes in RTE products, thereby

warranting effective post-processing interventions to control the pathogen on these foods.

In the present study, the antilisterial activity of GRAS (generally recognized as safe)-status

plant-derived antimicrobials (PDAs), namely β-resorcylic acid (BR), carvacrol (CR), and trans-

cinnamaldehyde (TC) either alone or in combination with hydrogen peroxide (HP) as post-

processing dip treatments on frankfurters was investigated. Frankfurters were surface inoculated

with a five-strain mixture of L. monocytogenes (~ 6.0 log CFU per frankfurter), followed by dip

treatment at 55°C for 60 s or 65°C for 30 s in sterile deionized water, or water containing BR

(1.5% w/v; 97 mM), CR (0.75% v/v; 50 mM), or TC (0.75% v/v; 56 mM) either alone or in

combination with HP (0.1% v/v; 29 mM). Treated frankfurters were vacuum-packaged, and

stored at 4°C for 70 days. Representative samples were analyzed on days 0, 1, 3, 7, 14, 28, 42,

56, and 70 of refrigerated storage for enumerating surviving L. monocytogenes on frankfurters.

Six frankfurters were sampled at each time point for each treatment.

On day zero, all PDAs reduced L. monocytogenes counts by > 2 log CFU/frankfurter at both temperatures (P < 0.05), compared to controls. From days 1 to 70, L. monocytogenes counts

on PDA-treated frankfurters were consistently lower (P < 0.05) and after 70 days of storage, the

pathogen counts were reduced to undetectable levels on frankfurters treated with PDA-HP

combinations at 65°C, and by combinations of BR and TC with HP at 55°C. Results suggest that

PDAs alone, or in combination with HP could be effectively used as post-processing dips to

108 reduce L. monocytogenes on frankfurters, although follow-up studies on sensory and quality characteristics of PDA-treated frankfurters are necessary.

109 1. Introduction

Listeria monocytogenes is a major foodborne pathogen responsible for causing listeriosis,

which accounts for approximately 19% of all foodborne disease related deaths in the United

States (Scallan et al., 2011). The widespread distribution of L. monocytogenes in the

environment (Chaturongakul et al., 2008, Donnelly, 2001 and Farber and Peterkin, 1991), along with its ability to form biofilms on food processing surfaces (Chavant et al., 2002, Lunden et al.,

2002 and Moretro and Langsrud, 2004), leads to recurring post-processing contamination of ready-to-eat (RTE) meat products, including frankfurters (Beresford et al., 2001 and

Chaturongakul et al., 2008). The psychrotrophic and halotolerant nature further enables L.

monocytogenes to proliferate on ready-to-eat (RTE) meat products during their long-term cold

storage (Brakat, 1999 and Farber and Peterkin, 1991). Since most RTE products, including

frankfurters do not receive significant heat treatments before consumption; these foods once

contaminated can potentially act as a source of infection to humans. The safety concerns are

further raised by reports of several outbreaks of listeriosis due to RTE meat products in the

United States. A multistate outbreak in 1998–1999 that infected 101 humans and caused 21

deaths was linked with the consumption of contaminated frankfurters and deli meats (CDC,

Centers for Disease Control and Prevention, 1999 and Mead et al., 2006). Similarly in 2000 and

2002, multistate outbreaks involving 11 deaths and 6 abortions were linked to consumption of

contaminated deli turkey meat (CDC, Centers for Disease Control and Prevention, 2000 and

CDC, Centers for Disease Control and Prevention, 2002). Because of the high case fatality rate

(~ 30%) associated with listeriosis, the United States Department of Agriculture-Food Safety

Inspection Service (USDA-FSIS) has established a zero tolerance policy for L. monocytogenes

on RTE meat products (Gerba et al., 1996 and U.S. Food Safety and Inspection Service, 1989).

110 Additionally, USDA-FSIS alternatives 1 and 2 of the L. monocytogenes regulations mandate that all RTE meat-processing plants apply post-lethality treatments of the products, which may include the use of antimicrobials to inactivate or suppress L. monocytogenes growth

(Anonymous, 2003 and U.S. Food Safety and Inspection Service, 2004).

Since ancient times, plant extracts have been used in foods as flavor enhancers and preservatives. In the past decade, numerous scientific studies have identified several plant- essential oils with antimicrobial properties (Ahmad and Beg, 2001, Burt, 2004, Holley and Patel,

2005, Tornuk et al., 2011 and Zeng et al., 2012) and various active components of these oils have been identified. Trans-cinnamaldehyde (TC) is an aldehyde extracted from bark of cinnamon tree

(Cinnamomum zeylandicum ). Carvacrol (CR) is a monoterpenoid phenolic compound present in oregano oil extracted from Origanum glandulosum . β-Resorcylic acid (BR; 2, 4 dihydroxy benzoic acid) is found in angiosperms, and is a secondary metabolite involved in key processes regulating plant growth (Friedman et al., 2003). All the aforementioned plant-derived antimicrobials (PDAs) are classified as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (Adams et al., 2004, Adams et al., 2005, Friedman et al., 2003 and

Knowles et al., 2005). Previous research conducted in our laboratory revealed that TC, CR, and

BR were effective in inactivating Salmonella spp. on tomatoes (Mattson et al., 2011). Moreover, these PDAs were also effective in killing major mastitis pathogens in milk (Ananda Baskaran et al., 2009). Hydrogen peroxide (HP) is a GRAS status compound, which has been shown to exert antimicrobial action against various pathogenic bacteria (Miyasaki et al., 1986 and Thomas et al.,

1994), including L. monocytogenes (Ukuku and Fett, 2002). Preliminary results conducted in our laboratory indicated that TC, CR and BR when used in combination with HP were more effective in inactivating L. monocytogenes on frankfurters than when used alone. Thus the objective of this

111 study was to investigate the efficacy of TC, CR, and BR either alone or in combination with HP

as post-processing antimicrobial dips for killing L. monocytogenes on frankfurters, and inhibiting its growth during subsequent storage of frankfurters at 4°C for 70 days.

2. Materials and Methods

2.1. Bacterial strains and culture conditions

All bacteriological media were purchased from Difco (Becton Dickinson, Sparks, MD). Five strains of L. monocytogenes from our culture collection, including ATCC Scott A, ATCC 19115,

101, 1 and Presque-598 were used in this study. Each L. monocytogenes strain was cultured separately in 10 ml of sterile tryptic soy broth with 0.6% yeast extract (TSBYE) in 50 ml screw- cap tubes, and incubated at 37°C for 24 h. Following incubation, the cultures were allowed to form sediments by centrifugation (3600 ×g for 15 min) at 4°C. The pellet was washed twice, resuspended in 10 ml of sterile phosphate buffered saline (PBS, pH 7.0) and serial ten-fold dilutions were inoculated on duplicate tryptic soy agar (TSA) and Oxford agar plates, followed by incubation at 37°C for 24 h. Equal portions of the five strains were combined, diluted appropriately, and the resulting suspension was used as the inoculum. The bacterial count of the five-strain mixture of L. monocytogenes was confirmed by plating 100 l of culture suspension from appropriate dilutions on TSA and Oxford agar plates with incubation at 37°C for 24 h.

2.2. Frankfurters

Fresh, skinless, pork–beef frankfurters (20% fat) were obtained from a local manufacturer. To rule out any inherent L. monocytogenes on the frankfurters, representative samples were placed in a sterile stomacher bag (one frankfurter/bag) (Fisher Scientific Co LLC, Hanover Park, IL) containing 50 ml of PBS, homogenized in a stomacher, and streaked on duplicate Oxford agar plates. In addition, the meat homogenate from the stomacher bag was enriched by transferring 1

112 ml of the sample to 20 ml of TSBYE, and incubating at 37°C for 24 h. The culture was then

streaked on Oxford agar, incubated at 37°C for 48 h and observed for typical L. monocytogenes colonies.

2.3. Inoculation and treatments

The frankfurters were equilibrated to room temperature (23 ± 2°C) and inoculated with the five- strain mixture of L. monocytogenes according to a previously published method (Bedie et al.,

2001 and Garcia et al., 2007). Briefly, each frankfurter was aseptically placed in a sterile stomacher bag and surface inoculated with 500 l of the five-strain mixture of L. monocytogenes

(~ 9.0 log CFU/ml) to obtain an inoculum level of 6.0 log CFU per frankfurter. The inoculum

was spread uniformly over the entire surface of the frankfurter by swirling the sample by hand

for 30 s in a stomacher bag. After inoculation, the frankfurters were placed in a sterile container

to facilitate bacterial attachment (30 min at 4°C), and were then immersed for 30 or 60 s in 100

ml of different dipping solutions held at 55 or 65°C, respectively. The dipping solutions included

0.75% (~ 56 mM) TC (Sigma-Aldrich Corp, St. Louis, MO), 0.75% (~ 50 mM) CR (Sigma-

Aldrich), or 1.5% (~ 97 mM) BR (Sigma-Aldrich) alone or in combination with 0.1% (~ 29 mM)

HP (Sigma-Aldrich). Sterile water containing only HP at 0.1% was also included. All PDAs

except BR were added directly to water to obtain the desired concentrations. BR was first

dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich), and subsequently added to sterile

deionized water to achieve the desired concentration. The concentrations of CR, TC, and BR

were selected based on preliminary experiments conducted in our laboratory. Treatment bags

containing only sterile deionized water without any antimicrobial served as controls. Moreover,

sterile deionized water containing 2.5% DMSO was also included as one of the controls to test if

DMSO by itself exerted any antimicrobial effect on L. monocytogenes. Moreover, inoculated, but

113 untreated frankfurters were included to determine the efficiency of L. monocytogenes inoculation and obtain baseline L. monocytogenes counts on frankfurter surface (baseline samples). After immersion in each dipping solution, the frankfurters were drained (~ 30 s), vacuum packed

(V2222 Vacuum food sealer, FoodSaver Co., Walmart) and stored at 4°C. L. monocytogenes counts on each frankfurter were determined on days 0, 1, 3, 7, 14, 28, 42, 56, and 70 of storage.

2.4. Enumeration of L. monocytogenes on frankfurters

On each sampling day, a frankfurter was transferred to a sterile stomacher bag containing 50 ml of sterile Dey–Engley neutralizing broth (D/E neutralizing broth; Difco, Becton Dickinson,

Sparks, MD), and homogenized in a stomacher for 1 min. The meat homogenate was serially diluted (1:10 in PBS) and plated (100 l) on duplicate Tryptic soya agar (TSA) and Oxford agar plates. The plates were incubated at 37°C for 48 h before counting the colonies. In addition, 1 ml of the meat homogenate was added to 20 ml TSBYE, followed by incubation at 37°C for 24 h.

Following enrichment in TSBYE, the culture was streaked on duplicate Oxford agar plates and incubated at 37°C for 24 h. Representative L. monocytogenes colonies from Oxford agar plates were confirmed by API-Listeria BioMeireux bacterial identification kit (BioMeireux).

2.5. pH determination

The pH of control and treated frankfurters was determined using an Accumet AR50 pH meter

(Fisher Scientific) standardized against pH 4.0 and pH 7.0 buffers. Each frankfurter was blended with 100 ml of sterile deionized water and the pH was measured (Garcia et al., 2007).

2.6. Statistical analysis

A completely randomized design with a 18 × 9 factorial treatment structure was used. Factors included 18 treatments and 9 sampling points (day 0, 1, 3, 7, 14, 28, 42, 56, 70). Six frankfurters

(n = 6) were used at each time point for each treatment. Data were analyzed using analysis of

114 variance and mean separation procedures of the Statistical Analysis Software (SAS Institute,

Inc., Cary, NC). The model statement accounted for variation due to different factors and interactions. Differences among means were detected at P < 0.05.

3. Results and Discussion

Since selective culture media such as Oxford agar may inhibit the growth of plant antimicrobial-stressed L. monocytogenes , we used the non-selective medium, TSA and Oxford agar for enumerating L. monocytogenes on frankfurters. However, no significant difference (P >

0.05) was found between L. monocytogenes counts recovered on TSA and Oxford agar plates

(data not shown). L. monocytogenes counts from Oxford agar plates were used for statistical analysis and discussion in the manuscript. Testing of frankfurters for the presence of inherent L. monocytogenes revealed that the frankfurters were not contaminated with the pathogen (data not shown).

The average L. monocytogenes population recovered from frankfurter surface after inoculation (baseline count) was ~ 6.5 ± 0.3 log CFU/frankfurter. The effect of dip treatments containing various PDAs alone (TC 0.75%, CR 0.75%, BR 1.5%), or with HP (0.1%) on L. monocytogenes at two different temperatures–time combinations is shown in Fig. 1 (55°C for

60 s) and Fig. 2 (65°C for 30 s). On control frankfurters dipped in sterile deionized water at 55°C for 60 s, the surviving L. monocytogenes population on day 0 was ~ 6.0 log CFU/frankfurter

(Fig. 1). Thereafter, during subsequent storage for 70 days at 4°C, the pathogen population increased by approximately 2.0 log CFU/frankfurter to reach a final count of

~ 8.0 log CFU/frankfurter ( P < 0.05) at end of 70 days (Fig. 1). However, on control frankfurters dipped in sterile deionized water at 65°C for 30 s, L. monocytogenes population did not change

(P > 0.05), and was ~ 6.0 log CFU/frankfurter on day 70 of storage (Fig. 2). This difference in L.

115 monocytogenes counts between the two control groups of water-treated frankfurters at the end of

70 day storage period could be due to the increased heat injury inflicted on L. monocytogenes at

65°C as against 55°C, and the consequent failure of the injured bacterial cells to grow

significantly at 4°C. Dimethyl sulfoxide (DMSO), which was used as a solvent for the BR did

not exert any inhibitory effect on L. monocytogenes , and no significant difference (P > 0.05) was

observed in L. monocytogenes counts between control samples and 2.5% DMSO treated

frankfurters on day 0 in both the temperature–time combinations ( Fig. 1 and Fig. 2). Moreover,

after 70 day storage period at 4°C, the surviving L. monocytogenes counts obtained on 2.5%

DMSO-treated frankfurters were similar to that on control samples (P > 0.05), suggesting that

2.5% DMSO did not exert any antimicrobial effect on L. monocytogenes . Similar results were

obtained by other researchers who found that DMSO does not exert significant antimicrobial

effect at 2.5% concentration (Ahameethunisa and Hopper, 2010, Nair et al., 2005 and Nakamura

and Hatanaka, 2002).

The dipping of frankfurters in treatment solutions containing the PDAs alone or in

combination with HP resulted in a rapid reduction (day 0) of L. monocytogenes populations by approximately 1 to 2 log CFU/frankfurter at both temperature–time combinations tested (55°C for 60 s or 65°C for 30 s) (Fig. 1 and Fig. 2). The combinations of PDAs with HP were more effective in killing L. monocytogenes on frankfurter than individual treatments (P < 0.05) at both

temperature–time combinations investigated (Fig. 1 and Fig. 2). After 70 days of storage, L.

monocytogenes was reduced to undetectable levels (enrichment negative) on frankfurters treated

with all combination treatments at 65°C (Fig. 2) and by combinations of BR and TC with HP at

55°C (Fig. 1) (P < 0.05). Among the various treatments, BR (1.5%) in combination with HP

(0.1%) was the most effective treatment, which decreased L. monocytogenes to undetectable

116 levels on day 0 (Fig. 2) and day 1 (Fig. 1) on frankfurters dipped at 65°C and 55°C, respectively.

The dipping of frankfurters in water containing TC (0.75%) with HP (0.1%) consistently reduced

L. monocytogenes populations (P < 0.05) during storage as compared to controls and decreased the pathogen to undetectable levels on day 70 at both temperature–time combinations (Fig. 1 and

Fig. 2). Likewise, CR (0.75%) with HP (0.1%) significantly reduced L. monocytogenes counts on day 70 by ~ 5.0 log CFU/frankfurter and to undetectable counts at 55°C and at 65°C, respectively (Fig. 1 and Fig. 2). The enhanced killing effect of the combination treatments on L. monocytogenes could be attributed to the multiple modes of action of the PDAs and hydrogen peroxide on bacterial cells. The hydrophobicity of the PDAs facilitates in targeting lipid- containing bacterial cell membrane (Knobloch et al., 1986 and Sikkema et al., 1995), making them more permeable with leakage of ions and other cell contents (Carson et al., 2002, Cox et al., 2000 and Ultee et al., 2002). However, the mechanism of antimicrobial action of hydrogen peroxide is by generation of peroxide radicals, which causes oxidation of bacterial macromolecules such as proteins and lipids, thereby destroying the cell (Finnegan et al., 2010).

Thus, the PDA-induced damage of bacterial plasma membrane potentially allows rapid accumulation of HP within the cells, thereby resulting in an enhanced bactericidal effect.

Previously, Venkitanarayanan et al. (1999) observed that a combination of hydrogen peroxide with lactic acid or other antimicrobial chemicals at moderate temperatures could rapidly inactivate large populations of Escherichia coli O157:H7 in comparison to the antibacterial effect when these chemicals were present individually.

Among the individual antimicrobial treatments, BR (1.5%) was the most effective in killing L. monocytogenes on frankfurter surface. When applied at 65°C for 30 s, BR (1.5%) consistently inhibited L. monocytogenes growth during the refrigerated storage period and

117 reduced the pathogen counts to undetectable levels at the end of storage on day 70 (Fig. 2),

whereas, at 55°C for 60 s, the treatment decreased pathogen counts on frankfurter surface to

approximately 1.0 log CFU/frankfurter at end of 70 days of storage at 4°C (P < 0.05) (Fig. 1).

Dipping in carvacrol (0.75%) was the least effective PDA treatment, which reduced L. monocytogenes counts by ~ 1.5 to 2.0 log CFU/frankfurter on day 0. Further at any of the time points, no significant reduction in the counts of L. monocytogenes was observed on storage of

CR-treated frankfurters until 70 days at 4°C (Fig. 1 and Fig. 2). Hydrogen peroxide (0.1%) when applied alone decreased L. monocytogenes population by approximately 2.5 to 3.0 log

CFU/frankfurter at the end of storage period.

The pH of various dipping solutions and frankfurters after treatment is provided in Table

1. The mean pH of control water solution with no added antimicrobials was 6.8. The pH of

dipping solutions was affected by the addition of the antimicrobials (P < 0.05) except carvacrol.

The treatment solution containing BR had the lowest pH (2.1 to 2.2), followed by HP and TC.

However, the pH of frankfurters was not affected after dipping treatment with the chemicals (P >

0.05) except for CR (0.75%), which had minimal effect on the pH of frankfurters in comparison

to control.

In conclusion, the PDAs, when applied as antimicrobial dips, were found to be very

effective in inactivating and inhibiting the growth of L. monocytogenes on frankfurters during refrigerated storage. Further, the combinations of PDAs with HP were more effective and rapid in killing L. monocytogenes on frankfurters than single PDA or HP treatment. Results suggest that the PDAs alone, or in combination with HP could be effectively used as post-processing dips to reduce L. monocytogenes on frankfurters. However, follow-up studies on the sensory and quality characteristics of PDA-treated frankfurters are necessary.

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122 Fig. 1. Inactivation of Listeria monocytogenes on frankfurters by dip treatment with β-resorcylic acid (BR), Carvacrol (CR), or Trans -cinnamaldehyde (TC) alone o r in combination with hydrogen peroxide (HP) at 55°C for 60 s. The treatments included Control, DMSO 2.5%, BR

1.5%, CR 0.75%, TC 0.75%, HP 0.1%, BR 1.5% and HP 0.1%, CR 0.75% and HP 0.1%, TC

0.75% and HP 0.1%. Error bars represent SEM (n = 6). Appropriate legends are included in the graph.

123 Fig. 2. Inactivation of Listeria monocytogenes on frankfurters by dip treatment with β-resorcylic acid (BR), Carvacrol (CR), or Trans -cinnamaldehyde (TC) alone or in combination with hydrogen peroxide (HP) at 65°C for 30 s. The treatments included Control, DMSO 2.5%, BR

1.5%, CR 0.75%, TC 0.75%, HP 0. 1%, BR 1.5% and HP 0.1%, CR 0.75% and HP 0.1%, TC

0.75% and HP 0.1%. Error bars represent SEM (n = 6). Appropriate legends are included in the graph.

124 Table 1. Effect of PDA and HP dip treatments on pH of frankfurters a

Treatments pH of dip treatment solution pH of frankfurters

Control 6.80 ±0.07 a 5.83 ± 0.05 a

DMSO (2.5%) 4.27 ± 0.10 b 5.66 ± 0.12 a

BR (1.5%) 2.20 ± 0.08 b 5.84 ± 0.09 a

CR (0.75%) 6.80 ± 0.04 a 6.10 ± 0.08 b

TC (0.75%) 3.77 ± 0.09 b 5.94 ± 0.04 a

HP (0.1%) 3.35 ± 0.05 b 5.54 ± 0.10 a

BR (1.50%) + HP (0.1%) 2.13 ± 0.11 b 5.53 ± 0.09 a

CR (0.75%) +HP (0.1%) 6.40 ± 0.08 b 5.65 ± 0.07 a

TC (0.75%) + HP (0.1%) 3.36 ± 0.10 b 5.91 ± 0.05 a

a Values are given as means ± standard errors. Means within a column with different letters are significantly different (P < 0.05).

125

Chapter V

Efficacy of plant-derived compounds combined with hydrogen peroxide as antimicrobial

wash and coating treatment for reducing Listeria monocytogenes on cantaloupes

(Under Review in Food Microbiology )

126

Abstract

The efficacy of four plant-derived antimicrobials (PDAs), namely carvacrol (CR), thymol

(TY), β-resorcylic acid (BR), and caprylic acid (CA), either alone or in combination with

hydrogen peroxide (HP), as antimicrobial wash and coating treatments for reducing Listeria monocytogenes on cantaloupes was investigated. Further, the efficacy of wash treatments in reducing transfer of L. monocytogenes from the fruit surface to the interior was examined.

Cantaloupe rind plugs inoculated with L. monocytogenes (10 7 CFU/cm 2; five-strain cocktail)

were washed for 3, 6, and 10 min at 25°C or 1, 3, and 5 min at 55 or 65°C in sterile deionized

water (control), or water containing 2% PDAs with or without 2% HP. Additionally, inoculated

whole cantaloupes (10 8 CFU/fruit) washed with PDA-HP combinations at 55 or 65°C for 5 min were cut into rindless pieces, stored at 4°C for 7 days and sampled for L. monocytogenes .

Furthermore, to test the antilisterial efficacy of PDAs while used as fruit coating, inoculated rind plugs coated with PDAs and stored at 4°C for 7 days were sampled for surviving L. monocytogenes . Individual PDA washes reduced L. monocytogenes on rinds by ≥ 2.5 log

CFU/cm 2 by 3 min at all temperatures (P < 0.05). Moreover, PDA-HP combinations decreased L. monocytogenes to undetectable levels by 5 min at 55 or 65°C, and 10 min at 25°C (P<0.05). In addition, PDA-HP washes reduced L. monocytogenes transfer from cantaloupe surface to interior

(P<0.05). All coating treatments reduced L. monocytogenes on cantaloupe surfaces to undetectable levels by 7 days (P < 0.05). Results indicate that the PDAs alone, or with HP could be used to reduce L. monocytogenes on cantaloupes.

127 1. Introduction

Listeria monocytogenes is a major foodborne pathogen in United States implicated in several multi-state outbreaks characterized by high mortality rate (CDC 2012a; Scallan et al.,

2011). A majority of these outbreaks has been primarily due to the consumption of dairy and ready-to-eat (RTE) meat products contaminated with L. monocytogenes (CDC 1999; CDC 2000;

CDC 2002; Mead et al., 2006). However, fresh produce has been increasingly associated with L. monocytogenes outbreaks in the last decade (Beuchat, 1996; CDC 2012b; FDA 2010). In 2011, a nation-wide outbreak of listeriosis affecting 146 people from 28 states with 30 deaths and 1 miscarriage was linked to the consumption of cantaloupes contaminated with the pathogen

(CDC, 2012a). In investigations following the outbreak, a number of deficiencies in cantaloupe packing and processing plant sanitation were observed (U.S. FDA, 2011). Analysis of foodborne pathogens such as L. monocytogenes and Salmonella spp. previously implicated in disease outbreaks associated with cantaloupes have shown that these pathogens can survive in soil for long duration (Islam et al., 2003; Watkins and Sleath, 1981; Welshimer, H.J. 1960), thereby potentially contaminating fruit rinds (Mohle-Boetani et al., 1999), irrigation (Steele and

Odumeru, 2004) and wash water (Parnell et al., 2005). L. monocytogenes can also be introduced in the fields by infected animals such as ruminants, reptiles, rodents and birds (Geldreich and

Bordner, 1971). Postharvest shipping and storage can lead to cross-contamination between cantaloupes (Hedberg et al., 1994; Tauxe, 1997), and contamination of cut pieces of the fruit

(Parnell et al., 2005).

In the United States, the cantaloupe industry has traditionally relied on good agriculture practices, implementation of hazard analysis critical control points, and the use of various chemical wash treatments to reduce pathogenic and spoilage microbial load on the fruit (Ray,

128 1992; Wei et al., 1985). The United States Food and Drug Administration alternatives 3 (Federal

Register Interim Final Rule 9 CFR Part 430) of the L. monocytogenes regulations recommends processing plants to implement sanitization program(s) for low risk foods such as vegetables

(US-FDA, 2006). Chlorine (200 ppm) has been commonly used for washing cantaloupes, but results in only 1-2 log CFU reduction in pathogen counts (Richards & Beuchat, 2004; Rodgers et al., 2004; Ukuku et al., 2006; Wei et al., 1995). Moreover, the potential generation of harmful chemical byproducts such as chloramines, trihalomethanes and other organochlorine compounds from the interaction of chlorine with organic materials is of concern due to associated health risks, including cancer (Donato and Zani, 2010, Richardson et al., 1998; Zhang & Farber, 1996).

The efficacy of a variety of antimicrobial chemicals including hydrogen peroxide (Thomas et al.,

1994; Ukuku et al., 2004), nisin (Ukuku & Fett, 2004), acidified calcium sulfate, acidified sodium chlorite and peroxyacetic acid (Fan et al., 2008), or treatments such as hot water (Fan et al., 2008; Ukuku et al., 2004), irradiation (Lamikanra et al., 2005), chitosan coating (Chen et al.,

2012) or their combinations in controlling foodborne pathogens on cantaloupes has been investigated with varying degrees of success. With limitations in the efficacy of chlorine-based sanitizers on fresh produce, extensive research is being conducted to find safe, effective and economical antimicrobial wash treatments to kill pathogens on fresh produce.

Since ancient times, plant extracts have been used as food preservatives, flavor enhancers and dietary supplements to prevent food spoilage, and maintain human health. The antimicrobial activity of several plant-derived compounds has been documented (Burt, 2004; Holley and Patel,

2005), and an array of active components has been identified (Dixon, 2001). Caprylic acid (CA;

Octanoic acid) is an eight-carbon medium chain fatty acid found in coconut oil (Sprong et al.,

2001) and palm oil (Rodger et al., 2001). Carvacrol [CR; 2-methyl-5-(1-methylethyl)-phenol]

129 and thymol (TY; 2-isopropyl-5-methylphenol) are monoterpenoid phenolic compounds present in oregano oil obtained from Origanum glandulosum. β-resorcylic acid (BR; 2, 4 dihydroxy benzoic acid) found in angiosperms, is a secondary metabolite involved in key processes of plant physiology (Friedman et al., 2003). All the aforementioned compounds are classified as generally recognized as safe (GRAS) by the FDA (Adams et al., 2004; Arrebola et al., 1994;

Knowles et al., 2005; Leriche and Carpentier, 1995). Hydrogen peroxide (HP) is another GRAS compound, which has been shown to exert antimicrobial effect against various pathogenic bacteria (Miyasaki et al., 1986; Thomas et al., 1994), including L. monocytogenes (Ukuku &

Fett, 2002). Previously, Mattson and coworkers (2011) found that carvacrol and β-resorcylic acid wash treatments were effective in inactivating Salmonella spp. on tomatoes. In addition,

Venkitanarayanan et al. (1999) observed that combination of hydrogen peroxide with lactic acid inactivated Escherichia coli O157:H7 population rapidly as compared to single compound treatments.

The objective of the present study was to investigate the efficacy of carvacrol, thymol, β- resorcylic acid and caprylic acid as antimicrobial wash and coating treatments, either alone or in combination with hydrogen peroxide, in reducing L. monocytogenes on cantaloupe surface. In addition, the efficacy of wash treatment of PDAs in combination with hydrogen peroxide in reducing L. monocytogenes transfer from cantaloupe surface to the fruit mesocarp was studied.

2. Materials and Methods

2.1. Bacterial strains, growth conditions and inoculum preparation

All bacteriological media were purchased from Difco (Becton Dickinson, Sparks, MD).

Five strains of L. monocytogenes , including Scott A (ATCC), 19115 (ATCC), 101 (meat isolate),

1 (apple isolate) and 598 (Presque Cultures Inc., Erie, PA) were used in this study. Each L.

130 monocytogenes strain was cultured separately in 10 ml of sterile tryptic soy broth with 0.6% yeast extract (TSBYE) in 50 ml sterile tubes (Fisher Scientific Co LLC, Hanover Park, IL) and incubated at 37°C for 24 h. Following incubation, the bacterial cultures were harvested by centrifugation (3600 × g for 15 min) at 4°C. The pellet was washed twice in 20 ml of sterile phosphate buffer saline (PBS, pH 7.0) and resuspended in 10 ml of PBS. The centrifugation parameters for washing the pellet were the same as used for harvesting the bacterial culture.

Equal portions of the five strains were combined, diluted appropriately, and the resulting suspension was used as inoculum (final bacterial concentration ~ 8.5 log CFU/ml). The bacterial count in the five-strain L. monocytogenes cocktail was confirmed by plating 100 l of culture suspension from appropriate dilutions on modified oxford agar (MOA) plates, followed by incubation at 37°C for 48 h.

2.2. Preparation and inoculation of cantaloupe rind plugs

Fresh, whole cantaloupes, free of visual blemishes, were purchased from a local grocery store in Storrs, Connecticut. Cantaloupes were placed on laboratory bench top for approximately

12 h to allow the fruit to attain room temperature (~20°C). Cantaloupe rind plugs were prepared from whole cantaloupes, as described previously (Sapers et al., 2001). Briefly, circular rind plugs

(2.5 cm diameter) with attached edible flesh were obtained from cantaloupes using a sterile stainless steel cork borer. The flesh portions were cut off using a sterile knife to minimize organic matter contamination in treatment solution. The final thickness of the rind plugs was ~

0.5 cm. To represent a focal point of contamination, each cantaloupe rind plug was spot- inoculated with 200 µl of the 5-strain cocktail of L. monocytogenes, as previously described

(Beuchat et al., 2001; Chen et al., 2012). Briefly, the inoculum was spotted (~ 10 spots, 20 µl

131 each) evenly on the rind surface. The rind plugs were then air-dried for 2 h at 20°C in a biosafety cabinet to facilitate bacterial attachment onto cantaloupe surfaces.

Prior to inoculation, representative rind plug samples were placed in sterile stomacher bags (10 cantaloupe rind plug/bag) containing 100 ml of sterile TSBYE, and incubated at 37°C for 24 h for enrichment. The enriched culture was streaked on MOA plates, incubated at 37°C for

48 h and observed for typical L. monocytogenes colonies. This was done to determine if the cantaloupes were naturally contaminated with L. monocytogenes.

2.3. PDA wash treatment of cantaloupe rind plugs

In order to represent postharvest washing of cantaloupes in a dump tank, each cantaloupe rind plug was placed in a sterile Whirl-Pak TM bag (Nasco, Fisher Scientific) containing 50 ml of sterile deionized water (control) or water containing 4% dimethyl sulfoxide

(DMSO, diluent control), chlorine (200 ppm), or PDAs (2%) (Sigma Aldrich, St. Louis, MO) either alone or in combination with 2% hydrogen peroxide (Sigma Aldrich). The various treatments applied on cantaloupe surfaces are provided in Table 1. The concentration of the

PDAs and hydrogen peroxide were selected based on preliminary experiments in which PDA concentrations ranging from 0.75 to 2% were investigated for antilisterial efficacy on cantaloupe surfaces (data not shown). All the PDAs except β-resorcylic acid and thymol were added directly to water at the desired concentrations, whereas β-resorcylic acid and thymol were dissolved in

4% DMSO (Sigma Aldrich) and subsequently added to deionized water. The final concentration of DMSO in the wash solution was ≤ 4%. Cantaloupe rind plugs inoculated with L. monocytogenes , but not subjected to wash treatment served as baseline to determine the efficiency of L. monocytogenes inoculation. Moreover, sterile deionized water containing 4%

DMSO was included as one of the controls to test if DMSO alone exerted any antimicrobial

132 effect on L. monocytogenes . Each inoculated cantaloupe rind plug was subjected to PDA wash

treatments at the three temperature-time combinations (25°C for 3, 6, 10 min; 55°C for 1, 3, 5

min; 65°C for 1, 3, 5 min) in a reciprocal water bath shaker (Model R76; New Brunswick

Scientific, Edison, NJ). After treatment, each cantaloupe rind plug was aseptically transferred to

a second Whirl-Pak TM bag containing 50 ml of Dey-Engley neutralizing broth (Singh et al.,

2002) and subjected to stomaching for 2 min. The broth was serially diluted (1:10 in PBS) and

plated (100 l) on MOA plates. The plates were incubated at 37°C for 48 h before counting the

colonies. In addition, 1 ml of neutralizing broth was added to 20 ml of TSBYE, and incubated at

37°C for 24 h. Following enrichment in TSBYE, the culture was streaked on MOA plates,

incubated at 37°C for 48 h, and observed for L. monocytogenes colonies. The limit of detection of L. monocytogenes on MOA plates was 500 cells/cantaloupe rind plug whereas the limit of

detection in enrichment was 50 cells/rind plug.

2.4. L. monocytogenes transfer from cantaloupe surface to inside while cutting

This experiment was conducted to determine the efficacy of wash treatments of PDAs in

combination with hydrogen peroxide in reducing L. monocytogenes transfer from cantaloupe

surface to inside while cutting the fruit. The inoculation of whole cantaloupes with L.

monocytogenes was done as described previously (Ukuku & Fett, 2002; Ukuku et al., 2012;

Vadlamudi et al., 2012). Briefly, fresh whole cantaloupes (n = 6) maintained at room temperature

(20°C) were submerged in 3 liters of L. monocytogenes inoculum (~ 7.5 log CFU/ml) and rotated

with a sterile glass rod for 10 min to facilitate uniform inoculation. The cantaloupes were

allowed to dry for 2 h in a laminar flow hood followed by dipping (5 min at 55 and 65°C) in 3 L

of sterile deionized water (Control) or water containing DMSO, Chlorine (200 ppm) or 2%

PDAs in combination with 2% hydrogen peroxide. The washed cantaloupes were allowed to air

133 dry in a biosafety cabinet at 20°C for 2 h before preparing fresh cut pieces. The treated cantaloupes were cut into 4 equal parts using a sterile knife. Each cut section was further divided into 6 rindless pieces (~ 50 g) and each piece was stored in a sterile Whirl-Pak TM bag at 4°C. On days 1, 3, 5, and 7 of storage, representative samples were tested for L. monocytogenes presence by enrichment in 200 ml of TSBYE at 37°C for 24 h. The enriched samples were streaked on

MOA plates and incubated 37°C for 48 h before observing for typical L. monocytogenes colonies.

2.5. Preparation of PDA coating solution and application

The coating of cantaloupe rind plugs with PDAs, either alone or in combination with hydrogen peroxide was done according to a previously published method (Chen et al., 2012).

Two grams of low molecular weight chitosan (~5 to 15 kDa) (Sigma Aldrich) was dissolved in sterile deionized water containing 1% acetic acid (Sigma Aldrich), and stirred at room temperature for 12 h to obtain a concentration of 2% (pH 4.6) (Chen et al., 2012). Subsequently, each PDA was added to the chitosan solution at the desired concentrations, and the solution was stirred for another 6 h to facilitate proper mixing of the PDAs. The various treatments used for coating cantaloupes are given in Table 1. Cantaloupe rind plugs were prepared from fresh whole cantaloupes as described previously, and divided into two groups; pre-inoculated and pre-coated, respectively. The cantaloupe rind plugs from the pre-inoculated group were inoculated with L. monocytogenes (7 log CFU/cm 2) as previously mentioned, followed by PDA coating treatments, whereas the rind plugs in the pre-coated group were first applied with the PDA coating solution and subsequently subjected to L. monocytogenes inoculation. For coating surface of cantaloupe rind plugs, 200 µl of each PDA coating solution was applied and spread uniformly over the rind using a sterile L- spreader. Samples were then allowed to dry in a laminar hood for 2 h at 20°C

134 (Chen et al., 2012). After coating treatment, the cantaloupe rind plugs were stored in sterile

Whirl-Pak TM bags at 4°C for 7 days. The enumeration of L. monocytogenes on cantaloupe rind plugs was done as previously described on days 1, 3, 5, and 7 of storage.

2.6 Statistical analyses

For the PDA wash experiment, a completely randomized design (CRD) was used with 12

X 3 X 3 factorial treatment structure. The factors included 12 treatments at 3 temperatures sampled at three time points (3, 6, 10 min for 25°C; 1, 3, 5 min for 55 and 65°C) (Table 1). In the case of PDA coating experiment, a CRD with a 12 x 4 factorial treatment structure [12 treatments at 4 time points (days 1, 3, 5, and 7)] was followed. Five cantaloupe rind plugs were included for each treatment/time point/trial and the study was repeated three times (n=15).

Pooled data were analyzed using the mixed procedure of SAS (Statistical Analysis Software) ver.

9.2. Differences among the means were analyzed at P < 0.05 using Fisher’s least significance difference test with appropriate corrections for multiple comparisons. For the experiments investigating the effect of PDAs in reducing L. monocytogenes transfer from cantaloupe surface to inside while cutting, a CRD was used with 7 X 2 X 4 factorial treatment structure. The factors included 7 treatments at 2 temperatures sampled at 4 time points (days 1, 3, 5, 7). This experiment had duplicate samples and the study was replicated three times (n = 6). The enrichment data were analyzed using Poisson regression procedure using treatment and day of treatment as main effects. Treatment to day interaction was also tested.

3. Results

Testing of cantaloupe rind plugs for the presence of inherent L. monocytogenes revealed that the cantaloupes were devoid of L. monocytogenes (data not shown).

135 The efficacy of wash treatments at the three temperature-time combinations with 2% of

caprylic acid, carvacrol, thymol, or β-resorcylic acid alone or with HP (2%), in reducing

L. monocytogenes on cantaloupe is presented in Figs. 1A-C. The average L. monocytogenes

population (baseline bacterial count) recovered from the undipped cantaloupe rind plugs was ~

7.5 ± 0.2 log CFU/cm 2. On cantaloupe rind plugs washed with sterile deionized water (control) at

25°C (Fig. 1A) and 55 oC (Fig. 1B), ~ 6 to 6.5 log CFU L. monocytogenes /cm 2 was recovered, and this did not decrease with increase in wash time (3, 6, 10 min) (P > 0.05). However, when the rind plugs were subjected to water wash treatment at 65°C (Fig. 1B), L. monocytogenes was reduced by ~ 1.5 log CFU/cm 2 with increase in treatment time from 1 to 5 min.

Washing of cantaloupe rind plugs in PDA treatment solutions with or without hydrogen peroxide resulted in a significant reduction of L. monocytogenes counts compared to controls at the three temperature-time combinations (Figs. 1A-C). The dipping of rind plugs in treatment solutions containing only the PDAs or hydrogen peroxide for 1 and 3 min decreased L. monocytogenes population by > 2.0 to 2.5 log CFU at the temperature-time combinations tested

(55°C for 60 s or 65°C for 30 s). However, the PDA-HP combinations were more effective than the individual treatments, especially with the 5 min dipping time at 55 and 65°C (P<0.05). For example, after 10 min of wash treatment at 25°C, all PDA-HP combinations reduced L. monocytogenes to undetectable levels on cantaloupe rind plugs ( P<0.05), whereas the same magnitude of reduction was observed with 5 min of treatment time at 55 and 65°C. Moreover, no

L. monocytogenes was detected in the wash water containing the PDA, HP or chlorine at any of the treatment temperatures, whereas ~ 2.0 ± 0.35 log CFU/ml of the pathogen was recovered from the control wash solution after dipping the inoculated rind plugs.

136 Since the PDA-HP combinations at 55 and 65°C for 5 min were found to be more effective in killing L. monocytogenes on cantaloupe surface, the efficacy of these treatments in reducing pathogen transfer from the fruit surface to interior while cutting the whole cantaloupe was investigated (Table 2). It was observed that more than 90% of the cut fruit samples (n = 36) from the cantaloupes subjected to water, chlorine and DMSO washes tested positive for L. monocytogenes at both temperatures (P < 0.05). However, all samples from PDA-HP treatments except β-resorcylic acid-HP and carvacrol-HP combination were devoid of the pathogen

(enrichment negative). All treatments differed significantly from the controls ( P<0.0001).

Moreover, only less than 12% of the fruit pieces from cantaloupes washed with β-resorcylic acid-HP and carvacrol-HP treatments yielded L. monocytogenes after enrichment. These results indicate that L. monocytogenes transfer from the fruit surface to inside was significantly reduced following the washing of cantaloupes in PDA-HP treatments.

The efficacy of PDA coating with and without hydrogen peroxide in reducing L. monocytogenes survival on pre-inoculated cantaloupes, and L. monocytogenes persistence on cantaloupe surface pre-coated with the compounds are presented in Table 3A and 3B, respectively. Approximately 7 log CFU/cm 2 of L. monocytogenes was recovered from the control rind surface, whereas coating of chitosan or hydrogen peroxide alone reduced the pathogen population by 3 log and 5 log CFU/cm 2, respectively on day 7 of storage ( P<0.05). However, from the first day of storage L. monocytogenes was not detected (enrichment negative) on rind surfaces, which were subjected to PDA-HP coating either before or after pathogen inoculation.

4. Discussion

Since cantaloupes can potentially get contaminated with pathogens preharvest in the field, postharvest in the processing plants, and during shipping, there is a need for decontamination

137 strategies, which are effective, and easily implemented at various stages of the cantaloupe processing, handling and distribution system.

Washing of cantaloupe rind plugs in sterile deionized water and water containing chlorine or DMSO (diluent control) had a minimal effect in reducing L. monocytogenes counts. Although there was a trend of increasing reductions in L. monocytogenes counts with increase in temperature, no significant differences in pathogen counts were observed among the samples subjected to washing in sterile water and water containing chlorine or DMSO (Fig. 1A, B, C).

Similar results were reported by other investigators, who found that a wash water treatment at

70°C for 1 min resulted in a reduction of only 2.0 log CFU/cm 2 Salmonella on cantaloupe surface (Ukuku et al., 2004). This could be attributed to the rough surface characteristic of the cantaloupes, where the microscopic lenticellar netting present on cantaloupe surface facilitates strong bacterial attachment by increasing the attachment sites besides providing a safe niche that protects bacteria from aqueous disinfectants (Annous et al., 2004; Wang et al., 2009; Ukuku and

Fett, 2002).

Temperature had a significant impact on the effect of various PDA treatments in reducing

L. monocytogenes population on the rind plugs. For example, the magnitude of L. monocytogenes reductions on cantaloupe surface brought about by the various PDA treatments after 10 min at 25 oC was achieved with 5 min treatment time at 55°C (Fig 1B). Similarly, all the PDA treatments with and without hydrogen peroxide applied at 65°C for 5 min (Fig 1C) reduced L. monocytogenes to undetectable levels (negative by enrichment), whereas the same degree of reduction in the pathogen counts was brought about at 55°C only by the PDA-HP treatments. The greater reduction in L. monocytogenes counts observed at 55°C and 65°C could

138 be attributed to the increased heat injury inflicted on L. monocytogenes at the higher temperatures compared to 25°C.

The combination of PDAs with hydrogen peroxide was more effective in reducing L. monocytogenes on cantaloupe surface than the individual treatments ( P < 0.05) (Fig. 1A and 1B).

For example, all the PDA-HP treatments applied at 25°C for 10 min or 55°C for 5 min decreased

L. monocytogenes population on the rind plugs to undetectable levels (negative by enrichment), whereas more than 4.0 log CFU/cm 2 of the pathogen was recovered from samples subjected to dipping in PDA alone. The higher inactivation efficacy of the PDA-HP treatments on L. monocytogenes could be attributed to the multiple modes of action of the PDAs and hydrogen peroxide on bacterial cells. The hydrophobicity of the PDAs enables them to target lipid- containing bacterial cell membrane (Knobloch et al., 1986; Sikkema et al., 1995), making them more permeable and leading to leakage of ions and other cell contents (Carson et al., 2002, Ultee et al., 2002). However, the mechanism of antimicrobial action of hydrogen peroxide is by generation of peroxide radicals, which cause oxidation of bacterial macromolecules such as proteins and lipids, thereby destroying the cell (Finnegan et al., 2010; Linley et al., 2012). Thus, the PDA-induced damage of bacterial plasma membrane potentially allows rapid accumulation of hydrogen peroxide within the cells, thereby resulting in an enhanced bactericidal effect. An increased antibacterial effect of combining hydrogen peroxide with lactic acid or other antimicrobial chemicals was previously observed against E. coli O157:H7 in comparison to treatments containing the chemicals individually (Venkitanarayanan et al., 1999).

Although on-farm washing of cantaloupes is critical for improving its microbiological safety and shelf life, the fruit can potentially get recontaminated during storage and transport with other batches of cantaloupes. Moreover, researchers have found that there is an equal or

139 even greater risk of post-wash recontamination of fruit due to persisting pathogen load, and/or

damaged integrity of surface rind during washing steps, thereby augmenting pathogen entry into

the fruit (Bennik et al., 1996; Ukuku, 2006). In order to prevent this, edible coatings and films

that can act as antimicrobial carriers have been investigated on foods, including fruits (Chen et al

2012; Vargas et al., 2006). Chitosan is an organic, GRAS-status, non-toxic polymer derived from

the deacetylation of chitin, a natural polysaccharide present as the main component of

exoskeletons of crustaceans (Ravi Kumar, 2000). Chitosan is biodegradable and has been widely

used as an antimicrobial coating and film-forming polymer on food products (Li et al., 1992;

Shahidi et al., 1999). Therefore, we investigated the efficacy of PDA treatments delivered

through a chitosan based coating in controlling L. monocytogenes on cantaloupe surface. All the

PDA coating treatments (with or without hydrogen peroxide) were found to be very effective in

killing L. monocytogenes on cantaloupes. On cantaloupe plugs coated with the PDAs before L.

monocytogenes inoculation (to control L. monocytogenes where cantaloupes would be exposed to the pathogen during post-washing storage and transport), no L. monocytogenes was detected (by plating and enrichment) on caprylic acid and PDA-HP coated plugs during the entire storage period (Table. 3A). Similar results were also observed on the cantaloupe plugs that were inoculated with L. monocytogenes and subsequently subjected to PDA coating (to control L.

monocytogenes surviving the chemical wash treatments on cantaloupes and re-contaminating

fresh fruits) (Table. 3B). This enhanced efficacy of PDA coating treatments against L.

monocytogenes could be due to the increased contact time between the antimicrobials and the

pathogen on cantaloupe surface. In addition, the gradual release of the PDAs from the coating

(active packaging) could have contributed to the extended antimicrobial protection provided by

the coating (Chang et al., 2000).

140 In conclusion, the PDAs alone or in combination with hydrogen peroxide were found to be effective ( P<0.05) in reducing L. monocytogenes on cantaloupe surface, and preventing L. monocytogenes transfer to inside of the fruit. The PDAs were also effective in killing L. monocytogenes when applied as a coating on cantaloupe surface. The PDAs, especially with hydrogen peroxide could potentially be used to decontaminate cantaloupes, but follow up studies on the sensory and quality characteristics of PDA-treated cantaloupes are warranted.

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147 Fig.1. Inactivation of L. monocytogenes on cantaloupe surface by wash treatment with caprylic acid (CA), carvacrol (CR), thymol (TY ), β-resorcylic acid (BR) alone or in combination with hydrogen peroxide (HP) at (A) 25°C for 3, 6, and 10 min (B) 55°C for 1 , 3, and 5 min (C) 65°C for 1, 3, and 5 min. ***All treatments at all sampling time points were significantly different from baseline, positive control and DMSO control at P < 0.05. * Enrichment negative.

A)

148 B)

149 C)

150 Table 1. PDA wash and coating treatments.

Wash treatments Coating treatments

Control (wash with sterile deionized water) Control (No coating)

Dimethyl sulfoxide (DMSO; 4%) DMSO (4%)

Hydrogen peroxide (HP 2%) HP (2%)

Caprylic acid (CA; 2%) CA (2%)

Carvacrol (CR; 2%) CR (2%)

Thymol (TY; 2%) TY (2%)

β-resorcylic acid (BR, 2%) BR (2%)

CA (2%) + HP (2%) CA (2%) + HP (2%)

CR (2%) + HP (2%) CR (2%) + HP (2%)

TY (2%) + HP (2%) TY (2%) + HP (2%)

BR (2%) + HP (2%) BR (2%) + HP (2%)

Chlorine (200ppm) Chitosan (2%)

151 Table 2. Effect of PDA-HP wash treatments at (A) 55°C for 5 min and (B) 65°C for 5 min on

transfer of L. monocytogenes from cantaloupe surface to inside during cutting. Treatments included caprylic acid (CA), carvacrol (CR), thymol (TY), β-resorcylic acid (BR) in combination with hydrogen peroxide (HP). All PDA-HP treatments at all time points were significantly different from control, DMSO and chlorine treatments at P < 0.0001.

2A)

Number of samples positive by enrichment Treatments Day 1 Day 3 Day 5 Day 7 Control 36/36 36/36 36/36 36/36 DMSO 4% 36/36 36/36 36/36 36/36 Chlorine (200 ppm) 36/36 36/36 36/36 36/36 BR 2% + HP 2% 1/36 0/36 0/36 0/36 TY 2% + HP 2% 0/36 0/36 0/36 0/36 CR 2% + HP 2% 4/36 2/36 2/36 2/36 CA 2% + HP 2% 0/36 0/36 0/36 0/36

(2B)

Number of samples positive by enrichment Treatments Day 1 Day 3 Day 5 Day 7 Control 36/36 36/36 36/36 36/36 DMSO 4% 36/36 36/36 36/36 36/36 Chlorine (200 ppm) 36/36 36/36 36/36 36/36 BR 2% + HP 2% 1/36 0/36 0/36 0/36 TY 2% + HP 2% 0/36 0/36 0/36 0/36 CR 2% + HP 2% 2/36 0/36 0/36 0/36 CA 2% + HP 2% 0/36 0/36 0/36 0/36

152 Table 3A. Effect of chitosan based coating of cantaloupe surface with caprylic acid (CA),

carvacrol (CR), thymol (TY), β-resorcylic acid (BR) alone or in combination with hydrogen

peroxide (HP) on L. monocytogenes . All PDA treatments either alone or in combination with HP at all time points were significantly different from control and chitosan treatments at P < 0.05.

Surviving L. monocytogenes counts (Log CFU/cm 2) on cantaloupe after coating treatment at

25°C

Coating treatments Day 1 Day 3 Day 5 Day 7 Control 7.02±0.39 7.09±0.80 7.08±0.22 7.13±0.32 Chitosan 2% 6.00±0.41 5.87±0.55 4.85±0.19 4.80±0.41 DMSO 4% 6.50±0.25 6.00±0.80 4.55±0.65 4.60±0.83 HP 2% 5.30±0.44 5.24±0.39 2.70±0.80 2.60±0.50 CA 2% 0.00±0.00* 0.00±0.00* 0.00±0.00* 0.00±0.00* CR 2% 3.54±0.64 3.17±0.14 0.00±0.00* 0.00±0.00* TY 2% 4.00±0.80 2.69±0.56 0.00±0.00* 0.00±0.00* BR 2% 3.87±0.70 2.80±0.60 0.00±0.00* 0.00±0.00* BR 2% + HP 2% 0.00±0.00* 0.00±0.00* 0.00±0.00* 0.00±0.00* TY 2% + HP 2% 0.00±0.00* 0.00±0.00* 0.00±0.00* 0.00±0.00* CR 2% + HP 2% 0.00±0.00* 0.00±0.00* 0.00±0.00* 0.00±0.00* CA 2% + HP 2% 0.00±0.00* 0.00±0.00* 0.00±0.00* 0.00±0.00*

*Enrichment negative

153 Table. 3B. Survival of L. monocytogenes on cantaloupe surface pre-coated with caprylic acid

(CA), carvacrol (CR), thymol (TY), β-resorcylic acid (BR) alone or in combination with hydrogen peroxide (HP). All PDA treatments either alone or in combination with HP at all time points were significantly different from control and chitosan treatments at P < 0.05.

Surviving L. monocytogenes counts (Log CFU/cm 2) on cantaloupe after coating treatment at

25°C.

Coating treatments Day 1 Day 3 Day 5 Day 7 Control 7.09±0.50 7.09±0.33 7.08±0.41 7.25±0.26 Chitosan 2% 5.80±0.36 5.35±0.65 4.65±0.21 3.60±0.34 DMSO 4% 4.80±0.50 5.50±0.80 4.20±0.64 3.50±0.40 HP 2% 4.65±0.49 4.35±0.74 2.70±0.80 2.50±0.14 CA 2% 3.20±0.66 3.50±0.35 0.00±0.00* 0.00±0.00* CR 2% 3.65±0.58 3.95±0.40 0.00±0.00* 0.00±0.00* TY 2% 4.00±0.50 2.75±0.35 0.00±0.00* 0.00±0.00* BR 2% 3.50±0.47 2.65±0.50 0.00±0.00* 0.00±0.00* BR 2% + HP 2% 0.00±0.00* 0.00±0.00* 0.00±0.00* 0.00±0.00* TY 2% + HP 2% 0.00±0.00* 0.00±0.00* 0.00±0.00* 0.00±0.00* CR 2% + HP 2% 0.00±0.00* 0.00±0.00* 0.00±0.00* 0.00±0.00* CA 2% + HP 2% 0.00±0.00* 0.00±0.00* 0.00±0.00* 0.00±0.00*

*Enrichment negative.

154

Chapter VI

Effect of plant-derived antimicrobials and probiotic bacteria on

Listeria monocytogenes virulence in vitro and in an invertebrate model, Galleria mellonella

155 Abstract

Listeria monocytogenes is a foodborne pathogen of significant concern in the United

States. Since crossing the intestinal barrier is the first critical step in listeriosis, reducing L. monocytogenes invasion of the intestinal epithelium, and production of its virulence factors could potentially improve the disease outcome in humans.

In this study, the efficacy of sub-inhibitory concentrations (SICs, concentrations not inhibiting bacterial growth) of four plant-derived antimicrobials (PDAs; trans-cinnamaldehyde, carvacrol, thymol, eugenol), either alone, or in combination with five probiotic bacteria, namely

Bifidobacterium bifidum (NRRL-B41410), Lactobacillus reuteri (B-14172), L. fermentum (B-

1840), L. plantarum (B-4496), and Lactococcus lactis subspecies lactis (B-633) in reducing L.

monocytogenes adhesion to and invasion of human intestinal epithelial cells (Caco-2) was investigated. Additionally, the effect of aforementioned PDAs and probiotics on L. monocytogenes motility, listeriolysin production, epithelial E-cadherin binding, and expression of virulence genes was investigated. Moreover, the in vivo efficacy of PDA-probiotic treatments in reducing L. monocytogenes virulence in the invertebrate model, Galleria mellonella was studied. Plant compounds and probiotics, either alone or in combination, significantly reduced L. monocytogenes adhesion to and invasion of intestinal cells (P < 0.05). Moreover, PDA-probiotic treatments decreased L. monocytogenes motility, hemolysin production, E-cadherin binding, and virulence gene expression (P < 0.05). In addition, the PDA-probiotic treatments significantly enhanced the survival rates of G. mellonella infected with lethal doses of L. monocytogenes (P <

0.05). The results highlight the antilisterial effect of aforementioned PDAs and probiotics, and justify further investigations in a mammalian model.

156 1. Introduction

Listeria monocytogenes (LM) has emerged as one of the major foodborne pathogens responsible for causing listeriosis, a severe, invasive, multi-systemic infection, characterized by high hospitalization (90%) and case fatality rates (20-30%) (Cartwright, 2012;

Rocourt and Brosch, 1992; Schuchat et al., 1991). Being a foodborne pathogen, the gastrointestinal tract is the major portal of entry for L. monocytogenes in the host (Vazquez-

Boland et al., 2001; Farber and Peterkin, 1991). In the human gastrointestinal tract, L. monocytogenes attaches and invades the intestinal epithelium, followed by systemic spread to other internal organs (Parida et al., 1998). An array of cell surface proteins, virulence factors and transcriptional regulators critical for attachment to and invasion of intestinal epithelium, intracellular proliferation, macrophage survival, and cell-to-cell spread in the host have been characterized in L. monocytogenes (Vazquez-Boland et al., 2001). These factors include internalins (Lingnau et al., 1995; Southwick and Purich, 1996), listeriolysin O (LLO)

(Greiffenberg et al., 1998; Smith-Palmer et al., 2002), phospholipases (Grundling et al., 2003;

Marquis and Hager, 2000) and actin polymerization protein, ActA (Alvarez-dominguez et al.,

1997; Kocks et al., 1992). Moreover, flagella-based motility increases the efficiency of epithelial cell invasion and contributes to the virulence of L. monocytogenes (Bigot et al. 2005; Dons et al.

2004). Thus, reducing L. monocytogenes motility, invasion of host tissue, and production of virulence factors that facilitate its intracellular survival and systemic spread could potentially control listeriosis in humans. Antibiotics are commonly used for the treatment of listeriosis; however, there have been recent reports of development of antibiotic resistance in L. monocytogenes (Krawczk-Balska et al., 2012; Cekmez et al., 2012). This increasing antibiotic resistance in L. monocytogenes and growing concerns over the reports of adverse drug reactions

157 in patients (Thong and Tan, 2011) has ignited an interest in exploring the potential of various

natural approaches as an alternative strategy to combat foodborne infections.

Since ancient times, plant extracts have been used as food preservatives, flavor enhancers

and dietary supplements to prevent food spoilage, and maintain human health. In addition,

fermented foods containing beneficial microbiota have been a part of traditional diets for their

health benefits. The antimicrobial activity of several plant-derived antimicrobials (PDAs) has

been documented (Ahmad and Beg, 2001; Bhatt and Negi, 2012; Kubo et al., 1993; Negi et al.,

1999, 2003a, 2003b, 2005, 2010; Silva et al., 1996; Zeng et al., 2012). A great majority of these

compounds are secondary metabolites, and are produced as a result of reciprocal interactions

between plants, microbes, and animals (Reichling, 2010), thereby facilitating plants to survive

local environments (Harborne, 1993) and microbial infections (Kennedy and Wightman, 2011).

Trans-cinnamaldehyde (TC) is an aldehyde present in the extract of cinnamon bark

(Cinnamomum zeylandicum ). Carvacrol (CR) and thymol (TY) are antimicrobial compounds in oregano oil obtained from Origanum glandulosum. Eugenol (EG) is an active ingredient in the oil of clove ( Eugenia caryophyllus ). All the aforementioned plant compounds are categorized as generally regarded as safe (GRAS) by the Food and Drug Administration (Arrebola et al., 1994;

Leriche and Carpentier, 1995).

The Food and Agriculture organization (FAO) and World Health Organization (WHO) define probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The major health benefits exerted by probiotic bacteria include nutrient assimilation (Sonnenburg et al. 2005; Yatsunenko et al. 2012), potentiating host immune function (Olszak, T. et al. 2012), and protection against enteric pathogens (Candela et al., 2008;

Fukuda et al., 2011). Although several species of bacteria have been identified, the majority of

158 probiotic bacteria supplemented in human diet belong to Lactobacillus and Bifidobacterium

species. Interestingly, recent studies have shown that PDAs that are highly bactericidal towards

enteric pathogens exert low antimicrobial effects against commensal gut microbiota (Hawrelak et

al., 2009; Di Pasqua et al., 2005). Since PDAs and probiotics exert their antimicrobial action by

different mechanisms (Shipradeep et al., 2012), a combinatorial approach using PDAs and

probiotics could be more effective in controlling L. monocytogenes . However, studies eliciting

their synergistic interactions on bacterial virulence are lacking.

The present study investigated the efficacy of sub-inhibitory concentrations (SICs,

concentrations not inhibitory to bacterial growth) of trans-cinnamaldehyde, carvacrol, thymol,

and eugenol, either alone or in combination with five probiotic bacteria, namely Bifidobacterium bifidum (NRRL-B41410), Lactobacillus reuteri (B-14172), L. fermentum (B-1840), L. plantarum

(B-4496), or Lactococcus lactis subspecies lactis (B-633) in reducing L. monocytogenes adhesion to and invasion of human intestinal epithelial cells (Caco-2), and production of virulence factors in vitro. In addition, the in vivo efficacy of PDA-probiotic treatments in reducing L. monocytogenes virulence in the invertebrate model, Galleria mellonella was investigated.

2. Materials and methods

2.1 Bacterial strains and growth conditions

All bacteriological media were purchased from Difco (Becton Dickinson, Sparks, MD).

Three strains of L. monocytogenes , including ATCC Scott A, 19115, and Presque-598, and five probiotic bacteria, namely Bifidobacterium bifidum (NRRL-B41410), Lactobacillus reuteri (B-

14172), L. fermentum (B-1840), L. plantarum (B-4496), and Lactococcus lactis subspecies lactis

(B-633) were used in this study. Each L. monocytogenes strain was cultured separately in 10 ml

159 of sterile tryptic soy broth with 0.6% (wt/vol) yeast extract (TSBYE) in 50 ml screw-cap tubes,

and incubated at 37°C for 24 h. Following incubation, the cultures were sedimented by

centrifugation (3600 × g for 15 min) at 4°C. The pellet was washed twice, resuspended in 10 ml

of sterile phosphate buffered saline (PBS, pH 7.0), and serial ten-fold dilutions were cultured on

duplicate tryptic soy agar (TSA) and modified oxford agar plates, followed by incubation at

37°C for 48 h. The probiotic bacteria were cultured in De Man-Rogosa-Sharpe (MRS) broth

supplemented with 0.05% (wt/vol) L-cysteine hydrochloride (Sigma Aldrich Chemical Co., St

Louis, MO), and incubated in an anaerobic incubator (88% Nitrogen, 10% carbon dioxide, 2%

oxygen) at 37°C for 24 h. Following incubation, the probiotic cultures were serially diluted in

PBS and plated on MRS agar plates, followed by incubation at 37°C for 48 h for bacterial

enumeration.

2.2 Plant-derived antimicrobials, probiotics and SIC determination

The SIC of each plant compound was determined using a standard protocol (Amalaradjou et al.,

2011; Johny et al., 2010). Sterile 24-well polystyrene tissue culture plates (Costar, Corning

Incorporated, Corning, NY) containing TSBYE were inoculated with ~6.0 log CFU of L. monocytogenes , followed by the addition of 1 to 10 l of trans-cinnamaldehyde, carvacrol,

thymol, or eugenol (Sigma-Aldrich) with an increment of 0.5 l. The plates were incubated at

37°C for 24 h, and bacterial growth was determined by culturing on modified oxford agar plates.

For establishing the SICs of probiotic culture supernatant, the cell-free probiotic culture

supernatant was serially diluted (100, 50, 25, 12.5, 6.25%) in 100 l MRS broth, followed by

addition of an equal volume of TSBYE containing ~10 6 L. monocytogenes. The plates were

incubated as described above, followed by bacterial enumeration. The two highest concentrations

of each plant compound or probiotic culture supernatant that did not inhibit L. monocytogenes

160 growth after 24 h of incubation were selected as the SICs for this study. Duplicate samples were

included and the experiment was repeated three times.

2.3 Motility assay

The effect of trans-cinnamaldehyde, carvacrol, thymol, and eugenol either alone or in

combination with probiotic culture supernatant of B. bifidum , L. reuteri , L. fermentum , L. plantarum , and L. lactis subspecies lactis on L. monocytogenes motility was determined according to a previously published method (Niu and Gilbert, 2004). Separate Luria Bertani (LB) agar (0.3%) plates containing the respective SICs of each plant compound either alone or in combination with probiotic culture supernatants were prepared. Luria Bertani agar not supplemented with the PDAs, probiotic supernatants or their combinations served as controls. An overnight culture of L. monocytogenes was centrifuged at 4000 ×g for 10 min, and washed three times with PBS. Twenty l of resuspended culture (~8 log CFU/ml) was spot-inoculated at the center of agar plates. The plates were incubated the plates at 37°C for 12 h, and the zone of motility was measured.

2.4 Cell culture

Human enterocyte-like Caco-2 cells (HTB-27) were obtained from the American Type Culture

Collection (Manassas, VA). Caco-2 cells were cultured in 25-cm 2 tissue culture flasks (Falcon,

Becton and Dickinson Company, Franklin Lakes, NJ) with minimum essential medium (MEM)

(Gibco, Invitrogen, Carlsbad, CA) containing 10% (vol/vol) fetal bovine serum (Invitrogen). All

cell cultures were incubated at 37°C in a 5% (vol/vol) CO 2 atmosphere.

2.4.1 Adhesion and invasion assay

The effect of SICs of plant compounds, either alone or in combination with each probiotic

bacterium on L. monocytogenes adhesion and invasion of Caco-2 cells, was determined as

161 previously reported (Moroni et al., 2006). A monolayer of Caco-2 cells was grown in 24-well tissue culture plates (Costar) at ~10 5 cells per well in whole media, and inoculated with L. monocytogenes ~ 6 log CFU/well (MOI-1:10) together with PDA-probiotic treatments (~10 6

CFU/well; MOI-1:10). The L. monocytogenes -inoculated monolayers were incubated at 37 oC for 1 h in a humidified, 5% CO 2 incubator. For the adhesion assay, the infected monolayers were rinsed three times in PBS after 1 h of incubation, and the cells were lysed with 0.1% Triton X-

100 (Invitrogen, Carlsbad, CA). The number of viable adherent L. monocytogenes was determined by serial dilution and culturing on TSA and modified oxford agar plates. For the invasion assay, the Caco-2 monolayer after 1 h of incubation following L. monocytogenes inoculation was rinsed three times in minimal media and incubated for another 2 h in cell culture medium containing gentamicin (100 g/ml) (Invitrogen) to kill the extracellular bacteria.

Subsequently, the wells were washed with PBS three times, followed by addition of 1 ml of PBS containing 0.1% Triton X-100 and incubation at 37°C in 5% CO 2 for 15 min to lyse the eukaryotic cells and release the intracellular L. monocytogenes . The cell lysates were serially diluted, cultured on TSA and modified oxford agar plates, and incubated at 37°C for 24 to 48 h before bacterial enumeration. The experiment was run in duplicate and replicated three times.

2.5 E-Cadherin binding assay

This assay was conducted as described previously, with slight modifications (Tzouvelekis et al.,

1991; Prakash et al., 2011). Briefly, microtiter plates (Corning, Costar) were coated with human recombinant E-Cadherin (5 g/well; Advanced Biomatrix, San Diego, CA) suspended in coating buffer (KPL Inc, Gaithersburg, MA), and incubated overnight at 4°C. The unbound E-cadherin was removed by three washes using washing buffer (KPL). Residual binding sites were blocked with PBS-2% BSA and then incubated at 37°C for 1 h. After removing unbound BSA by three

162 washes (100 l) with washing buffer, L. monocytogenes (~9 log CFU) either alone (control) or with probiotic bacteria (~ 9 log CFU) was added to each well and incubated at 37°C for 4 h. The unbound bacteria were removed by washing three times with wash buffer. One-hundred l of mouse monoclonal antibody (1:100 in PBS-1% BSA; Pierce Thermo Scientific, Rockford, IL) against L. monocytogenes was added to each well and incubated at 37°C for 1 h. The unbound antibody was removed by washing three times. Subsequently, 100 l of anti-mouse HRP conjugated secondary antibody (1:2000 in PBS-1% BSA; KPL) was added to each well and incubated at 37°C for 1 h. The unbound antibody was removed by washing three times with washing buffer. Then, the wells were incubated in the dark with 100 l of substrate-H2O2 for 15 min. One-hundred l of 1 M H 2SO 4 was added to arrest the reaction. The color developed was measured at 450 nm in a micro-plate reader. All experiments were done in triplicate and repeated three times. Specific binding to E-Cadherin was calculated by subtracting the value of bacteria bound to BSA from the values of bacteria bound to E-Cadherin. A value of 0.2 or higher was used as an indicator of specific binding (Prakash et al., 2011).

2.6 Hemolysis assay

Listeriolysin O activity in the supernatant of L. monocytogenes culture grown in the presence or absence of SICs of PDAs, either alone or in combination with probiotic culture supernatant, was quantified by a hemolysis assay described previously (Sampathkumar et al., 1998; Smith-Palmer et al., 2002). Briefly, an overnight culture of L. monocytogenes was centrifuged at 4000 ×g for

10 min and washed three times with PBS. The washed pellet was resuspended in TSBYE at a concentration of ~ 6.0 log CFU/ml, and incubated with or without the SICs of PDA-probiotic supernatant at 37°C for 12 h. One ml of a 12 h L. monocytogenes culture was centrifuged at

12,000 ×g in 1.5 ml sterile tubes, and the supernatant was collected. Defibrinated sheep blood

163 (Quad five, Ryegate, MT) was centrifuged at 600 ×g for 10 min and the supernatant was

discarded. Sheep red blood cells (SRBC) were washed three times and resuspended at 3%

concentration in PBS. Serial 2-fold dilutions were made by mixing 100 l of the L.

monocytogenes culture supernatant with 100 l of PBS, and 100 l of freshly prepared 3%

SRBC was added to each well. The plates were incubated for 30 min at 37°C. Saline controls

(0% hemolysis) obtained by addition of 100 l of PBS to 100 l of 3% SRBC and positive hemolysis controls (100% hemolysis) obtained by addition of 100 l of sterile distilled water and

100 l of 3% SRBC were also included for appropriate comparison. The pellets in the microtiter

plate were mixed using a vortex mixer, the absorbance was measured at 600 nm and percentage

hemolysis was estimated according to the formula % hemolysis = (1 − OD s / OD t) × 100, where

OD s refer to the differences in optical density (600 nm) between the sample and positive control, and OD t refers to the differences in optical density (600 nm) between the saline control and positive control respectively (Bhakdi et al., 1984).

2.7 RNA isolation and real-time quantitative PCR

The effect of PDAs either alone or in combination with probiotic supernatants on the expression of L. monocytogenes virulence genes was investigated using RT-qPCR, as described previously

(Ollinger et al., 2009). Each L. monocytogenes strain was grown separately with the respective

SICs of plant compounds, probiotic supernatants or their combinations at 37°C in TSBYE to

mid-log phase (8 h), and total RNA was extracted using RNeasy RNA isolation kit (Qiagen,

Valencia, CA). Complementary DNA (cDNA) synthesis was performed using the Superscript

Reverse transcriptase kit (Invitrogen). The cDNA synthesized was used as the template for RT-

qPCR. The amplification product was detected using SYBR Green reagents. The primers for

each gene (Table 1) were designed from published GenBank L. monocytogenes sequences using

164 Primer Express® software (Applied Biosystems, Foster city, CA). Relative gene expression was

determined using the comparative critical threshold (Ct) real-time PCR using a 7500 Step One

Real Time PCR system (Applied Biosystems, Carlsbad, CA). Data were normalized to the

endogenous control (16S rRNA), and the level of candidate gene expression between treated and

untreated samples was compared to study relative gene expression, and the effect of PDA-

probiotic treatments on each gene.

2.8 In vivo studies using Galleria mellonella larvae

Larvae of the Greater Wax moth, Galleria mellonella, in the final-instar stage (Mealworms by

the pound, Boerne, TX) were maintained in dark at room temperature, and used within 7 days

from shipment. Prior to experiment, ten larvae were randomly selected and sacrificed to confirm

that the larvae were initially devoid of L. monocytogenes.

2.8.1 Galleria mellonella injection and determination of L. monocytogenes infectious dose

G. mellonella injections were carried out using standard protocol (Mylonakis et al., 2005; Peleg et al., 2006). Three strains (Scott A, 19115, Presque-598) of L. monocytogenes were washed and resuspended separately in PBS at appropriate cell density as described previously. Five l of bacterial inoculums (10 7 to 10 3 CFU/larvae) were injected dorsolaterally into the hemocoel of last-instar larvae using a Hamilton syringe (capacity 50 l; 24G; point style 2 with beveled tip).

After injection, the larvae were incubated at 37°C in sterile petri plates supplemented with sterile bedding material, and the number of dead larvae was scored at 24 h intervals for 7 days. Larvae were considered dead when they displayed no movement in response to touch. For all experiments, the following control groups were used: negative control (larvae inoculated with

PBS), trauma control (larvae administered blank injection to monitor killing due to physical trauma), and positive control (larvae inoculated L. monocytogenes ). In addition, treatment

165 control groups were maintained to ensure that the PDA-probiotic supernatant treatments were not lethal to the larvae.

2.8.2 Galleria mellonella survival assay

The efficacy of SICs of plant compounds (TC, CR, TY, EG) and probiotic culture supernatant either alone or in combination in protecting G. mellonella against L. monocytogenes was investigated according to a previously published protocol (Mukharjee et al., 2010). Six separate experiments were conducted, wherein ten G. mellonella larvae each (200 to 250 mg) were randomly allocated to different PDA-probiotic treatment groups, and injected with 5 l of PBS containing plant compounds, probiotic culture supernatants or combination, followed by L. monocytogenes inoculation (5 l volume; 5 log CFU/larvae). After injection, the larvae were incubated at 37°C in dark, and the larval death time was recorded for a period of 7 days.

2.8.3 G. mellonella antimicrobial peptide assay

The effect of PDAs or probiotic culture supernatant or their combination on the expression of antimicrobial peptides (gallerimycin, lysozyme) of G. mellonella was investigated using RT-

PCR, as described previously (Mukherjee et al., 2010). Groups of 10 larvae each were injected with SIC concentrations of PDAs, either alone or in combination with probiotic culture supernatant. After 1 h of incubation at 37°C, the larvae were challenged with L. monocytogenes

(5 log CFU/larvae) and incubated for 24 h. At 6 h post L. monocytogenes inoculation, 3 larvae from each treatment group were homogenized in 1 ml of Trizol reagent (Sigma), and whole insect RNA was extracted according to the manufacturer’s recommendations. Quantitative real- time RT-PCR was performed using specific published primers (Altincicek and Vilcinskas, 2006;

Table 1) for expression of antimicrobial peptide genes in G. mellonella .

166 2.8.4 Statistical analysis:

A completely randomized design was used for the study. All experiments had duplicate samples and were repeated three times. For each treatment and control, the data from independent replicate trials were pooled and analyzed using the PROC MIXED subroutine of the Statistical

Analysis Software (SAS ver. 9.2; Institute, Inc, Cary, NC). Data comparisons for the gene expression study were made by using Student’s t test. Differences were considered significant at

P < 0.05. For the larvae experiments, significant differences between the two values were compared with a paired student’s t test. A P value of < 0.05 was considered to be statistically significant. All G. mellonella experiments were performed at least six times.

3. Results

Since the results from the tests conducted were not significantly different among the three L. monocytogenes strains studied (P > 0.05), only the results obtained with Scott A strain are presented here.

3.1 Determination of SICs of plant compounds and probiotic culture supernatant

The two highest SICs of the plant compounds that did not inhibit L. monocytogenes growth as compared to control were 0.0075% (0.50 mM) and 0.01% (0.75 mM) for TC; 0.0075% (0.50 mM) and 0.01% (0.65 mM) for CR; 0.005% (0.33 mM) and 0.0075% (0.50 mM) for TY; and

0.03% (1.8 mM) and 0.04% (2.5 mM) for EG. The aforementioned concentrations of PDAs were not inhibitory to the growth of probiotic strains as well (data not shown). Similarly, since 6.25% and 12.5% of cell-free probiotic culture supernatant were found to be sub-inhibitory to the growth of L. monocytogenes , they were selected for further experiments. The SICs of PDAs or probiotic culture supernatants did not change the pH of the culture media (P < 0.05).

167 3.2 Effect of SICs of PDAs with or without probiotic supernatant on L. monocytogenes

motility

All PDAs and probiotic treatments either alone or in combination decreased L. monocytogenes motility (P<0.05) (Fig. 1A-D). The zone of motility of L. monocytogenes treated with PDA or probiotic treatments was reduced to < 3.5 cm compared to control, which had a zone of nearly 6 cm after 12 h incubation at 37°C. The combination treatments of PDAs with probiotic supernatants were found to be more effective than single treatments, and reduced the zone of L. monocytogenes motility to less than 2 cm (P<0.05).

3.3 Effect of SICs of PDAs either alone or in combination with probiotic bacteria on L. monocytogenes adhesion to and invasion of Caco-2 cells

The efficacy of PDAs either alone or in combination with probiotic bacteria in reducing L. monocytogenes adhesion and invasion of Caco-2 cells is presented in Fig 2A-D and Fig. 3A-D, respectively. Results revealed that the PDAs and probiotic bacteria either alone or in combination significantly reduced L. monocytogenes adhesion to and invasion of Caco-2 cells,

compared to controls. For example, the single treatment of each PDA or probiotic bacterium

reduced L. monocytogenes adhesion to (Fig.2A-D) and invasion of (Fig. 3A-D) Caco-2 cells by

~1.5 to 2 log CFU (P<0.05). However, the combination treatments of PDA and probiotic bacteria

were generally more effective in reducing the adhesion-invasion capacity of L. monocytogenes

than the single treatments. This was particularly pronounced in the treatments combining EG and

B. bifidum, L. reuteri , and L. plantarum, where L. monocytogenes adhesion to Caco-2 cells was

decreased by ~2.5 log CFU ml (Fig. 2D). Similarly, the combination treatments of EG with all

probiotic bacteria completely inhibited bacterial invasion of the intestinal epithelial cells

(P<0.05) (Fig. 3D).

168 3.4 Effect of SICs of PDAs either alone or in combination with probiotic bacteria on L.

monocytogenes binding to human epithelial E-Cadherin.

The binding of increasing concentrations of L. monocytogenes to E-Cadherin is presented in Fig.

4. The binding of L. monocytogenes to E-Cadherin was concentration-dependent, and maximum binding of L. monocytogenes (9 log CFU/well) to E-cadherin was equivalent to ~0.35 absorbance units (Fig 4, 5). The PDAs and probiotic treatments, either alone or in combinations reduced L.

monocytogenes binding to E-cadherin by ~ 0.15 absorbance units (P < 0.05), which corresponded

to a reduction in binding of ~ 2 log CFU L. monocytogenes /well (Prakash et al., 2012). However, the combination treatments did not differ significantly in their efficacy for reducing L. monocytogenes binding to E cadherin compared to the corresponding single PDA or probiotic treatments (P > 0.05).

3. 5 Effect of SICs of PDAs either alone or in combination with probiotic culture supernatant on L. monocytogenes hemolysin production

All PDAs and probiotic culture supernatants either alone or in combination reduced L. monocytogenes hemolysin production (Fig. 6A-C) compared to controls (P < 0.05). However, the magnitude of reduction was found to be greater in PDA treatments compared to probiotic supernatants (P<0.05). For example, the PDAs reduced hemolysin production by ~ 50%, whereas the maximum reduction in hemolysin production due to probiotic supernatant treatments was

30% (Fig 6A-B). Moreover, the reduction in hemolysis production by TC, CR and TY was concentration-dependent, where the higher SIC was more inhibitory than the lower SIC on hemolysis. The higher SIC of CR was the most effective PDA treatment which reduced hemolysin production by ~ 70% (P<0.05). The combination treatments of PDAs and probiotics were equally effective as the single PDA treatments (P>0.05).

169 3.6 Effect of SICs of PDAs and probiotic supernatants on expression of L. monocytogenes

virulence genes and G. mellonella antimicrobial peptide genes

Real-Time quantitative PCR results revealed that trans-cinnamaldehyde (0.01%), carvacrol

(0.01%), thymol (0.0075%), and eugenol (0.04%) either alone or in combination with probiotic

culture supernatants down-regulated the expression of the majority of virulence genes of L. monocytogenes tested (Tables 2A-C). In general, the magnitude of down-regulation was greater in combination treatments as compared to single PDA or probiotic treatment (P<0.05). Similarly,

RT-qPCR results revealed that the PDAs, probiotic supernatants and their combinations significantly up-regulated the expression of G. mellonella genes encoding the antimicrobial

peptides, gallerimycin and lysozyme in infected larvae (Fig. 9 A-C). It was also observed that the

combination of PDAs with B. bifidum supernatant was more inhibitory on the aforementioned

genes than the PDA or probiotic treatment alone (P < 0.05). Moreover, among the various PDA-

B. bifidum supernatant combinations, the treatment containing EG down-regulated gallerimycin

and lysozyme genes more than the other PDAs (P < 0.05).

3.7 Effect of PDAs and probiotic culture supernatant on Galleria mellonella survival

The inoculation of G. mellonella with L. monocytogenes resulted in a dose-dependent killing of

the larvae (Fig. 7A) (P < 0.05). For example, 100% of G. mellonella larvae were killed within 24

h of infection with ~ 7 log CFU of L. monocytogenes /larvae, whereas only 10% mortality was

recorded at 24 h in the group inoculated with 4 log CFU of L. monocytogenes /larvae (P < 0.05).

A moderate L. monocytogenes infectious dose of 5 log CFU/larvae that resulted in 70% mortality by 2 days was selected as the challenge dose for subsequent experiments. The sub-inhibitory concentrations of all PDAs except thymol significantly enhanced the survival rates of infected G.

170 mellonella larvae by at least 40% on day 7 (P < 0.05). Among the PDAs, CR was found to be the most effective compound in protecting the larvae, where the survival rate was 80% compared to control larvae (Fig. 7B). Although thymol was effective in protecting the larvae until day 5 (P

< 0.05), all the thymol-treated larvae died on day 7 similar to control. Similar to the PDAs, the cell-free supernatants from all the five probiotic bacteria increased larval survival by at least 30% on day 7 compared to controls (Fig. 7C). Amongst the various treatments, B. bifidum supernatant

(12.5%) was most effective in protecting the larvae, where ~ 60% survival was observed. The combination treatments of PDAs and B. bifidum supernatant were more effective in protecting G. mellonella against L. monocytogenes than the single treatments of PDA or supernatant (P < 0.05)

(Fig. 7D). For example, all combinations of PDAs and B. bifidum supernatant enhanced the survival rates of worms by more than 50% compared to control (P < 0.05). Among the various treatments, the combination of B. bifidum supernatant with 0.01% CR and 0.03% EG were more effective in increasing larval survival than others (P < 0.05).

4. Discussion

Upon entry in the host, L. monocytogenes attaches and invades the intestinal epithelium followed by colonization of the liver, spleen and systemic spread to various organs, leading to meningitis and placental infections (Jaradat and Bhunia, 2003). This is mediated by an array of virulence factors that contribute to various stages of L. monocytogenes pathophysiology inside the host. Therefore, reducing L. monocytogenes attachment to and invasion of host tissue as well as production of critical virulence factors could potentially control listeriosis in humans. With increasing antibiotic resistance in L. monocytogenes (Pesavento et al., 2010; Sakaridis et al.,

2011; Yucel et al., 2005; Wieczorek, et al., 2012), there is a need for novel strategies for controlling listeriosis. Therefore, we investigated the efficacy of four GRAS-status, plant-derived

171 antimicrobials and five probiotic bacteria in reducing L. monocytogenes virulence in vitro and in the invertebrate host, Galleria mellonella.

Since adhesion to and invasion of intestinal epithelium is augmented by bacterial

motility (Bigot et al., 2005; O’Neil and Marquis, 2006), a reduced bacterial motility could

potentially reduce L. monocytogenes virulence. Our results revealed that PDAs and probiotic

supernatants not only significantly decreased L. monocytogenes motility (Fig. 1A-D), but also

reduced the attachment and invasive abilities of the pathogen on intestinal epithelial cells (Figs.

2A-D and 3A-D). Adhesion to and internalization of L. monocytogenes within epithelial cells is

mainly mediated by two bacterial surface proteins, Internalin A (InlA) and Internalin B (InlB)

that bind to host cell receptors E-cadherin and Met, respectively (Lingnau et al., 1995; Bonazzi et

al., 2009). Since the bacterial binding assay revealed that plant compounds and probiotics

significantly reduced binding of L. monocytogenes to E-cadherin, the decreased attachment and

invasion of L. monocytogenes observed in cell culture assays could partially be attributed to its

decreased binding to the receptor.

After receptor-mediated internalization in the intestinal epithelium, L. monocytogenes is

engulfed in a phagocytic vacuole (Gaillard et al., 1991), from where the pathogen escapes into

the cell cytoplasm by disrupting the vacuolar membrane (Kreft and Vazquez-Boland, 2001). This

is mediated by pore-forming toxin, listeriolysin O (Vazquez-Boland et al., 2001; Greiffenberg et

al., 1998; Smith-Palmer et al., 2002). The plant compounds either alone or in combination with

probiotic culture supernatants significantly decreased the production of listeriolysin O by the

pathogen, as evident from the diminished hemolysis on blood agar plates.

Sub-inhibitory concentrations (SICs) of antimicrobials, including antibiotics are

reported to modulate transcription of bacterial genes (Goh et al., 2002; Tsui et al., 2004). We

172 previously observed that sub-inhibitory concentrations of TC inhibited biofilm synthesis of

Cronobacter sakazakii on abiotic surfaces, by modulating the expression of several genes,

including those associated with quorum sensing (Amalaradjou and Venkitanarayanan, 2011).

Additionally, research conducted in our laboratory revealed that the SICs of TC suppressed

attaching and invading abilities of uropathogenic Escherichia coli in human urinary tract

epithelial cells, by down-regulating the genes critical for host tissue colonization (Amalaradjou

et al., 2010). Since the SICs of the four plant compounds and probiotics were used in the

experiments, the reduction observed in L. monocytogenes virulence properties was not due to the

killing of the bacterium by the plant compounds and probiotics, but could be attributed to their

potential effect in modulating the expression of virulence genes associated with its pathogenesis.

Therefore, we used RT-qPCR to determine the effect of PDAs and probiotic culture supernatants

on the transcription of major genes that are previously reported to play a role in L.

monocytogenes virulence and infection in humans. Of the various genes tested, the

transcriptional modulator PrfA, positively regulates the expression of genes coding for L.

monocytogenes virulence, thus contributing to colonization, intracellular survival, and

dissemination in the host (Brehm et al., 1996; Renzoni et al., 1997). In addition, flaA, fliP, fliG,

flgE along with motA and motB encode for a part of the flagellar structure, biosynthesis and motor responsible for flagellar movement contributing to bacterial motility. A motAB mutant of

L. monocytogenes displayed impaired flagellar motion and reduced motility (Gorski et al., 2009).

The gene lmo1847 transcribe Listeria adhesion binding protein, which plays a role in L.

monocytogenes adhesion to host tissue (Pandiripally et al., 1999; Reis et al., 2010). Similarly,

inlA, inlB, and iap also encode for proteins assisting in L. monocytogenes invasion of host tissue

(Cabanes et al., 2004; Gaillard et al., 1991; Park et al., 2000). The gene hly encodes listeriolysin

173 production, while plcA, plcB genes produce phospholipase (Boland et al., 2001; Cossart et al.,

1989). Genes hly and plcA, plcB along with dnaK (encodes major stress protein DnaK) are critical for phagosomal escape and intracytoplasmic survival. Once free in the cytoplasm of the host cell, L. monocytogenes uses the protein ActA to harness the host cell’s actin polymerization machinery and facilitate its intracellular movement and cell-to-cell spread in the host tissue

(Alvarez-dominguez et al., 1997; Kocks et al., 1992; Lambrechts et al., 2008). RT-qPCR results revealed that the four plant compounds and probiotic bacterial supernatant either alone or in combination significantly down-regulated the expression of all of the aforementioned virulence genes compared to control (Tables 2A-C). However, the magnitude of down-regulation of the genes by the various treatments differed by plant compound and probiotic bacterium. For example, CR and TY down-regulated the expression of prfA by approximately 9 folds, whereas the magnitude of down-regulation due to TC was ~ 1.5 folds (Table 2). Similarly, L. plantarum culture supernatant (12.5%) reduced the expression of inlA by nearly 5-fold compared to other probiotic treatments, which reduced the expression by only 1 to 2 folds (Table 2B). This suggests that the plant compounds and probiotics may act through different mechanisms. In general, the reduction in expression of virulence genes was more significant (P < 0.05) due to plant compound treatments compared to probiotic culture supernatants. Among the probiotic bacteria, since B. bifidum was generally more inhibitory on the various virulence factors tested, it was used in further experiments in combination with the PDAs. The combination treatments containing the PDAs and B. bifidum supernatant were found to be significantly more effective in down-regulating the expression of critical virulence genes compared to PDA or supernatant alone (Table 2C). Among the various combination treatments, EG and B. bifidum supernatant was generally found to be more inhibitory on majority of the genes tested. The enhanced

174 inhibitory effect of PDA-probiotic combinations on L. monocytogenes virulence could be attributed to the reciprocal interaction of the PDAs with probiotic bacteria. Several researchers have highlighted the prebiotic potential of dietary bioactive compounds such as phytochemicals in influencing gut microbiota physiology (Brown et al., 2012) and composition (Ley et al., 2006;

Duncan et al., 2007), which in turn has been associated with attenuated pathogen virulence and reduced susceptibility of the host to foodborne infections (Dominguez-Bello and Blaser, 2008;

Ghosh et al., 2011). Bomba and coworkers (2002) observed that the administration of poly- unsaturated fatty acids positively influenced the adhesion of Lactobacillus spp. to the jejunal mucosa of gnotobiotic piglets, suggesting that intake of such fatty acids may influence the intestinal levels of beneficial gut microbiota (Bomba et al., 2002). In another study, low dose of dietary resveratrol, a potent antioxidant found in grapes, was found to increase the counts of

Bifidobacterium and Lactobacillus spp. in the colon microbiota of rats (Larrosa et al., 2009).

These studies suggest that PDAs, in addition to exerting a direct inhibitory effect on foodborne pathogens, may potentiate probiotic bacteria in conferring protection to the host. Another mechanism by which the PDA-probiotic interaction could confer an enhanced protection against pathogens is by the action of probiotic bacteria on the bioavailability and chemical transformation of PDAs (Aura, 2008). The microbiota-mediated metabolites of polyphenols are better absorbed in the intestine, leading to prolonged entero-hepatic circulation, and resident time in the plasma and gut, thereby enhancing their antimicrobial efficacy (Laparra and Sanz, 2010).

However, indepth research is needed to fully elucidate the interactive mechanisms behind the enhanced antivirulence effect of PDAs and probiotic bacteria.

The use of animal models to assess human listeriosis is critical for understanding the disease pathology and testing new therapies. Although appropriate mammalian models are

175 primarily employed for such studies, their use poses significant practical, financial, and ethical

concerns. Insects are increasingly being used as surrogate hosts because they share with

mammals, the essential aspects of the innate immune response to microbial infections

(Mukherjee et al., 2010). Consequently, insect models have been proposed as cost-effective, and

easy alternatives that generate valid data on microbial virulence (Jander et al., 2000; Mylonakis

et al., 2005). Galleria mellonella is the larva of the greater wax moth that has been utilized to study microbial pathogenesis in a wide array of organisms, including Pseudomonas aeruginosa

(Jander et al., 2000; Miyata et al., 2003), Burkholderia cepacia (Seed and Dennis, 2008),

Burkholderia mallei (Schell et al., 2008), Bacillus cereus (Fedhila et al., 2006), Acinetobacter baumannii (Peleg et al., 2009), Klebsiella pneumoniae (Insua et al., 2013) several pathogenic fungi (Cotter et al., 2000; Mylonakis et al., 2005; Reeves et al., 2004) and L. monocytogenes

(Joyse et al., 2010; Mukherjee et al., 2010, 2011). The innate immunity of Galleria is a multi- component response involving hemolymph coagulation, cellular phagocytosis, and phenol oxidase-based melanization. Additionally, killing of pathogens in G. mellonella is achieved similar to that in mammals, i.e., by lysozyme, reactive oxygen species, and antimicrobial peptides such as gallerimycin (Mukherjee et al., 2010). Therefore we utilized G. mellonella as an in vivo model to determine the efficacy of PDA and probiotic bacteria in attenuating L. monocytogenes virulence. The results from G. mellonella survival curves revealed that although the PDAs and probiotic supernatants were effective in protecting the larvae from L. monocytogenes compared to control (P < 0.05), the combination of PDAs with B. bifidum supernatant resulted in an enhanced larval survival compared to the single treatment of PDA or probiotic supernatant (Fig. 7D). Since G. mellonella ’s innate immune response against pathogens includes the production of lysozyme and gallerimycin, we determined the effect of PDA and

176 probiotic treatments on the expression of genes encoding these defensins in the larva. The RT-

qPCR data from G. mellonella gene expression revealed that the PDAs, probiotic supernatants

(except L. plantarum) , and their combinations significantly down-regulated G. mellonella genes

encoding lysozyme and gallerimycin, which could have played a role in increasing the survival

of infected larvae compared to control.

In conclusion, this study demonstrated that the plant compounds, probiotics, and their

combinations were effective in significantly reducing the virulence properties of L.

monocytogenes in vitro . In addition, the aforementioned treatments were effective in vivo , as

evident from the enhanced survival of L. monocytogenes -infected G. mellonella . The PDAs and

probiotics could potentially be used as an oral supplement to control L. monocytogenes infection.

However, additional in vivo studies in an appropriate mammalian model are necessary to validate the efficacy and safety of the aforementioned treatments.

177 Table 1. List of primers used in this study

Gene with accession number Primer Sequence (5'- 3')

16S-rRNA* Forward 5'-AGCTTGCTCTTCCAAAGT- 3' (Somer and Kashi, 2003; Reverse 5'-AAGCAGTTACTCTTATCCT- 3' Burkholder et al. 2009) flaA Forward 5'-GACTTGTTACAAACAGCGGATTCA- 3' (NC_003210.1) Reverse 5'-ATTGACGCATACGTTGCAAGAT- 3' fliP Forward 5'-TTGGCCGGGTGTGAATGT- 3' (NC_003210.1) Reverse 5'-CCATTTACACCAAGCGAATCC- 3' fliG Forward 5'-AATCGCGACCGAAGTGGTT- 3' (NC_003210.1) Reverse 5'-CTCGTGCAAGGCGTTGTTT- 3' flgE Forward 5'-CAGCAGGTTCCCCGACTTC- 3' (NC_003210.1) Reverse 5'-CGGCCTTGTAGTGCTGCAT- 3' motA Forward 5'-CAACGCTCGGTGTACTTGGA- 3' (NC_003210.1) Reverse 5'-TTTCGCCCATCGCATGA- 3' motB Forward 5'-TGCAAAAAAATTCGAACAAATGG- 3' (NC_003210.1) Reverse 5'-CTGCCGCGCCTTCCT- 3' prfA Forward 5'-TGAGCAAGAATCTTACGCACTTTT- 3' ( NC_012488.1) Reverse 5'-GCTAGGCTGTATGAAACTTGTTTTTG- 3' plcA Forward 5′-TCGGACCATTGTAGTCATCTTGA-3′ (NC_003210.1) Reverse 5′-CACAAATTCGGCATGCAGTT-3′ plcB Forward 5′-CGCAGCTCCGCATGATATT-3′

(NC_003210.1) Reverse 5′-GATTATCCGCGGACCAACTAAG-3′ dnaK Forward 5'-CAACACCTTCTGTAGTTGGTTTCAA- 3'

(NC_003210.1) Reverse 5'-TGCAGCACGTTTCGCTACTT- 3'

178 hly Forward 5′-TCTCCGCCTGCAAGTCCTA-3′

(NC_002973.6) Reverse 5′-TCGATTTCATCCGCGTGTT-3′

actA Forward 5′-CGTCGTCATCCAGGATTGC-3′

(NC_003210.1) Reverse 5′-TGCTATGGCTTTCCTTCTTTTTTT-3′

inlA Forward 5′-AATGTAACAGACACGGTCTCACAAA-3′

(NC_003210.1) Reverse 5′-TCCCTAATCTATCCGCCTGAAG-3′

inlB Forward 5′-CGAAAGTACAAGCGGAGACTATCA-3′

(NC_003210.1) Reverse 5′-GTTTCTGCAAAAGCATCATCTGA-3′

lmo1634 Forward 5′-GTTGTTGCCGGCGTTACAC-3′

(NC_003210.1) Reverse 5′-CGCGATAATTGCTTTGAAAAGA-3′

Lmo1647 Forward 5′-GCGTGGATCCGCATGAAT-3′

(NC_003210.1) Reverse 5′-GCATCCGCAGCACTTTGAAT-3′

iap Forward 5'-TATTTTGCTAGATAATGCGGTTGAA- 3'

(NC_003210.1) Reverse 5'-CGCGATTCGAATAACTGGATTT- 3'

Galleria Actin* Forward 5’-ATCCTCACCCTGAAGTACCC-3’ Reverse 5’-CCACACGCAGCTCATTGTA-3’

Gallerimycin Forward 5’-CGCAATATCATTGGCCTTCT-3’ Reverse 5’CCTGCAGTTAGCAATGCACTC-3’

Lysozyme Forward 5’-TCCCAACTCTTGACCGACGA-3’ Reverse 5’-AGTGGTTGCGCCATCCATAC-3’

* Endogenous control for L. monocytogenes gene expression studies.

* Endogenous control for Galleria mellonella antimicrobial peptides.

179 Table 2. Relative fold change in the expression level of Listeria monocytogenes virulence genes in response to SICs of (A) TC, CR, TY, and EG (B) probiotic cultures supernatant (12.5%) or

(C) their combinations. Control had a basal level of expression of 1.

(A) Relative fold change a

Gene Gene product or TC CR TY EG Function (0.01%) (0.01%) (0.0075%) (0.04%) flaA Structural flagella protein -2.50±0.6* -6.85±2.0* -5.50±2.5* -3.43±2.4* fliP Flagellar biosynthesis -0.05±0.2 -3.20±2.2* -4.58±1.9* -2.15±0.5* protein fliG Flagellar motor switch -2.40±0.4* -4.55±0.4* -3.54±0.5* -2.13±0.4* protein flgE Flagellar hook protein -0.05±0.2 -1.50±0.5 -7.45±1.5* -0.99±1.1 motA Flagellar motor protein -3.80±0.5* -9.50±2.5* -1.44±0.5* -4.20±0.4* motB Flagellar motor protein -5.05±0.2* -3.24±0.4* -1.60±0.8* -1.50 ±0.4

prfA Transcriptional regulator -1.50±0.5 -10.12±2.5* -9.50±2.5* -4.40±0.8* plcA Intracellular survival -4.65±0.4* -3.52±1.50* -2.55±0.1* -7.55±1.5* plcB Intracellular survival -5.25±0.2* -4.56±1.50* -4.50±1.2* -8.64±2.5* dnaK Intracellular survival -5.64±2.3* -3.50±1.0* -4.45±0.4* -3.54±1.5* hly Intracellular survival -6.50±1.1* -4.40±0.8* -2.35±0.4* -6.55±0.4* actA Cell-to-cell spread -3.55±1.5* -5.72±0.4* -6.55±0.6* -3.42±1.4* inlA Adhesion-invasion -8.50±1.2* -5.36±0.8* -3.40±1.2* -5.05±0.4* inlB Adhesion-invasion -4.25±0.4* -2.15±0.24* -1.22±0.7 -2.22±0.3* Lmo Adhesion -2.43±0.4* -4.22±0.43* -2.71±0.6* -3.11±0.4* 1847 iap Invasion -4.37±1.50 -7.22±0.58 -2.50±1.11 -2.12±0.2*

aFold change in gene expression relative to control (no PDA).

*L. monocytogenes virulence genes significantly down-regulated as compared to control

(P<0.05).

180

(B) Relative fold change a

Gene B. bifidum L. reuteri L. fermentum L. plantarum L. lactis 12.5% 12.5% 12.5% 12.5% 12.5% flaA -3.55±0.5* -1.20±0.6 -1.25±0.8 -1.20±1.0 -1.55±0.7 fliP -2.42±0.9* -1.45±0.2 -2.23±0.5* -2.15±0.4* -2.05±0.4* fliG -1.80±0.3 -1.11±0.4 -1.35±0.4 -1.15±0.3 -2.17±0.5* flgE -1.55±0.4 -1.82±0.2 -2.20±0.5* -2.0±0.5* -0.55±0.9 motA -2.60±0.2* -1.11±0.7 -3.30±0.9* -1.55±0.4 -2.50±0.7* motB -1.20±0.7 -2.28±0.6* -1.14±0.2 -1.60±0.1 -0.58±0.6

prfA -2.41±0.5* -1.23±0.3 -1.85±1.2 -2.30±0.6* -3.25±0.3* plcA -1.50±0.9 -2.12±0.5* -2.27±1.5* -1.70±0.3 -1.34±0.5

plcB -3.10±0.8* -1.14±0.1 -2.15±0.3* -0.85±0.2 -2.50±0.2*

dnaK -1.50±0.5 -2.42±1.3* -1.18±1.0 -1.60±0.2 -6.20±1.5*

hly -2.88±0.4* -1.34±1.0 -1.11±0.8 -0.50±0.5 -3.50±0.3*

actA -2.45±0.2* -2.15±1.0* -3.25±0.6* -7.50±0.3* -2.22±0.5* inlA -1.05±0.5 -2.73±1.2* -1.13±0.1 -5.40±0.4* -1.45±0.4 inlB -2.11±0.2* -6.47±2.0* -4.25±0.2* -2.05±0.9* -1.15±0.1 Lmo 1847 -3.60±0.7* -1.02±0.5 -2.51±0.3* -4.21±0.7* -2.65±0.5* iap -2.40±0.5* -2.31±1.4* -2.40±0.3* -1.49±0.5 -3.40±0.4*

aFold change in gene expression relative to control (no PDA). Control had a basal level of expression of 1.

*L. monocytogenes virulence genes significantly down-regulated as compared to control

(P<0.05).

181 (C) Relative fold change a

Gene Gene product or B. bifidum 12.5% Function TC CR TY EG (0.01%) (0.01%) (0.0075%) (0.04%) flaA Structural flagella protein -6.50±0.2* -9.40±1.0* -8.30±0.8* -5.40±0.6* fliP Flagellar biosynthesis -3.40±0.7* -7.60±1.5* -9.10±0.5* -8.60±0.3* protein fliG Flagellar motor switch -5.25±0.3* -9.60±0.4* -6.30±0.8* -6.50±0.9* protein flgE Flagellar hook protein -2.36±0.1* -6.35±0.5* -10.6±0.9* -3.50±0.6* motA Flagellar motor protein -6.42±0.6* -10.50±0.5* -5.90±0.5* -9.45±0.4* motB Flagellar motor protein -8.40±0.1* -6.80±0.6* -8.65±0.5* -7.90 ±0.2* prfA Transcriptional regulator -6.37±0.2* -9.45±1.0* -10.1±1.0* -6.85±0.5* plcA Intracellular survival -7.50±0.3* -6.85±1.0* -8.30±0.1* -9.35±0.3* plcB Intracellular survival -9.68±0.5* -8.50±0.4* -6.50±0.2* -9.10±0.6* dnaK Intracellular survival -7.50±2.9* -5.65±0.6* -9.35±0.2* -7.60±0.5* hly Intracellular survival -8.22±0.6* -8.20±0.6* -8.50±0.4* -8.40±0.4* actA Cell-to-cell spread -7.38±0.9* -9.35±0.2* -10.5±0.2* -7.69±2.0* inlA Adhesion-invasion -9.44±0.6* -8.44±0.7* -7.20±0.8* -10.5±0.4* inlB Adhesion-invasion -9.37±0.9* -9.70±0.2* -6.82±0.9* -9.50±0.7* Lmo Adhesion -5.61±0.8* -9.65±0.4* -7.75±0.8* -8.50±0.4* 1647 iap Invasion -3.73±0.1* -9.70±0.5* -8.50±0.5* -2.50±0.2* a Fold change in gene expression relative to control (no treatment). Control had a basal level of expression of 1.

L. monocytogenes virulence genes significantly down-regulated as compared to control (P<0.05)

182 Fig.1. Effect of SICs of Trans-cinnamaldehyde (TC), carvacrol (CR), thymol (TY) and eugenol

(EG) either alone or in combination with probiotic bacterial supernatant (6.25, 12.5%) on L. monocytogenes (Scott A) motility. Error bars represent SEM (n=6). Bars with different letters represent a significant difference between treatments (P < 0.05).

(A) Trans-cinnamaldehyde (TC)

183

(B) Carvacrol (CR)

184

(C) Thymol (TY)

185

(D) Eugenol (EG)

186 Fig. 2. Effect of SICs of trans-cinnamaldehyde (TC), carvacrol (CR), thymol (TY) and eugenol

(EG) either alone or in combination with probiotic bacteria on Listeria monocytogenes (Scott A) adhesion to Caco-2. Error bars represent SEM (n=6). Bars with diffe rent letters represent a significant difference between treatments (P < 0.05) .

(A) Trans-cinnamaldehyde (TC)

187 (B) Carvacrol (CR)

188 (C) Thymol (TY)

189 (D) Eugenol (EG)

190 Fig. 3. Effect of SICs of trans-cinnamaldehyde (A) (TC), (B) carvacrol (CR), (C) thymol (TY) and (D) eugenol (EG) either alone or in combination with probiotic bacteria on Listeria monocytogenes (Scott A) invasion of Caco -2. Error bars represent SEM (n=6). Bars with different letters represent a significant difference between treatments (P < 0.05) .

(A) Trans-cinnamaldehyde (TC)

191 (B) Carvacrol (CR)

192 (C) Thymol (TY)

193 (D) Eugenol (EG)

194 Fig. 4. Effect of Listeria. monocytogenes (Scott A) concentration on E-Cadherin binding. Error bars represent SEM (n=6).

195

Fig. 5. Effect of (A) TC, (B) CR (C) TY (D) EG either alone or in combination with probiotic bacteria on E-cadher in binding of Listeria monocytogenes (Scott A). Error bars represent SEM

(n=6). Bars with different letters represent a significant difference between treatments (P < 0.05).

(A) Trans-cinnamaldehyde (TC)

196 (B) Carvacrol (CR)

197 (C) Thymol (TY)

198 (D) Eugenol (EG)

199 Fig. 6. Effect of (A) PDAs, (B) probiotic supernatant or (C) combination on hemolysis of 3% sheep RBC by Listeria monocytogenes (Scott A). Error bars represent SEM (n=6). Bars with different letters represent a significant difference between treatments (P < 0.05) .

(A)

(B)

200 (C) Trans-cinnamaldehyde (TC)

201 Carvacrol (CR)

202 Thymol (TY)

203 Eugenol (EG)

204 Fig. 7A. Dose-dependent survival of Galleria mellonella larvae inoculated with L. monocytogenes (Scott A). Results represent means of at least three independent determinations ± standard deviations for 10 larvae per treatment.

205 Fig. 7B. Effect o f SICs of PDAs on survival of Galleria mellonella larvae inoculated with

L. monocytogenes (Scott A). Results represent means of at least three independent determinations ± standard deviations for 10 larvae per treatment.

206 Fig. 7C. Effect of SICs of probiotic culture supernatant on survival of Galleria mellonella larvae inoculated with L. monocytogenes

(Scott A). Results represent means of at least three independent determinations ± standard deviations for 10 larvae per treatment.

207 Fig. 7D. Effect of SICs of plant-derived antimicrobials (PDAs) either alone or in combination with probiotic culture supernatant on survival of Galleria mellonella larvae inoculated with L. monocytogenes (Scott A). Results represent means of at least three independent determinations ± standard deviations for 10 larvae per treatment.

208 Fig. 8. Relative fold change in the expression level of antimicrobial peptide g enes (gallerimycin, lysozyme) of uninoculated G. mellonella in response to SICs of (A) TC, CR, TY, and EG (B) probiotic cultures supernatant (12.5%) or (C) their combinations. Error bars represent SEM

(n=6). Bars with different letters represent a signific ant difference between treatments (P < 0.05).

Control had a basal level of expression of 1.

(A)

(B)

209 (C)

210 Fig 9. Relative fold change in the expression level of antimicrobial peptide genes (gallerimycin, lysozyme) of G. mellonella inoculated with L. monocytogenes in response to SICs of (A) TC,

CR, TY, and EG (B) probiotic cultures supernatant (12.5%) or (C) their combinations. Error bars represent SEM (n=6). Bars with different letters represent a significant difference betw een treatments (P < 0.05). Control had a basal level of expression of 1.

(A)

(B)

211

(C)

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221

Chapter VIII

Summary

222 Listeria monocytogenes has emerged as a significant foodborne pathogen causing life-

threatening infections in susceptible populations. The widespread presence of L. monocytogenes

in the environment such as soil, irrigation water, and sewage along with its ability to form

sanitizer-tolerant biofilms result in its persistence in food processing and packaging

environments, thereby contaminating foods. In the host, L. monocytogenes infections can result in high hospitalization and case-fatality rates. Growing concerns on the use of synthetic chemicals in the food industry and emerging antibiotic resistance in L. monocytogenes underscore the need for alternative approaches for controlling this pathogen in food processing environments, high-risk foods, and humans.

This study was undertaken to investigate the potential of plant-derived antimicrobials

[PDAs; trans-cinnamaldehyde (TC), carvacrol (CR), thymol (TY), eugenol (EG), β-resorcylic acid (BR), caprylic acid (CR)], and probiotic bacteria, namely Bifidobacterium bifidum (NRRL-

B41410), Lactobacillus reuteri (B-14172), L. fermentum (B-1840), L. plantarum (B-4496), and

Lactococcus lactis subspecies lactis (B-633) for controlling L. monocytogenes biofilms on food processing surfaces, survival on high-risk foods previously implicated in outbreaks (frankfurters, cantaloupes), and attenuating major virulence factors responsible for pathogenesis in humans.

To control L. monocytogenes’ biofilms in food processing environments, the first objective of the study investigated the efficacy of sub-inhibitory concentrations (SICs, concentrations not inhibiting bacterial growth) and bactericidal concentrations (MBCs) of PDAs in inhibiting

Listeria monocytogenes biofilm formation and inactivating mature L. monocytogenes biofilms at

37°, 25° and 4°C on polystyrene plates and stainless-steel coupons. In addition, the effect of

SICs of PDAs on the expression of L. monocytogenes genes critical for biofilm synthesis was determined by real-time quantitative PCR. The PDAs and their SICs used for biofilm inhibition

223 were TC at 0.0075% (0.50mM) and 0.01% (0.75 mM); CR at 0.0075% (0.50 mM) and 0.01%

(0.65 mM); TY at 0.005% (0.33 mM) and 0.0075% (0.50 mM); and EG at 0.03% (1.8 mM), and

0.04% (2.5 mM) , whereas the PDA concentrations used for inactivating mature biofilms were

0.1% (5.0 mM) and 0.2% (10.0 mM) for TC, 0.1% (5.0 mM) and 0.2% (10.0 mM) for CR, 0.1

(3.3 mM) and 0.2% (5.0 mM) for TY; and 0.3% (18.5 mM) and 0.4% (25.0 mM) for EG. All

PDAs inhibited biofilm synthesis and inactivated fully formed L. monocytogenes biofilms on both matrices at three temperatures tested (P < 0.05). Real- time quantitative PCR data revealed that the PDAs down-regulated critical L. monocytogenes biofilm-associated genes (P < 0.05).

Results suggest that TC, CR, TY, and EG could potentially be used to control L. monocytogenes

biofilms in food processing environments, although further studies under commercial settings are

necessary.

Being a robust foodborne pathogen, L. monocytogenes is able to survive on a variety of

RTE food products, including frankfurters. The USDA-FSIS has established a zero tolerance

policy for L. monocytogenes in RTE products, thereby warranting effective post-processing

interventions to control the pathogen on these foods. Hence the antilisterial efficacy of PDAs,

namely BR, CR, and TC either alone or in combination with hydrogen peroxide (HP) as post-

processing dip treatments on frankfurters was investigated. Frankfurters were surface-inoculated

with a five-strain mixture of L. monocytogenes (~ 6.0 log CFU per frankfurter), followed by dip

treatment at 55°C for 60 s or 65°C for 30 s in sterile deionized water (control), or water

containing BR (1.5% w/v; 97 mM), CR (0.75% v/v; 50 mM), or TC (0.75% v/v; 56 mM) either

alone or in combination with HP (0.1% v/v; 29 mM). Treated frankfurters were vacuum-

packaged, and stored at 4°C for 70 days. Representative samples were analyzed on days 0, 1, 3,

224 7, 14, 28, 42, 56, and 70 of refrigerated storage for enumerating surviving L. monocytogenes on frankfurters.

On day zero of storage, all PDAs reduced L. monocytogenes counts by > 2 log

CFU/frankfurter at both temperatures (P < 0.05), compared to controls. From days 1 to 70, L. monocytogenes counts on PDA-treated frankfurters were consistently lower (P < 0.05), and after

70 days of storage, the pathogen counts were reduced to undetectable levels on frankfurters treated with PDA-HP combinations at 65°C, and by combinations of BR and TC with HP at

55°C. Results suggest that PDAs alone, or in combination with HP could be effectively used as post-processing dips to reduce L. monocytogenes on frankfurters, although follow-up studies on the sensory and quality characteristics of PDA-treated frankfurters are necessary.

In the past three decades, a majority of L. monocytogenes outbreaks has been primarily due to the consumption of dairy and ready-to-eat (RTE) meat products contaminated with L. monocytogenes . However, fresh produce has been increasingly associated with L. monocytogenes outbreaks in the last decade. In 2011, a nation-wide outbreak of listeriosis affecting 146 people from 28 states with 30 deaths and 1 miscarriage was linked to the consumption of cantaloupes contaminated with the pathogen. A variety of FDA-approved disinfectants, including chlorine have been evaluated for decontaminating cantaloupes, but are minimally effective in rendering the fruit safe for consumption.

Therefore, the efficacy of four PDAs, namely CR, TY, BR, and CA, either alone or in combination with HP, as antimicrobial wash and coating treatments for reducing Listeria monocytogenes on cantaloupes was investigated. Further, the efficacy of wash treatments in reducing the transfer of L. monocytogenes from the fruit surface to the interior was examined.

Cantaloupe rind plugs inoculated with L. monocytogenes (10 7 CFU/cm 2; five-strain cocktail)

225 were washed for 3, 6, and 10 min at 25°C, or 1, 3, and 5 min at 55 or 65°C in sterile deionized

water (control), or water containing 2% PDAs with or without 2% HP. Additionally, inoculated

whole cantaloupes (10 8 CFU/fruit) washed with PDA-HP combinations at 55 or 65°C for 5 min were cut into rindless pieces, stored at 4°C for 7 days and sampled for L. monocytogenes .

Furthermore, to test the antilisterial efficacy of PDAs used as a fruit coating, inoculated rind plugs coated with PDAs and stored at 4°C for 7 days were sampled for surviving L. monocytogenes . Individual PDA washes reduced L. monocytogenes on rinds by ≥ 2.5 log

CFU/cm 2 by 3 min at all temperatures (P < 0.05). The PDA-HP combinations decreased L. monocytogenes to undetectable levels by 5 min at 55 or 65°C, and 10 min at 25°C (P < 0.05). In addition, PDA-HP washes reduced L. monocytogenes transfer from cantaloupe surface to the interior (P < 0.05). All coating treatments reduced L. monocytogenes on cantaloupe surfaces to

undetectable levels by 7 days (P < 0.05). Results indicate that the PDAs alone, or with HP could

be used to reduce L. monocytogenes on cantaloupes.

Being a food-borne pathogen, crossing the intestinal barrier is the first critical step in L.

monocytogenes infection in humans. Reducing L. monocytogenes invasion of intestinal epithelium and production of virulence factors can potentially reduce risk of infection in humans.

In this study, the efficacy of SICs of four PDAs (TC, CR, TY, EG), either alone or in combination with five probiotic bacteria, namely Bifidobacterium bifidum (NRRL-B41410),

Lactobacillus reuteri (B-14172), L. fermentum (B-1840), L. plantarum (B-4496), and

Lactococcus lactis subspecies lactis (B-633) in reducing L. monocytogenes adhesion to and invasion of human intestinal cells (Caco-2) was investigated. Additionally, the effect of aforementioned PDAs and probiotics on L. monocytogenes motility, listeriolysin production, epithelial E-cadherin binding and expression of virulence genes was investigated. Moreover, the

226 in vivo efficacy of PDA-probiotic treatments in reducing L. monocytogenes virulence in the invertebrate model, Galleria mellonella was studied. The PDAs and probiotics, either alone or in combination, significantly reduced L. monocytogenes adhesion to and invasion of cultured human intestinal epithelial cells (P < 0.05). Moreover, PDA-probiotic treatments decreased L. monocytogenes motility, hemolysin production, E-cadherin binding, and virulence gene expression (P < 0.05). In addition, the PDA-probiotic treatments significantly enhanced the survival rates of G. mellonella infected with lethal doses of L. monocytogenes (P < 0.05). The results highlight the antilisterial effect of aforementioned PDAs and probiotics, and justify further investigations in a mammalian model.

In conclusion, the results of this study indicate the potential of PDAs and probiotics for controlling L. monocytogenes on abiotic surfaces, high-risk food products and in humans thereby enhancing food safety and public health.

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