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Theses and Dissertations

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Phenotypic, Physiological and Growth Interactionsamong Salmonella SerovarsJuliany Rivera CaloUniversity of Arkansas, Fayetteville

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Phenotypic, Physiological and Growth Interactions among Salmonella Serovars

Phenotypic, Physiological and Growth Interactions among Salmonella Serovars

A thesis submitted in partial fulfillment

of the requirements for the degree of

Master of Science in Food Science

by

Juliany Rivera Calo

University of Puerto Rico at Mayagüez

Bachelors of Science in Industrial Microbiology, 2005

December 2013

University of Arkansas

This thesis is approved for recommendation to the Graduate Council.

___________________________________

Dr. Steven C. Ricke

Thesis Director

___________________________________ ___________________________________

Dr. Young Min Kwon Dr. Steven L. Foley

Committee Member Committee Member

ABSTRACT

This thesis consists of four research parts: a literature review that covers Salmonella spp.,

one of the more prominent foodborne pathogens that represents a major risk to humans (chapter

1). Understanding the growth of Salmonella serovars and strains is an important basis for more in

depth research. In this case we studied a) the aerobic and anaerobic growth responses of multiple

strains from six different serovars, b) how the spent media from different serovars, more

importantly S. Heidelberg, affect the growth of S. Typhimurium, and c) determined whether or

not two different serovars undergo competitive interactions when they were grown together

(chapter 2). Growth responses of four Salmonella serovars, Heidelberg, Kentucky,

Typhimurium, and Enteritidis in enrichment and non-enrichment media were compared to

determine the effectiveness of a non-selective enrichment media, such as Luria Bertani broth

over selective enrichment methods (chapter 3). Since antimicrobial resistance continues to be

one of the major concerns about Salmonella, and consumers are demanding more natural

antimicrobials we studied the effectiveness of five essential oils against Salmonella Heidelberg.

Besides being one of the top five Salmonella enterica serovars associated with human infections,

S. Heidelberg is the primary focus for our research (chapter 4). Given that Salmonella enterica

are important facultative intracellular pathogens that are widely known to be isolated from

poultry and to cause infections in humans, understanding the ability of this pathogen to attach

and invade different cell types is of great importance. We studied the adhesion and invasion of

four different strains to chicken macrophage (HD11), and human epithelial (Caco-2) cells

(chapter 5).

ACKNOWLEDGMENTS

With deep gratitude and sincerity, I would like to thank my advisor, Dr. Steven C. Ricke,

for his guidance, support, mentorship, and expertise during my masters program in the

University of Arkansas. He provided me with constant motivation to do research with great

understanding. The scientific discussions with him were very fruitful. I also thank my committee

members, Dr. Steven L. Foley and Dr. Young Min Kwon for their contributions and support on

my thesis and research.

Special thanks to former and current members of the Center for Food Safety, Dr. Philip

G. Crandall, Dr. Corliss O’Bryan, Candy, specially Michelle, Dr. Si Hong Park, Nathan, Peter,

and Adam for their friendship, insights, support and all their help. I am very thankful to Dr. Ok

Kyung Koo for her mentorship, experimental advice and her infinite help during this process. I

also thank the Department of Food Science for providing graduate stipend support. Thanks to the

Food Science and Poultry Science staff and graduate students for their kindness, friendship and

all their help.

I thank all my family and friends, especially Mika, Jonah, Sofia, and Rafael for their

company, love, support, words of encouragement and constant motivation to keep going.

DEDICATION

I dedicate this thesis to my parents, Julie and Rafy, and my brother Rafael. Without their

infinite support, encouragement, words of advice and love this would not have been possible. I

thank them for their unconditional love and constant motivation in every step of the way and

everything I have ever wanted to accomplish. I sincerely dedicate this thesis to them, for being

instrumental in inspiring me at every step throughout my life on both personal and professional

fronts.

TABLE OF CONTENTS

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

CHAPTER 1: Salmonella spp. - Literature Review .......................................................................3

1. Abstract ...............................................................................................................................4

2. Introduction ..........................................................................................................................5

3. Foodborne Pathogen, Salmonella enterica ..........................................................................9

3.1. Epidemiology of Salmonella .......................................................................................11

3.2. Salmonella and Food Contamination ..........................................................................12

3.3. Pathogenesis of Salmonella ........................................................................................13

3.4. Competitive Interactions among Serovars and Spent Media ......................................16

3.5. Salmonella in Poultry ..................................................................................................17

3.6. Salmonella Drug-resistant Strains ...............................................................................17

3.7. Mechanisms of Antimicrobial Action .........................................................................20

4. Natural Antimicrobials against Salmonella spp. ...............................................................22

4.1. Antimicrobials from Plant Sources ............................................................................23

5. Essential Oils ....................................................................................................................26

5.1. Properties ...................................................................................................................26

5.2. Mode of Action of Essential Oils ...............................................................................28

5.3. Essential Oils as a Pre-harvest Intervention ...............................................................32

5.4. Essential Oils as a Post-harvest Intervention .............................................................33

6. Hurdle Technology ............................................................................................................35

7. Conclusions ........................................................................................................................36

8. References ..........................................................................................................................39

9. Authorship Statement for Chapter 1 ..................................................................................66

CHAPTER 2: Growth Responses, Spent Media, and Competitive Interactions among

Salmonella Serovars ......................................................................................................67

1. Abstract .............................................................................................................................68

2. Introduction ........................................................................................................................69

3. Materials and Methods .......................................................................................................71

3.1. Salmonella spp. growth responses after aerobic and anaerobic incubation from

poultry Salmonella isolates ........................................................................................71

3.1.1. Bacterial strains ................................................................................................71

3.1.2. Aerobic and anaerobic growth responses ........................................................71

3.2. Effect of Salmonella spent media on the growth of S. Typhimurium .....................72

3.2.1. Aerobic and anaerobic spent media growth responses ....................................72

3.2.2. Salmonella Heidelberg and S. Typhimurium spent media ...............................72

3.2.3. Salmonella Heidelberg spent media .................................................................73

3.2.4. Salmonella Heidelberg spent media comparison between two different media

and an alternate temperature ....................................................................................73

3.3. Competitive growth between Salmonella Typhimurium ATCC 14028 and

Salmonella Heidelberg ARI-14 ..................................................................................74

3.3.1. Growth responses from mixed cultures ...........................................................74

3.3.2. DNA extraction ................................................................................................74

3.3.3. Bacterial quantification by real time quantitative PCR (qPCR) ......................75

3.4. Competitive growth between Salmonella Heidelberg ARI-14 and Salmonella

Enteritidis ATCC 13076 ............................................................................................75

3.4.1. Growth responses .............................................................................................75

3.5. Statistical analysis ....................................................................................................76

4. Results ................................................................................................................................76

4.1. Salmonella spp. growth responses after aerobic and anaerobic incubation from

poultry Salmonella isolates ........................................................................................76

4.2. Effect of Salmonella spent media on the growth of S. Typhimurium .....................77

4.2.1. Aerobic and anaerobic spent media growth responses ...................................77

4.2.2. Salmonella Typhimurium growth in S. Heidelberg spent media ....................77

4.3. Competitive growth between (1) S. Typhimurium and S. Heidelberg, and (2) S.

Heidelberg and S. Enteritidis .....................................................................................79

5. Discussion ..........................................................................................................................80

6. Acknowledgments ..............................................................................................................83

7. References ..........................................................................................................................84

8. Authorship Statement for Chapter 2 ................................................................................113

9. Appendix ..........................................................................................................................114

CHAPTER 3: Salmonella spp. Growth Rate Comparisons in Selective and Non-selective

Enrichment Media Methods ........................................................................................130

1. Abstract ...........................................................................................................................131

2. Introduction ......................................................................................................................132

3. Materials and Methods .....................................................................................................133

3.1. Bacterial cultures for non-selective enrichment media method ................................133

3.2. Bacterial cultures selective enrichment media method .............................................134

4. Results and Discussion ....................................................................................................134

5. Acknowledgments ............................................................................................................138

6. References ........................................................................................................................139


8. Appendix ..........................................................................................................................152

CHAPTER 4: Antimicrobial Activity of Essential Oils against Salmonella Heidelberg Strains

from Multiple Sources.................................................................................................163

1. Abstract ...........................................................................................................................164

2. Introduction ......................................................................................................................165


3.1. DNA extraction .........................................................................................................166

3.2. Confirmatory PCR for Salmonella Heidelberg strains .............................................167

3.3. Essential oils and cultures .........................................................................................167

3.4. Modified tube dilution assay .....................................................................................168

3.5. Statistical analysis .....................................................................................................168



6. References ........................................................................................................................172


8. Appendix ..........................................................................................................................186

CHAPTER 5: Salmonella spp. Adhesion and Invasion Comparisons to Caco-2 and HD11 Cell

Lines ............................................................................................................................197

1. Abstract ...........................................................................................................................198

2. Introduction ......................................................................................................................199


3.1. Bacterial strains for antibiotic susceptibility testing .................................................200

3.2. Antibiotic susceptibility testing ................................................................................200

3.3. Cell cultures ..............................................................................................................201

3.4. Bacterial cultures for adhesion and invasion assays .................................................202

3.5. Adhesion assays ........................................................................................................202

3.6. Invasion assays ..........................................................................................................203

3.7. Statistical analysis .....................................................................................................204



6. References ........................................................................................................................208


8. Appendix ..........................................................................................................................223

CONCLUSIONS ........................................................................................................................234

LIST OF FIGURES

CHAPTER 2

Figure 2.1. Salmonella Typhimurium average from a) aerobic growth response, b) anaerobic

growth response ............................................................................................................91

Figure 2.2. Salmonella Heidelberg average from a) aerobic growth response, b) anaerobic

growth response ............................................................................................................92

Figure 2.3. Salmonella Kentucky average from a) aerobic growth response, b) anaerobic growth

response .........................................................................................................................93

Figure 2.4. Salmonella Newport average from a) aerobic growth response, b) anaerobic growth

response .........................................................................................................................94

Figure 2.5. Salmonella Enteritidis average from a) aerobic growth response, b) anaerobic growth

response .........................................................................................................................95

Figure 2.6. Salmonella Montevideo average from a) aerobic growth response, b) anaerobic

growth response ............................................................................................................96

Figure 2.7. Average aerobic spent media growth response ..........................................................99

Figure 2.8. Average anaerobic spent media growth response.....................................................100

Figure 2.9. Combinations of S. Heidelberg and S. Typhimurium spent media and inoculum ...101

Figure 2.10. Salmonella Typhimurium ATCC 14028 in S. Heidelberg spent media from

multiple sources ..........................................................................................................103

Figure 2.11. Confirmation that the drop showed during growth response only occurs when

growing S. Typhimurium on S. Heidelberg spent media ............................................104

Figure 2.12. Growth kinetic comparison for S. Typhimurium on S. Heidelberg spent media

growing at 42°C on LB and BPW ...............................................................................105

Figure 2.13. Growth kinetic comparison for S. Typhimurium on S. Heidelberg spent media

growing at 37°C on LB and BPW ...............................................................................106

Figure 2.14. Salmonella Typhimurium and Salmonella Heidelberg growth kinetic from a mixed

culture ..........................................................................................................................108

Figure 2.15. Salmonella Typhimurium and S. Heidelberg competitive interaction ...................109

Figure 2.16. Log CFU/mL for S. Typhimurium and S. Heidelberg from mixed culture ............110

Figure 2.17. Log CFU/mL from qPCR and plate counts from mixed culture ............................111

Figure 2.18. Growth of S. Heidelberg ARI-14 and S. Enteritidis ATCC 13076 individually and

as a mixed culture........................................................................................................112

CHAPTER 3

Figure 3.1. Optical density at 600 nm for each strain from overnight cultures (16 h) on LB and

BPW broth prior to enrichment ...................................................................................146

Figure 3.2. Optical density at 600 nm from each strain inoculated into 10 mL LB broth starting

on the first detectable time point .................................................................................147

CHAPTER 4

Figure 4.1. Gel electrophoresis of PCR products to confirm Salmonella Heidelberg strains .....178

Figure 4.2. Modified tube dilution assay for trans-cinnamaldehyde ..........................................180

Figure 4.3. Modified tube dilution assay for carvacrol ...............................................................181

Figure 4.4. Modified tube dilution assay for orange terpenes.....................................................182

Figure 4.5. Modified tube dilution assay for R-(+)-limonene .....................................................183

Figure 4.6. Modified tube dilution assay for cold compressed orange oil ..................................184

CHAPTER 5

Figure 5.1. Antibacterial activity of gentamicin against strains of S. Typhimurium, S. Heidelberg

and E. coli....................................................................................................................214

Figure 5.2. Gentamicin minimum inhibitory concentration against Salmonella Typhimurium

ATCC14028 ................................................................................................................216

Figure 5.3. Gentamicin minimum inhibitory concentration against S. Heidelberg ARI-14 .......217

Figure 5.4. Gentamicin minimum inhibitory concentration against E. coli 25922 .....................218

Figure 5.5. MIC of gentamicin based on CFU/mL for each strain .............................................219

LIST OF TABLES

CHAPTER 1

Table 1.1. The 10 most frequently reported Salmonella serovars from human infections reported

to CDC, 1998-2011 .......................................................................................................64

Table 1.2. The 10 most frequent serovars from analyzed PR/HACCP verification samples for

young chicken (broilers), 1998–2011 ...........................................................................65

CHAPTER 2

Table 2.1. Bacterial strains used in this study ...............................................................................90

Table 2.2. Optical density at 600 nm for Salmonella serovars under aerobic conditions .............97

Table 2.3. Optical density at 600 nm for Salmonella serovars under anaerobic conditions .........98

Table 2.4. Salmonella Heidelberg strains used in this study .......................................................102

Table 2.5. Primer pairs used in competitive growth studies .......................................................107

CHAPTER 3

Table 3.1. List of components for each media broth used for non-selective and selective

enrichment studies .......................................................................................................145

Table 3.2. Average CFU/mL for each Salmonella serovar inoculated on Luria Bertani broth ..148

Table 3.3. Average CFU/mL for each Salmonella serovar inoculated on Rappaport –Vassiliadis

broth ............................................................................................................................149

Table 3.4. Average CFU/mL for each Salmonella serovar inoculated on tetrathionate broth ....150

CHAPTER 4

Table 4.1. Salmonella Heidelberg specific primer pair used in this study ..................................176

Table 4.2. Salmonella Heidelberg strains used in this study .......................................................177

Table 4.3. Minimum inhibitory concentrations (in μg/mL) of five different EOs against

Salmonella Heidelberg strains .....................................................................................179

CHAPTER 5

Table 5.1. Salmonella strains used in this study .........................................................................212

Table 5.2. Zones of inhibition (in millimeters) of gentamicin against S. Heidelberg and S.

Typhimurium ..............................................................................................................213

Table 5.3. MIC of gentamicin against strains of S. Heidelberg, S. Typhimurium, and E. coli

examined .....................................................................................................................215

Table 5.4. Salmonella Typhimurium and S. Heidelberg adhesion and invasion to human

epithelial, Caco-2, cells ...............................................................................................220

Table 5.5. Adhesion and invasion of four Salmonella strains from different serovars to chicken

macrophage, HD11, cells ............................................................................................221

1

INTRODUCTION

Foodborne illnesses continue to be one of the primary public health concerns in the

United States. Annually, it is estimated that over 1 million Americans contract Salmonella

infections, resulting in approximately 35% of hospitalizations and 28% of deaths attributed to

foodborne pathogens (Scallan et al., 2011). Salmonella infections are typically contracted

through the consumption of contaminated food, water, or by direct contact with an infected host

(Alcaine et al., 2007). Eggs, poultry, poultry products, meat and meat products have all been

classified as common food vehicles of salmonellosis to humans (Ricke, 2003b; Patrick et al.,

2004; Braden, 2006; Dunkley et al., 2009; Foley et al., 2011; Finstad et al., 2012; Howard et al.,

2012; Koo et al., 2012; Alali et al., 2013). Salmonella are also associated with fresh fruits and

vegetables (Pui et al., 2011; Ganesh et al., 2010; Hanning et al., 2009). Almost all of the

Salmonella serovars that have been identified are of major concern to most sectors of the food

industry (Bell and Kyriakides, 2002).

The emergence and prevalence of Salmonella serovars in chickens has been studied, but

the predominant serovars have varied somewhat over the years. Understanding the basic growth

kinetics of Salmonella serovars and their respective strains is of great importance and serves as a

basis for in depth research. In this research, we studied a) the aerobic and anaerobic growth

responses of multiple strains from six different serovars, b) how the spent media from different

serovars, particularly S. Heidelberg, affect the growth of S. Typhimurium, and c) determined

whether or not two different serovars experience a competitive relationship when they are grown

together. Growth responses of four Salmonella serovars, Heidelberg, Kentucky, Typhimurium,

and Enteritidis in selective enrichment (Rappaport-Vassiliadis and Tetrathionate broth) and non-

2

selective enrichment (Luria Bertani broth) media were compared to determine the relative

effectiveness of the methods.

Historically, a number of antibiotics have been used to control foodborne pathogens but

the emergence of multidrug resistance pathogens has increased concerns over the consequences

and the impact in human health due to antibiotic resistance (Jones and Ricke, 2003; Kim et al.,

2005; Boerlin, 2010; Muthaiyan et al., 2011; Solórzano-Santos and Miranda-Novales, 2012).

Salmonella enterica serovar Heidelberg has become an important public health concern, since it

has been identified as one of the primary Salmonella serovars responsible for human outbreaks.

Because of this, we studied the effectiveness of five essential oils, orange terpenes, orange oil,

carvacrol, cinnamaldehyde, and R-(+)-limonene, against Salmonella Heidelberg strains from

multiple sources.

Adhesion of Salmonella to the intestinal epithelial surface is an important first step in

pathogenesis and is central to its colonization of the intestine. Understanding the ability of this

pathogen to attach and invade different cell types is of great importance. As a result, since

Salmonella is commonly known to be isolated from poultry and to cause human infections, we

studied the adhesion and invasion of four different strains to chicken macrophage (HD11), and

human epithelial (Caco-2) cells.

3

CHAPTER 1

Salmonella spp.

- Literature Review

Juliany Rivera Calo1,2

and Steven C. Ricke1,2

1Department of Food Science, University of Arkansas, Fayetteville, AR

2Center for Food Safety and Department of Food Science, University of Arkansas,

Fayetteville, AR

4

1. Abstract

Salmonella spp. are one of the more prominent foodborne pathogens that represent a

major health risk to humans, given they are facultative intracellular pathogens that cause

gastroenteritis. Many Salmonella serovars have been associated with the consumption of poultry

products, these being one of the main reservoirs of Salmonella (CDC, 2009). The emergence and

prevalence of Salmonella serovars in chickens has been studied but the predominant serovars

have varied somewhat over the years. Over time Salmonella spp. have developed resistance to

conventional and widely used antibiotics leading to considerable public health concerns.

Consequently, interest in more natural, non-synthesized, antimicrobials as potential alternatives

to conventional antibiotics to treat Salmonella infections has heightened. Aromatic plants and

their components have been examined as potential growth and health promoters and most of their

properties have been linked to essential oils (EOs) and other secondary plant metabolites.

Historically, EOs from different sources have been widely promoted for their potential

antimicrobial capabilities against microorganisms. In this review, multi-drug resistant

Salmonella strains, mechanisms of antimicrobial action, and the antimicrobial properties of plant

EOs, especially from citrus, are discussed, including their mode of action, effectiveness,

synergistic effects, major components and benefits.

5

2. Introduction


United States. Each year, it is estimated that over 1 million Americans contract Salmonella

infections (Scallan et al., 2011). Annual costs for Salmonella control efforts are estimated to be

up to $14.6 billion (Scharff, 2010; Heithoff, et al., 2012). Usually Salmonella infections are self-

limited in healthy individuals, only causing mild gastroenteritis with recovery occurring

anywhere from 4 to 7 days after initial exposure. Symptoms of salmonellosis include diarrhea,

fever, and abdominal cramps occurring 12 to 72 h after consumption of food containing the

pathogen (Finstad et al., 2012). Salmonella infections are typically contracted through the

consumption of contaminated food, water, or through direct contact with an infected host

(Alcaine et al., 2007). Eggs, poultry, poultry products, meat and meat products have all been

classified as common food vehicles of salmonellosis in humans (Ricke, 2003b; Patrick et al.,

2004; Braden, 2006; Dunkley et al., 2009; Hanning et al., 2009; Foley et al., 2011; Finstad et al.,

2012; Howard et al., 2012; Koo et al., 2012). Salmonella are also associated with contamination

of fresh fruits and vegetables (Pui et al., 2011; Ganesh et al., 2010; Hanning et al., 2009). Almost

all of the Salmonella serovars that have been identified are of major concern to most sectors of

the food industry (Bell and Kyriakides, 2002).

Historically, a number of antibiotics have been used to control foodborne pathogens but

the emergence of multidrug resistant pathogens has increased concerns over the consequences

and the impact in human health due to antibiotic resistance (Jones and Ricke, 2003; Kim et al.,

2005; Boerlin, 2010; Muthaiyan et al., 2011; Solórzano-Santos and Miranda-Novales, 2012).

Antibiotic resistance essentially confers the capacity of bacteria to inactivate or exclude

antibiotics or a mechanism that blocks the inhibitory or lethal effects of antibiotics (FDA, 2002).

6

Based on the definitions set by the FDA, antibiotics are drugs produced by a microorganism that

has the capacity, in dilute solutions, to inhibit the growth of or to kill other microorganisms;

while an antimicrobial compound is any substance that kills bacteria (bactericidal) or suppresses

(bacteriostatic) their multiplication or growth, including antibiotics and synthetic agents (FDA,

2002). Antimicrobials are probably one of the most successful forms of chemotherapy in the

history of medicine (Aminov, 2010).

Salmonella is not only a public health concern due to the significant number of cases per

year, but also because many strains are becoming more resistant to antimicrobial agents (Kim et

al., 2005; Alcaine et al., 2007; Foley and Lynne, 2008; Bajpai et al., 2012; Kollanoor-Johny et

al., 2012) due to the previous relatively unrestricted use of antimicrobials in feeds and increased

therapeutic use (Sørum and L’Abée-Lundm 2002; Su et al., 2004; Kim et al., 2005). Since their

discovery, antimicrobials have been extensively used in poultry and livestock for the treatment,

prevention and/or control of animal diseases, as well as for production purposes, for example to

improve feed efficiency and growth enhancement (Hur et al., 2012). The two mechanisms known

to be critical for the spread of antimicrobial resistance in Salmonella are 1) horizontal transfer of

antibiotic resistance genes and 2) clonal spread of antimicrobial drug-resistant Salmonella

isolates (Mølbak 2005; Alcaine et al., 2007). A major concern has been the acquired resistance to

cephalosporins (ceftriaxone and ceftiofur), since ceftriaxone is required to treat severe

salmonellosis in children and ceftiofur is the only antibiotic approved for veterinary use in the

U.S. (Bradford et al., 1999; Rabsch et al., 2001). Because of this, the development of strategies

that focus less on specific antibiotic administration to control pathogens is of major interest from

both a food safety and a medical perspective (Burt, 2004; Bajpai et al., 2012).

In recent years, aromatic plants and their extracts have been examined for their

7

effectiveness for food safety and preservation applications (Fisher and Phillips, 2008) and have

received attention as growth and health promoters (Brenes and Roura, 2010). Most of their

properties are due to their EOs and other secondary plant metabolite components (Brenes and

Roura, 2010). Phytochemicals, such as EOs, are naturally occurring antimicrobials found in

many plants that have shown to be effective in a variety of applications by decreasing growth

and survival of microorganisms (Callaway et al., 2011a). EOs exhibit antimicrobial properties

that may make them suitable alternatives to antibiotics (Chaves et al., 2008). These potential

attributes and an increasing demand for natural medicinal treatment options have led to an

interest in the use of EOs as potential alternative antimicrobials (Fisher and Phillips, 2008;

Solórzano-Santos and Miranda-Novales, 2012). Essential oils derived as by-products of the

citrus industry have been screened for antimicrobial properties against common foodborne

pathogens and several have been shown to possess antimicrobial properties (Dabbah et al.,

1970).

Historically, citrus oils have been incorporated into human diets (Dabbah et al., 1970;

Kim et al, 1995) because of their flavor and beneficial health properties and are approved as

generally recognized as safe (GRAS) compounds by the Food and Drug Administration (FDA),

due to their natural role as flavoring agents in citrus juices (Chalova et al., 2010; Hardin et al.,

2010; Callaway et al., 2011a). Their use as alternatives to chemical-based antimicrobials is not a

new concept as they have been used for medicinal purposes from ancient times into present day

applications and considerable research has been conducted to demonstrate their bioactivity

against bacteria, yeasts and molds (Fisher and Phillips, 2008). Approximately 400 compounds of

citrus oils have been identified from citrus and their content depends on the specific citrus

cultivar, separation and extraction methods (Caccioni et al., 1998; Robinson, 1999; Tao et al.,

8

2009). The most well known and characterized of the EOs from citrus products include citrullene

and limonene (Dabbah et al., 1970; Caccioni et al., 1998), which can exert potent, broad-

spectrum antimicrobial activity (Di Pasqua et al., 2006).

Citrus fruits, including oranges, contain a number of compounds, such as EOs, that are

toxic to bacteria, can exert antimicrobial activity and can alter the microbial ecology of the

gastrointestinal tract (Fisher and Phillips, 2006; Kim et al., 1995; Callaway et al., 2011a). In their

research, Callaway et al., (2008) reported that Salmonella populations were reduced by more

than 2 log colony forming units (CFU) per gram by the addition of up to 2% orange peel and

pulp in in vitro mixed ruminal microorganism fermentations. In addition to possessing

antimicrobial activity, citrus EOs can serve as optimal sources of antioxidants (Chalova et al.,

2010; Callaway et al., 2011a). Due to the antioxidant effect, the antimicrobial activity and other

health benefits of citrus consumption, there has been increased interest in these compounds for

additional applications (Callaway et al., 2011a). Little research has been done on how these

compounds affect the microbial ecosystem of the human gut or food animals, and the subsequent

impact in human health (Callaway et al., 2011a). In this review, multi-drug resistant Salmonella

strains, mechanisms of antimicrobial action, and the antimicrobial properties of plant EOs,

especially from citrus, are discussed, including their mode of action, effectiveness, synergistic

effects, major components and benefits. Since there have been numerous studies in terms of

Salmonella genetics and physiology, the stress and virulence characteristics of this organism

under antimicrobial treatment conditions has been well documented (Foley et al., 2011; 2013; Li

et al., 2013) and is beyond the scope of this review.

9

3. Foodborne Pathogen, Salmonella spp.

The genus name Salmonella was first suggested by Lignieres in 1900 in recognition of

the work carried out by the American bacteriologist, D.E. Salmon, who, with T. Smith in 1886,

described the hog cholera bacillus causing ‘swine plague’ (Topley and Wilson, 1929; D’Aoust,

1989). Salmonella species are Gram-negative, facultative anaerobic, flagellated, rod-shaped,

motile bacteria of the enterobacteria group, of approximately 2-3 x 0.4-0.6μm in size (Pui et al.

2011; Li et al., 2013). Salmonella can multiply under various environmental conditions outside

their respective living host (Park et al., 2008; Pui et al., 2011). Most Salmonella serovars grow at

a temperature range of 5 to 47°C with an optimum temperature of 35 to 37°C, but some can grow

at temperatures as low as 2 to 4°C and as high as 54°C (Gray and Fedorka-Cray, 2002). Given

the ability of Salmonella spp. to grow under a variety of different growth conditions and their

well-characterized genetics, they are a fairly ideal foodborne pathogen model for developing an

understanding of the mechanism(s) involved in the efficacy of antimicrobial compounds.

Classification of Salmonella is based on their susceptibility to different bacteriophages, as

well as grouping based on their somatic (O), flagellar (H), and capsular (Vi) antigenic patterns

(Dunkley et al., 2009; Pui et al., 2011). Salmonella H and O surface antigens can serve as typing

characteristics for potential reservoirs for each serovar (Hur et al., 2012). Currently the

Salmonella genus is composed of three species: S. enterica, S. bongori and S. subterranea

(Brenner, 1998; Su and Chiu, 2007; Foley et al., 2013). The species S. enterica is divided into six

subspecies: I (enterica), II (salamae), IIIa (arizonae), IIIb (diarizonae), IV (houtenae), and VI

(indica) based on biochemical and genetic properties (Le Minor and Popoff, 1987; Brenner,

1998; 2000). Salmonella bongori contains the members of subspecies V, and S. enterica consists

10

of members of the remaining six subgenera (Brenner, 1998; Foley et al., 2013). Salmonella

subterranea was described as a species in 2005 (Su and Chiu, 2007; Foley et al., 2013).

The Salmonella genus is widely heterogeneous, currently comprised of over 2,500

serovars (Popoff et al., 2004). The majority of the serovars that are pathogenic to human belong

to S. enterica, which contains over 99% of the serovars (Brenner et al., 2000; Tindall et al., 2005;

Hur et al., 2012). Members of this species have usually been named based on the isolation

geographical location of the serovar (Alakomi, 2007). Salmonella enterica subspecies I is mainly

isolated from warm-blooded animals and more than 99% of clinical isolated pertain to this

group, whereas the remaining subspecies are mostly isolated from cold-blooded animals and

account for less than 1% of clinical isolates (Pui et al., 2011; Bell and Kyriakides, 2002; Brenner

et al., 2000). Almost all of the Salmonella serovars that have been identified are of major

concern to nearly all sectors of the food industry (Bell and Kyriakides, 2002).

Salmonella cause gastroenteritis commonly referred to as salmonellosis, which can result

in a much more systemic infection, depending on several factors including susceptibility of the

host (Owens and Warren, 2012). Salmonella enterica serovars Typhimurium and Enteritidis

cause gastroenteritis and these two are the most common serovars responsible for human

infections (Olsen et al., 2001a; CDC, 2011; Dunkley et al., 2009; Foley et al., 2011; Finstad et

al., 2012; Howard et al., 2012). S. Enteritidis cases have often been associated with consumption

of contaminated eggs (Guard-Petter, 2001; Cogan et al., 2004; Howard et al., 2012). S.

Typhimurium cases are usually linked to the consumption of contaminated poultry, swine and

bovine meat (Jorgensen et al., 2000). Salmonella enterica serovar Typhi is the most invasive,

causing typhoid fever in humans. Some Salmonella strains may exhibit low infectious doses (500

or less) while others require 100,000 or more organisms to cause infection depending on the

11

virulence of the strain (Finstad et al., 2012). At least 106 to 10

9 cells are required to cause

salmonellosis in healthy human adults (Nester et al., 2009). Antibiotic therapy is not

recommended for simple cases of gastroenteritis (Hohmann, 2001). However for more systemic

infections, treatment with chloramphenicol may be needed (Li et al., 2013). Even though

chloramphenicol is used as treatment for systemic infections, a large number of cultures taken

from the bone marrow over the first week of treatment remained positive for Salmonella (Gasem

et al., 1995; Hussein Gasem et al., 2003). This persistence of Salmonella, potentially surviving

inside macrophages, could be responsible for a relapse of the infection (Sanders, 1965; Hussein

Gasem et al., 2003). In invasive life-threatening infections, the use of antimicrobial drugs is

required, but the efficacy of these drugs is decreasing due to the emergence of antimicrobial

resistant Salmonella strains (Shaw et al., 1993; Angulo et al., 2000; Winokur et al., 2000; Varma

et al, 2005a; Chen et al., 2007). Administration of antimicrobial agents such as ampicillin,

chloramphenicol, and tetracycline has become limited due to the increased resistance of

Salmonella to these agents. Fluoroquinolones, such as ciprofloxacin, or extended-spectrum

cephalosporins are commonly used to treat adult patients infected with Salmonella enterica or to

treat a severe case of gastroenteritis (Mølbak, 2005; Chen et al., 2007; Hur et al., 2012; Schmidt

et al., 2012). The emerging resistance to fluoroquinolones, the main treatment of severe

salmonellosis, has become a greater public health concern more recently (Olsen et al., 2001b;

Mølbak, 2005; Alcaine et al., 2007; Chen et al., 2007; Brichta-Harhay et al., 2011).

3.1. Epidemiology of Salmonella

Salmonella is a foodborne pathogen that causes gastroenteritis known as salmonellosis,

often resulting in systemic infection. Typhoid cases are stable with low numbers in developed

12

countries, but nontyphoidal salmonellosis has increased worldwide (Pui, et al., 2011). Annually,

it is estimated that over 1 million Americans contract Salmonella (Scallan, et al., 2011).

Epidemiologically Salmonella is classified into three groups based on the host

preferences that may include, 1) host-restricted, which are the serotypes capable of causing a

typhoid-like disease in a single host species (for example Salmonella Typhi in humans), 2) host-

adapted, which are serotypes associated with one host species, but also able to cause disease in

other hosts; some of these are human pathogens and may be contracted from foods (for example

S. Choleraesuis in swine and S. Gallinarum in poultry) and 3) unadapted serovars (no host

preference), which are pathogenic for humans and other animals, and they include most

foodborne serovars, for example S. Typhimurium (Boyen et al., 2008; Pui et al., 2011; Li et al.,

2013). Depending on the host and serotype, Salmonella can cause diseases ranging from mild

gastroenteritis to typhoid fever (Chiu et al., 2004; Humphrey, 2004). Antibiotic therapy is not

recommended for simple cases of gastroenteritis (Alcaine et al., 2007). In case of a systemic

infection, treatment with chloramphenicol may be needed (Shaw et al., 1993; Alcaine et al.,

2007).

3.2. Salmonella and Food Contamination

The primary sources of Salmonella are the gastrointestinal tract of humans, domestic and

wild animals, birds and rodents (Bell and Kyriakides, 2002). Because of this, they are

widespread in the natural environment including waters and soil, where they do not usually

multiply in a significant way but may survive for long periods (Bell and Kyriakides, 2002).

Salmonella enterica species are normally orally acquired pathogens, which cause one of four

major syndromes: enteric fever (typhoid), enterocolitis (gastroenteritis) or diarrhea, bacteremia

13

and chronic asymptomatic carriage (Coburn et al. 2007). Although the primary habitat of

Salmonella is the intestinal tract, it may also be found in other parts of the body and can also be

found in water. The organisms are excreted through the feces from which they can then be

transmitted by insects and others to a wide range of places (CDC, 2012).

Salmonella is among the most commonly isolated foodborne pathogens that are

associated with fresh fruits and vegetables (Pui et al., 2011). Eggs, poultry, meat and meat

products are the most common food vehicles of salmonellosis to humans. From 1996 to 1997,

four of the top-ranked Salmonella serovars were Typhimurium, Enteritidis, Heidelberg, and

Newport respectively (Olsen et al., 2001a).

After ingestion with contaminated food, symptoms usually develop in 12 to 14 hours, but

this time has been reported to be shorter or longer, up to 72 hours. Prevention of Salmonella can

be achieved by proper food handling, avoiding cross-contamination, personal hygiene and

sanitation and education to the public. Proper cooking following the recommended minimum

internal temperatures followed by prompt cooling to 3 to 4°C or freezing within 2 h would

eliminate Salmonella from foods (USDA/FSIS, 2013a).

3.3. Pathogenesis of Salmonella

Salmonella growth and virulence responds to a series of factors such as pH, oxygen

availability and osmolarity. When there is a lack of nutrients, such as carbon sources, this can

trigger global stress responses in Salmonella and a change in gene expression patterns can be

seen toward a more increased virulence and survivability (Altier, 2005; Kundinger et al., 2007).

Some of the genes necessary for invasion of intestinal epithelial cells and induction of

intestinal secretory and inflammatory responses are encoded in Salmonella Pathogenicity Island

14

1 (SPI-1) (Watson et al., 1995; Hur et al., 2012). Salmonella Pathogenicity Island 2 (SPI-2) is

key for the establishment of systemic infection beyond the intestinal epithelium; it also encodes

essential genes for intracellular replication (Cirillo et al., 1998; Ohl and Miller, 2001; Hur et al.,

2012). Adhesion of Salmonella to the intestinal epithelial surface is an important first step in

pathogenesis and is central to its colonization of the intestine. Once Salmonella is attached to the

intestinal epithelium, normally it expresses a multiprotein complex, known as T3SS, that

facilitates endothelial uptake and invasion (Foley et al., 2008, 2013; Winnen et al., 2008). This

type III secretion system (T3SS) is associated with SPI-1, which contains virulence genes

associated in Salmonella adhesion, invasion and toxicity (Foley et al., 2013). The human

intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the

intestinal barrier. The parental cell line, originally obtained from a human colon

adenocarcinoma, goes through a spontaneous differentiation process that leads to the formation

of a monolayer of cells, expressing several morphological and functional characteristics of the

mature enterocyte (Sambuy et al., 2005). The underlying mechanisms used by intracellular

pathogens such as Salmonella to penetrate the host epithelium are not well understood. As a

result, researchers have used cultured mammalian cells as in vitro models to study interaction

and internalization of Salmonella (Gianella et al., 1973; Finlay et al., 1988; Durant et al., 1999).

Invasion in cultured epithelial cells is commonly used to measure the pathogenicity of

Salmonella (van Asten et al., 2000, 2004; Shah et al., 2011). Previous studies have reported that

Salmonella invasion into cultured mammalian cells can be influenced by several environmental

stimuli such as osmolarity (Galán and Curtis, 1990; Tartera and Metcalf, 1993) carbohydrate

availability (Schiemann, 1995), and oxygen availability (Ernst et al., 1990; Lee and Falkow,

1990; Francis et al., 1992). In their research, Durant et al., (1999) reported that Salmonella can

15

encounter high concentrations of short-chain fatty acids (SCFA) in the lower regions of the

gastrointestinal tract, this in addition to changes in pH, oxygen tension, and osmolarity. Since a

combination of environmental conditions are what regulate virulence gene expression, SCFA

may contribute to the environmental stimuli that regulate cell-association and invasion of S.

Typhimurium epithelial cells (Durant et al., 1999). Shah et al., (2011) reported that in cultured

Caco-2 cells, isolates with high invasiveness were able to invade and/or survive in significantly

higher numbers within chicken macrophage cells than other isolates with low invasiveness.

HD11 cells are a macrophage-like immortalized cell line derived from chicken bone

marrow and transformed with the avian myelocytomatosis type MC29 virus (Beug et al., 1979).

In their research, He et al., (2012) compared Salmonella cell invasion and intracellular survival

of five different poultry-associated serovars (S. Heidelberg, S. Enteritidis, S. Typhimurium, S.

Senftenberg, and S. Kentucky) to HD11 cells. Their results showed that S. Enteritidis was more

resistant to intracellular killing, leading them to believe that the intracellular survival ability of

this serovar may be related with systemic invasion in chickens (He et al., 2012). Shah et al.,

(2011) suggested that not all isolates of S. Enteritidis recovered from poultry might be equally

pathogenic or have similar potential to invade cells; and that S. Enteritidis pathogenicity is

related to both the secretion of T3SS effector proteins and motility. Saeed et al., (2006) reported

that compared to isolates of S. Enteritidis recovered from chicken ceca, isolates recovered from

eggs or from human clinical cases exhibited a greater adherence and invasiveness of chicken

ovarian granulosa cells. Several pathogens and host factors may be an important key to

determine the mechanisms for the differences in Salmonella responses within different cell types

(Bueno et al., 2012; Foley et al., 2013). Understanding the ability and mechanisms of this

16

pathogen to attach and invade different cell lines could be helpful for the reduction of Salmonella

infections.

3.4. Competitive Interactions among Serovars and Spent Media

A significant shift in the predominant Salmonella serovars associated with poultry and

human infections had been seen over the last several decades. S. Enteritidis and S. Heidelberg are

among the top five serovars associated with human infections and some of the most commonly

detected serovars in chickens over the last 25 years. S. Enteritidis remains as a significant

problem in commercial poultry, but its prevalence has declined in chickens in the U.S. since the

mid 1990s. From 1997 to 2006 S. Heidelberg supplanted S. Enteritidis as the predominant

serovar, but for 2007 S. Kentucky became the most commonly isolated serovar (Foley et al.,

2008). This serovar is less commonly identified as a source of human salmonellosis and it is

unclear why it has become a predominant colonizer in chicken ceca but has not been a significant

threat to humans as are S. Typhimurium, S. Enteritidis or S. Heidelberg. Although S. Kentucky is

not among the most common serovars causing human diseases, it has a significant prevalence of

multidrug resistance isolates from poultry and humans in other parts of the world (Foley et al.,

2011).

Spent media is a growth medium exhausted of nutrients due to extensive growth of

bacteria species and is known to influence the gene expression patterns of S. Typhimurium. The

filtered supernatant of this media has the potential to serve as an in vitro screen of ecosystems

after growth of either specific microorganisms or a microbial consortium from a specific in vivo

site such as the gastrointestinal tract (Nutt et al., 2002; Kundinger et al., 2007). The use of spent

17

media has been previously applied to simulate chicken gastrointestinal tract conditions after feed

withdrawal (Nutt et al., 2002).

3.5. Salmonella in Poultry

Numerous Salmonella serovars have been associated with the consumption of poultry

products, these being one of the main reservoirs of Salmonella (Shah et al., 2011; He et al.,

2012). Contaminated poultry, meat, and eggs are important vehicles of Salmonella infections,

especially when the organism is in the egg contents (Foley et al., 2011). Kuehn (2010) reported

this contamination problem when a salmonellosis outbreak caused by S. Enteritidis was traced

back to contaminated eggs from Iowa. Some of the factors that can affect the colonization of

Salmonella in poultry include the age and genetic susceptibility of the birds, bird stress due to

overcrowding or underlying illness, level of exposure to the pathogen, competition with gut

microflora for the colonization sites, infecting Salmonella serovar, and whether or not the strains

carry genetic factors that may facilitate the attachment to the gastrointestinal tract of the bird or

evade host defenses (Bailey, 1988). In the past few years, the emergence and prevalence of

Salmonella serovars in chickens has been studied but the predominant serovars have varied

somewhat over the years (Foley et al., 2008; 2011; 2013).

3.6. Salmonella Drug-resistant Strains

A microbial strain is considered resistant to a particular antimicrobial when the

antimicrobial is no longer efficacious for treatment of a clinical disease caused by a particular

bacterial pathogen (Alcaine et al., 2007). Extensive research with the purpose of characterizing

antimicrobial resistance strains of Salmonella enterica from different food and animal sources

18

has revealed that numerous serotypes may have multiple antimicrobial resistance determinants

(White et al., 2001; Zhao et al., 2007; Brichta-Harhay et al., 2011). Multi-drug resistant

Salmonella (MDR) strains are defined as those strains which are resistant to two or more

antimicrobial agents. These may carry their resistance determinants on chromosomal sites,

resistance gene carrying plasmids or on both (Chen et al., 2004; Alcaine et al., 2007; Lindsey et

al., 2009). Resistance to antimicrobial compounds can result from one or more target gene

mutations (Condell et al., 2012b). Overall the human clinical Salmonella isolates resistant to one

or more drug classes declined from 1996 to 2003 in the United States (CDC, 2004b). Moreover,

in 2011 the CDC reported that resistance to one or more drug classes was lower than during the

period of 2003 to 2007 (CDC, 2013).

However, infections by MDR Salmonella remain a concern due to potential treatment

difficulties resulting in human infections with these strains being more severe and patients in

turn, are at greater risk of bacteremia, hospitalization and death compared to patients infected

with antibiotic susceptible strains (Helms et al., 2002; CDC, 2004b; Mølbak, 2005; Talbot et al.,

2006). Eight of the ten serotypes most commonly reported by the CDC, include at least some

isolates that displayed resistance to at least five or more antimicrobial drugs (CDC, 2004a;

2004b). Salmonella serotypes reported with the highest multidrug resistance were Typhimurium,

Heidelberg, and Newport (CDC, 2004a). The frequency of MDR in Typhimurium and Newport

is reportedly increasing (Zhao et al., 2008; Hur et al., 2012). Additionally, serovars such as

Agona, Anatum, Cholerasuis, Derby, Dublin, Kentucky, Pullorum, Schwarzengrund, Seftenberg,

and Uganda also contain MDR Salmonella strains (Chen et al., 2004; Zhao et al., 2008; Hur et

al., 2012).

19

There are two common resistance patterns for MDR Salmonella Typhimurium isolates:

1) resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline or 2)

resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, the

resistance profile most commonly associated with S. Typhimurium DT104 (CDC, 2004a).

Salmonella Heidelberg isolates were typically characterized by resistance to ampicillin,

amoxicillin-clavulanic acid, ceftiofur, and cephalothin (CDC, 2004a).

The difference between these two resistance patterns for MDR S. Typhimurium is that

one has kanamycin and the other one has chloramphenicol. These antibiotics pertain to different

families of antibiotics, and each of them possesses a specific mechanism of action. Streptomycin

and kanamycin belong to the aminoglycosides family, which bind to conserved sequences within

the 16S rRNA of the 30S ribosomal subunit, leading to a codon misreading and translation

inhibition (Alcaine et al., 2007). Aminoglycoside resistance in Salmonella has been associated

with the expression of aminoglycoside-modifying enzymes. Ampicillin belongs to the beta-

lactam family, where the ability to interfere with the penicillin-binding proteins (involved in the

synthesis of peptidoglycan) confer antimicrobial effects on the target microorganism (Alcaine et

al., 2007). In Salmonella, the mechanism of resistance to beta-lactams that has been most

commonly described involves the secretion of beta-lactamases (Alcaine et al., 2007).

Chloramphenicol belongs to the phenicols family and its mode of action is via the

prevention of peptide bond formation. There are two resistance mechanisms in Salmonella from

this antibiotic: 1) enzymatic inactivation of the antibiotic and 2) removal of the antibiotic via an

efflux pump. The mechanism of action for tetracyclines is by inhibiting protein synthesis; and its

resistance to Salmonella is conferred by production of an energy-dependent efflux pump, which

excretes the antibiotic from the bacterial cell (Alcaine et al., 2007). Sulfamethoxazole belongs to

20

the sulfonamides family of antibiotics, which act by inhibiting enzymes that are involved in the

synthesis of tetrahydrofolic acid. Resistance in Salmonella is due to the presence of an extra sul

gene, which expresses an insensitive form of dihyrdropteroate synthetase (DHPS) (Alcaine et al.,

2007).

Varma et al., (2005b) reported that antimicrobial resistance in nontyphoidal Salmonella is

characterized by an increased frequency of bloodstream infections and hospitalizations of the

corresponding patients. Bloodstream infections have been reported to occur more frequently

among people infected with a nontyphoidal Salmonella isolate resistant to more than one

antimicrobial agent (CDC, 2003a; CDC, 2003b). Human-health consequences of increasing

resistance may be substantial if antimicrobial-resistant infection increases the risk of bloodstream

infection (Varma et al., 2005b). Previous research has revealed a strong association between

resistance, bloodstream infection, and hospitalization (Holmberg et al., 1984; 1987; Lee et al.,

1994; Helms et al., 2002; WHO, 2005; Varma et al., 2005b). Patients who become infected with

resistant Salmonella isolates are more prone to experience failure when administered

antimicrobial therapy, resulting in a more invasive illness.

3.7. Mechanisms of Antimicrobial Action

Antimicrobial agents are not essential to treat most cases of Salmonella infections, but

they are more likely to be essential at times of severe or systemic infection (Chen, et al., 2007).

In bacteria, resistance to antimicrobial agents may be mediated by a variety of mechanisms such

as (1) energy-dependent removal of antimicrobials via membrane-bound efflux pumps, (2)

changes in bacterial cell permeability, which restricts the access of antibiotics to target sites

(Ravishankar et al., 2010) (3) modifications of the site targeted by drug action, and (4)

21

inactivation or destruction of antimicrobials (Chen et al., 2004). It has been suggested that the

high resistance level of Salmonella to fluoroquinolones is due to the combination of two major

resistance mechanisms, active efflux mediated by AcrAB-Tolc and multiple target gene

mutations (Velge et al., 2005; Hur et al., 2012). In the case of tetracyclines, bacteria normally

acquire resistance to these antibiotics through an efflux mechanism by which the drug is pumped

out from the cell (Kim et al., 2005). In their research, Hur et al., 2012 described the resistance to

tetracycline and chloramphenicol to be highly associated with the acquisition and expression of

efflux pumps, which are responsible of reducing toxic levels of the drug in the bacterial cells.

To inhibit microbial growth, antimicrobials tend to act on several specific target sites

such as inhibition of synthesis of specific proteins, DNA and RNA synthesis, interference with

cell wall synthesis, disruption of membrane permeability structure and inhibition of essential

metabolite synthesis (Hughes and Mellows, 1978; Reynolds, 1989; Falla et al., 1996; Hooper,

2001). For example, antibiotics such as penicillins and cephalosporins inhibit bacterial cell wall

synthesis by interference with the enzymes associated with peptidoglycan layer synthesis; while

tetracyclines and streptogramins work by disrupting protein synthesis (Tenover, 2006). Due to

their different biological structures and properties, target sites for each antimicrobial could vary

(Li et al., 2011) depending on the type of microorganism that is being exposed to the respective

antimicrobial agent. The potential target site for Gram-positive cocci involves the cell wall,

cytoplasmic membrane, functional proteins, DNA and RNA. In the case of Gram-negative

bacteria they share the same target sites, except that the target is the outer membrane instead of

the cell wall (Li et al., 2011).

The continuous and widespread use of antibiotics are considered two of the important

factors that have led to the rapid increase of antibiotic resistance; resulting in more demand to

22

either replace or at least reduce the use of antibiotics by using more natural, non synthesized,

alternatives (Bajpai et al., 2012; O’Bryan et al., 2008b; Li et al., 2011).

4. Natural Antimicrobials against Salmonella

Control of food spoilage and pathogenic bacteria is mainly achieved by chemical control

but the use of synthetic chemicals is limited due to undesirable aspects including carcinogenicity,

acute toxicity, teratogenicity and slow degradation periods, which could lead to environmental

problems, such as pollution (Faleiro, 2011). The negative public perception of of industrially

synthesized food antimicrobials has generated interest in the use of more naturally occurring

compounds (Sofos et al., 1998; Faleiro, 2011). There has been an extensive search for potential

natural food additive candidates that retain a broad spectrum of antioxidant and antimicrobial

activities while possessing the ability to improve the quality and shelf life of perishable foods

(Fratianni et al., 2010). The emergence of bacterial antibiotic resistance and the negative

consumer attitudes toward food preservatives have led to an increased interest in the use of plant

components that contain EOs and essences as alternative agents for the control of food spoilage

and harmful pathogens (Shelef, 1983; Smith-Palmer et al., 1998; 2001; Burt, 2004; Nostro et al.,

2004; Fisher and Phillips, 2008). There are certain limitations to the use of antimicrobial plant

oils in livestock waste, for example their cost effectiveness is not clear, there could be potential

environmental effects and/or there could be little to no degradation of the waste (Varel, 2002).

Despite these limitations, Varel (2002) described some potential advantages of using natural

antimicrobial chemicals such as EOs including: 1) inhibition of microbial fermentation of waste,

hence reducing the rate of odor emissions as well as the production of global warming gases, and

thus conserve nutrients in the waste for increased fertilizer value; 2) destruction of pathogenic

23

fecal coliforms; 3) the phenolic plant oils are stable under anaerobic conditions, although they

degrade under aerobic environments; 4) these products are classified as GRAS; and 5) these oils

can also be used as pesticides, which could play a significant role in controlling flies in livestock

waste.

Natural compounds with animal, plant or microbiological origins have been used in order

to kill or at least prevent the growth of pathogenic microorganisms (Roller and Lusengo, 1997;

Li et al., 2011, Muthaiyan et al., 2011; Juneja et al., 2012). A number of naturally occurring

antimicrobial agents are present in animal and plant tissues, where they probably evolved as part

of their hosts’ defense mechanisms against microbiological invasion and exist as natural

ingredients in foods (Sofos et al., 1998).

4.1. Antimicrobials from Plant Sources

Substances that are naturally occurring and directly derived from biological systems

without alteration or modification in a laboratory setting are recognized as natural antimicrobials

(Lopez-Malo et al., 2000; Sirsat et al., 2009; Li et al., 2011). An ideal antimicrobial would be

one that is available in large volumes as a co-product and one that has GRAS status because it

has already been part of the typical human diet for years (Friedly et al., 2009; Nannapaneni et al.,

2009b; Chalova et al., 2010; Callaway et al., 2011a). There is great potential for new drug

discoveries based on collecting and characterizing traditional medicinal plants throughout the

world (Lewis and Elvin-Lewis, 1995). Some plant extracts such as Garcinia kola and Vernonia

amygdaina have previously been investigated for their phytochemical properties and

antibacterial effects on bacterial pathogens such as Escherichia coli, Klebsiella pneumonia,

Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus.

24

Chemicals deployed by plants against bacteria to obtain an advantage in their ecosystem include:

EOs, organic acids, and specific toxins (Friedman et al, 2002; Callaway et al., 2011a).

The use of pesticides in farming and the use of growth hormones and antibiotics in

livestock production (Magkos et al., 2003; Van Loo et al., 2010), lower environmental impact

and animal welfare practices (Magnusson et al., 2003; Van Loo et al., 2010); and quality and

safety of organic foods are some of the factors that meet consumers concerns and increase the

demand of organic foods (Magkos et al., 2006; Van Loo et al., 2010). Most of the research on

organic foods has concluded that there is no evidence that organic food is safer, healthier, or

more nutritious (Magkos et al. 2003; Van Loo et al., 2012). Organic foods are required to meet

the same food safety standards as foods that are conventional; there are no stricter food safety

requirements (Van Loo et al., 2012).

The United States Department of Agriculture (USDA) defines organic food as: “Organic

food is produced by farmers who emphasize the use of renewable resources and the conservation

of soil and water to enhance environmental quality for future generations. Organic meat, poultry,

eggs, and dairy products originate from animals that are given no antibiotics or growth

hormones. Organic food is produced without using most conventional pesticides; fertilizers made

with synthetic ingredients or sewage sludge; bioengineering; or ionizing radiation. Before a

product can be labeled ‘organic,’ a government-approved certification agent inspects the farm

where the food is grown to confirm that the farmer is following all the rules necessary to meet

USDA organic standards. Companies that handle or process organic food before it gets to your

local supermarket or restaurant must be certified, too” (USDA, 2013).

In their survey, Van Loo et al., (2010) reported that consumers leaned towards buying

organic chicken due to their perception that organic chicken is safer, healthier and has fewer

25

pesticides, antibiotics and hormones. Van Loo et al., (2011) assessed consumer’s willingness to

pay for organic chicken, and their results demonstrated that consumers were willing to pay 35%

premium for a general organic labeled chicken breast and 104% for a USDA certified organic

labeled based on an overview of current published research reports. Van Loo et al., (2012)

concluded that consumers should not assume that chickens labeled as organic, all natural, or free

range are any less contaminated with Salmonella than conventionally raised chickens. Crandall

et al., (2011) surveyed a total of 305 consumers to determine their concerns and beliefs about the

safety of foods obtained at three Arkansas farmers’ markets. Results showed that 36% of the

consumers preference for organic foods was that they wanted food free of chemicals; 45% of the

consumers biggest safety concerns were pesticides; and only 2 to 6% were actually concerned

about foodborne pathogens. A total of 76% of the surveyed consumers believed that organic

foods were safer than conventional foods (Crandall et al., 2011).

Based on health, economic and environmental issues, natural plant extracts represent a

safer choice as an alternative for synthetic antimicrobials (Burt, 2004; Jo et al., 2004; Callaway

et al., 2011a; Muthaiyan et al., 2011; Bajpai et al., 2012). Citrus products and other EOs sources

can be used as a green alternative to reduce antibiotic use (Callaway et al., 2011a; Warnke et al.,

2009). Including essential citrus oils in animal feeds and human foods was reported to be a

means to improve human health, also to be a green solution and economically feasible (Callaway

et al., 2011a). Jo et al., (2004) reported that lyophilized citrus extracts might be useful as natural

antioxidants to benefit health and also have the potential to be environmentally friendly and cost

effective. These naturally occurring antimicrobials have extensive histories of their use in foods

and can be identified from various components of the plants leaves, barks, stems, roots, flowers

and fruits (Rahman and Gray, 2002; Erasto et al., 2004; Zhu et al., 2004).

26

A wide range of products derived from plant sources (e.g., fruit preparations, vegetable

preparations or extracts and spices) have been used throughout history for the preservation and

extension of the shelf life of foods (Billing and Sherman, 1998; Cutter, 2000; McCarthy et al.,

2001; Islam et al., 2002; Draughon, 2004; Kim et al., 2011). Plant EOs have been widely used

for a variety of medicinal and cosmetic purposes (Crandall et al., 2012). It has been recognized

that certain plant extracts, including their EOs and essences possess antimicrobial properties

(Smith-Palmer et al., 1998; 2001; Brenes et al., 2010; Kim et al., 2011; Shannon et al., 2011b;

Solórzano-Santos and Miranda Novales, 2012) against bacteria, yeast and moulds (Smith-Palmer

et al., 1998).

5. Essential Oils

5.1. Properties

Historically, EOs and their components isolated from aromatic and medicinal plants

possess antimicrobial properties when used for health and food preservation (Faleiro, 2011).

Essential oils are natural, complex, multi-component systems composed mainly of terpenes in

addition to other non-terpene components (Edris, 2007). Essential oils are concentrated oils

containing volatile aroma compounds such as terpenes of plant material that are obtained by

solvent extraction, or fermentation; but the most common method used is steam distillation

(Burt, 2004; Kelkar et al., 2006; Edris, 2007; Faleiro, 2011; Shannon et al., 2011a; Solórzano-

Santos and Miranda Novales, 2012). During the juice extraction from the oranges, cold pressed

oils are recovered from the orange peels. The essence oils are what are collected during the

process while the orange juice is being evaporated into concentrate (Crandall and Hendrix 2001).

27

Bakkali et al., (2008) defined EOs as liquid, volatile, limpid and rarely colored, lipid

soluble and soluble in organic solvents with a usually lower density than that of water. The

composition of EOs is influenced by the respective extraction methods and the antimicrobial

properties of these are determined by the components recovered in the particular extraction

process (Burt, 2004). They can also act as biopreservatives, reducing or eliminating pathogenic

bacteria and increasing the overall quality of animal and vegetable food products (Solórzano-

Santos and Miranda-Novales, 2012).

Essential oils are primarily used for fragrances, flavors as well as pharmaceuticals due to

their chemopreventive and chemotherapeutic activities, antioxidants activities against low

density lipoproteins, antiviral and antidiabetic activities and even potential as insect repellents

(Burt, 2004; Edris, 2007; Nannapaneni et al., 2008; Chalova et al., 2010; Callaway et al., 2011a;

Jacob and Pescatore, 2012). Essential oils also retain characteristic odor and flavor of the plant

they were extracted from (Jacob and Pescatore, 2012). Approximately 3000 EOs are known, of

which approximately 300 are commercially important and used for the flavours and fragrances

market (Burt, 2004). Essential oils have been documented to be effective antimicrobials against

several foodborne pathogens including Escherichia coli O157:H7, Salmonella Typhimurium,

Staphylococcus aureus, Listeria monocytogenes, Campylobacter and others (Friedman et al.,

2002; Moreira et al., 2005; Callaway et al., 2008; Nannapaneni et al., 2008; O’Bryan et al.,

2008a; Friedly et al., 2009; Nannapaneni et al., 2009a; 2009b; Soković et al., 2010; Pittman et

al., 2011b; Shannon et al., 2011a; 2011b; Callaway et al., 2011b; Callaway et al., 2011c;

Muthaiyan et al., 2012; Pendleton et al., 2012).

28

5.2. Mode of Action of Essential Oils

The antimicrobial effects of EOs have been constantly screened in a wide range of

microorganisms, but their mechanism(s) of action are still not completely understood. Several

mechanisms have been proposed to explain the chemical compounds contained in the EOs (Cox

et al., 2000; Burt, 2004). Because EOs contain several components, their antimicrobial activity

cannot be confirmed based on the action of only one compound (Bajpai et al., 2012). Several

researchers have proposed that the antimicrobial action of EOs may be attributed to their ability

to penetrate through bacterial membranes to the interior of the cell and exhibit inhibitory activity

on the functional properties of the cell, and to their lipophilic properties (Smith-Palmer et al.,

1998; Fisher and Phillips, 2009; Guinoiseau et al., 2010; Bajpai et al., 2012). The phenolic nature

of EOs also elicits an antimicrobial response against foodborne pathogen bacteria (Shapira and

Mimran, 2007; Bajpai et al., 2012). Phenolic compounds disrupt the cell membrane resulting in

the inhibition of the functional properties of the cell, and eventually cause leakage of the internal

contents materials of the cell (Bajpai et al., 2012). The mechanisms of action may be the ability

of phenolic compounds to alter microbial cell permeability, damage cytoplasmic membranes,

interfere with cellular energy (ATP) generation system, and disrupt the proton motive force

(Burt, 2004; Friedly et al., 2009; Li et al., 2011; Bajpai et al., 2012). The disrupted permeability

of the cytoplasmic membrane can result in cell death (Li et al., 2011). An important

characteristic of EOs and their components is hydrophobicity, allowing the EOs to separate the

lipids of the bacterial cell membrane and mitochondria and in the process causing the bacterial

cell to become more permeable (Burt, 2004; Friedly et al., 2009).

The interaction of EOs with microbial cell membranes results in the growth inhibition of

some Gram-positive and Gram-negative bacteria (Calsamiglia et al., 2007). Gram-positive

29

bacteria such as Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus are more

susceptible to EOs than Gram-negative bacteria such as E. coli and S. Enteritidis

(Chorianopoulos et al., 2004). It is generally believed that EOs mechanistically should be more

effective against Gram-positive bacteria due to the direct interaction of the cell membrane with

hydrophobic components of the EOs (Shelef, 1983; Smith-Palmer et al., 1998; Chao and Young,

2000; Cimanga et al., 2002; Soković et al., 2010). Conversely, based on this premise, Gram-

negative cells should be more resistant to plant oil because they possess a hydrophilic cell wall

(Kim et al., 2011). This outer layer helps to prevent the penetration of hydrophobic compounds

(Calsamiglia et al., 2007; Ravichandran et al., 2011). Deans and Ritchie (1987) concluded that

Gram-positive and Gram-negative bacteria were equally sensitive to citrus EOs and their

components.

In a study by Dorman and Deans (2000) carvacrol and thymol acted differently against

Gram-positive and Gram-negative bacteria. Carvacrol and thymol were able to cause

disintegration of the outer membrane of Gram-negative bacteria, releasing lipopolysaccharides

(LPS) and increasing the permeability of the cytoplasmic membrane to ATP (Burt, 2004).

Previous studies by Ravishankar et al., (2010) suggested that the various S. enterica isolates may

not be equally susceptible to inactivation, and that the antimicrobials may act by similar

mechanisms against resistant and susceptible S. enterica isolates. In their research, Ravishankar

et al., (2010) stated that the use of cinnamaldehyde did not cause the bacterial outer membrane to

disintegrate and suggested that instead, cinnamaldehyde may interfere with the activity of some

enzymes. When comparing inactivation susceptibility of the bacteria because of the EOs,

Ravishankar et al., (2010) found that antibiotic-resistant isolates exhibited similar susceptibility

to inactivation as the susceptible isolates.

30

Factors present in complex food matrices such as fat content, proteins, water activity, pH,

and enzymes can potentially diminish the efficacy of EOs (Burt, 2004; Firouzi et al., 2007;

Friedly et al., 2009). According to Vigil (2001) low pH can increase the solubility and stability

of EOs, enhancing the antimicrobial activity. Additional methods to enhance EOs activity

include increasing salt content, and decreasing storage temperatures (Burt, 2004; Friedly et al.,

2009). Sivropoulou et al. (1996) reported that the antimicrobial effect of EOs is also

concentration dependent. Ravishankar et al., (2010) observed that at a 0.2% concentration,

antimicrobials, carvacrol and cinnamaldehyde completely inactivated the antibiotic-resistant and

-susceptible isolates of S. enterica. The antibacterial effects of organic acids increase as the

concentration increases (Dickson and Anderson, 1992). Organic acids, chlorine dioxide, and

trisodium phosphate are classified as safe (GRAS) and effective sanitizers (Cutter, 2000).

Besides being used as food additives and preservatives, organic acids and their salts also have

food processing applications (Ricke, 2003a; Ricke et al., 2005; Milillo et al., 2011). Organic

acids have several different antimicrobial mechanisms, such as disruption of intracellular pH,

osmotic stress, and membrane perturbation (Russell, 1992; Hirshfield et al., 2003; Ricke, 2003a).

Friedly et al., (2009) observed significant synergistic antimicrobial properties against Listeria by

combining citrus EOs with four different organic acids, citric, malic, ascorbic and tartaric acid,

making citrus EOs a more attractive antimicrobial control measure. Their study revealed that low

concentrations of citrus EOs in combination with organic acids could be effective to control

Gram-positive microbial growth, in this case Listeria. By combining organic acids and EOs in

Listeria, Friedly et al., (2009) concluded that their findings would allow for a reduction of the

concentration of EOs by more than 10-fold. Zhou et al., (2007b) reported that the combination of

different antimicrobials such as thymol or carvacrol with EDTA, thymol or carvacrol with acetic

31

acid, and thymol or carvacrol with citric acid all resulted in significantly reduced populations of

S. Typhimurium. In samples treated with combinations, these antimicrobials exhibited

synergistic effects compared with samples treated with thymol, carvacrol, EDTA, acetic acid, or

citric acid alone (Zhou et al., 2007b).

The production of off-flavor or strong odor limits the use of EOs as food preservatives to

increase the safety and shelf life of food products (Friedly et al., 2009; Tiwari et al., 2009;

Soković et al., 2010; Bajpai et al., 2012; Solórzano-Santos and Miranda-Novales, 2012). As a

result of this, an inhibitory dose (minimum concentration at which no bacterial growth will be

observed) instead of a bactericidal dose (which will kill the bacteria) is usually applied

(Chorianopoulos et al., 2006, Li et al., 2011). McMahon et al., (2007) reported that the increased

use of bacteriostatic (sublethal), rather that bactericidal (lethal), food preservation systems may

contribute to the development and dissemination of antibiotic resistance in food related

pathogens. High doses of EOs (0.05% v/v) have been shown to have a cytotoxic effect on Caco-2

cells and actually increase the damage to the tissue cell population caused by E. coli, although

medium to low doses (≤ 0.01%) of thyme, oregano and carvacrol over a short period cause no

detectable damage, which emphasizes the necessity to find a balance between the risk of food

poisoning and/or food spoilage and the required dose of EOs to have an effect (Fisher and

Phillips, 2008). In their research, Firouzi et al., (2007) reported that although in vitro work with

EOs and their components indicated that compounds such as oregano and nutmeg possessed

substantial antimicrobial activity, when used in food systems the amounts required were

approximately 1 to 3% higher, often higher than what would normally be organoleptically

acceptable (Firouzi et al., 2007).

32

Tassou and Nychas (1996) found that it required 10 times as much mastic gum to achieve

the same results in pork sausages as compared to in vitro. Mastic gum is a white,

semitransparent, natural resin obtained as a trunk exudate from mastic trees Pistacia lentiscus

(Koutsoudaki et al., 2005). Mastic gum possesses numerous qualities, such as antimicrobial,

antioxidant, anti-inflammatory, anticancer; and used as a health promoter, used in cosmetic

industry, culinary, toothpaste and lotions, among others (Koutsoudaki et al., 2005; Giaginis and

Theocharis, 2011; Dimas et al., 2012). The in vitro antimicrobial activity of P. lentiscus extracts

has also been tested on bacteria and fungi (Koutsoudaki et al., 2005). Huwez et al., (1998)

reported that 1 mg of mastic gum per day could cure peptic ulcers and that mastic gum could kill

Helicobacter pylori, which was in turn correlated with their effect on peptic ulcers. Al-Said et

al., (1986) reported that an oral dose of 500 mg/kg significantly reduced gastric secretions,

protected cells and the intensity of gastric mucosal damage. Mastic gum can also act as an oral

antiseptic, tighten gums and prevent or reduce plaque formation (Topitsoglou-Themeli et al.,

1984). Moreover, mastic gum has also been applied for culinary uses, for example, in biscuits,

ice cream, and mastic “sweets of the spoon” (Koutsoudaki et al., 2005). Smith-Palmer et al.

(1998) found that foods with higher lipid content required more EOs to inhibit bacterial growth.

Further studies will be needed to confirm the effects of these orange EOs in vivo as well as

incorporating them into multiple-hurdle approaches to improve food safety for meat products

(Ricke et al., 2005; Ganesh et al., 2010; Sirsat et al., 2011).

5.3. Essential Oils as a Pre-harvest Intervention

The use of antimicrobials as a pre-harvest intervention, before crops or livestock products

are sold, is an important part of animal production and food safety. These interventions have

33

been examined as feed additives to improve feed characteristics, increase digestion and

performance if the animal is being fed these compounds, or to enhance desirable characteristics

of the meat produced (Jacob and Pescatore, 2012). Alali et al., (2013) determined the effect of

non-pharmaceuticals (a blend of organic acids, a blend of EOs, lactic acids, and a combination of

levulinic acid and sodium dodecyl sulfate) on weight gain, feed conversion ratio, and mortality

of broilers and their ability to reduce colonization and fecal shedding of Salmonella Heidelberg

following S. Heidelberg challenge and feed withdrawal. Their results showed that the broilers

that received the EOs had significantly increased weight gain and mortality was lower compared

to the other treatments. Salmonella Heidelberg contamination in crops was significantly lower in

challenged and unchallenged broilers that received EOs and lactic acids in drinking water, than

any of the other treatments (Alali et al., 2013).

Although orange peel and pulp feeding will not prevent all human foodborne illnesses,

including citrus oils and products in the diet can be utilized as a part of a multiple-hurdle system

with the objective of reducing the passage of foodborne pathogens from farm to human

consumers (Callaway et al., 2011b). In their research, Callaway et al., (2011b) showed that

including orange peel and pulp as dietary components reduced populations of Salmonella

Typhimurium in the gut of experimentally inoculated sheep.

5.4. Essential Oils as a Post-harvest Intervention

The antimicrobial activity of several EOs against foodborne pathogens have been studied

in the laboratory. Orange and lemon oil are known to have in vitro antibacterial effects on

Salmonella and other foodborne microorganisms (Subba et al., 1967). Components of citrus EOs

such as carvacrol, citral, and geraniol exhibit antimicrobial activity against S. Typhimurium and

34

its rifampicin resistant strain (Kim et al., 1995). In this research they inoculated S. Typhimurium

onto fish cubes and reported that carvacrol at 3% w/v completely killed the inoculated bacteria.

Pittman et al., (2011a) studied the use of naturally occurring compounds citrus EOs

extracted from orange peel to reduce or kill Salmonella spp. and E. coli. They found that 3%

cold-pressed terpeneless Valencia orange oil could be used as an additional intervention against

these two foodborne pathogens at refrigerated storage stages of processing. Dabbah et al. (1970)

assessed the antibacterial properties of orange, lemon, grapefruit, and mandarine citrus oils and

their derivatives, lime terpeneless oil, orange terpeneless oil, lemon terpeneless oil, d-limonene,

terpineol, and geraniol in vitro against S. Seftenberg, E. coli, Staphylococcus aureus, and

Pseudomonas spp. In their findings they observed the fractions of citrus oil were more active

against all bacteria than the composite oils themselves. The citrus oils used in this research did

not reduce the populations of S. Seftenberg, but lime terpeneless, terpineol, and geraniol reduced

corresponding populations by 100%.

Some studies have reported that it would require a much higher concentration of EOs in

actual food systems to inhibit bacterial growth (O’Bryan et al., 2008a). In their research,

O’Bryan et al., (2008a) reported that differences were observed between serotypes of Salmonella

in response to the antibacterial activity of various fraction orange EOs, with S. Enteritidis having

a minimal inhibitory concentration of 0.13% v/v for terpenes from orange juice essence as

compared to 0.5% v/v for S. Typhimurium. Ravishankar et al., (2010) reported that S. Newport

was found to be resistant to six of the seven EOs tested, exhibiting different susceptibilities to

carvacrol and cinnamaldehyde than the other strains demonstrated. Carvacrol and

cinnamaldehyde antimicrobial activity against antibiotic-resistant and antibiotic-susceptible

Salmonella strains on PBS, and S. Newport on celery and oysters were tested. Results from

35

inoculation of S. enterica in PBS buffer showed that at low concentrations (0.1 to 0.2% v/v)

carvacrol exhibited stronger antimicrobial activity than cinnamaldehyde (Ravishankar et al.,

2010). Carvacrol also showed to have greater effectiveness against antibiotic-resistant

Salmonella Newport on celery; while carvacrol and cinnamaldehyde were equally effective

against antibiotic-resistant S. Newport on oysters (Ravishankar et al., 2010).

6. Hurdle Technology

The hurdle technology developed by Leistner and Gorris (1995) sought to prevent the

survival and regrowth of pathogens in food by using a series of combinations of preservation

techniques. A hurdle intervention is any treatment, physical, chemical or biological that is used

to reduce or eliminate the presence of pathogens on a food surface (Leistner, 1985; Leistner and

Gorris, 1995).

When used in combination of more than one component, EOs could work differently than

when they are used as independent compounds. Combinations of more than one antimicrobial

intervention treatment in lower doses have been often found to increase bactericidal activity

more than any single treatment, working synergistically (Leistner and Gorris 1995; Leistner,

2000; Ricke et al., 2005; Tiwari et al., 2009; Li et al., 2011). Zhou et al., (2007a) investigated the

antimicrobial activity of cinnamaldehyde, thymol and carvacrol alone or in combinations against

S. Typhimurium, and reported the lowest concentrations of cinnamaldehyde, thymol and

carvacrol inhibiting the growth of S. Typhimurium significantly were 200, 400 and 400 mg/L,

respectively. Results from the combinations, cinnamaldehyde/thymol, cinnamaldehyde/carvacrol

and thymol/carvacrol, demonstrated that the concentration of cinnamaldehyde, thymol and

36

carvacrol could be decreased from 200, 400 and 400 mg/L to 100, 100 and 100 mg/L,

respectively (Zhou et al., 2007a).

Barnhart et al., (1999) studied the potential to eliminate Salmonella in the presence of

organic matter and reported a synergistic combination of d-limonene (DL) and citric acid (CA).

Individually combinations of less than 10% DL or less than 2% CA did not reduce Salmonella

recovery, but when used in combination, 2% CA with 0.5% DL, completely eliminated

detectable Salmonella (Barnhart et al., 1999). Their observations suggested that, in nature, the

unknown anti-Salmonella action of DL/CA in combination might be bactericidal, rather than

bacteriostatic.

The application of more than one hurdle treatment at a time is advantageous because it

may decrease the requirement for a higher dose of the specific hurdle (Leistner, 2000). Multiple

hurdle technologies could be more exploited in order to control contaminant bacteria. Instead of

using high doses of a single antimicrobial compound, combinations of more than one

antimicrobial at lower concentrations will effectively inhibit the contaminants to the same

degree, and due to synergism, result in minimal development of antimicrobial resistance (Sirsat

et al., 2009; Ricke, 2010; Milillo et al., 2011; Muthaiyan et al., 2011).

7. Conclusions

Through the years, the pharmaceutical industry has heavily invested in the research and

discovery of natural products with the end goal of producing therapeutic antibiotic formulations.

Today more than two-thirds of the antibiotics in clinical use are natural products or a result of

their semisynthetic derivatives (Benowitz et al., 2010). The constant problem of increasing drug

resistance has created a more urgent demand to develop new and improved antimicrobials

37

against resistant microorganisms (Spellberg et al., 2004; Berghman et al., 2005). Unfortunately

at the moment the status of potential novel antibacterial drugs indicates that there are only a few

compounds in development by the large pharmaceutical companies (Overybye et al., 2005).

There are approximately 10,000 plants in the world that have documented medicinal use.

In western medicine it is reported that only around 150 to 200 of such agents are in use

(McChesney et al., 2007). Understanding the mechanisms involved in bacterial response, could

facilitate the refining and optimization of antimicrobial formulations or find new ways to

overcome potential tolerance mechanisms, leading to an improvement in biosecurity measures

and to enhance the protection of farm-animal and as a result, public health (Condell et al.,

2012a).

Foodborne illnesses continue to be one of the primary public health concerns in the U. S.

Salmonella is one of the major foodborne pathogens responsible for foodborne illnesses. The

potential use of EOs as natural antimicrobial agents is less exploited than their uses as flavoring

and antioxidant compounds (Chorianopoulos et al., 2006). Previous research confirmed that EOs

from citrus could be used as effective antimicrobial agents against Salmonella (O’Bryan et al.,

2008a). For an optimally targeted use of EOs and organic acids against Salmonella, more

research is needed for a better understanding of the synergestic antimicrobial mechanisms and

effects when natural antimicrobials, such as citrus EOs are used in combinations with multiple

hurdle technologies.

As we previously discussed, one of the previous concerns about Salmonella is its

antimicrobial resistance. In this research we studied the effectiveness of five essential oils against

Salmonella Heidelberg, the primary focus for our research. Understanding the growth of

Salmonella serovars and strains serves as an important basis for our research. In this case we

38

studied a) the aerobic and anaerobic growth responses of multiple strains from six different

serovars, b) how the spent media from different serovars, more importantly S. Heidelberg, affect

the growth of S. Typhimurium, and c) determined whether or not two different serovars undergo

competitive exclusion when are grown together. The growth rates of four Salmonella serovars,

Heidelberg, Kentucky, Typhimurium, and Enteritidis in enrichment and non-enrichment media

were compared to determine the effectiveness of a non-enrichment media, such as Luria Bertani

broth over enrichment methods. Given that Salmonella enterica are important facultative

intracellular pathogens that are widely known to be isolated from poultry and to cause infections

in humans, understanding the ability of this pathogen to attach and invade different cell types is

of great importance. We studied the adhesion and invasion of four different strains to chicken

macrophage (HD11), and human epithelial (Caco-2) cells.

39

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Table 1.1. The 10 most frequently reported Salmonella serovars from human infections reported to CDC, 1998-2011. (CDC, 2011)

Rank 1998 2001 2006 2011

1 Typhimurium Typhimurium Typhimurium Enteritidis

2 Enteritidis Enteritidis Enteritidis Typhimurium

3 Newport Newport Newport Newport

4 Heidelberg Heidelberg Heidelberg Javiana

5 Javiana Javiana Javiana I 4,[5],12:1:-

6 Agona Montevideo I 4,[5],12:1:- Montevideo

7 Montevideo Oranienburg Montevideo Heidelberg

8 Oranienburg Muenchen Muenchen Muenchen

9 Muenchen Thompson Oranienburg Infantis

10 Infantis Saintpaul Mississippi Braenderup

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Table 1.2. The 10 most frequent serovars from analyzed PR/HACCP verification samples for young chicken (broilers), 1998–2011.

(USDA/FSIS, 2013b).

* Typhimuriu

Rank 1998 2001 2006 2011

1 Kentucky Kentucky Kentucky Kentucky

2 Heidelberg Heidelberg Enteritidis Enteritidis

3 Typhim* var. Copenhagen Typhimurium Heidelberg Typhim* var. 5-

4 Typhimurium Typhim* var. Copenhagen 4,[5],12:I:- Infantis

5 Hadar Montevideo Typhim* var. Copenhagen Heidelberg

6 Schwarzengrund Schwarzengrund Typhimurium Typhimurium

7 Montevideo Hadar Montevideo Johannesburg

8 Enteritidis Thompson Schwarzengrund 4,[5],12:1:-

9 Thompson Enteritidis Infantis 8,20:-:z6

10 Infantis Berta Mbandaka Mbandaka

66

9. Authorship Statement for Chapter 1

Juliany Rivera Calo is the first author of the paper and completed at least 51% of the studies

among coauthors, which the title is “Salmonella spp. – Literature Review ” in chapter 1.

Major Advisor: Dr. Steven C. Ricke

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CHAPTER 2

Growth Responses, Spent Media, and Competitive Interactions among Salmonella Serovars


Ok Kyung Koo3 and Steven C. Ricke

1,2



Fayetteville, AR

3Food Safety Research Group, Korea Food Research Institute, Seongnam-si, Gyeonggi-do,

Republic of Korea

68

1. Abstract

Salmonella enterica is one of the most prevalent pathogens responsible for foodborne

illness worldwide. Many of its serovars have been associated with the consumption of poultry

products, these being one of the main reservoirs of Salmonella. The emergence and prevalence of

Salmonella serovars in chickens has been studied but the predominant serovars have varied

somewhat over the years. The purpose of this research was to a) understand the aerobic and

anaerobic growth responses of predominant Salmonella serovars b) study how spent media from

different serovars affect the growth of S. Typhimurium, and c) determine if there is serovar

interactions as a result of growing two Salmonella strains from independent serovars on the same

culture. Results showed that among all six serovars, the growth under aerobic conditions was

comparable; while under anaerobic conditions the growth in LB broth was slower. For spent

media, results were similar for most strains, except from S. Heidelberg ARI-14, where a decrease

in the growth of S. Typhimurium ATCC 14028 was observed. Competitive growth studies were

conducted to determine if there was any inhibition from one serovar against the other. These

studies will provide a better understanding of the differences in growth among Salmonella

serovars versus S. Typhimurium in chickens.

69

2. Introduction


illness worldwide. Annually, an estimated 1,027,561 Americans contract Salmonella (Scallan, et

al., 2011). Yearly costs for Salmonella control efforts are estimated to be up to $14.6 billion

(Scharff, 2010; Heithoff, et al., 2012). Salmonella can multiply under various environmental

conditions outside their respective living host (Park et al., 2008; Pui et al., 2011). Most

Salmonella serovars grow at a temperature range of 5 to 47°C, with an optimum temperature of

35 to 37°C, although some can grow at temperatures as low as 2 to 4°C and as high as 54°C

(Gray and Fedorka-Cray, 2002).

More than 99% of Salmonella strains causing human infections belong to Salmonella

enterica. These are mainly isolated from warm-blooded animals and account for more than 99%

of clinical isolates (Pui, et al., 2011). Many of its serovars have been associated with the

consumption of poultry products, these being one of the main reservoirs of Salmonella (CDC,

2009). The emergence and prevalence of Salmonella serovars in chickens has been studied but

the predominant serovars have varied somewhat over the years. Salmonella enterica serovars

Typhimurium, Enteritidis, and Heidelberg cause gastroenteritis and these are the most common

serovars responsible for human infections (Olsen et al., 2001; Dunkley et al., 2009; CDC, 2011;

Foley et al., 2008; 2011; 2013; Finstad et al., 2012; Howard et al., 2012). These three serovars

along with S. Kentucky are the most common serovars found in poultry.

Salmonella Heidelberg foodborne illnesses have been found to be associated with several

foods such as chicken (Snoeyenbos et al., 1969; Mahony et al., 1990; Layton et al., 1997;

Hennessy et al., 2004), pork (O’Mahoney et al., 1983; Di Guardo et al., 1992), as well as eggs

and eggshells (Jones et al., 1995; Schonei et al., 1995). Salmonella Enteritidis cases have often

70

been associated with consumption of contaminated eggs (Guard-Petter, 2001; Cogan et al., 2004;

Howard et al., 2012). Salmonella Typhimurium cases are usually linked to the consumption of

contaminated poultry, swine and bovine meat (Jorgensen et al., 2000). S. Typhimurium is an

invasive enteric pathogen that is remarkably adaptable to diverse hosts including humans,

poultry, rodents, cattle, sheep and horses (Luo et al., 2012).

Spent media is defined as a bacterial growth medium that has been exhausted of nutrients

due to extensive growth of the microorganism and is then filter sterilized (Nutt et al., 2002). One

of the benefits from spent media is that it has the potential to serve as an in vitro screen of

ecosystems after growth of the microorganism (Nutt, et al., 2002). Competitive interaction

phenomena have been studied with homologous as well as heterologous bacteria, and we

hypothesize whether competition exists between Salmonella serovars. Therefore, the objectives

of this research were to a) understand the aerobic and anaerobic growth responses of several

Salmonella serovars, b) study how spent media from different serovars affect the growth of S.

Typhimurium, and c) determine if there is serovar interaction as a result of growing two

Salmonella strains from independent serovars on the same culture. These studies will provide a

better understanding of the effect of spent media from multiple Salmonella serovars and the

competitive growth between different Salmonella serovars versus S. Typhimurium in chickens.

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3. Materials and Methods

3.1. Salmonella spp. growth responses after aerobic and anaerobic incubation from poultry

Salmonella isolates.

3.1.1. Bacterial strains

A total of 21 strains from six of the most predominant Salmonella serovars, S.

Typhimurium, S. Kentucky, S. Heidelberg, S. Newport, S. Enteritidis and S. Montevideo isolated

from poultry were studied and are listed in Table 2.1. Growth responses were generated under

aerobic and anaerobic conditions.

3.1.2. Aerobic and anaerobic growth responses

One colony of each strain was inoculated in 5 mL of Luria Bertani (LB) broth and

incubated for 16 h at 37°C, 190 rpm shaking using a C76 Water Bath Shaker (New Brunswick

Scientific, Edison, NJ, USA). Using a Nunclon 96-well plate, 200 μL of fresh LB broth and 2 μL

(1%) of each Salmonella overnight culture were added to each well. Optical density (OD) was

measured at 600 nm every hour over 24 h using a TECAN Infinite M200 (Tecan Systems Inc.,

San Jose, CA, USA). For the anaerobic growth response, the 96-well plate was stored in an

anaerobic box (BD Biosciences, Franklin Lakes, NJ, USA), argon gas was used to provide

anaerobic conditions, and incubated at 37°C. The OD values were measured at 600 nm at 0, 1, 2,

4, 8, 12, 16 and 24 h using the TECAN Infinite M200.

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3.2. Effect of Salmonella spent media on the growth of S. Typhimurium

3.2.1. Aerobic and anaerobic spent media growth responses

One colony from 11 Salmonella strains from six different serovars was inoculated into 16

mL of LB broth and incubated for 16 h at 37°C. Tubes were centrifuged at 7000 rpm for 10 min

using the Centrifuge 5804 R (Eppendorf, Westbury, NY, USA), and supernatant was filter

sterilized using 0.2 μm sterile syringe filters (VWR, Radnor, PA, USA) in 15 mL tubes. Eight

milliliters of each spent media were used to generate growth curves; the other 8 mL were saved

for further analysis. One percent (80 μL) of S. Typhimurium ATCC 14028 culture was

inoculated into each spent media sample. Optical density (OD) at 600 nm was measured at 0, 1,

2, 3, 4, 5, 6, 7, 8, 12, 14 and 24 h using a Spectronic 20D+ Spectrophotometer (Thermo

Scientific, Rockford, IL, USA). For the anaerobic conditions, the tubes were stored in an

anaerobic jar (BD Biosciences, Franklin Lakes, NJ, USA) and GasPak (BD Biosciences) were

used to provide anaerobic conditions. Optical density at 600 nm was measured at 0, 24, 32, 48,

60, 72, 84 and 96 h using Spectronic 20D+ Spectrophotometer (Thermo Scientific).

3.2.2. Salmonella Heidelberg and S. Typhimurium spent media

Salmonella Heidelberg and S. Typhimurium were selected to conduct further spent media

studies. One colony of S. Heidelberg ARI-14 and S. Typhimurium ATCC 14028 was inoculated,

per duplicate, into 8 mL of LB broth and incubated for 16 h at 37°C. One tube from each strain

was centrifuged at 7000 rpm for 10 min and supernatant was filter sterilized using 0.2 μm sterile

syringe filter (VWR). One percent (80 μL) of S. Typhimurium ATCC 14028 culture was

inoculated into S. Typhimurium and S. Heidelberg spent media and incubated for 24 h at 37°C.

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Optical density (OD) at 600 nm was measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 14 and 24 h using a

Spectronic 20D+ Spectrophotometer (Thermo Scientific).

3.2.3. Salmonella Heidelberg spent media

Based on the results from growing S. Typhimurium on multiple Salmonella spent media

from different serovars, several S. Heidelberg strains were selected for spent media growth

curves with S. Typhimurium ATCC 14028. One colony of 15 different S. Heidelberg strains and

one colony of S. Typhimurium ATCC 14028 was inoculated into 8 mL of LB broth and

incubated for 16 h at 37°C. All S. Heidelberg cultures were centrifuged at 7000 rpm for 10 min

and supernatant was filter sterilized using 0.2 μm sterile syringe filter (VWR). One percent (80

μL) of S. Typhimurium ATCC 14028 culture was inoculated into all 15 S. Heidelberg spent

media and incubated for 24 h at 37°C, 190 rpm shaking using a C76 Water Bath Shaker (New

Brunswick Scientific). Optical density (OD) at 600 nm was measured at 0, 2, 4, 6, 7:45, 8, 12

and 24 h using Spectronic 20D+ Spectrophotometer (Thermo Scientific).

3.2.4. Salmonella Heidelberg spent media comparison between two different media and an

alternate temperature

One colony of S. Heidelberg ARI-14 and S. Typhimurium ATCC 14028 was inoculated

into 8 mL of LB broth and 8 mL of BPW and incubated for 16 h at 37°C, 190 rpm shaking using

a C76 Water Bath Shaker (New Brunswick Scientific). S. Heidelberg overnight cultures were

centrifuged at 7000 rpm for 10 min and supernatant was filter sterilized using 0.2 μm sterile

syringe filter (VWR). One percent (80 μL) of S. Typhimurium ATCC 14028 culture was

inoculated into both S. Heidelberg spent media and incubated for 24 h at 42°C, 190 rpm shaking

74

using a C76 Water Bath Shaker (New Brunswick Scientific). The same was repeated on BPW

and incubated at 37°C. Optical density (OD) at 600 nm was measured at 0, 2, 4, 6, 7, 8, 8:30; 12

and 24 h using Spectronic 20D+ Spectrophotometer (Thermo Scientific).

3.3. Competitive growth between S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14.

3.3.1. Growth response from mixed cultures

One colony of S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14 respectively,

were inoculated into 8 mL of LB broth, individually, and incubated for 16 h at 37°C, 190 rpm

shaking using a C76 Water Bath Shaker (New Brunswick Scientific). From each strain, 500 μL

(1% total) were inoculated into 100 mL of LB broth, individually, and as a mix culture, and

incubated at 37°C for 24 h, 190 rpm shaking. Optical density values at 600 nm were measured at

0, 2, 4, 6, 8, 12, 14 and 24 h. At each time point, 1 mL of culture was collected for genomic

DNA extraction and qPCR; and 1 mL collected for serial dilutions and plating on LB agar plates

to determine colony forming units per mL (CFU/mL).

3.3.2. DNA extraction

Samples were centrifuged at 14,000 x g for 10 min and supernatant was discarded.

Genomic DNA was extracted using the Qiagen DNeasy Blood Tissue kit (Qiagen, Valencia, CA,

USA) according to the manufacturer’s instruction. The isolated genomic DNA concentration and

purity were measured using a NanoDrop ND-1000 (Thermo Scientific) and DNA samples were

stored at -20°C until used.

75

3.3.3. Bacterial quantification by real time quantitative PCR (qPCR)

Quantification of bacteria was performed using qPCR and strain-specific primers,

STM4497M2-f and STM4497M2-r (310 bp) for S. Typhimurium; and SH_SHP-2f and SH_SHP-

1r (180 bp) for S. Heidelberg (Table 2.5). The qPCR was conducted using an Eppendorf

Masterplex thermocycler ep (Eppendorf, Westbury, NY, USA). Real Time PCR assays were

conducted as three independent experiments and triplicate samples in each experiment. A 20 μL

total reaction volume composed of 1 μL of template DNA, 1 μL of each primer (IDT, Coralville,

IA, USA), 10 μL of SYBR Green SYBR Green Premix Ex Taq (Takara, Clontech, Mountain

View, CA, USA), and 7 μL of DNase-RNase free water. The PCR conditions consisted of pre-

denaturation at 94°C for 5 min, then 35 cycles of denaturation at 94°C for 30 s, annealing at

65°C for 30 s, extension at 72°C for 30 s, with fluorescence being measured during the extension

phase. Because SYBR green was being used, a subsequent melting curve was required and

consisted of 94°C for 30 s, 65°C for 20 min increasing in 0.5°C increments to 95°C.

3.4. Competitive growth between Salmonella Heidelberg ARI-14 and Salmonella Enteritdis

ATCC 13076.

3.4.1. Growth responses

One colony of S. Heidelberg ARI-14 and S. Enteritidis ATCC 13076 was inoculated in 8

mL of LB broth, individually, and incubated for 16 h at 37°C, 190 rpm shaking using a C76

Water Bath Shaker (New Brunswick Scientific). From each strain, 500 μL were inoculated

individually into 100 mL of LB broth, and 500 μL of each strain into 100 mL LB broth as a mix

culture; flasks were incubated at 37°C for 24 h, 190 rpm shaking. Optical density values at 600

76

nm were measured at 0, 2, 4, 6, 8, 12 and 24 h. At each time point, 1 mL collected for serial

dilutions and plating on LB agar plates to determine CFU/mL.

3.5. Statistical analysis

All experiments were repeated at least three times. Statistical comparisons were carried

out using the Student t-test comparison of means at P < 0.05 to show significant differences. All

statistical analyses were conducted using JMP Pro Software Version 11.0 (SAS Institute Inc.,

Cary, NC).

4. Results

4.1. Salmonella spp. growth responses after aerobic and anaerobic incubation from poultry

Salmonella isolates.

Among all six serovars, the growth under aerobic conditions was comparable, while

under anaerobic conditions the growth in LB broth was slower (Figures 2.1 to 2.6). Optical

density values at different time points are listed on tables 2.2 (aerobic) and 2.3 (anaerobic). In

general, under aerobic conditions the fastest growing serovars were S. Montevideo followed by

S. Kentucky; while under anaerobic conditions the fastest growing was S. Heidelberg followed

by S. Montevideo. Under aerobic conditions, the range in optical density (600 nm) at 24 h for S.

Typhimurium was between 0.913 to 0.964, for S. Heidelberg 0.732 to 0.864, 0.831 to 1.003 for

S. Kentucky, S. Newport 0.770 to 0.968, S. Enteritidis 0.564 and 0.869, and S. Montevideo

between 0.915 and 0.994. Compared to the anaerobic conditions the ranges in OD values at 600

nm were different. S. Typhimurium was between 0.807 and 0.833, S. Heidelberg 0.677 and

0.928, S. Kentucky 0.719 and 0.904, S. Newport 0.751 and 0.919, S. Enteritidis 0.483 and 0.747

77

and S. Montevideo ranged between 0.761 and 0.871. Among all serovars, strain differences were

observed based on the optical density. Based on these results, 11 strains from the six different

serovars were selected for further studies using spent media from these strains.

4.2. Effect of Salmonella spent media on the growth of S. Typhimurium

4.2.1. Aerobic and anaerobic spent media growth responses

In general, under anaerobic conditions, after 96 h no growth was observed (Figure 2.8).

As a control on this experiment we included S. Typhimurium ATCC 14028 growing on LB broth

and growth under anaerobic conditions was observed. Under aerobic conditions the growth of S.

Typhimurium ATCC 14028 in different Salmonella strains spent media was slower (Figure 2.7).

When grown in LB, these strains reach exponential phase faster. Results were similar for most of

the spent media from different strains, except from S. Heidelberg ARI-14. A decrease in the

growth of S. Typhimurium ATCC 14028 was observed between 6 to 12 h after inoculating it on

S. Heidelberg ARI-14 spent media. Based on this effect we selected these two strains to conduct

more experiments using different combinations of spent media and inoculum to confirm and

better describe this effect.

4.2.2. Salmonella Typhimurium growth in S. Heidelberg spent media

In order to confirm the phenomena observed when S. Typhimurium ATCC 14028 was

grown on S. Heidelberg ARI-14 spent media, we repeated the experiment inoculating S.

Typhimurium in S. Typhimurium and S. Heidelberg spent media (Figure 2.9). As controls, we

grew (1) S. Typhimurium in LB broth and (2) both S. Typhimurium and S. Heidelberg in spent

media. Results showed that only when S. Typhimurium is grown on S. Heidelberg spent media a

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decrease in growth between 6 to 12 h is observed. No growth was observed on S. Typhimurium

and S. Heidelberg spent media used as a control.

To further confirm the previous results and determine if the effect being observed was

specific to S. Heidelberg ARI-14 spent media, we screened 14 additional S. Heidelberg strains

from multiple sources (Table 2.4). Results showed that the growth of S. Typhimurium on 12 of

the 15 S. Heidelberg strains spent media decreased at different points between hours 6 and 12

after inoculation (Figure 2.10). The three strains that did not show this effect are from different

sources, two from turkey (163 and 824) and one from cattle (114). This suggests there are strain

differences. We also inoculated S. Heidelberg strains on S. Heidelberg spent media (Figure 2.11)

to further confirm the effect is observed specifically for S. Typhimurium on S. Heidelberg spent

media. When inoculating the strains on BPW prior to spent media, the drop on the growth of S.

Typhimurium was observed.

The same experiment was conducted at 37°C and 42°C, and using two different media,

LB broth and BPW. Growth of S. Typhimurium was faster on LB broth than BPW, but there

appeared to be no effect caused by the spent media when incubated at 42°C (Figure 2.12). When

incubated at 37°C, the drop in the growth of S. Typhimurium was observed for both media used

(Figure 2.13). These results suggested that the previous effect caused by spent media could be

temperature dependent and media independent. Further studies are needed to have a better

understanding of the growth differences at different temperatures.

79

4.3. Competitive growth between (1) S. Typhimurium and S. Heidelberg, and (2) S.

Heidelberg and S. Enteritidis

The above spent media growth studies showed an interesting phenomenon when growing

S. Typhimurium ATCC 14028 on S. Heidelberg spent media. Competitive growth studies were

performed where the two Salmonella strains were grown together as a mixed culture to observe

any serovar interaction (Figure 2.14). Quantitative PCR was used to determine the cell number of

each strain at specific time points over 24 h. Growth responses showed similar initial

concentrations for the mixed culture containing S. Typhimurium ATCC 14028 and S. Heidelberg

ARI-14, of approximately 6 log CFU/mL (Figure 2.16). Salmonella Heidelberg appeared to

grow at a faster rate than S. Typhimurium. Positive controls for both strains showed an initial

cell concentration of 7 log CFU/mL for S. Typhimurium ATCC 14028 and 8 log CFU/mL for S.

Heidelberg ARI-14 (Figure 2.16). Growth of S. Heidelberg ARI-14 is not affected by the

presence of S. Typhimurium ATCC 14028, instead the growth of S. Typhimurium was inhibited

by S. Heidelberg, as the growth response was reduced after the 6 h time point. CFU/mL growth

determination on LB agar plates was chosen to verify the results from the qPCR experiments

(Figure 2.15).The qPCR results were compared to CFU/mL obtained from the mixed culture

(Figure 2.17). These results correlate with results of previous spent media results; the growth of

S. Typhimurium also decreases when it grows in a mixed culture with S. Heidelberg. This effect

is not limited just to spent media.

Preliminary studies were conducted to determine if there was any serovar exclusion

between S. Heidelberg ARI-14 and S. Enteritidis ATCC 13076. In a growth curve we compared

each strain grown by itself as well as both strains as a mixed culture. The growth for all three

cultures appears to be similar until they reached stationary phase (Figure 2.18). At 24 h the

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OD600 nm for individual S. Heidelberg ARI-14 was slightly higher than S. Enteritidis and the

mixed culture respectively. Further studies, such as qPCR of the mixed culture, should be

conducted to determine if the differences in OD600 nm observed in the growth curve are due to

serovar interactions, where S. Heidelberg is inhibiting the growth of S. Enteritidis as it does with

S. Typhimurium.

5. Discussion

Growth responses for 22 strains from 6 different Salmonella serovars, Typhimurium,

Heidelberg, Kentucky, Newport, Enteritidis, and Montevideo (Figures 2.1 to 2.6) were

conducted. Under aerobic conditions S. Montevideo and S. Kentucky appeared to not be

significantly different between each other and were the fastest growing strains. Growth of S.

Heidelberg strains was faster than S. Typhimurium, but slower than S. Kentucky strains. In

general, both S. Typhimurium strains showed slower growth. Under anaerobic conditions most

of the serovars were not significantly different. S. Enteritidis ARS-50 appeared to be the only

one significantly different from the rest of the strains and exhibited slower growth under both

conditions. Based on these results, 11 strains from these six serovars were selected for further

research using spent media, in order to study the effect on the growth of S. Typhimurium ATCC

14028 caused by spent media from multiple strains.

Spent media, which contains unspecified and autostimulatory compounds may be used to

initiate or promote the growth of uncultivable microorganisms (Weichart and Kell, 2001; Yang

et al., 2006; Brehm-Stecher et al., 2009). Our results showed that S. Typhimurium grows slower

on spent media than it does on Luria Bertani (LB) broth. Previous research have found that spent

culture supernatant of the probiotic Lactobacillus rhamnosus GG exert antimicrobial activity

81

against Salmonella Typhimurium and other intestinal pathogens (Silva et al., 1987; Hudault et

al., 1997; Lehto and Salminen, 1997; De Keersmaecker et al., 2005, 2006). In their research, De

Keersmaecker et al., (2006) reported that antimicrobial activity of spent culture supernatant of

Lactobacillus rhamnosus GG against Salmonella Typhimurium to be mediated by lactic acid. In

contrast, Silva et al., (1987) reported the produced antimicrobial substance to be different than

lactic acid, and to have inhibitory activity against other bacteria within a pH from 3 to 5. Stevens

et al., (1991) reported the inhibitory activity against S. Typhimurium was not due to the

production of bacteriocin. Alverdy and Stern, (1998) reported growth environment and the

composition of the growth media to be important factors in regulating constitutive expression of

several virulence factors. Salmonella expression of invasion genes is regulated by several factors,

such as the HilA regulator and environmental conditions. Durant et al., (2000) in an attempt to

mimic the effects of Lactobacillus on the crop environment, studied if the growth of a poultry

probiotic lactobacilli strain can influence S. Enteritidis hilA expression. This was conducted by

growing S. Enteritidis in spent media from a 24 h growth of a poultry Lactobacillus sp. strain.

Their data suggested that the pH of the medium could be a key factor for the expression of hilA

(Durant et al., 2000). Durant et al., (2000) discussed possible explanations for less hilA

expression in Lactobacillus spent media compared to Salmonella spent media, 1) the possibility

that Salmonella spent media encompass a completely depleted nutrient source than Lactobacillus

spent media; 2) the growth of Lactobacillus may produce soluble signals that are specifically

inhibitory to the virulence of S. Enteritidis; 3) compounds could be present in Salmonella spent

media which serve as a signal to enhance the virulence expression of Salmonella. Nutt et al.,

(2002) studied the ability of spent media from both a pure culture and a cecal mixed culture to

influence virulence expression in a Salmonella Typhimurium hilA:lacZY fusion strain. Results

82

showed a dramatic increase in the virulence expression of the Salmonella Typhimurium strain

when exposed to spent media recovered after 23 h growth, where nutrients are depleted, as

compared to 2 h of growth (Nutt et al., 2002). This data suggests that during the first hours of

exposing the bacteria to spent media, the organism uses all the nutrients for growth and

reproduction; once the nutrients are exhausted the virulence expression increases.

Competitive interactions have been studied with bacteria from the same species as well as

using different species, but the mechanisms causing the inhibition are poorly understood.

Inhibition of colonization has been demonstrated with E. coli and Salmonella in chickens (Linton

et al. 1978; Barrow et al., 1987; Berchieri and Barrow, 1990). Berchieri and Barrow (1990)

reported that an explanation for the inhibition occurring between Salmonella and E. coli could be

that one strain, which acts as a protector, is occupying the niche required by the challenge

organism. That same mechanism is the one that takes place for the inhibition of K88+

enterotoxigenic E. coli by K88+ non-enterotoxigenic E. coli, which involves the occupation of an

attachment site (Davidson et al., 1976). Crhanova et al., (2011) presented some possible effects

of competitive exclusion products, such as the ability of the bacteria to compete directly with

pathogens and also the possibility of stimulating the gut immune system of newly hatched

chickens to mature. Our results showed that the growth of S. Typhimurium decreases when it

grows in a mixed culture with S. Heidelberg. This effect is not limited just to spent media.

Results suggest that the inhibition is not dependent on live cells, but on some factor that is

filterable within the spent medium of S. Heidelberg. To the best of our knowledge this is the first

experiment that studies the effect of S. Heidelberg spent media on the growth of S. Typhimurium

and whether there is competitive interaction between these two serovars.

83

6. Acknowledgments

This research was funded by the National Integrated Food Safety Initiative (NIFSI)

(2008-51110-04339), the Institute of Food Science and Engineering (IFSE), and the U.S. Poultry

& Egg Association. We thank Dr. Si Hong Park, Center for Food Safety, University of Arkansas,

Fayetteville, AR, for providing Salmonella strain specific primers and for his training to conduct

the quantitative PCR.

84

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Handley. 1978. Attempts to displace the indigenous antibiotic resistance gut flora of chickens by

feeding sensitive strains of Escherichia coli prior to slaughter. J. Appl. Bacteriol. 45:239-247.

Luo, Y., Q. Kong, J. Yang, A. Mitra, G. Golden, S. Y. Wanda, K. L. Roland, R. V.

Jensen, P. B. Ernst, and R. Curtis III. 2012. Comparative genome analysis of the high

pathogenicity Salmonella Typhimurium strain UK-1. PLoS One. 7:e40645.

Lynne A. M., P. Kaldhone, D. David, D. G. White, and S. L. Foley. 2009.

Characterization of antimicrobial resistance in Salmonella enterica serotype Heidelberg isolated

from food animals. Foodborne Pathog. Dis. 6:207-215.

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outbreak of Salmonella Heidelberg infection associated with a long incubation period. J. Public

Health Med. 12:19-21.

Nutt, J., L. Kubena, D. J. Nisbet, and S. C. Ricke. 2002. Virulence response of a

Salmonella Typhimurium hila:laczy fusion strain to spent media from pure cultures of selected

bacteria and poultry cecal mixed culture. J. Food Saf. 22:169-181.

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humans in the United States, 1987-1997. J. Infect. Dis. 183:753-761.

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of British fresh sausages and ingredients with salmonellas. J. Hygiene. 90:213-223.

Park, S. H., H. J. Kim, W. H. Cho, J. H. Kim, M. H. Oh, S. H. Kim, B. K. Lee, S. C.

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Review article. Int. Food Res. J. 18:465-473.





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Food Microbiol. 24:385-396.

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from a human Lactobacillus strain. Antimicrob. Agents Chemother. 31:1231-1233.

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ovary and peritoneum of chickens. Avian Dis. 13:668-670.

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treatment for inactivation of Salmonella species and other gram-negative bacteria. Appl.

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innocua stimulates emergene from a resting state: not a response to E. coli quorum sensing

autoinducer AI-2. Biotechnol. Prog. 22:387-393.

90

Table 2.1. Bacterial strains used in this study.

Salmonella strain Source

S. Typhimurium

ATCC 14028 tissue, animal

LT2 wild type from natural source

S. Heidelberg

ATCC 8326

ARI-14 poultry product in Arkansas



S. Kentucky turkey as a source

WVN-216 left over feed

WVN-222 turkey ceca

WVN-343 litters

WVN-352 drinkers

S. Newport

ARS-14 retail

ARA-2 retail

282 retail

S. Enteritidis

ARS-49 farm

ARS-50 farm

ARS-58 retail

ATCC 13076

S. Montevideo

G4639

ARS-11 farm

ARS-12 farm

ARS-13 farm

91

a)

b)

Figure 2.1. Salmonella Typhimurium average from a) aerobic growth response, b) anaerobic

growth response.

92

a)

b)

Figure 2.2. Salmonella Heidelberg average from a) aerobic growth response, b) anaerobic

growth response. Strains labeled as ARI were isolated from poultry products in Arkansas.

93

a)

b)

Figure 2.3. Salmonella Kentucky average from a) aerobic growth response, b) anaerobic growth

response. WVN strains have Turkey as a source.

94

a)

b)

Figure 2.4. Salmonella Newport average from a) aerobic growth response, b) anaerobic growth

response. Strains ARS-14 and ARA-2 are from retail.

95

a)

b)

Figure 2.5. Salmonella Enteritidis average from a) aerobic growth response, b) anaerobic growth

response. ARS-49 and ARS-50 are strains from the farm and ARS-58 from retail.

96

a)

b)

Figure 2.6. Salmonella Montevideo average from a) aerobic growth response, b) anaerobic

growth response. The source of ARS strain is the farm.

97

Table 2.2. Optical density at 600 nm for Salmonella serovars under aerobic conditions.

Isolate

2 h 8 h 12 h 24 h

S. Typhimurium

ATCC 14028 0.156 ± 0.01c,d,e,f

0.539 ± 0.01h,i

0.752 ± 0.02d,e,f

0.964 ± 0.02a,b,c

LT2 0.157 ± 0.02c,d,e

0.517 ± 0.02i,j

0.797 ± 0.04c,d

0.913 ± 0.03c,d,e

S. Heidelberg

ATCC 8326 0.131 ± 0.01e,f

0.750 ± 0.02a

0.891 ± 0.03a,b

0.801 ± 0.06f,g

ARI-14 0.158 ± 0.01c,d,e

0.558 ± 0.01g,h

0.692 ± 0.01f,g,h

0.864 ± 0.04d,e,f

ARI-15 0.120 ± 0.01f

0.461 ± 0.02k,l

0.554 ± 0.05i

0.732 ± 0.02h

ARI-16 0.184 ± 0.01b,c,d

0.560 ± 0.01g,h

0.702 ± 0.03f,g

0.847 ± 0.06e,f

S. Kentucky

WVN-216 0.185 ± 0.01b,c,d

0.776 ± 0.01a

0.940 ± 0.02a

1.003 ± 0.01a

WVN-222 0.166 ± 0.03b,c,d,e

0.571 ± 0.03e,f,g,h

0.733 ± 0.03e,f,g

0.924 ± 0.03b,c,d

WVN-343 0.170 ± 0.02b,c,d

0.571 ± 0.02e,f,g,h

0.717 ± 0.02e,f,g

0.834 ± 0.02f,g

WVN-352 0.169 ± 0.02b,c,d

0.561 ± 0.01f,g,h

0.709 ± 0.03f,g

0.831 ± 0.02f,g

S. Newport

ARS-14 0.151 ± 0.01d,e,f

0.529 ± 0.01h,i

0.641 ± 0.03h

0.770 ± 0.03g,h

ARA-2 0.157 ± 0.01c,d,e

0.648 ± 0.02b,c

0.837 ± 0.02b,c

0.968 ± 0.03a,b,c

282 0.186 ± 0.02b,c,d

0.589 ± 0.01d,e,f,g

0.720 ± 0.03e,f,g

0.850 ± 0.03e,f

S. Enteritidis

ARS-49 0.159 ± 0.02c,d,e

0.547 ± 0.01g,h,i

0.681 ± 0.05g,h

0.820 ± 0.04f,g

ARS-50 0.153 ± 0.02d,e,f

0.434 ± 0.05l 0.511 ± 0.07

i 0.564 ± 0.09

j

ARS-58 0.158 ± 0.01c,d,e

0.582 ± 0.01d,e,f,g

0.725 ± 0.03e,f,g

0.869 ± 0.04d,e,f

ATCC 13076 0.160 ± 0.02c,d,e

0.481 ± 0.04j,k

0.558 ± 0.04i

0.638 ± 0.05i

S. Montevideo

G4639 0.225 ± 0.06a

0.658 ± 0.02b

0.864 ± 0.02b

0.994 ± 0.01a,b

ARS-11 0.196 ± 0.01a,b,c

0.604 ± 0.02d,e,f

0.735 ± 0.02d,e,f,g

0.915 ± 0.02b,c,d,e

ARS-12 0.203 ± 0.02a,b

0.608 ± 0.01c,d,e

0.744 ± 0.01d,e,f,g

0.938 ± 0.02a,b,c,d

ARS-13 .0182 ± 0.03a,b,c,d

0.621 ± 0.03b,c,d

0.775 ± 0.04c,d,e

0.958 ± 0.03a,b,c

Values labeled with same letters are not significantly different between each time point.

98

Table 2.3. Optical density at 600 nm for Salmonella serovars under anaerobic conditions.

Isolate

2 h 8 h 12 h 24 h

S. Typhimurium

ATCC 14028 0.162 ± 0.01a,b,c,d

0.607 ± 0.07c,d

0.658 ± 0.03d,e,f

0.807 ± 0.07b,c,d,e

LT2 0.137 ± 0.01c,d

0.584 ± 0.04d,e

0.756 ± 0.03b,c

0.833 ± 0.05a,b,c,d

S. Heidelberg

ATCC 8326 0.160 ± 0.02a,b,c,d

0.735 ± 0.06a,b

0.812 ± 0.02b

0.928 ± 0.03a

ARI-14 0.152 ± 0.03a,b,c,d

0.587 ± 0.05d,e

0.611 ± 0.04e,f,g

0.681 ± 0.05f

ARI-15 0.127 ± 0.01d

0.495 ± 0.07e,f

0.564 ± 0.05g

0.677 ± 0.07f

ARI-16 0.176 ± 0.03a,b

0.591 ± 0.06d,e

0.629 ± 0.02d,e,f,g

0.741 ± 0.07d,e,f

S. Kentucky

WVN-216 0.171 ± 0.02a,b,c

0.784 ± 0.06a

0.882 ± 0.04a

0.904 ± 0.08a,b

WVN-222 0.183 ± 0.04a,b

0.632 ± 0.06b,c,d

0.638 ± 0.02d,e,f

0.819 ± 0.05b,c,d,e

WVN-343 0.170 ± 0.03a,b,c

0.635 ± 0.04b,c,d

0.676 ± 0.06d,e

0.751 ± 0.05d,e,f

WVN-352 0.185 ± 0.04a

0.649 ± 0.04b,c,d

0.691 ± 0.06c,d

0.719 ± 0.03e,f

S. Newport

ARS-14 0.146 ± 0.01b,c,d

0.612 ± 0.05c,d

0.614 ± 0.04e,f,g

0.768 ± 0.03d,e,f

ARA-2 0.161 ± 0.02a,b,c,d

0.702 ± 0.05a,b,c

0.771 ± 0.04b

0.919 ± 0.03a,b

282 0.169 ± 0.02a,b,c

0.660 ± 0.06b,c,d

0.686 ± 0.03d

0.751 ± 0.04d,e,f

S. Enteritidis

ARS-49 0.157 ± 0.02a,b,c,d

0.598 ± 0.07d,e

0.601 ± 0.03f,g

0.713 ± 0.04e,f

ARS-50 0.167 ± 0.03a,b,c

0.443 ± 0.12f 0.447 ± 0.08

h 0.673 ± 0.02

f

ARS-58 0.163 ± 0.02a,b,c,d

0.629 ± 0.07c,d

0.669 ± 0.03d,e

0.747 ± 0.04d,e,f

ATCC 13076 0.169 ± 0.02a,b,c

0.497 ± 0.06e,f

0.482 ± 0.04h

0.483 ± 0.06g

S. Montevideo

G4639 0.176 ± 0.03a,b

0.660 ± 0.05b,c,d

0.769 ± 0.02b

0.871 ± 0.05a,b,c

ARS-11 0.163 ± 0.02a,b,c,d

0.618 ± 0.06c,d

0.630 ± 0.03d,e,f,g

0.761 ± 0.06d,e,f

ARS-12 0.159 ± 0.02a,b,c,d

0.620 ± 0.06c,d

0.639 ± 0.03d,e,f

0.766 ± 0.06c,d,e,f

ARS-13 0.166 ± 0.01a,b,c

0.623 ± 0.05c,d

0.644 ± 0.03d,e,f

0.816 ± 0.05b,c,d,e

Values labeled with same letters are not significantly different between each time point.

99

Figure 2.7. Average aerobic spent media growth response.

100

Figure 2.8. Average anaerobic spent media growth response. No growth was observed when S.

Typhimurium ATCC 14028 was grown on spent media from multiple serovars and strains.

Growth observed represents the control, S. Typhimurium ATCC 14028 on LB broth.

101

Figure 2.9. Combinations of S. Heidelberg and S. Typhimurium spent media and inoculum.

Confirmation that the decrease in the growth of S. Typhimurium is observed only when this

strain is grown on S. Heidelberg ARI-14 spent media.

102

Table 2.4. Salmonella Heidelberg strains used in this study.

Strain Source Reference

ARI-14 poultry products In this study

SL 476 ground turkey Fricke et al., 2011

SL 486 human Fricke et al., 2011

692 chicken egg house Lynne et al., 2009

945 human Han et al., 2011

114 cattle Lynne et al., 2009

163 turkey Kaldhone et al., 2008

136 swine Lynne et al., 2009



130 chicken Lynne et al., 2009





103

Figure 2.10. Salmonella Typhimurium ATCC 14028 in S. Heidelberg spent media from multiple sources. From all strains, thirteen of

them showed the drop in the growth.

104

Figure 2.11. Confirmation that the drop showed during growth response only occurs when

growing S. Typhimurium on S. Heidelberg spent media. This effect is not observed when

Salmonella Heidelberg strains are inoculated on any S. Heidelberg spent media.

105

Figure 2.12. Growth response for S. Typhimurium on S. Heidelberg spent media growing at

42°C on LB and BPW. When incubated at 42°C, S. Typhimurium ATCC 14028 growing on S.

Heidelberg spent media did not show the drop that was observed at 37°C.

106

Figure 2.13. Growth response for S. Typhimurium on S. Heidelberg spent media growing at

37°C on LB and BPW. The drop in the growth of S. Typhimurium ATCC 14028 growing on S.

Heidelberg spent media was observed when strains were incubated at 37°C.

107

Table 2.5. Primer pairs used in competitive growth studies.

Primer Target Sequences PCR product

size (bp)

Reference

STM4497M2-f Salmonella

Typhimurium

5’-AACAA CGGCT CCGGT

AATGA GATTG-3’

310 Park et al., 2009

STM4497M2-r 5’-ATGAC AAACT CTTGA

TTCTG AAGAT CG-3’

SH_SHP-2f Salmonella

Heidelberg

5’-GCATA GTTCC AAAGC

ACGTT-3’

180 In this study

SH_SHP-1r 5’-GCTCA ACATA AGGGA

AGCAA-3’

108

Figure 2.14. Salmonella Typhimurium and Salmonella Heidelberg growth responses from a

mixed culture. Growth response of S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14

growing together in LB broth for 24 h.

109

Figure 2.15. Salmonella Typhimurium and S. Heidelberg competitive interaction.

110

Figure 2.16. Log CFU/mL for S. Typhimurium and S. Heidelberg from mixed culture.

111

Figure 2.17. Log CFU/mL from qPCR and plate counts from mixed culture.

112

Figure 2.18. Growth of S. Heidelberg ARI-14 and S. Enteritidis ATCC 13076 growth as

individual and mixed cultures.

113



among coauthors, which the title is “Growth Responses, Spent Media, and Competitive

Interactions among Salmonella Serovars” in chapter 2.


114

9. Appendix

9.1. Motility of S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14 on different agar

concentrations.

9.1.1 Materials and Methods

One colony of S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14 was inoculated

into 8 mL of Luria Bertani (LB) Broth (Alfa Aesar, Ward Hill, MA, USA) and incubated for 16

h at 37°C, 190 rpm shaking. A sterile cotton swab was dipped into the overnight bacterial culture

and gently squeezed against the tube to remove any excess fluid. The cotton swab subsequently

streaked on three different LB agar concentrations, 0.3, 0.5 and 1.5%, to observe the motility of

these strains and their ability to move through the filter papers, 4.25 cm (Whatman, Sigma

Aldrich, St. Louis, MO, USA), inserted on the center of the agar plate. For the plates where spent

media was used, 100 μL of spent media were inoculated on the center of the plate and streaked

evenly across the plate using a sterile cotton swab. One μL of bacteria was then inoculated on the

center of the plate.

115

a)

b)

c) (Photo taken by author)

Figure 1. Motility of S. Heidelberg and S. Typhimurium at different agar concentrations a) 1.5%,

b) 0.5%, and c) 0.3%. As the agar concentration decreases, S. Heidelberg and S. Typhimurium

were able to move across the plate and in some cases, cross through the filter paper. Plate F

reperesents S. H. inoculated on S. T. spent media; plate G is S. T. inoculated on S. H. spent

media.

116

(Photo taken by author)

Figure 2. When inoculating S. Heidelberg on S. Typhimurium spent media it was observed how

the growth of S.T. spread across the plate.


Figure 3. The plate on the left represents S. T. inoculated on S. H. spent media; and the plate on

the right is S. H. inoculated on S. T. spent media, at 0.3% agar concentration. We can observe

how the growth is different in both cases.

117


Figure 4. Filter papers were cut and used as paper disk. We dip the paper disk in 100 μL of

overnight bacterial strain and placed them on the corner of the plates. On the other side we used

a sterile cotton swab to streak bacteria on the plate. Without filter papers we can observe how the

two strains, S. T. and S. H. do not cross between each other. On the plate at the top it is shown a

a line dividing the two strains.

118


Figure 5. Multiple combinations of S. Typhimurium and S. Heidelberg, filter papers in the center

of the plate, paper disks on the corner of the plate, and spent media were conducted.

119

9.2. Institutional Biosafety Committee (IBC) Number

120


IBC#: 13008

Please check the boxes for each of the forms that are applicable to the research project you

are registering. The General Information Form - FORM 1 (this form) MUST be completed

on all submitted project registrations, regardless of the type of research.

Recombinant DNA (EVEN IF IT IS EXEMPT from the NIH Guidelines.) (FORM 2) Pathogens (human/animal/plant) (FORM 3) Biotoxins (FORM 4) Human materials/nonhuman primate materials (FORM 5) Animals or animal tissues and any of the above categories; transgenic animals or tissues; wild vertebrates or tissues (FORM 6)

Plants, plant tissues, or seed and any of the above categories; transgenic plants, plant tissues, or seeds (FORM 7)

CDC regulated select agents (FORM 8)

Notice to Pat Walker Health Center (FORM 9)

To initiate the review process, you must attach and send all completed registration forms

via email to [email protected]. All registration forms must be submitted electronically. To

complete the registration, print page 1 of this form, PI sign, date, and mail to: Compliance

Coordinator-IBC, 210 Admin. Building, Fayetteville, AR 72701, or FAX it to 479-575-3846.

As Principal Investigator:

I attest that the information in the registration is accurate and complete and I will submit changes to the Institutional Biosafety Committee (IBC) in a timely manner.

I am familiar with and agree to abide by the current, applicable guidelines and regulations governing my research, including, but not limited to: the NIH Guidelines for Research Involving Recombinant DNA Molecules and the Biosafety in Microbiological and Biomedical Laboratories manual.

I agree to accept responsibility for training all laboratory and animal care personnel involved in this research on potential biohazards, relevant biosafety practices, techniques, and emergency procedures.

If applicable, I have carefully reviewed the NIH Guidelines and accept the responsibilities described therein for principal investigators (Section IV-B-7). I will submit a written report to the IBC and to the Office of Recombinant DNA Activities at NIH (if applicable) concerning: any research related accident, exposure incident, or release of rDNA materials to the environment; problems implementing biological and physical containment procedures; or violations of NIH Guidelines. I agree that no work will be initiated prior to project approval by the IBC.

I will submit my annual progress report to the IBC in a timely fashion.

mailto:[email protected]

121

Principal Investigator Typed/Printed Name: Steven C. Ricke

Signature (PI): ____________________________________ Date: ____________________

CONTACT INFORMATION:

Principal Investigator:

Name: Steven C. Ricke

Department: FDSC

Title: Professor

Campus Address: FDSC E 27

Telephone: 575-4678

*After Hours Phone:

Fax: 575-6936

E-Mail: [email protected]

Co-Principal Investigator:

Name: Philip G. Crandall

Department: FDSC

Title: Professor

Campus Address: FDSC N221

Telephone: 575-7686

*After Hours Phone:

Fax: 575-6936


*Required if research is at Biosafety Level 2 or higher

PROJECT INFORMATION:

Have you registered ANY project previously with the IBC? Yes

Is this a new project or a renewal?

Project Title: Production of a New Vaccine for Poultry to Prevent Salmonella

Project Start Date: 11/1/2012

Project End Date: 10/31/2013

Granting Agency: USDA/SBIR

Indicate the containment conditions you propose to use (check all that apply):

Biosafety Level 1 Ref: 1

2

Biosafety Level 1A Ref: 1

2

Biosafety Level 1P Ref: 1

2


2


2


2

http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-052.html











122




References:

1: Biosafety in Microbiological and Biomedical Laboratories (BMBL) 4th Edition

2: NIH Guidelines for Research Involving Recombinant DNA Molecules

3: University of Arkansas Biological Safety Manual

If you are working at Biosafety Level 2 or higher, has your laboratory received an onsite

inspection by the Biosafety Officer or a member of the IBC?

If yes, enter date if known: 1/9/2012

If no, schedule an inspection with the Biological Safety Officer.

Please provide the following information on the research project (DO NOT attach or insert

entire grant proposals unless it is a Research Support & Sponsored Programs proposal).

Project Abstract:

Avirulent live Salmonella vaccines are considered to be more effective for preventing the spread

of Salmonella in poultry flocks. A live Salmonella vaccine should be completely genetically

stable and avirulent for both animals and humans. We have developed a double deletion mutant

that is deficient in its ability to synthesize lysine (lysA-) and is defective in its virulence

properties (hilA-).The goal of this project is to determine the survival and control of this vaccine

strain and differences in attachment or invasiveness in human Caco-2 cell model.

Specific Aims:

a. Determine motility of the vaccine strain.

b. Determine survival of vaccine strain under acid conditions.

c. Determine the ability of the vaccine strain to withstand thermal treatment.

d. Evaluate the attachment and invasiveness of the vaccine strain in human Caco-2 cell

model.

Relevant Materials and Methods (this information should be specific to the research

project being registered and should highlight any procedures that involve biohazardous or

recombinant materials):

Motility (Shah et al, 2011; Vikram et al., 2011; Wang et al. 2007): Grow Salmonella overnight

(~16 h). Stab-inoculate 1 µl of culture in the middle of semi-solid media (0.3% LB agar).

Incubate at 37 °C for 7-8 hr and measure the diameter of the halo of growth.

Survival under acid conditions (Malheiros et al., 2008): Prepare overnight Salmonella culture in

LB broth. Wash the cells with PBS three times and add the cells to PBS acidified to pH 3.5, 4.0,






http://ehs.uark.edu/DocumentPages/BiosafetyManual04.pdf

123

4.5, and 5.0 with HCl. Incubate the cells in 30°C water bath and take 1 ml at each interval time

(20~30 min, up to 3 h) to plate on LB agar with appropriate dilutions for enumeration.

Survival under thermal condition (Malheiros et al., 2008; Alvarez-Ordonez et al., 2008): Prepare

overnight bacteria culture. Wash the cells with PBS three times and add the bacterial cells to

PBS. Incubate the cells in 52, 56, 60°C water bath and take 1 ml at each interval time (10 – 20

min up to 2h and 5 min up to 30 min for 60°C) to plate on LB agar with appropriate dilution for

enumeration.

Adhesion and Invasion:

Caco-2 cells preparation: Media: Dulbecco’s modified Eagle medium (DMEM) (Invitrogen)

supplemented with 10 % (v/v) fetal bovine serum (FBS; Invitrogen) in 95 % air/5 % CO2 at

37°C. Cell condition: Caco-2 human colon adenocarcinoma cells with passage of 20-35. When

confluence is achieved (~10 days), harvest the cells by trypsinization (add trypsin for 10-15min,

disrupt the cells) and resuspend in D10F. Plate the resuspeneded cells onto 12-well plates

(Cellstar) at 1x10E5 ~10E6 cells per well. Change the medium every other day until confluency

(10~12 or up to 14 days until differentiated). Cell differentiation can be monitored by estimation

of the production of intestinal alkaline phosphatase using SensoLyte pNPP alkaline phosphatase

assay kit (AnaSpec). (Intestinal alkaline phosphatase is a brush border enzyme expressed

exclusively in villus-associated enterocytes, and expression indicates the development of

digestive and absorptive function (Goldberg et al., 2008; Hara et al., 1993).)

Adhesion assay: Prepare overnight bacteria culture (~16 h, grown in LB broth). Wash the cells

with PBS three times and dilute the bacterial cells to MOI ration of 10: 1 (10E6 Salmonella:

10E5 Caco-2 cells) Add the bacteria to Caco-2 cells and incubate for 2 h. Wash the cells with

cell-PBS (137 mM NaCl, 5.4 mM KCl, 3.5 mM Na2HPO4, 4.4 mM NaH2PO4, 11 mM glucose,

pH 7.2) four times. Treat the adherent cells with 0.1% Triton-X 100 (Sigma, St. Louis, MO) in

cell-PBS for 10 min at 37°C and collect the disrupted cells. Plate the bacteria on LB agar with

appropriate dilution for enumeration.

Invasion assay: Prepare overnight bacteria culture (~16 h, grown in LB broth). Wash the cells

with PBS three times and dilute the bacterial cells to MOI ration of 10: 1 (Salmonella: Caco-2

cells). Add the bacteria to Caco-2 cells and incubate for 2 h. Wash the cells with cell-PBS for

four times. Add 100 µg gentamicin ml/1 in D10F in each well and incubate for 2 h to kill

extracellular bacteria. Wash the cells with cell-PBS four times, add 0.1% Triton-X 100 in cell-

PBS to the Caco-2 cells for 10 min at 37°C and collect the disrupted cells. Plate the bacteria on

LB agar with appropriate dilution for enumeration (Shah et al, 2011).

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PERSONNEL QUALIFICATIONS & FACILITY INFORMATION:

List all personnel (including PI and Co-PI) to be involved in this project:

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previous work or training with biohazardous and/or

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Example: Bob Biohazard -

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BL2, 8 yrs working with transgenic mice.

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Star Int., PhD Cell Biology

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126

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Liquid biological waste will always be discarded into freshly made 10% bleach and then

autoclaved for decontamination treatment before it is discarded. Other biological waste will be

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Describe the procedures/equipment that will be used to prevent personnel exposure during

aerosol-producing procedures:

All pipetting of infectious material will take place in the biological safety cabinet. Mechanical

pipetting devices will be used. Lab coats buttoned over street clothes, gloves and goggles will be

worn. All materials needed will be placed in the biological safety cabinet before work begins.

Sash of the cabinet will be lowered and all movements will be slow to avoid disruption of the air

currents. Centrifuged cultures will be contained in a closed Eppendorf tube or contained in

screw-capped polypropylene or polystyrene tubes with gasket seals to prevent aerosol exposure.

Cultures to be vortexed will be contained in screw-capped polypropylene or polystyrene tubes,

and vortexing will be done within the biological safety cabinets. Sonicating will be done within

the biosafety cabinet or within an enclosure on the bench top.

EMERGENCY PROCEDURES:

128

In the event of personnel exposure (e.g. mucous membrane exposure or parenteral

inoculation), describe what steps will be taken including treatment, notification of proper

supervisory and administrative officials, and medical follow up evaluation or treatment:

In the event of accidental exposure of personnel the person exposed should notify the laboratory

supervisor immediately. Treatable exposures will be treated by use of the first aid kit containing

antimicrobial agents. Mucous membrane exposure or puncture with contaminated material will

result in the person being taken to the Health Center for prophylactic antibiotic therapy. In the

event of environmental contamination, describe what steps will be taken including a spill

response plan incorporating necessary personal protective equipment (PPE) and decontamination

procedures.

In the event of environmental contamination, describe what steps will be taken including a

spill response plan incorporating necessary personal protective equipment (PPE) and

decontamination procedures.

For a spill inside the biological safety cabinet, alert nearby people and inform laboratory

supervisor. Safety goggles, lab coat buttoned over street clothes and latex gloves should be worn

during clean up. If there are any sharps they will be picked up with tongs, and the spill covered

with paper towels. Carefully pour disinfectant (freshly made 10% bleach) around the edges of

the spill, then into the spill without splashing. Let sit for 20 minutes. Use more paper towels to

wipe up the spill working inward from the edge. Clean the area with fresh paper towels soaked in

disinfectant. Place all contaminated towels in a biohazard bag for autoclaving. Remove personal

protective clothing and wash hands thoroughly. For a spill in the centrifuge turn off motor, allow

the machine to be at rest for 30 minutes before opening. If breakage is discovered after the

machine has stopped, re close the lid immediately and allow the unit to be at rest for 30 minutes.

Unplug centrifuge before initiating clean up. Wear strong, thick rubber gloves and other personal

protective equipment (PPE) before proceeding with clean up. Flood centrifuge bowl with

disinfectant. Place paper towels soaked in a disinfectant over the entire spill area. Allow 20

minute contact time. Use forceps to remove broken tubes and fragments. Place them in a sharps

container for autoclaving and disposal as infectious waste. Remove buckets, trunnions and rotor

and place in disinfectant for 24 hours or autoclave. Unbroken, capped tubes may be placed in

disinfectant and recovered after 20 minute contact time or autoclaved. Use mechanical means to

remove remaining disinfectant soaked materials from centrifuge bowl and discard as infectious

waste. Place paper towels soaked in a disinfectant in the centrifuge bowl and allow it to soak

overnight, wipe down again with disinfectant, wash with water and dry. Discard disinfectant

soaked materials as infectious waste. Remove protective clothing used during cleanup and place

in a biohazard bag for autoclaving. Wash hands whenever gloves are removed.

For a spill outside the biological safety cabinet or centrifuge have all laboratory personnel

evacuate. Close the doors and use clean up procedures as above.

TRANSPORTATION/SHIPMENT OF BIOLOGICAL MATERIALS:

Transportation of Biological Materials: The Department of Transportation regulates some

biological materials as hazardous materials; see 49 CFR Parts 171 - 173. Transporting any of

these regulated materials requires special training for all personnel who will be involved in the

http://www.access.gpo.gov/nara/cfr/waisidx_06/49cfrv2_06.html

129

shipping process (packaging, labeling, loading, transporting or preparing/signing shipping

documents).

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Cultures of Human or Animal Pathogens Environmenatl samples known or suspected to contain a human or anumal pathogen Human or animal material (including excreta, secreta, blood and its components, tissue, tissue fluids, or cell lines) containing or suspected of containing a human or animal pathogen.

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130

CHAPTER 3

Salmonella spp. Growth Responses Comparisons in Selective and Non-selective

Enrichment Media Methods





Fayetteville, AR

131

1. Abstract

The use of enrichment culture is one of the most common methods to recover Salmonella

from food samples. Growth rate and recovery of different Salmonella strains on Luria Bertani

broth versus buffered peptone water (BPW) followed by selective enrichment on Rappaport-

Vassiliadis (RV) broth and Tetrathionate (TT) broth were compared. One strain from Salmonella

serovars Typhimurium, Enteritidis, Heidelberg and Kentucky was tested. Strains were grown

overnight on LB broth and BPW at 35°C. Approximately 100 colony forming units per mL

(CFU/mL) of each strains was then sub cultured on LB, TT and RV broth and incubated at 42°C

for 24 hours. CFU/mL were determined by serial dilutions and plating on LB agar. Strains

inoculated on LB broth exhibited a faster growth rate than those on RV and TT broth, and were

detectable at an earlier time point. At 8 hours of incubation, approximately 30 to 60 CFU/mL

were recovered from samples on LB broth while no CFU/mL were recovered from samples on

RV or TT broth at that time point. The use of a non-selective enrichment media such as LB broth

could potentially be more effective for earlier quantification and recovery of different Salmonella

serovars present in samples.

132

2. Introduction


illness worldwide. Salmonella has been a leading cause of risk to humans with 1.0 million cases

occurring that represents 11% of all foodborne pathogens (Moore-Neibel et al., 2011;

Ravichandran et al., 2011). Annual costs for Salmonella control efforts are estimated to be up to

$14.6 billion (Scharff, 2010; Heithoff, et al., 2012). Many of its serovars have been associated

with the consumption of poultry products, these being one of the main reservoirs of Salmonella

(CDC, 2009). The emergence and prevalence of Salmonella serovars in chickens has been

studied but the predominant serovars have varied somewhat over the years. Salmonella

Enteritidis, S. Typhimurium and S. Heidelberg are the three most frequent serovars recovered

from humans. These three serovars along with S. Kentucky are the most common serovars found

in poultry.

Isolation and identification of Salmonella through conventional methods includes five

steps: pre-enrichment, selective enrichment, plating on selective media, biochemical screening,

and serotyping (Afflu and Gyles, 1997). The use of enrichment culture is one of the most

common methods to recover Salmonella from food samples, faeces, and other material (Banič,

1964; Afflu and Gyles, 1997). Theoretically, enrichment methods are used to detect very low

levels of Salmonella, while these may not be detectable without the use of enrichment broth.

Brehm-Stecher et al., (2009) reported that as generally used, enrichment culture effectively

erases valuable information about initial microbial numbers within a sample, downgrading a

potentially quantitative test into a qualitative one. Singer et al., (2009) reported that the

probability of detecting a specific Salmonella strain had little to do with its starting concentration

in the sample. The bias introduced by culture growth-based systems could be dramatically

133

biasing Salmonella surveillance systems (Singer et al., 2009). We hypothesized that the use of a

non-selective enrichment media such as Luria Bertani (LB) broth could potentially be more

effective and equally representative for earlier quantification and recovery of different

Salmonella serovars present in samples. In this project, the growth rate and recovery of four

Salmonella strains on LB broth versus Buffered Peptone water (BPW) followed by selective

enrichment on Rappaport-Vassiliadis (RV) broth and Tetrathionate (TT) broth were compared.

RV broth serves as a base for the selective enrichment of Salmonella; and TT broth is a base for

enrichment medium for isolation of Salmonella. The composition of each broth media is listed

on Table 1.


3.1. Bacterial cultures for non-selective enrichment media method

One colony of S. Typhimurium ATCC 14028, S. Heidelberg SL 476, S. Enteritidis ATCC

13076 and S. Kentucky WVN-222 was inoculated in 8 mL of LB broth and incubated for 16 h at

35°C, 190 rpm shaking using a C76 Water Bath Shaker (New Brunswick Scientific, Edison, NJ,

USA). Overnight cultures were diluted to 10-3

CFU/mL and 100 μL of each strain were

inoculated in 10 mL LB broth. Tubes were incubated for 24 h at 42°C, 190 rpm shaking. Optical

density at 600 nm was measured at 0, 2, 4, 6, 8, 12, and 24 h. From the 10-3

dilutions, 1 mL of

each strain was inoculated into 100 mL of LB broth. One mL of culture was collected at 0, 2, 4,

6, 8, 12, and 24 h for serial dilutions and plating, per duplicate, on LB agar plates for

enumeration, colony forming units per mL (CFU/mL).

134

3.2. Bacterial cultures for selective enrichment media method

One colony of S. Typhimurium ATCC 14028, S. Heidelberg SL 476, S. Enteritidis ATCC

13076 and S. Kentucky WVN-222 was inoculated in 8 mL of BPW broth and incubated for 16 h

at 35°C, 190 rpm shaking using a C76 Water Bath Shaker (New Brunswick Scientific). Optical

density at 600 nm was measured for all four overnight cultures. These were then diluted to 10-3

CFU/mL and 1 mL of each strain were inoculated in 100 mL RV broth, and 5 mL of each strain

inoculated in 100 mL TT broth. Flasks were incubated for 24 h at 42°C, 190 rpm shaking. One

milliliter of culture was collected at 0, 2, 4, 6, 8, 12, and 24 h for serial dilutions and plating, per

duplicate, on LB agar plates for enumeration, CFU/mL. This experiment was conducted

following the flow chart specific for FSIS laboratory Salmonella analyisis (USDA/FSIS, 2013).

4. Results and Discussion

Many researches have concluded that enrichment media are of great importance for the

isolation of Salmonella (Banič, 1964; Palumbo and Alford, 1970; Harvey et al., 1979; Harvey

and Price, 1980; 1981; Afflu and Gyles, 1997; AOAC, 1995; FDA, 1995; Hammack et al., 1999;

2001). Through history, the most commonly used enrichment media have been TT broth (Bohls,

1950; Palumbo and Aflord; 1970; Harvey et al., 1979; Harvey and Price, 1981; Beckers et al.,

1986; Hammack et al., 1999; 2001), selenite F broth (Leifson, 1936; Palumbo and Alford, 1970;

Harvey et al., 1979; Hammack et al., 1999) and RV broth (Beckers et al., 1986; Harvey et al.,

1979; Harvey and Price, 1980; 1981; Fricker et al., 1985; Hammack et al., 1999; 2001). These

media have been introduced with the purpose of inhibiting non-pathogenic enteric bacteria, while

allowing for Salmonella to grow (Iveson et al., 1964; Palumbo and Alford, 1970).

135

Tetrathionate broth was first used by Mueller (1923); later Kauffmann (1941) modified

the medium by the addition of bile and brilliant green. It is believed that most Salmonella and

Proteus reduce tetrathionate to thiosulfate in the media, while species such as E. coli and

Shigella do not, which may explain why Salmonella is able to flourish in the media (Knox et al.,

1942; 1945; Pollock et al., 1942; Knox et al., 1943; Pollock and Knox, 1943; Knox and Pollock,

1944; Jeffries, 1959; Palumbo and Alford, 1970).

In 1956, Rappaport et al., (1956) published a new medium for isolating Salmonella from

human faeces, claiming it to be better and more efficient than selenite and TT broth. In 1976,

Vassiliadis et al., (1979) modified the medium in order to make it more suitable for incubation at

43°C, this is why the media is known as Rappaport-Vassiliadis. The ability of the RV medium

for the isolation of Salmonella has been said to depend on the ability of the pathogen to multiply

at low pH-value, high osmotic pressure, and also on the resistance of Salmonella to malachite

green (Peterz et al., 1989). Previous researches have reported that RV media is more effective

than TT and selenite media when recovering Salmonella from different samples, including food

products (Harvey et al., 1979; Harvey and Price, 1980; 1981; Vassiliadis et al., 1981; Fricker et

al., 1983; Kalapothaki et al., 1983; van Schothorst and Renaud, 1983; Vassiliadis, 1983; Fricker,

1984; Tongpim et al., 1984; Vassiliadis et al., 1985; Rhodes and Quesnel, 1986; Allen et al.,

1991a; 1991b; June et al., 1995; 1996; Hammack et al., 1999; 2001). Brest Nielsen and Grunnet,

(1976) reported no difference when isolating Salmonella from meat and bone-meal, sewage or

receiving waters, while isolates were significantly less frequent from RV than from TT. In order

to be effective, the Rappaport medium should be very lightly inoculated; otherwise the media

loses its sensitivity and selectivity to isolate Salmonella (Rappaport et al., 1956; Collard and

136

Unwin, 1958; Hooper and Jenkins, 1965; Vassiliadis et al., 1985). Contrary to the case of

tetrathionate broth where the initial inoculation is required to be higher than RV broth.

We hypothesized that the use of a non-enrichment media such as LB broth could

potentially be more effective for earlier quantification and recovery of different Salmonella

serovars present in samples. To prove this, we compared the growth responses and recovery of

four Salmonella strains on LB broth versus BPW followed by enrichment on RV and TT broth.

Results showed that all four strains inoculated on LB broth exhibited a faster growth response

than those on RV and TT broth, and were detectable at an earlier time point. At 8 hours of

incubation, approximately 20 to 70 CFU/mL were recovered from samples on LB broth at a 10-5

dilution (Table 3.2), while no CFU/mL were recovered from samples on RV or TT broth at that

time point. Using enrichment methods, RV and TT broth, Salmonella numbers were lower at

every time point studied. Rappaport-Vassiliadis showed no detectable levels of Salmonella at

dilutions higher than 10-6

CFU/mL (Table 3.3); while using tetrathionate broth, no growth was

detected at dilutions higher than 10-4

(Table 3.4). For Salmonella strains inoculated on LB broth,

Salmonella was detected as early as 4 h at 10-2

and 10-3

dilutions, while the recovery was 0

CFU/mL for RV and TT broth at the same time point. At initial time points, the growth of

Salmonella on TT broth was higher than on RV, while at 24 h the growth on RV was

significantly higher. An explanation for this could be the fact that for enrichment on TT broth it

is required for the initial inoculum to be higher, 5 mL, while it was only 1 mL for RV broth.

Zhang et al., (2013) reported tryptic soy broth (TSB) to be significantly more efficient

than nutrient broth and BPW. Buffered peptone water is the most commonly used by food

industries before conducting enrichment, and was used for our experiment before inoculating on

RV and TT broth. After enrichment in RV broth, Zhang et al., (2013) observed S. Enteritidis

137

population to be 0.40 to 1.11 log CFU/mL lower than those in preenrichment cultures. Afflu and

Gyles, (1997) reported that enrichment in TT broth appears not to be necessary for enrichment.

Their findings showed the same results when inoculating directly from pre-enrichment or after

enrichment with TT broth (Afflu and Gyles, 1997). Because the BAM Method (FDA, 1995;

Hammack et al., 1999; 2001) recommends using selective enrichment pairs, TT at 35°C and RV

at 42°C are used from foods with low microbial load. June et al., (1995 1996) recommended

incubating TT broth at 42°C for the recovery of Salmonella with high microbial loads. In our

experiment, we incubated both enrichment media at 42°C.

In general, out of all four serovars studied, Salmonella Kentucky was the fastest growing

on every single media tested. A reason for this could be the hypothesis where S. Kentucky may

devote all metabolic efforts toward growth. González-Gil et al., (2012), studied virulence

expression of the hilA gene in S. enterica serovars in response to acid stress. Their results

showed no significant changes in hilA expression in S. Kentucky, which supports the idea of

focusing on growth rather than virulence (González-Gil et al., 2012). This could explain why S.

Kentucky is one of the most commonly isolated from poultry but so far is not of public health

concern in the United States. According to the CDC (2011), S. Kentucky is one of the most

common serovars isolated from broilers in the US and commonly found in dairy catle as well

(Foley et al., 2011; 2013). Several factors could contribute for the increase in isolating this

serovar from chickens, such as flock immunity, genetic changes in the organism, and

management practices (Foley et al., 2013). Several researchers have reported that it is most likely

for S. Kentucky to lack genes necessary to cause disease in humans, but that it does possess

characteristics that allow it to be a competitive colonizer in chicken cecum, such as having a

better ability to grow under moderately acidic conditions (pH 5.5) (Joerger et al., 2009;

138

González-Gil et al., 2012). It has been reported that S. Kentucky possess plasmids with factors

associated with antimicrobial and disinfectant resistance, bacteriocin production, iron

acquisition, and complement resistance, which could benefit and enhance their abilities to

survive in chickens (Han et al., 2012; Foley et al., 2013).

In conclusion, comparing the growth of different Salmonella serovars, Typhimurium,

Heidelberg, Enteritidis and Kentucky, it appears that the type of media used to recover

Salmonella serovars can influence the population levels detectable by culturing. Further

examination of additional serovars and more strains within serovars is needed to further

differentiate differences in recovery media. This may be critical for assessing the original

distribution of serovars in a sample prior to enrichment for detection and quantification.

5. Acknowledgments

This research was funded with a grant obtained from the U.S. Poultry & Egg Association.

We thank Dr. Corliss O’Bryan, Center for Food Safety, University of Arkansas, Fayetteville,

AR, for sharing her expertise about enrichment protocols of Salmonella and providing insights to

conduct this research.

139

6. References

Afflu, L., and C. L. Gyles. 1997. A comparison of procedures involving single step

Salmonella, 1-2 test, and modified semisolid rappaport-vassiliadis medium for detection of

Salmonella in ground beef. Int. J. Food Microbiol. 37:241-244.

Allen, G., V. R. Bruce, W. H. Andrews, F. B. Satchell, and P. Stephenson. 1991a.

Recovery of Salmonella from frozen shrimp: evaluation of short term selective enrichment,

selective media, post-enrichment, and a rapid immunodiffusion method. J. Food Prot. 54:22-27.

Allen, G., V. R. Bruce, P. Stephenson, F. B. Satchell, and W. H. Andrews. 1991b.

Recovery of Salmonella from high-moisture foods by abbreviated selective enrichment. J. Food

Prot. 54:492-495.

AOAC International. 1995. Official methods of analysis. 16th

ed., 967.25, 967.26, 967.28,

and 995.20. AOAC International, Gaithersburg, Md.

Banič, S. 1964. A new enrichment medium for salmonellae. J. Hyg. Camb. 62:25-28.

Beckers, H. J., F. M. Van Leusden, and R. Peters. 1986. Comparison of Muller-

Kauffmann’s tetrathionate broth and modified Rappaport’s medium for isolation of Salmonella.

J. Food Saf. 8:1-9

Bohls, S. W., and L. H. Mattman. 1950. Use of tetrathionate broth in isolation of

salmonella, shigella, and paracolon bacilli. J. Lab. Clin. Med. 35:654-657.

Brehm-Stecher B, C. Young, L. A. Jaykus, and M. L. Tortorello. 2009. Sample

preparation: the forgotten beginning. J. Food Prot. 72:1774-1789.

Brest Nielsen, B., and K. Grunnet. 1977. A comparison of tetrathionate broth and

rappaport’s medium as enrichment media for Salmonella. Nord. Vet.-Med. 29:141-143.



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Centers for Disease Control and Prevention (CDC). 2011. Salmonella surveillance:

annual summary, 2009. Centers for Disease Control and Prevention, Atlanta, GA.

Collard, P., and M. Unwin. 1958. A trial of Rappaport’s medium. J. Clin. Pathol. 11:426-

427.

Foley, S. L., R. Nayak, I. B. Hanning, T. J. Johnson, J. Han, and S. C. Ricke. 2011.


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77:582-607.

Fricker, C. R., R. W. A. Girdwood, and D. Munro. 1983. A comparison of enrichment

media for the isolation of salmonellae from seagull cloacal swabs. J. Hyg. (Camb). 91:53-58.

Fricker, C. R., 1984. A comparison of methods for the isolation of salmonellae from

sewage sludge. Zentralbl Bakteriol. Mikrobiol. Hyg. B. 179:170-178.

Fricker, C. R., E. Quail, L. McGibbon, and R. W. A. Girdwood. 1985. An evaluation of

commercially available dehydrated Rappaport-Vassiliadis medium for the isolation of

salmonellae from poultry. J. Hyg. Camb. 95:337-344.

González-Gil, F., A. Le Bolloch, S. Pendleton, N. Zhang, A. Wallis, and I. Hanning.

2012. Expression of hilA in response to mild acid stress in Salmonella enterica is serovar and

strain dependent. J. Food Sci. 77:M292-M296.

Hammack, T. S., R. M. Amaguaña, G. A. June, P. S. Sherrod, and W. H. Andrews. 1999.

Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis

medium for the recovery of Salmonella spp. from foods with a low microbial load. J. Food Prot.

62:16-21.

Hammack, T. S., R. M. Amaguaña, and W. H. Andrews. 2001. Rappaport-Vassiliadis

medium for recovery of Salmonella spp. from low microbial load foods: collaborative study. J.

AOAC Int. 84:65-84.

141




Harvey, R. W. S., T. H. Price, and E. Xirouchaki. 1979. Comparison of selenite F,

Muller-Kauffmann tetrathionate and Rappaport’s medium for the isolation of salmonellas from

sewage-polluted natural water using a pre-enrichment technique. J. Hyg. 83:451-460.

Harvey, R. W. S., and T. H. Price. 1980. Salmonella isolation with Rappaport’s medium

after pre-enrichment in buffered peptone water using a series of inoculum ratios. J. Hyg. 85:125-

128.

Harvey, R. W. S., and T. H. Price. 1981. Comparison of selenite F, Muller-Kauffmann

tetrathionate and Rappaport’s medium for salmonella isolation from chicken giblets after pre-

enrichment in buffered peptone water. J. Hyg. Camb. 87:219-224.




Hooper, W. L., and H. P. Jenkins. 1965. An evaluation of Rappaport’s magnesium

chloride/malachite green medium in the routine examination of faeces. J. Hyg. (Camb). 63:491-

495.

Iveson, J. B., N. Kovacs, and W. M. Laurie. 1964. An improved method of isolating

salmonellae from contaminated desiccated coconut. J. Clin. Path. 17:75-78.

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Typhimurium in broth and chicken meat system. J. Food Saf. 31:462-471.

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broh with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage

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145

Table 3.1. List of components for each media broth used for non-enrichment and enrichment

studies.

Media Ingredient g/liter

Luria Bertani (LB) broth Tryptone 10.0

Yeast extract 5.0

Sodium chloride 10.0

Buffered peptone water

(BPW)

Peptone from casein 10.0

Sodium chloride 5.0

Disodium hydrogen phosphate dodecahydrate 9.0

Potassium dihydrogen phosphate 1.5

Tetrathionate (TT) Broth

Base

Yeast extract 2.0

Tryptose 18.0

Dextrose 0.5

D-Mannitol 2.5

Sodium desoxycholate 0.5

Sodium chloride 5.0

Sodium thiosulfate 38.0

Calcium carbonate 25.0

Brilliant green 0.01

Rappaport-Vassiliadis (RV)

R10 Broth

Pancreatic Digest of Casein 4.54

Sodium chloride 7.2

Monopotassium Phosphate 1.45

Magnesium chloride 13.4

Malachite green oxalate 0.036

146

Figure 3.1. Optical density at 600 nm for each strain from overnight cultures (16 h) on LB and

BPW broth prior to enrichment. When compared, OD values were higher for S. Typhimurium

and S. Kentucky when grown on LB broth; and values were similar for S. Enteritidis and S.

Heidelberg.

147

Figure 3.2. Optical density at 600 nm from each strain inoculated into 10 mL LB broth starting

on the first detectable time point. Salmonella growth is not detected until the 8 h.

148

Table 3.2. Average CFU/mL for each Salmonella serovar inoculated on Luria Bertani broth.

Time (h) Dilution S. Typhimurium S. Heidelberg S. Enteritidis S. Kentucky

0

Inoculum 2 2 1 1

10-1 BD

* BD* BD

* BD*

10-2 BD

* BD* BD

* BD*

2

Inoculum 24 27 27 45

10-1 3 2 3 2

10-2 BD

* BD* BD

* 1

10-3 BD

* BD* 1 BD

*

4

Inoculum TNTC+

TNTC+ TNTC

+ TNTC

+

10-1 104 101 55 TNTC

+

10-2 9 12 6 21

10-3 1 1 2 2

6

10-2

TNTC+ TNTC

+ TNTC

+ TNTC

+

10-3 85 41 84 160

10-4 10 3 6 10

10-5 2 BD

* BD* 2

8

10-4 TNTC

+ 64 239 TNTC

+

10-5 38 24 24 72

10-6 7 3 2 11

12

10-6 123 137 113 115

10-7 13 16 9 9

10-8 2 1 2 3

24 10

-7 25 23 27 22

10-8 2 2 1 3

TNTC+ (too numerous too count) - number of colonies over 250

BD* - below detection level. Detection level was estimated to be approximately 10 CFU/mL.

149

Table 3.3. Average CFU/mL for each Salmonella serovar inoculated on Rappaport –Vassiliadis

broth.


0 Inoculum 1 1 1 2

10-1 BD

* BD* BD

* BD*

2

Inoculum 1 2 1 2

10-1 BD

* BD* BD

* BD*

10-2 BD

* BD* 1 BD

*

4

Inoculum 3 7 2 27

10-1 BD

* 4 BD* 4

10-2 BD

* 1 BD* 1

10-3 BD

* BD* BD

* BD*

6

10-2 BD

* 2 BD* 4

10-3 BD

* BD* BD

* 1

10-4 BD

* BD* BD

* BD*

8

10-3 BD

* 11 BD* 11

10-4 BD

* 1 BD* 1

10-5 BD

* BD* BD

* BD*

10-6 BD

* BD* BD

* BD*

12

10-3 BD

* TNTC+ BD

* TNTC+

10-4 BD

* TNTC+ BD

* TNTC+

10-5 BD

* TNTC+ BD

* TNTC+

10-6 BD

* 20 BD* 17

24

10-2 9 TNTC

+ BD

* TNTC+

10-4 1 TNTC

+ BD

* TNTC+

10-6 BD

* 62 BD

* 103

TNTC+ (too numerous too count) - number of colonies over 250


150

Table 3.4. Average CFU/mL for each Salmonella serovar inoculated on Tetrathionate broth.


0 Inoculum 6 2 4 5

10-1 BD

* BD* BD

* BD*

2

Inoculum 5 9 6 7

10-1 BD

* 2 2 BD*

10-2 BD

* BD* BD

* BD*

4

Inoculum 2 13 17 18

10-1 BD

* 1 1 BD*

10-2 BD

* BD* BD

* BD*

6 Inoculum 19 32 26 20

10-2 1 1 1 1

8

Inoculum 20 45 28 106

10-2 BD

* 3 2 4

10-3 BD

* BD* BD

* BD*

12 10

-2 60 15 2 4

10-4 BD

* BD* BD

* BD*

24

10-2 230 28 2 130

10-4 4 1 BD

* 4

10-6 BD

* BD* BD

* BD*


151


Juliany Rivera Calo is the first author of the paper and completed at least 51% of the studies among

coauthors, which the title is “Salmonella spp. Growth Responses Comparisons in Selective and

Non-selective Enrichment Media Methods” in chapter 3.


152

8. Appendix


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163

CHAPTER 4

Antimicrobial Activity of Essential Oils against Salmonella Heidelberg Strains from

Multiple Sources





Fayetteville, AR

164

1. Abstract

Salmonella spp. is one of the more prominent foodborne pathogens that represent a major

health risk to humans. Salmonella serovar Heidelberg strains are increasingly becoming an

important public health concern, since they have been identified as one of the primary

Salmonella serovars responsible for human outbreaks. Over the years, Salmonella Heidelberg

isolates have exhibited higher rates of resistance to multiple antimicrobial agents compared to

other Salmonella serovars. Essential oils (EOs) have been widely used as alternatives to

chemical-based antimicrobials. In the current research, five EOs were screened to determine their

antimicrobial activity against 15 Salmonella Heidelberg strains from different sources. Oils

tested were R(+)-limonene, orange terpenes, cold compressed orange oil, trans-cinnamaldehyde

and carvacrol. EOs were stabilized in nutrient broth by adding 0.15% (w/v) agar. Tube dilution

assays and minimal inhibitory concentrations (MIC) were determined by observing any color

change in the samples. Carvacrol and trans-cinnamaldehyde completely inhibited the growth of

Salmonella Heidelberg strains, while R(+)-limonene and orange terpenes did not show any

inhibitory activity against these strains. Cold compressed orange oil only inhibited growth of two

of the strains exhibiting a MIC of 1%. No relationship was found between the activity of the EOs

tested and the sources of the strain. The use of all natural antimicrobials, such as EOs, offers the

potential for improving the safety of foods contaminated with S. Heidelberg, but there are strain

differences, which must be taken into account.

165

2. Introduction


United States. Annually, it is estimated that over 1 million Americans contract Salmonella

(Scallan et al., 2011). Yearly costs for Salmonella control efforts are estimated to be up to $14.6

billion (Scharff, 2010; Heithoff, et al., 2012). Salmonella is not only a public health concern due

to the significant number of cases per year, but also because many strains have developed

resistance to antimicrobial agents (Kim et al., 2005; Foley and Lynne, 2008; Bajpai et al., 2012)

due to the unrestricted use of antimicrobials in feeds and increased therapeutic use (Su et al.,

2004; Kim et al., 2005).

Salmonella enterica serovar Heidelberg (S. Heidelberg) ranks fourth among the top five

serovars associated with human infections and is responsible for causing an estimated 84,000

illnesses in the United States annually (CDC, 2008; FDA, 2009; Foley et al., 2011; Han et al.,

2011). This serovar is one of the most commonly isolated in the United States and Canada from

clinical cases of salmonellosis, retail meats and livestock (Zhao et al., 2008; Hur et al., 2012).

While most Salmonella infections are self-limiting and become resolved within a few days, S.

Heidelberg tends to cause a significantly higher percentage of invasive infections (Vugia et al.,

2004; Han et al., 2011). As a result, antimicrobial therapy is often necessary, making

antimicrobial resistance a significant concern. Because of the tendency of this serovar to cause

severe extra-intestinal infections (Wilmshurst and Sutcliffe, 1995) such as myocarditis and

septicemia (Vugia et al., 2004), the occurrence of S. Heidelberg MDR strains is of extreme

importance. Salmonella Heidelberg strains exhibiting antimicrobial resistance have been isolated

from humans, retail meats and food animals (Logue et al., 2003; Nayak et al., 2004; Kaldhone et

al., 2008; Zhao et al., 2008; Lynne et al., 2009; Oloya et al., 2009; Han et al., 2011). Studies have

166

shown poultry-associated Salmonella Heidelberg strains to harbor IncFIB, IncA/C, IncH2, and

IncI1 plasmids, which may contain genes that confer resistance to several antibiotics such as

tetracycline, kanamycin, streptomycin, and sulfonamides (Han et al., 2012). Because S.

Heidelberg is responsible for causing more invasive infections when compared to other serovars,

it is important to monitor its prevalence and resistance. Novel and unique intervention strategies

are a priority to reduce or eliminate its presence.

Aromatic plants and their extracts have been examined for their effectiveness in food

safety and preservation applications (Fisher and Phillips, 2008). Essential oils for example,

exhibit antimicrobial properties that may make them suitable alternatives to antibiotics (Chaves

et al., 2008). These potential attributes and an increasing demand for natural medicinal treatment

options have brought the attention to the use of EOs as potential alternative antimicrobials

(Fisher and Phillips, 2008; Solórzano-Santos and Miranda-Novales, 2012). Essential oils derived

as by-products of the citrus industry have been screened for antimicrobial properties against

common foodborne pathogens and several have been shown to possess antimicrobial properties

(Dabbah et al., 1970). This study reports the results of testing various EOs against several

Salmonella Heidelberg strains from different sources using tube dilution assays.


3.1. DNA extraction

One colony of each of the 15 S. Heidelberg strains were inoculated in 5 mL Luria Bertani

(LB) broth and incubated at 37°C for 16 h, shaking at 190 rpm using a C76 Water Bath Shaker

(New Brunswick Scientific, Edison, NJ, USA). One mL of bacterial cells was transferred to a

microcentrifuge tube, samples were centrifuged at 14,000 x g for 10 min and supernatant was

167

discarded. Genomic DNA was extracted using the Qiagen DNeasy Blood Tissue kit (Qiagen,

Valencia, CA, USA) according to the manufacturer’s instruction. The isolated genomic DNA

concentration and purity were measured using a NanoDrop ND-1000 (Thermo Scientific,

Wilmington, DE, USA) and DNA samples were subsequently stored at -20°C until used.

3.2. Confirmatory PCR for S. Heidelberg strains

The conventional PCR assay was conducted using MJ Mini Personal Thermal Cycler

(Bio-Rad, Hercules, CA, USA). Salmonella Heidelberg specific primers were used to confirm

that all 15 strains belong to the serovar Heidelberg (Table 4.1). A 25 μL total reaction volume

composed of 1 μL of template DNA, 1 μL of each primer (IDT, Coralville, IA, USA), 12.5 μL of

SYBR Green (Cambrex Bioscience, Walkersville, MD, USA), and 9.5 μL of DNase-RNase free

water. The PCR conditions consisted of pre-denaturation at 94°C for 5 min, then 35 cycles of

denaturation at 94°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 30 s, with a final

extension cycle at 72°C for 5 min. The PCR products were confirmed on 1.5% of agarose gel

and visualized on a transilluminator (Bio-Rad, Hercules).

3.3. Essential oils and cultures

All of the EOs tested, R(+)-limonene, orange terpenes, cold compressed orange oil, trans-

cinnamaldehyde and carvacrol, were obtained from Sigma Aldrich (St. Louis, MO, USA).

Fifteen S. Heidelberg strains from different sources were examined (Table 4.2). One colony of

each strain was inoculated into Luria-Bertani broth and incubated for 18 h at 42°C, shaking at

190 rpm. This experiment was repeated growing S. Heidelberg strains at 37°C.

168

3.4. Modified tube dilution assay

To maintain the EOs in a homogeneous mixture, 0.15% agar was added to nutrient broth

(NB), and boiled for 1 min before autoclaving. When the media reached room temperature

triphenyl tetrazolium chloride (TTC) was added at a rate of 1mL/100mL to act as a growth

indicator. As previously described by O’Bryan et al., (2008), serial dilutions were made by

placing 10 mL of the NB with 0.15% agar (NBA) in the first tube and 5 mL in the remaining

tubes for a total of four tubes. One hundred μL of oil was added to the 1st (10 mL) tube for an

initial concentration of 10 μL/mL. Serial dilutions were done by transferring 5 mL of the

emulsion to the next tube, the procedure was repeated for a total of four dilutions. Five mL were

removed from the last tube and discarded so that all tubes had the same volume (5 mL). All tubes

were inoculated with 50 μL of an overnight culture of each S. Heidelberg strain. All tubes were

incubated for 24 h at 37°C. A change in color from light yellow to pink/red indicated growth.

The MIC was determined to be the lowest concentration of EOs that showed no growth, no color

in the medium.


MIC tests were repeated as three independent experiments. Mean ± standard deviation

was conducted using JMP Pro Software Version 11.0 (SAS Institute Inc., Cary, NC).


We used S. Heidelberg specific primers to confirm that all 15 strains used in this study

(Table 4.1) belong to S. Heidelberg (Figure 4.1). Minimum inhibitory concentrations (Table 4.3)

were determined by observing the tubes for any color change to pink or red. When S. Heidelberg

169

strains were grown at 42°C, results showed that trans-cinnamaldehyde (Figure 4.2) and carvacrol

(Figure 4.3) exhibited MICs of 131 μg/mL and 122 μg/mL respectively, by completely inhibiting

the growth of each S. Heidelberg strain, while orange terpenes (Figure 4.4) and R-(+)-limonene

(Figure 4.5) did not exhibit any inhibitory activity against any of these strains. Cold compressed

orange oil only inhibited growth on two of the strains (945 and 114), with MIC’s of 843 μg/mL

(Figure 4.6). No relationship was observed between the source of the strain and the EOs tested.

Similar results were obtained when strains were grown at 37°C, although under this condition

cold compressed orange oil showed no antimicrobial activity for all strains, while at 42°C it

inhibited strains 945 and 114 with an MIC of 843 μg/mL. Temperature did not seem to have an

impact in terms of the effectiveness of each EOs as an antimicrobial.

Studies have shown the existence of differences between serovars as well as strains

within the same serovar of Salmonella enterica. In their research, González et al., (2012)

compared hilA gene expression in response to acid stress different Salmonella serovars and

strains. Results showed that there are serovar and strain differences in virulence gene expression

and acid tolerance; regulation of hilA showed to be serovar and strain dependent as well as

dependent on acid type (González et al., 2012). In an extensive genome analysis comparison

between UK-1 and other S. Typhimurium strains, Luo et al., (2012) reported that virulence

factors pertaining to one strain might increase or decrease virulence when present in a different

strain. Shah et al., (2011) reported that isolates from S. Enteritidis that have been recovered from

poultry or poultry environment are not equally pathogenic, nor do they have similar

invasiveness.

Previous research has shown that different EOs confer antimicrobial activity pre and post

harvest on Salmonella. The antimicrobial effect of EOs has been reported to be concentration

170

dependent (Sivropoulou et al. 1996). Ravishankar et al., (2010) reported that at a 0.2%

concentration, antimicrobials, carvacrol and cinnamaldehyde completely inactivated the

antibiotic-resistant and -susceptible isolates of S. enterica. Zhou et al., (2007) investigated the

antimicrobial activity of cinnamaldehyde, thymol and carvacrol individually and in combination

against S. Typhimurium, and reported the lowest concentrations of cinnamaldehyde, thymol and

carvacrol inhibiting the growth of S. Typhimurium significantly were 200, 400 and 400 mg/L,

respectively. When combined, cinnamaldehyde/thymol, cinnamaldehyde/carvacrol and

thymol/carvacrol showed the concentration of cinnamaldehyde, thymol and carvacrol could be

decreased from 200, 400 and 400 mg/L to 100, 100 and 100 mg/L, respectively (Zhou et al.,

2007). In this project we used cinnamaldehyde in its trans isomer, which is the form that is

present as a major component of bark extract of cinnamon (Kollanoor Johny et al., 2008).

Using the MIC method to study the effectiveness of orange essential oils against different

Salmonella serovars, O’Bryan et al., (2008) reported that it appeared not to be a significant

difference in response between strains of the same serovar or between different Salmonella

serovars tested. From seven citrus EOs tested in their research, three of them: high purity orange

terpenes, d-limonene and terpenes from orange essence, showed antimicrobial activity against

Salmonella. Despite the fact that limonene is one of the most well known and characterized of

the EOs from citrus products (Dabbah et al., 1970; Caccioni et al., 1998), which can exert potent,

broad-spectrum antimicrobial activity (Di Pasqua et al., 2006), this essential oil, in the form of

R-(+)-limonene did not exhibit any antimicrobial activity in this study (Figure 4.5).

Essential oils have also been studied to have antimicrobial activity against Salmonella

when used post harvest. In their research, Alali et al., (2013) determined the effect of non-

pharmaceuticals (a blend of organic acids, a blend of EOs, lactic acids, and a combination of

171

levulinic acid and sodium dodecyl sulfate) on weight gain, feed conversion ratio, mortality of

broilers and their ability to reduce colonization and fecal shedding of Salmonella Heidelberg.

Their results showed that the broilers that received the EOs had significantly increased weight

gain and mortality was lower compared to other treatments. Salmonella Heidelberg

contamination in crops was significantly lower in challenged and unchallenged broilers that

received EOs and lactic acids in drinking water, when compared to other treatments (Alali et al.,

2013). Results of our study shows that some EOs could be used as an antimicrobial agent against

the foodborne pathogen Salmonella. Further studies will be needed to confirm the antimicrobial

effect of these EOs in vivo.

5. Acknowledgments

We thank Dr. Steven L. Foley, Division of Microbiology, NCTR, U.S. Food and Drug

Administration, Jefferson, AR, and Dr. W. Florian Fricke, University of Maryland, School of

Medicine, Baltimore, MD for providing Salmonella Heidelberg strains used in this study. Dr.

Young M. Kwon, Poultry Science Department, University of Arkansas, Fayetteville, AR for

providing the EOs for the study. We also thank the Department of Food Science, University of

Arkansas, Fayetteville, AR for providing graduate stipend support.

172

6. References

Alali, W. Q., C. L. Hofacre, G. F. Mathis, G. Faltys, S. C. Ricke, and M. P. Doyle. 2013.

Effect of non-pharmaceutical compounds on shedding and colonization of Salmonella enterica

serovar Heidelberg in broilers. Food Control. 31:125-128.

Bajpai, V. K., K.-H. Baek, and S. C. Kang. 2012. Control of Salmonella in foods by

using essential oils: a review. Food Res. Int. 45:722-734.

Caccioni, D. R. L., M. Guizzardi, D. M. Biondi, A Renda, and G. Ruberto. 1998.

Relationship between volatile components of citrus fruit essential oils and antimicrobial action

on Penicillium digitatum and Penicillium italicum. Int. J. Food Microbiol. 43:73-79.

Chaves, A. V., M. L. He, W. Z. Yang, A. N. Hristov, T. A. McAllister, and C. Benchaar.

2008. Effects of essential oils on proteolytic, deaminative and methanogenic activities of mixed

ruminal bacteria. Can. J. Anim. Sci. 88:117-122.

Centers for Disease Control and Prevention (CDC). 2008. Salmonella Surveillance:

Annual Summary, 2006. Centers for Disease Control and Prevention.

Dabbah, R., V. M. Edwards, and W. A. Moats. 1970. Antimicrobial action of some citrus

fruit oils on selected food-borne bacteria. Appl. Microbiol. 19:27-31.

Di Pasqua, R., N. Hoskins, G. Betts, and G. Mauriello. 2006. Changes in membrane fatty

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176

Table 4.1. Salmonella Heidelberg specific primer pair used in this study.

Primer Target Sequence PCR product

size (bp)

Reference

SH_SHP-2f Salmonella Heidelberg 5’-GCATA GTTCC AAAGC

ACGTT-3’ 180 In this study

SH_SHP-1r 5’-GCTCA ACATA AGGGA

AGCAA-3’

177

Table 4.2. Salmonella Heidelberg strains used in this study

Strain Source Reference

ARI-14 poultry products In this study

SL 476 ground turkey Fricke et al., 2011

SL 486 human Fricke et al., 2011

692 chicken egg house Lynne et al., 2009







130 chicken Lynne et al., 2009





178

Figure 4.1. Gel electrophoresis of PCR products to confirm Salmonella Heidelberg strains. Lane

1: DNA ladder, lanes 2 to16: Salmonella Heidelberg strains, and NC: negative control.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 NC

180bp

179

Table 4.3. Minimum inhibitory concentrations (in μg/mL) of five different EOs against

Salmonella Heidelberg strains.

Strain Essential oils tested

carvacrol

R-(+)-

limonene

orange

terpenes orange oil

trans-

cinnamaldehyde

SL 486 122 ± 0 No effect No effect No effect 131 ± 0

SL 476 122 ± 0 No effect No effect No effect 131 ± 0

ARI-14 122 ± 0 No effect No effect No effect 131 ± 0

692 122 ± 0 No effect No effect No effect 131 ± 0

945 122 ± 0 No effect No effect 843 ± 0 131 ± 0

114 122 ± 0 No effect No effect 843 ± 0 131 ± 0










180


Figure 4.2. Modified tube dilution assay for trans-cinnamaldehyde. This EO exhibited MICs of

131 μg/mL for all 15 strains, inhibiting their growth. Tubes 1 through 4 represent treatment with

different EOs concentrations. Tube 5 contains NAB with the correspondent Salmonella

Heidelberg strain (positive control), and tube 5 contains NAB with trans-cinnamaldehyde

(negative control).

1 2 3 4 5 6

181


Figure 4.3. Modified tube dilution assay for carvacrol. For all 15 strains carvacrol exhibited

MICs of 122 μg/mL. Tubes 1 through 4 represent treatment with different EOs concentrations

Tube 5 contains NAB with the correspondent Salmonella Heidelberg strain (positive control),

and tube 5 contains NAB with carvacrol (negative control).

1 2 3 4 5 6

182


Figure 4.4. Modified tube dilution assay for orange terpenes. This EO did not exhibit any

antimicrobial activity on these strains. Tubes 1 through 4 represent treatment with different EOs

concentrations. Tube 5 contains NAB with the correspondent Salmonella Heidelberg strain

(positive control), and tube 5 contains NAB with orange terpenes (negative control).

1 2 3 4 5 6

183


Figure 4.5. Modified tube dilution assay for R-(+)-limonene. No antimicrobial activity was

observed for any of the strains. Tubes 1 through 4 represent treatment with different EOs

concentrations. Tube 5 contains NAB with the correspondent Salmonella Heidelberg strain

(positive control), and tube 5 contains NAB with R-(+)limonene (negative control).

1 2 3 4 5 6

184


Figure 4.6. Modified tube dilution assay for cold compressed orange oil. Orange oil did not

exhibit any antimicrobial activity for 13 of the Salmonella Heidelberg strains, but strains 945

(human) and 114 (cattle) showed an MIC of 843 μg/mL. Tubes 1 through 4 represent treatment

with different EOs concentrations. Tube 5 contains NAB with the correspondent Salmonella

Heidelberg strain and no EOs (positive control), and tube 5 contains NAB with orange oil, no S.

Heidelberg (negative control).

1 2 3 4 5 6

1 2 3 4 5 6

185



among coauthors, which the title is “Antimicrobial Activity of Essential Oils Against

Salmonella Heidelberg Strain from Multiple Sources” in chapter 4.


186

8. Appendix


187


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CHAPTER 5

Salmonella spp. Adhesion and Invasion Comparisons to Caco-2 and HD11 Cell Lines





Fayetteville, AR

198

1. Abstract

Salmonella enterica are important facultative intracellular pathogens that cause

gastroenteritis in humans. Four different strains from three of the most predominant Salmonella

serovars in poultry, Typhimurium, Enteritidis, and Heidelberg were studied. Agar disc diffusion

test and tube dilution assays were used to determine gentamicin susceptibility of S. Typhimurium

ATCC 14028 and S. Heidelberg ARI-14. Both strains were susceptible to the presence of

gentamicin disc on the plate. Minimum inhibitory concentration (MIC) of gentamicin was 1% for

the strains tested. The adhesion and invasion abilities of these strains were determined using two

different cell lines: HD11, a chicken macrophage cell line and Caco-2, a human intestinal

epithelial cell line. Results showed that attachment percentages for each Salmonella strain were

higher than the ability of the strain to invade the cells. For Caco-2 cells, Salmonella

Typhimurium and Salmonella Heidelberg showed similar attachment percentages. For HD11

cells, attachment percentages were lower than for Caco-2 but Salmonella exhibited higher

percentages for invasion, presumably due to phagocytosis by HD11. Salmonella Enteritidis

exhibited lower percentages for adhesion and invasion in HD11. The type of cell line and the

different serovars studied appeared to be a factor for the differences in adhesion and invasion.

Understanding the ability and mechanisms of this pathogen to attach and invade different cell

lines could be helpful for the reduction of Salmonella infections.

199

2. Introduction

Infections by Salmonella enterica subspecies enterica are one of the leading causes of

food borne gastroenteritis in humans (WHO, 2007). Annually, it is estimated that over 1 million

Americans contract Salmonella (Scallan et al., 2011). Yearly costs for Salmonella control efforts

are estimated to be up to $14.6 billion (Scharff, 2010; Heithoff, et al., 2012). Many of its

serovars have been associated with the consumption of poultry products, one of the main

reservoirs of Salmonella (CDC, 2009). Salmonella Enteritidis, S. Typhimurium and S.

Heidelberg are the three most frequent serovars recovered from humans and isolated from

poultry (Foley et al., 2011; Finstad et al., 2012; Howard et al., 2012).

Some of the genes necessary for invasion of intestinal epithelial cells and induction of

intestinal secretory and inflammatory responses are encoded in Salmonella Pathogenicity Island

1 (SPI-1) (Watson et al., 1995; Hur et al., 2012). Salmonella Pathogenicity Island 2 (SPI-2) is

key for the establishment of systemic infection beyond the intestinal epithelium; it also encodes

essential genes for intracellular replication (Cirillo et al., 1998; Ohl and Miller, 2001; Hur et al.,

2012). Adhesion of Salmonella to the intestinal epithelial surface is an important first step in

pathogenesis and is central to its colonization of the intestine. Once Salmonella is attached to the

intestinal epithelium, normally it expresses a multiprotein complex, known as T3SS, that

facilitates endothelial uptake and invasion (Foley et al., 2008, 2013; Winnen et al., 2008). This

T3SS is associated with SPI-1, which contains virulence genes involved in Salmonella adhesion,

invasion and toxicity (Foley et al., 2013). The human intestinal Caco-2 cell line has been

extensively used over the past twenty years as a model of the intestinal barrier. The parental cell

line, originally obtained from a human colon adenocarcinoma, goes through a spontaneous

differentiation process that leads to the formation of a monolayer of cells, expressing several

200

morphological and functional characteristics of the mature enterocyte (Sambuy et al., 2005).

HD11 cells are a macrophage-like immortalized cell line derived from chicken bone marrow and

transformed with the avian myelocytomatosis type MC29 virus (Beug et al., 1979). In this

experiment, adhesion and invasion of different Salmonella serovars to Caco-2 and HD11 cell

lines were studied. Several pathogens and host factors may be an important key to determine the

mechanisms for the differences in Salmonella responses within different cell types (Bueno et al.,

2012; Foley et al., 2013). Understanding the ability and mechanisms of this pathogen to attach

and invade different cell lines could be helpful for the reduction of Salmonella infections.


3.1. Bacterial strains for antibiotic susceptibility testing

One colony of S. Typhimurium ATCC 14028, S. Heidelberg ARI-14 and E. coli 25922

(negative control) was inoculated into 5 mL of Luria Bertani (LB) Broth (Alfa Aesar, Ward Hill,

MA, USA) and incubated for 16 h at 37°C, 190 rpm shaking.

3.2. Antibiotic susceptibility testing

Susceptibility of Salmonella Typhimurium ATCC 14028 and S. Heidelberg ARI-14 to

gentamicin was determined by two methods, the agar disc diffusion test and the modified tube

dilution assay. For the agar disc diffusion test, a sterile cotton swab was dipped into the

overnight bacterial culture and gently squeezed against the tube to remove any excess fluid. The

cotton swab subsequently streaked on Mueller Hinton Agar (BD Biosciences, Franklin Lakes,

NJ, USA) plate at different angles to provide even growth. One 6-mm gentamicin paper disc

(Becton Dickson, Sparks, MD, U.S.A.) was aseptically placed on the center of each agar plate,

201

followed by incubation for 16 to 24 h at 37°C. Zone inhibition diameters were measured for

each plate and averages were calculated. Plates were streaked per triplicate and each experiment

was repeated three times.

The minimum inhibitory concentration for each bacterial strain was determined using a

modified tube dilution assay method. Serial dilutions were made by placing 10 mL of nutrient

broth (EMD Millipore, Billerica, MA, USA) in the first tube and 5 mL in the remaining tubes for

a total of five tubes. One hundred μL of gentamicin (Life Technologies, Grand Island, NY, USA)

at a stock concentration of 500 μg/mL was added to the 1st (10 mL) tube and serial dilutions were

done by transferring 5 mL of the mixture to the next tube, the procedure was repeated for a total

of five dilutions. Five μL were removed and discarded from the last tube so that all tubes had the

same volume (5 mL). All tubes were inoculated with 50 μL of the bacterial overnight culture.

Fifty microliters of triphenyl tetrazolium chloride (TTC) was added to each tube to act as a

growth indicator. All tubes were incubated for 24 h at 37°C. A change in color from light yellow

to pink/red indicated growth. The MIC was determined to be the lowest concentration of

gentamicin that showed no growth, no color in the medium. After the 24 h incubation, 100 μL of

each dilution for each bacterial strain was inoculated into Mueller-Hinton agar (BD Biosciences)

plates to determine CFU/mL and confirm MIC results. Plates were done per triplicate and each

experiment was repeated three times.

3.3. Cell cultures

Human epithelial (Caco-2) cells and chicken macrophage (HD11) cells were maintained

in HyClone™ Classical Liquid Media: Dulbeccos Modified Eagles Medium (MEM), High

Glucose (Thermo Scientific, Logan, UT, USA) with 10% fetal bovine serum (FBS) and non

202

essential amino acids (NEAA) and grown routinely in a 75 cm2 flask at 37°C in a 5% CO2

incubator (New Brunswick, Eppendorf, Enfield, CT, USA). Once the cells in flask were

approximately 80% confluent, they were treated with 0.25% Trypsin-EDTA (Life Technologies)

to release the attached cells and new stock cultures were inoculated with 104

cells per mL. For

the adhesion and invasion assays, 104 Caco-2 and HD11 cells per mL were inoculated in 24-well

tissue culture plates (Greiner Bio-One, Monroe, NC, USA) and incubated at 37°C in a 5% CO2

incubator until a semi-confluent monolayer was obtained.

3.4. Bacterial cultures for adhesion and invasion assays

Salmonella strains used for this experiment are listed in Table 5.1. One colony of each

strain, S. Typhimurium ATCC 14028, S. Typhimurium UK-1, S. Heidelberg ARI-14 and S.

Enteritidis ATCC 13076, was inoculated on 8 mL LB broth and incubated for 16 to 18 h at 37°C.

3.5. Adhesion assays

Cells were enumerated in three representative wells by trypsinizing each well with 0.3

mL trypsin-EDTA, and incubating at 37°C in a 5% CO2 incubator for 5 min and adding 0.7 mL

D10F. Overnight bacterial cultures were washed three times with PBS (137 mM NaCl, 2.7 mM

KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). Bacterial cells were diluted to an MOI ratio of

10:1 (106

Salmonella: 105 HD11 or Caco-2 cells). Washed bacteria were diluted 10

-6 with PBS

and 100 μL were plated on LB agar plates for determining CFU/mL. The diluted bacteria were

added to the cell lines, Caco-2 or HD11, and plate was incubated at 37°C in a 5% CO2 incubator

(Thermo/Forma Scientific, Marietta, OH, USA) for 2 h. Cells were subsequently washed three

times with cell-PBS (137 mM NaCl, 5.4 mM KCl, 3.5 mM Na2HPO4, 4.4 mM NaH2PO4, 11 mM

203

glucose, pH 7.2), and treated with 1 mL of 0.1% Triton X-100 in cell-PBS. The plate was

incubated for 10 min, at 37°C in a 5% CO2 incubator. The disrupted cells were collected, serially

diluted and plated on LB agar plates, in duplicate, to determine adhesion percentage. Plates were

incubated at 37°C for 16 h. All LB agar plates to determine CFU/mL were inoculated per

duplicate. Each strain was tested in triplicate in three independent experiments.

3.6. Invasion assays

Cells were enumerated in three representative wells by trypsinizing each well with 0.3

mL trypsin-EDTA, and incubating at 37°C in a 5% CO2 incubator for 5 min and adding 0.7 mL

D10F. Overnight bacterial cultures were washed three times with PBS. Bacterial cells were

diluted to obtain an MOI ratio of 10:1 (Salmonella: animal cells). Washed bacteria were diluted

10-6

with PBS and 100 μL were plated on LB agar plates for determining CFU/mL. The diluted

bacteria was added to the cell lines, Caco-2 or HD11, and plate was incubated at 37°C in a 5%

CO2 incubator for 2 h. Cells were subsequently washed three times with cell-PBS and treated

with 1 mL DMEM with 100 μg/mL gentamicin per well to kill cell-adherent extracellular

bacteria. Plate was incubated for 2 h, at 37°C in a 5% CO2 incubator. Cells were then washed

three times with cell-PBS and treated with 1 mL of 0.1% Triton X-100 in cell-PBS. The plate

was incubated for 10 min, at 37°C in a 5% CO2 incubator. Serial dilutions of suspensions were

made in PBS and inoculated onto LB agar plates, in duplicate, to determine the number of

organisms that survived treatment with gentamicin and hence had invaded the Caco-2 or HD11

cells. Plates were incubated at 37°C for 16 h. Each strain was tested in triplicate in three

independent experiments.

204


All statistical analyses were conducted using JMP Pro Software Version 11.0 (SAS

Institute Inc., Cary, NC). Mean ± standard deviation was calculated for each antibiotic

susceptibility test. One-way ANOVA and Tukey-Kramer HSD test were performed on the

adhesion and invasion percentages of each bacterial strain to Caco-2 and HD11 cells. Statistical

significance was established at P < 0.05.


The disc diffusion assay results are presented in Table 5.2. Salmonella Heidelberg ARI-

14 and S. Typhimurium ATCC 14028 produced an average inhibition zone of 20.7 ± 1.1 and

20.7 ± 0.1 respectively. Escherichia coli strain 25922 used as a control, showed an inhibition

zone of 19.0 ± 0. All three strains appeared to be susceptible to the presence of gentamicin

(Figure 5.1). Results from the tube dilution method are listed in Table 5.3. For all three strains,

the MIC of gentamicin was 500 μg/mL; no change in color was observed (Figures 5.2 to 5.4). To

further confirm results from this method, CFU/mL was calculated for each strain. Contrary to

what was observed on the tubes, where there was no change in color that should have indicated

the presence of bacteria in the media, growth was observed on the agar plates for all three strains

after the third dilution (0.25%) (Figure 5.5). Based on the CFU/mL the MIC of gentamicin at

which no growth was observed for any of the strains was 250 μg/mL. Minimum inhibitory

concentrations results provide confirmation for using gentamicin during invasion assays to kill

cell-adherent extracellular bacteria. Andrews (2001) published expected MIC ranges for

determining the susceptibility of several bacteria to a wide selection of antibiotics, along with a

list of appropriate controls that must be included when determining MIC. The suggested MIC

205

range for Enterobacteriaceae susceptibility against gentamicin was 0.03 to 128 mg/L using E.

coli 25922 as a control (Andrews, 2001). Shah et al (2011) studied cell invasion of several

poultry-associated Salmonella Enteritidis isolates and they reported a MIC of gentamicin of

<0.125 μg for all the isolates used in the study.

The ability of several Salmonella strains from different serovars to attach and invade two

cell lines: Caco-2, a human intestinal epithelial cell line; and HD11, a chicken macrophage cell

line and was studied. As expected, attachment percentages for each Salmonella strain were

higher than the ability of the strain to invade the cells. Results showed that S. Heidelberg and S.

Typhimurium exhibited adhesion percentages of 28.8 ± 6.37 and 18.1 ± 6.25 respectively, to

Caco-2 cells (Table 5.4). In contrast, the ability of these strains to invade Caco-2 cells was lower,

1.37 ± 0.25 for S. Heidelberg and 1.52 ± 0.02 for S. Typhimurium (Table 5.4). The underlying

mechanisms used by intracellular pathogens such as Salmonella to penetrate the host epithelium

are not well understood. As a result, researchers have used cultured mammalian cells as in vitro

models to study interaction and internalization of Salmonella (Gianella et al., 1973; Finlay et al.,

1988; Durant et al., 1999). Invasion in cultured epithelial cells is commonly used to measure the

pathogenicity of Salmonella (van Asten et al., 2000, 2004; Shah et al., 2011). Previous studies

have reported that Salmonella invasion into cultured mammalian cells can be influenced by

several environmental stimuli such as osmolarity (Galán and Curtis, 1990; Tartera and Metcalf,

1993) carbohydrate availability (Schiemann, 1995), and oxygen availability (Ernst et al., 1990;

Lee and Falkow, 1990; Francis et al., 1992). In their research, Durant et al., (1999) reported that

Salmonella can encounter high concentrations of short-chain fatty acids (SCFA) in the lower

regions of the gastrointestinal tract, this in addition to changes in pH, oxygen tension, and

osmolarity. Shah et al., (2011) reported that in cultured Caco-2 cells, isolates with high

206

invasiveness were able to invade and/or survive in significantly higher numbers within chicken

macrophage cells than other isolates with low invasiveness.

For HD11 cells, attachment percentages were higher than for Caco-2 (Table 5.5), and

Salmonella exhibited higher percentages for invasion, ranging from 2.9 to 17.6%. Two other

strains, S. Typhimurium UK-1 and S. Enteritidis ATCC 13076, were used for experiments with

HD11 cells. From all strains, Salmonella Typhimurium strain UK-1 invaded HD11 cells with

high percentages, 17.6 ± 3.29, which is not surprising given the high virulence of this strain.

Salmonella Typhimurium UK-1, (UK stands for universal killer) is a chicken-passaged isolate of

a highly virulent strain that was originally isolated in 1991 from an infected horse (Curtis et al.,

1991). Invasion results from Salmonella Typhimurium ATCC 14028 (9.3 ± 0.57) and UK-1

(17.6 ± 3.29) to HD11 cells demonstrate that there are strain differences among the same

serovar; different strains can have different invasiveness and virulence. Luo et al., (2012)

conducted an extensive genome analysis comparison between UK-1 and other S. Typhimurium

strains and reported that virulence factors pertaining to one strain may increase or decrease

virulence when are found present in a different strain (Luo et al., 2012). Once the polymorphic

genomic regions of the strains are identified and analyzed, even highly similar strains of S.

Typhimurium could be differentiated (Luo et al., 2012).

From all three serovars studied, Salmonella Enteritidis showed the lowest adhesion and

invasion percentage, 2.9 ± 1.48. In their research, He et al., (2012) compared Salmonella cell

invasion and intracellular survival of five different poultry-associated serovars (S. Heidelberg, S.

Enteritidis, S. Typhimurium, S. Senftenberg, and S. Kentucky). Their results showed that

compared to the other four serovars, S. Enteritidis was more resistant to intracellular killing,

leading them to believe that the intracellular survival ability of this serovar may be related with

207

systemic invasion in chickens (He et al., 2012). In their research, Shah et al., (2011) tested the

ability of S. Enteritidis isolates with low and high invasiveness to invade chicken macrophage

HD11 cells. Results showed that invasion percentages of 4.88 ± 0.13 and 6.07 ± 0.08 for isolates

with low and high percentages respectively (Shah et al., 2011). These results are comparable to

our findings of low invasiveness of S. Enteritidis. Matulova et al., (2012) also reported lower

invasion after pre-treating the HD11 cells with avidin prior infection with S. Enteritidis. Shah et

al., (2011) suggested that not all isolates of S. Enteritidis recovered from poultry might be

equally pathogenic or have similar potential to invade cells; and that S. Enteritidis pathogenicity

is related to both the secretion of Type III secretion system effector proteins and motility. Saeed

et al., (2006) reported that compared to isolates of S. Enteritidis recovered from chicken ceca,

isolates recovered from eggs or from human clinical cases showed a greater adherence and

invasiveness of chicken ovarian granulosa cells.

In conclusion, the type of cell line and the different serovars studied appeared to be a

factor for the differences in adhesion and invasion. Further studies should be conducted in order

to understand the properties that make some serovars more able to attach and invade the cells

than others. Understanding the ability and mechanisms of this pathogen to attach and invade

different cell lines could be helpful for the reduction of Salmonella infections.

5. Acknowledgments

We thank Dr. Peter Rubinelli, Center for Food Safety, University of Arkansas,

Fayetteville, AR, for his training and insights in conducting adhesion and invasion assays. We

also thank the Department of Food Science, University of Arkansas, Fayetteville, AR for

providing graduate stipend support to Juliany Rivera Calo.

208

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212

Table 5.1. Salmonella strains used in this study.

Adhesion and Invasion to Caco-2 cells Adhesion and Invasion to HD11 cells

Salmonella Typhimurium ATCC 14028 Salmonella Typhimurium ATCC 14028

Salmonella Heidelberg ARI-14 Salmonella Typhimurium UK-1

Salmonella Heidelberg ARI-14

Salmonella Enteritidis ATCC 13076

213

Table 5.2. Zones of inhibition (in millimeters) of gentamicin against S. Heidelberg and S.

Typhimurium. (mean ± standard deviation).

Strain Zone Diameter

(mm) Susceptibility

S. Heidelberg ARI-14 20.7 ± 1.1 Susceptible

S. Typhimurium ATCC 14028 20.7 ± 0.9 Susceptible

E. coli 25922 (negative control) 19.0 ± 0 Susceptible

214


Figure 5.1. Antibacterial activity of gentamicin against strains of S. Typhimurium, S. Heidelberg

and E. coli. All three strains studied showed to be sensitive against gentamicin.

215

Table 5.3. MIC of gentamicin against strains of S. Heidelberg, S. Typhimurium, and E. coli

examined.

Bacterial strain MIC (μg/mL)

S. Heidelberg ARI-14

500 ± 0

S. Typhimurium ATCC 14028

500 ± 0

E. coli 25922 500 ± 0

216


Figure 5.2. Gentamicin minimum inhibitory concentration against Salmonella Typhimurium

ATCC 14028. No change in color was observed for any of the dilutions treated with gentamicin.

217


Figure 5.3. Gentamicin minimum inhibitory concentration against Salmonella Heidelberg ARI-

14. No change in color was observed for any of the dilutions treated with gentamicin.

Negative control:

no gentamicin

218


Figure 5.4. Gentamicin minimum inhibitory concentration against Escherichia coli 25922. No

change in color was observed for any of the dilutions treated with gentamicin. Tube labeled as

NB: nutrient broth (positive control, no bacteria).

219

a)

b)

c)


Figure 5.5. MIC of gentamicin based on CFU/mL for each strain. The minimum inhibitory

concentration for all three bacterial cultures showed to be 0.5% of the stock gentamicin

concentration (500 μg/mL). a) Salmonella Typhimurium ATCC 14028, b) Salmonella

Heidelberg ARI-14, and c) Escherichia coli 25922.

220

Table 5.4. Salmonella Typhimurium and S. Heidelberg adhesion and invasion to human epithelial, Caco-2, cells.

Bacterial strain Log

Dilution

# of colonies # bacteria

added

# bacteria adhering % Adhesion

S. Typhimurium

ATCC 14028

4 17.67 ± 2.31 9.83x106 ± 0 1.78x10

6 ± 0.25 18.1 ± 6.25

a

S. Heidelberg

ARI-14

4 26.0 ± 2.83 9.13x106 ± 0 2.63x10

6 ± 0.32 28.8 ± 6.37

a

# bacteria invading

% Invasion

S. Typhimurium

ATCC 14028

2

124.75 ± 10.69

8.26x106 ± 0

1.25x105 ± 0.01

1.52 ± 0.02a

S. Heidelberg

ARI-14

2

63.17 ± 17.54

5.24x106 ± 0

5.96 x104

± 1.42

1.37 ± 0.25a

*Percentages not connected by the same letter are significantly different between each assay (P < 0.05).

221

Table 5.5. Adhesion and invasion of four Salmonella strains from different serovars to chicken macrophage, HD11, cells.

Bacterial strain Log Dilution # of colonies # bacteria

added

# bacteria

adhering % Adhesion*

S. Heidelberg 4 59.6 ± 15.96 1.3x107 ± 0.47 5.5x10

6 ± 1.98 43.4 ± 7.46

a

S. Typhimurium

ATCC 14028 4 61.8 ± 17.58 1.6x10

7 ± 0.47 6.2x10

6 ± 1.15

38.7 ± 4.57a

S. Typhimurium

UK-1 4 69.2 ± 19.85 1.8x10

7 ± 0.71 7.0x10

6 ± 1.93 38.9 ± 9.24

a

S. Enteritidis 4 39.2 ± 9.24 2.1x107 ± 0 3.9x10

6 ± 0.50 18.6 ± 2.42

b

# bacteria

invading

% Invasion*

S. Heidelberg 4 33.2 ± 9.04 6.91x10

7 ± 0 3.3x10

6 ± 0.50 4.8 ± 0.73

c

S. Typhimurium

ATCC 14028

4 37.3 ± 4.19 4.0x107 ± 0.28 3.7x10

6 ± 0.08 9.3 ± 0.57

b

S. Typhimurium

UK-1 4 25.3 ± 4.99 1.4x10

7 ± 0.15 2.5x10

6 ± 0.40

17.6 ± 3.29a

S. Enteritidis 4 4.05 ± 3.06 1.7x107 ± 0.04 4.9x10

5 ± 2.30 2.9 ± 1.48

c

*Percentages not connected by the same letter are significantly different between each assay (P < 0.05).

222



among coauthors, which the title is “Salmonella spp. Adhesion and Invasion Comparisons to

Caco-2 and HD11 Cell Lines” in chapter 5.


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8. Appendix


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CONCLUSIONS

Salmonella spp. continues to be one of the major foodborne pathogens of great concern

for public health, for the food industry, and in general all around the world. Understanding the

basic growth, mechanisms, pathogenesis, and antimicrobial resistance of this pathogen is of great

importance in order to be able to develop effective, accurate and rapid ways for detection and

isolation, and being able to find ways to reduce the presence of Salmonella.

In this research we studied the basic growth of multiple Salmonella serovars, which were

found to be both serovar and strain dependent. We observed the effect of spent media, more

specifically from S. Heidelberg, to the growth of S. Typhimurium and found an intriguing effect

for which more studies will be needed in order to determine the cause of it. Several factors could

play a role into this, metabolism, nutrients, virulence, and genetic traits, among many others that

have yet to be determined. The idea of competitive interaction has been investigated by many

researchers. In this research we studied the ability of two Salmonella serovars to compete

directly with each other, for which we found that the growth of S. Typhimurium is inhibited by S.

Heidelberg, the same characteristic that we observed on our spent media studies. Selective and

non-selective enrichment media methods were tested to determine the more effective and rapid

way to detect and isolate Salmonella, for which non-selective enrichment methods seemed to be

more effective.

The constant problem of increasing drug resistance has created a more urgent demand to

develop new and improved antimicrobials against resistant microorganisms (Spellberg et al.,

2004; Berghman et al., 2005). We tested the antimicrobial activity of different EOs against S.

Heidelberg strains from multiple sources. Lastly, the ability of Salmonella serovars to attach and

invade human epithelial and chicken macrophage cell lines was studied. Understanding the

235

mechanisms involved in bacterial response, could facilitate the refining and optimization of

antimicrobial formulations or discover new ways to overcome potential tolerance mechanisms,

leading to an improvement in biosecurity measures and to enhance the protection of farm-animal

and as a result, public health (Condell et al., 2012a).

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