Identification, Properties, and Application of Enterocins Produced by Enterococcal Isolates from Foods

Home , Class II bacteriocin, Enterocin, Enterococcus, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum

IDENTIFICATION, PROPERTIES, AND APPLICATION OF ENTEROCINS PRODUCED BY ENTEROCOCCAL ISOLATES FROM FOODS

THESIS

Presented in Partial Fulfillment of the Requirement for

the Degree Master of Science in the Graduate

School of The Ohio State University

Xueying Zhang, B.S.

*****

The Ohio State University 2008

Master Committee: Approved by

Professor Ahmed E. Yousef, Advisor

Professor Hua Wang ______

Professor Luis Rodriguez-Saona Advisor Food Science and Nutrition

ABSTRACT

Bacteriocins produced by lactic acid bacteria have gained great attention because they have potentials for use as natural preservatives to improve food safety and stability.

The objectives of the present study were to (1) screen foods and food products for lactic acid bacteria with antimicrobial activity against Gram-positive bacteria, (2) investigate virulence factors and antibiotic resistance among bacteriocin-producing enterooccal isolates, (3) characterize the antimicrobial agents and their structural gene, and (4) explore the feasibility of using these bacteriocins as food preservatives.

In search for food-grade bacteriocin-producing bacteria that are active against spoilage and pathogenic microorganisms, various commercial food products were screened and fifty-one promising Gram-positive isolates were studied. Among them, fourteen food isolates with antimicrobial activity against food-borne pathogenic bacteria,

Listeria monocytogenes and Bacillus cereus, were chosen for further study. Based on 16S ribosomal RNA gene sequence analysis, fourteen food isolates were identified as

Enterococcus faecalis, and these enterococcal isolates were investigated for the presence of virulence factors and antibiotic resistance through genotypic and phenotypic screening.

Results indicated that isolates encoded some combination of virulence factors. The esp gene, encoding extracellular surface protein, was not detected in any of the isolates.

Phenotype of antibiotic resistance indicated that all isolates were resistant to kanamycin

ii (25 µg/ml). Some isolates were also resistant to tetracycline (16 µg/ml). All isolates were sensitive to ampicillin, erythromycin, and chloramphenicol.

Bacteriocins produced by E. faecalis OSY-RM1 and E. faecalis OSY-3E1, referred to as enterocins OSY-RM1 and OSY-3E1, respectively, were chosen for further characterization due to their strong antimicrobial activity against selected indicators.

Results indicated that both enterocins were relatively heat stable, sensitive to protease, and stable over a wide pH range.

Application of enterocin OSY-RM1 to inactivate selected spoilage and food-borne pathogenic bacteria was investigated. Enterocin OSY-RM1 (44.7 nisin-equivalents

IU/ml), extracted by ammonium sulfate precipitation, had a bactericidal effect on L. monocytogenes Scott A and bacteriostatic effect on B. cereus ATCC 11778 in sterile milk. Although enterocin OSY-RM1 was not active against Escherichia coli K12, combination treatment of high pressure processing and enterocin OSY-RM1 caused significant inactivation of E. coli K12 in phosphate buffer saline.

Through polymerase chain reaction (PCR) amplification and direct sequencing, the structural gene of enterocin OSY-RM1 was identified with similarity to published enterocin EJ-97. Unlike enterocin EJ-97 which is encoded on a plasmid, enterocin OSY-

RM1 is likely encoded on bacterial chromosome since plasmid-cured E. faecalis OSY-

RM1 retained its antimicrobial activity.

iii Gene encoding enterocin OSY-3E1 was also identified by PCR amplification and direct sequencing and result indicted that its structural gene was similar to that of enterocin AS-48. Unlike enterocin AS-48 that is active against Gram-negative bacteria

(i.e. E. coli and Shigella sonnei), enterocin OSY-3E1 has no inhibitory activity against

Gram-negative bacteria tested. Enterocin OSY-3E1 was further applied to inactivate spoilage bacterium in food system. Result showed that enterocin OSY-3E1 (25,600

AU/ml), obtained from modified MRS broth containing 5% glucose, can effectively inactivate the spoilage bacterium, Leuconostoc mesenteroides ATCC 14935, in sterile milk. In conclusion, bacteriocins produced by food isolates, E. faecalis OSY-RM1 and

OSY-3E1 have potential uses to inactivate spoilage and pathogenic bacteria.

To my family whom I honor and love

v ACKNOWLEDGMENTS

I first wish to thank my advisor, Professor Ahmed E. Yousef, for his guidance, encouragements, and supports throughout this project. I have learned from him about hard work and critical thinking. I would like to extend my sincere thanks to Professor

Hua Wang and Professor Luis Rodriguez- Saona, for serving as my committee members.

I am grateful to Dr. Joy Waite for the proofreading of my thesis. I also would like to thank Dr. Zengguo He, Dr. Yoon Chung, and Yuan Yan, for their exceptional and invaluable cooperation and help during my research. I am also grateful for the help and support from the other members of our laboratory, including Dr. Luis Rodriguez-Romo,

Mustafa Varma, Jennifer Perry, Joe Jones, and Amrish-Suresh Chawla.

Finally, I wish to thank my parents, my sisters and my brother for their constant support on my study. I also want to express my great appreciations to my husband for his encouragement and support.

vi VITA

December 11, 1973……………………………….….………Born, Inner Mongolia, China

1995………………….….………Associate degree, Shijiazhuang Medical College, China

2002………………B.S. The Medical Analysis, Third Military Medical University, China

2006 – present..………..…….…Graduate Research Associate, The Ohio State University

FIELDS OF STUDY

Major Field: Food Science and Nutrition Graduated Program

vii TABLE OF CONTENTS

Page

Abstract …………………………………………………………………………… ii

Dedication ………………………………………………………………………… v

Acknowledgement ………………………………………………………………... vi

Vita ………………………………………………………………………………… ii

List of Tables ……………………………………………………………………… x

List of Figures ……………………………………………………………………... xi

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

Chapters:

1. Literature Review ………………...... 8

Lactic acid bacteria (LAB) ...... 8 Enterococci ...... 8 Bacteriocins ……………………………………………………………...... 9 LAB bacteriocins ………………………………………………………….. 10 Class I bacteriocins ……………………………………………………….. 10 Nisin ……………………………………………………………………...... 12 Class II bacteriocins ………………………………………………………. 13 Class III bacteriocins ………………………………………………….….. 14 Class IV bacteriocins ……………………………………………………... 15 Regulation of bacteriocin production …………………………………….. 15 Regulation of nisin biosynthesis ………………………………………….. 16 Isolation and purification of LAB bacteriocins …………………………… 17 Food biopreservation with LAB bacteriocins …...... 17 References …………………………………………………………………. 19

viii 2. Investigation of virulence factors and antibiotic resistance among bacteriocin-producing enterococci isolated from food products …………...... 36

Abstract …………………………………………………………………….. 36 Introduction ………………………………………………………………… 37 Materials and Methods ……………………………………………………... 38 Results and Discussion ……………………………………………………… 44 References…………………………………………………………………… 48

3. Enterocin OSY-RM1, a bacteriocin from Enterococcus faecalis: isolation and characterization of the peptide and structural gene ……...... 57

Abstract ……………………………………………………………………... 57 Introduction …………………………………………………….…………… 58 Materials and Methods …………………………………………...... 59 Results and Discussion ……………………………………………………… 68 References …………………………………………………………………… 73

4. Characterization of a bactreriocin produced by Enterococcus faecalis OSY-3E1 isolated from comtė cheese ...... 89

Abstract ……………………………………………………………………... 89 Introduction …………………………………………………………………. 90 Materials and Methods ……………………………………………………… 91 Results and Discussion ……………………………………………………… 95 References……………………………………………………………………. 98

List of References …………………………………………………………………. 109

ix LIST OF TABLES

Table Page

1.1 Overview of biochemically and genetically characterized Class II bacteriocins……………………………………………………….……… 31

2.1 PCR primers and product size for detection of virulence factors in enterococcal isolates …………………………………………………. 52

2.2 Antimicrobial activities of supernatants from overnight culture of Enterococcus faecalis isolates ………………………………………….. 53

2.3 Identified enterococcal isolates and their sources ……………………... 54

2.4 Occurrence of virulence factors from genotypic and phenotypic screening among Enterococcus faecalis isolates ………………………... 55

2.5 Resistance of Enterococcus faecalis strains isolated from foods to selected antibiotics ……………………………………………………... 56

3.1 Primers used in the PCR reactions for attempted amplification of Enterococcus faecalis OSY-RM1 bacteriocin …………………………… 79

3.2 Susceptibility of enterocin OSY-RM1 to heat, enzyme, and wide pH value range treatments ……..………… ………………………. 80

3.3 Different properties between enterocin OSY-RM1 and enterocin EJ-97 ………………….………………………………….… 81

4.1 Primers used in the PCR reactions for attempted amplification of Enterococcus faecalis OSY-3E1 bacteriocins …………………………….. 103

4.2 Susceptibility of enterocin OSY-3E1 to heat, enzyme, and wide pH value range treatments …………………………………….. 104

x 4.3 Antimicrobial activity and pH value of supernatants from MRS broth supplemented with various concentration of glucose after inoculation with OSY-3E1 and incubation overnight ……………………………………… 105

xi LIST OF FIGURES

Figure Page

1.1 Lanthionine syntheses …………………………………………………….. 32

1.2 The structure of mature nisin ……………………………………………… 33

1.3 Organization of nisin biosynthesis gene cluster …………………………… 34

1.4 Model for nisin biosynthesis and regulation ………………………………. 35

3.1 Dose-response plot of the concentration of commercial nisin and determination of nisin-equivalent units of CE OSY-RM1 …………… 82

3.2 Analysis of peptide extracted from Enterococcus faecalis OSY-RM1 culture by 16% Tris-Tricine SDS-PAGE ………………………………….. 83

3.3 Changes in the populations of Listeria monocytogenes Scott A (A) and Bacillus cereus ATCC 11778 (B) in Parmalat® milk treated with enterocin OSY-RM1 crude extract (44.7 IU added /ml of milk) and incubated at 35°C for 24 hours ……………………………………….. 84

3.4 Survivor of Escherichia coli K12 (log CFU/ml) treated with CE OSY-RM1 (44.7 IU added/ ml) and/or HPP (500 MPa, 25oC ± 2oC for 1 min) …………………………………………. 85

3.5 PCR amplification of enterocin EJ-97 structural gene using total DNA of Enterococcus faecalis OSY-RM1 as template ……………………………. 86

3.6 Comparison of the nucleotide sequences of structural gene of enterocin OSY-RM1 and enterocin EJ-97, including the predicted amino acid sequences …………………………………………………….. 87

xii 3.7 Relation between plasmid-curing by acridine orange (AO) and persistence of antimicrobial activity in Enterococcus faecalis OSY-RM1 culture ……………………………………………………….. 88

4.1 PCR amplification of enterocin AS-48 structural gene using total DNA of Enterococcus faecalis OSY-3E1 as template …………………………… 106

4.2 Comparison of the nucleotide sequences of structural gene of enterocin OSY-3E1 and enterocin AS-48, including the predicted amino acid sequences ……………………………………………...... 107

4.3 The change in population of Leuconostoc mesenteroides ATCC 14935 in Parmalat® milk treated with enterocin OSY-3E1 (25,600AU added /ml of milk) and incubated at 16°C for 5 days ………….……….. 108

xiii INTRODUCTION

Bacteriocins are gene encoded, ribosomally synthesized peptides or proteins with a

bactericidal or bacteriostatic mode of action against closely related species (Jack et al.,

1995; Riley, 1998). According to biochemical and genetic characteristics, bacteriocins

are classified into four groups: the lantibiotics (Class I), heat-stable posttranslationally

unmodified nonlantibiotics (Class II), heat-labile proteins (Class III) and an undefined

mixture containing proteins, carbohydrates and lipids (Class IV) (Klaenhammer, 1993).

Bacteriocins produced by lactic acid bacteria (LAB) are investigated extensively due to

their potential use as biopreservatives to inactivate Gram-positive food-borne pathogenic

bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Clostridium

perfringens and Clostridium botulinum (Jack et al., 1995; Klaenhammer, 1988; Nes et al.,

1996).

Bacteriocin-producing LAB includes lactococci, lactobacilli, pediocococci,

leuconostoc, and enterococci; the latter are of particular relevance to the current

investigation. Enterococci are normal inhabitants of the gastrointestinal tract of warm-

blooded animals (Franz et al., 1999). They also can be found in various foods or food products, including milk, cheeses, and fermented sausages (Knudtson and Hartman,

1993; Litopoulout-Tzanetaki and Tzanetakis, 1992; Trovatelli and Schiesser, 1987). The

1 presence of high levels of enterococci in cheese is generally considered poor hygienic

quality, and often results in sensory defects in some cheeses (Litopoulout-Tzanetaki,

1990; Lopez Diaz et al., 1995). However, these bacteria play a significant role in the

ripening and aroma development of other cheeses due to their proteolytic and lipolytic

enzyme activity and citrate utilization (Foulquie-Moreno et al., 2006). Strains of

Enterococcus faecalis and E. faecium have been successfully used as starter or adjunct cultures in cheeses (Coppola et al., 1990; Hugas et al., 2003). These species are also considered as probiotics and have been used with success in commercial probiotic preparations, including foods and other dietary supplements (Fuller, 1989; Holzapfel et al., 1998; Lund et al., 2002; Pollman et al., 1980; O’Sullivan, et al., 1992). However, the uses of enterococci in food fermentation and as probiotics are being questioned due to the increasing number of nosocomial enterococcal infections. Virulence factors, including collagen-binding protein, endocarditic antigen, hemolysin activator, gelatinase, aggregation substances, and extracellular surface protein, have been identified in E. faecalis (Archimbaud et al., 2000; Mundy et al., 2000). Moreover, the resistance of some

Enterococcus spp. to antibiotics used in currently is reported (Low et al., 1994;

Moellering, 1982; Morrision et al., 1997).

Bacteriocins produced by enterococci are generally referred to as enterocins. As a group, enterocins have different characteristics, such as inhibitory activity spectrum, mode of action, molecular weights and chemical structures (Cintas et al., 2001; Foulquie-

Moreno et al., 2003). They span different subclasses of bacteriocin, most commonly in class II. Recently, enterocins have received much attention because they are active against food-borne pathogens, such as Staphylococcus spp., Clostridium spp., Bacillus

2 spp. and L. monocytogenes (Franz et al., 1996; Galvez et al., 1998; Giraffa, 1995; Jennes

et al., 2000).

The objectives of the present study were to (1) screen foods and food products for

lactic acid bacteria with antimicrobial activity against Gram-positive bacteria, (2) investigate virulence factors and antibiotic resistance among bacteriocin-producing E. faecalis isolates, (3) characterize the antimicrobial agent and its structural gene, and (4)

explore the feasibility of using these bacteriocins as food preservatives.

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7 CHAPTER 1

LITERATURE REVIEW

Lactic acid bacteria (LAB)

Lactic acid bacteria are a group of Gram-positive, non-spore-forming, fastidious,

fermentative bacteria that produce lactic acid as major end product during carbohydrate

fermentation (Stiles and Holzapfel, 1997). Most of LAB are generally recognized as safe

(GRAS) bacteria and play a significant role in food fermentation as natural or selective starter or adjunct cultures due to their ability to produce desirable changes in the taste, flavor, and texture of food (Cintas et al., 2001; Reddy et al., 2007; Stiles and Holzapfel,

1997). Metabolic products of LAB, such as lactic acid, acetic acid, hydrogen peroxide, carbon dioxide, and bacteriocins, can effectively inhibit the growth of some spoilage and food-borne pathogenic microorganisms and may enhance safety of food products with a prolonged shelf-life (Chen and Hoover, 2003; Cleveland et al., 2001; Drider et al., 2006).

Enterococci

Enterococcus is a genus that is grouped under LAB (Franz, et al., 2003; Devriese et al., 1993; Devriese and Pot, 1995; Schleifer and Kilpper-Balz, 1984). They have been used in some Mediterranean cheeses as either starter or adjunct cultures, contributing to

8 ripening and aroma development (Giraffa et al., 1997; Manolopoulou et al., 2003). Some

strains of Enterococcus faecalis and E. faecium have been used as probiotics in many

countries (Franz et al., 2003). However, the safe status of enterococci in foods is being

questioned because of the nosocomial infections caused by enterococci (Giraffa, 2002).

Several virulence factors have been identified in Enterococcus spp., and antibiotic resistance is prevalent in enterococci (Archimbaud et al., 2002; Eaton and Gasson, 2001;

Mundy et al., 2000). Studies suggest an antibiotic resistance gene encoded by enterococci might be transferred to pathogenic bacteria making them resistance to antibiotics; enterococci possess effective gene transfer mechanisms through plasmid or conjugative transposon (Clewell, 1990; Landman and Quale, 1997; Lukasova and Sustackova, 2003;

Simjee and Gill, 1997)

Bacteriocins

Bacteriocins are peptides or proteins produced by bacteria that are active against closely related species (Jack et al., 1995; Riley, 1998). The bacteriocins produced by

Gram-negative bacteria are different from those produced by Gram-positive bacteria in two ways. Typically, the secretion of bacteriocins produced by Gram-negative bacteria is through cell lysis and is dependent on host regulatory pathways (Braun et al., 1994;

Smarda and Smajs, 1998). The biosynthesis of bacteriocins of Gram-positive bacteria is self-regulated and release is through specific transport mechanisms (van der Wal et al.,

1995). Both types of bacteriocins share some properties that were identified from the well-studied bacteriocin of Escherichia coli, colicin (James et al., 1991; Pugsley, 1984;

Vuyst and Vandamme, 1994). Firstly, bacteriocins inactivate target cells either through

9 (i) the formation of ion-permeable channels in the cytoplasmic membrane, (ii) nonspecific degradation of cellular DNA, or (iii) inhibition of the synthesis of peptidoglycan or proteins (Riley, 1998). Secondly, the bacteriocin producers protect themselves from the action of their own bacteriocin by production of an immunity protein commonly linked to the C-terminal domain of the bacteriocin. Furthermore, genes for expressing similar functions, such as production, maturation, immunity, and regulation, are encoded in a gene cluster.

LAB bacteriocins

Bacteriocins produced by LAB are a heterogeneous group of gene-encoded, ribosomally-synthesized peptides or proteins with antimicrobial activity against other

Gram-positive bacteria. During the last decade, a great number of LAB bacteriocins have been identified and their potential application as biopreservatives in foods or food products has been explored (Cintas, et al., 2001).

According to biochemical and genetic properties, bacteriocins are categorized into four classes: class I-IV (Klaenhammer, 1993). Class I contains small (<5 kDa) and heat stable lantibiotics containing unusual amino acid, such as lanthionine (Lan) and/or β- methyl-lanthionine (MeLan). Class II contains the small (<10 kDa) and heat-stable nonlantibiotics. Class III contains large (>30 kDa) and heat-labile bacteriocins. Other undefined proteins, lipids, and carbohydrates are referred to as class IV bacteriocins.

Class I bacteriocins

10 The lantibiotics are synthesized as inactive precursors containing an N-terminal

leader peptide that is cleaved during the export process of the mature and fully active

bacteriocins (Kuipers et al., 2004; Sahl et al., 1995; de Vos et al., 1995). Due to posttranslational modification, class I lantibiotics contain unusual amino acids, such as

Lan or MeLan. The formation of Lan or MeLan follows the formation of unsaturated amino acids didehydroalanine (Dha) or 2, 3-didehydrobutyrine (Dhb) from dehydration reaction of serine or threonine, respectively. Subsequently, a free cysteine and Dha or

Dhb within the same peptide are covalently linked, resulting in the formation of either

Lan or Melan, bridged by thioether bond (Chatterjee et al., 2005; Twomey et al., 2002).

The position and size of thioether bond are demonstrated by the positions of Dha or Dhb

and cysteine (McAuliffe et al., 2005; Sahl et al., 1995; de Vos et al., 1995). The synthesis of lanthionine is presented in Figure 1.1.

According to structural and functional characteristics, lantibiotics are subdivided

into two groups, group A and group B, classified by Jung and Sahl (1991), although other

classifications have also been proposed (Cotter et al., 2005). Group A lantibiotics are

cationic, elongated, and flexible peptides, which depolarize the cytoplasmic membrane

(CM) resulting in the formation of pores in the CM and the efflux of small molecules

such as amino acids and ATP (Guder et al., 2000; de Vos et al., 1995). Lacticin 481,

nukacin ISK-1, and nisin belong to group A (Gross and Morell, 1971; van den Hooven et

al., 1996; Kimura et al., 1998; Piard et al., 1993; Xie et al., 2004). Group B lantibiotics are globular peptides that are anionic or possess no net charge. Group B lantibiotics disrupt the function of essential enzymes, leading to the death of cells. Mersacidin and actagardine belong to group B lantibiotics (Chatterjee et al., 1992; Vértesy et al., 1999).

11 Nisin

Nisin is the oldest known and the best characterized lantibiotics. Nisin was first

discovered in 1928 (Rogers and Whittier, 1928), and approved for use in food industry as

an antimicrobial in 1969 (Delves-Broughton, 1996). Currently, nisin is licensed for use as

a food preservative in over 50 countries (Delves-Broughton, 1996). Produced by several

strains of Lactococcus lactis, nisin is a 34-amino acid peptide with positive net charge and, and as mentioned above, it has a linear structure containing a series of five internal rings and a flexible central hinge region (Gross and Morell, 1971). The primary structure of nisin is shown in Figure 1.2.

The mechanism of permeabilizing cellular membranes by nisin may be accomplished through two different ways. One theory is through the pore-formation mechanism. Nisin attaches the bacterial CM by binding to lipid II, a membrane-bound cell-wall precursor that is necessary for bacterial cell-wall biosynthesis. When several nisin molecules aggregate, the pore forms in the CM (Brotz et al., 1998; Breukink et al.,

1999; Wiedemann et al., 2001). The second theory is the low–affinity permeation mechanism. After binding to the negative charged bacterial phospholipid, nisin inserts between the hydrophilic head groups of phospholipids. Nisin monomers aggregate in the outer lipid monolayer and form of a short-lived pore-like structure, allowing nisin to cross the lipid bilayer (Brogden, 2005). Both mechanisms can alter CM formation and inhibit cell-wall synthesis of target bacterial cells, leading to the growth inhibition. Some

Gram-positive genera, such as Lactococcus, Lactobacillus, Pediococcus, Micrococcus,

Staphylococcus, and Listeria, are sensitive to nisin (Cintas et al., 1998; Hurst 1983). In

addition, endospore outgrowth and vegetative growth of Bacillus spp. and Clostridium

12 spp. can be prevented or inhibited by nisin (Delves-Broughton et al., 1996). Gram-

negative bacteria are resistant to nisin due to their outer membrane acting as a

permeability barrier (Breukink and Kruijff, 2006).

Some factors of target cells, such as low cellular phospholipid contents, altered

membrane fatty acids composition, and changes in cell wall composition, can affect the

activity of nisin (Crandall and Montville, 1998; Davies et al., 1996; Mazzotta and

Montville, 1997; Ming and Daeschel, 1995). In addition, negatively charged

polysaccharides bound to the cell wall could increase the resistance of target cells to nisin

(Breuer and Radler, 1996).

Class II bacteriocins

Class II bacteriocins are a heterogeneous group of non-lantibiotics that do not

undergo extensive posttranslational modification (Cintas et al., 2001). Although the

amino acid sequences of class II bacteriocins are quite different, they share some of the

following properties (Klaenhammer, 1993; Moll et al., 1999; Nes et al., 1996). They are

small (<10 kDa), heat stable peptides with net positive charge. Most of class II

bacteriocins inactivate target cells by permeabilizing the cellular membrane, leading to

the leakage of small solutes (Moll et al., 1999; Nes et al., 1996). Class II bacteriocins are further subdivided into four subclasses: class IIa-IIb.

Class IIa bacteriocins are pediocin-like bacteriocins with strong anti-listerial properties, designated as pediocin-like bacteriocins, which refer to as pediocin PA-1, the first characterized class IIa bacteriocin (Henderson et al., 1992). Analysis of the amino

acid sequence reveals that N-terminal part of IIa bacteriocins contains a highly conserved

13 hydrophilic and charged consensus sequence: YGNGV(X)C(X)4C(X)V(X)4A (X denotes any amino acid) and the C-terminal part is less conserved and more hydrophobic

(Ennahar et al., 2000). In addition, class IIa bacteriocins have a broad antimicrobial

spectrum against Gram-positive spoilage and food-borne pathogenic bacteria, especially

L. monocytogenes (Farber and Peterkin, 1991; Gahan and Collins, 1991).

Class IIb bacteriocins, also called two-peptide bacteriocins, are formed by two

clearly distinct peptides, suggesting that they originated from two independent and

probably different bacteriocins, but their antimicrobial activity requires the

complementary action of both peptides (Nes and Holo, 2000). In addition, like other

bacteriocins, they have only one dedicated immunity proteins and the gene coding

immunity proteins is linked to two structural genes of the peptides, usually in an operon

structure (Nes et al., 1996).

Unlike other bacteriocins that are exported by ATP-binding cassette (ABC)

transporters and their accessory proteins (Franke et al., 1996; Havarstein et al., 1995),

class IIc bacteriocins are cleaved during export by a dedicated general secretory

dependent (sec-dependent) pathway, where the antimicrobial peptide is exported to the

cytoplasmic membrane and then processed during translocation across the cytoplasmic

membrane (Havarstein et al., 1995; Sahl et al., 1995).

The bacteriocins that are not included by the previous subgroups of class II

bacteriocins are referred to as class IId bacteriocins. An overview of biochemically and

genetically characterized Class II bacteriocins is presented in Table 1.1.

Class III bacteriocins

14 Class III bacteriocins are large and heat labile proteins (Cintas et al., 2001). They are sensitive to heat treatment (60-100oC for 10-15 min). Helveticin J and Helveticin V belong to this group (Joerger and Klaenhammer, 1986; Vaugham et al., 1992)

Class IV bacteriocins

Class IV bacteriocins are an undefined mixture of proteins, lipids, and carbohydrates

(Klaenhammer, 1993), including glycoproteins like leucocin S and lactocin 27 (Lewus et al., 1992; Upreti, 1994), lipoproteins like mesenterocin 52 (Sudirman et al., 1994), and glycolipoproteins like fermenticin (de Klerk and Smit, 1967). The antimicrobial activity of class IV bacteriocins requires the presence of glucidic and/or lipid moieties as well as the proteinaceous component.

Regulation of bacteriocin production

The production of bacteriocins of Gram-positive bacteria is regulated at the transcriptional level in a cell-density dependent manner, called quorum sensing. Quorum sensing bacteria have the ability to communicate and coordinate behaviors through signaling molecules (Kleerbezem et al., 1997; Quadri, 2002).

Two-component regulatory systems are involved in the bacteriocin production

(Stock et al., 1990). After synthesis and secretion through an ATP- dependent transport out of the cell, a signaling molecule (autoinducer) stimulates a membrane-located histidine protein kinase of surrounding cells, phosphorylating the downstream regulatory protein. Phosphorylated regulatory protein then binds to the promoter of bacteriocin

(Jack et al., 1995). This binding activates the transcription of target genes, regulates

15 expression of various genes, and results in the production of the bacteriocin (Kotelnikova and Gelfand, 2002).

Regulation of nisin biosynthesis

Eleven genes organized in a cluster, nisABTCIPRKFEG, are involved in the biosynthesis of nisin (Buchman et al., 1988; Ra et al., 1996; Siezen et al., 1996). Gene nisA encodes the nisin A precursor peptide (Kuipers et al., 1993). Gene nisB and nisC are involved in post-translational modification reaction (Kuipers et al., 1995). Gene nisT is involved in translocation of precursor nisin (Engelke et al., 1992; Kuipers et al., 1993).

Gene nisP is involved in precursor processing (van der Meer et al., 1993). Gene nisI and nisFEG are involved in self-protection and immunity against nisin, respectively (Kuipers et al., 1993; Siegers and Entian, 1995). Gene nisR and nisK are involved in the regulation of nisin biosynthesis (Engelke et al., 1994; van der Meer et al., 1993). The organization of gene cluster for nisin biosynthesis is shown in Figure 1.3.

Nisin biosynthesis is regulated by quorum sensing, where nisin itself act as a signal molecule. During the autoregulation of nisin biosynthesis, the two-component regulatory system, NisK and NisR, plays an important role in the transcriptional activation and downstream production of nisin (Engelke et al., 1994). NisK, a sensor histidine kinase, senses the presence of nisin and autophosphorylates (Kuipers et al., 1995). After phosphorylation, the phosphate group is transferred to NisR, a response regulator, to activate the induction of nisA and nisF promoters, leading to the synthesis of unmodified precursor nisin (de Ruyter et al., 1996). Unmodified precursor nisin is modified by the putative enzymes, NisB and NisC, and then translocated across the membrane by an ABC

16 transporter, NisT (Hess et al., 1988; Engelke et al., 1992). After modification of precursor nisin by NisP, nisin is activated and released (Hess et al., 1988; van der Meer et al.,

1993). In addition, NisIFEG work together to prevent the destruction of producing cells from the bactericidal action of nisin, confirming immunity to the host cell (Engelke et al.,

1994; Siegers and Entian, 1995). An overall model for nisin biosynthesis and regulation is shown in Figure 1.4.

Isolation and purification of LAB bacteriocins

The isolation of LAB bacteriocins from foods and food products involves the screening of LAB for antimicrobial activity and isolation and confirmation of the product as a bacteriocin. Generally, soft agar (0.75%) overlay method is used to isolate bacteriocinogenic LAB (Lewus et al., 1991). To exclude the possibility that the inhibition is produced by hydrogen peroxide, catalase is added to the culture medium; and carbonate or phosphate buffer is added to the solid agar to avoid antagonism by organic acids (Tagg et al., 1976). The inhibitory effect caused by bacteriophage also can be excluded by the absence of phage plaque on the agar seeded with sensitive indicators

(Lewus et al., 1991). The inhibitory activity is detected and confirmed through a spot-on- lawn method or a well diffusion assay on agar plates seeded with sensitive indicators

(Lewus et al., 1991).

Bacteriocins are proteinaceous compounds secreted into the culture medium. Before purification, bacteriocins need to be concentrated from cell-free supernatants of the bacteriocin-producing cultures (Muriana and Luchansky, 1993; Venema et al., 1997).

Normally, approaches including protein precipitation by ammonium sulfate (Holo et al.,

17 1991) and protein extraction with organic alcohols, such as butanol and ethanol (Piva and

Headon, 1994) are used to selectively extract bacteriocins. The bacteriocin-enriched fraction is then purified through several chromatographic steps, including cation exchange, gel filtration, hydrophobic-interaction, and reverse-phase liquid chromatography. The yield of purified bacteriocin is often poor, probably due to the numerous steps in the protocol. Therefore, the optimization of bacteriocin production is an important step for maximizing bacteriocin concentration.

Food biopreservation with LAB bacteriocins

Some of LAB bacteriocins have potential for use as food biopreservatives.

Advantages of LAB bacteriocins include (1) producers have GRAS status, (2) there are no known toxic effects on humans, (3) bacteriocins are sensitive to digestive proteases,

(4) they are resistant to heat, (5) they are effectively active against some of Gram-positive spoilage and food-borne pathogenic bacteria, (6) bacteriocins are generally encoded by plasmid, indicating ease of genetic regulation, manipulation to increase yield (Gálvez et al., 2007). Nisin, produced by several strains of L. lactis, is the only bacteriocin licensed for use as a food preservative in certain processed cheeses, dairy products, and canned foods (Delves-Broughton et al., 1996). Adding LAB bacteriocins to food products may enhance organoleptic and nutritional properties of processed products by decreasing the use of chemical preservatives and/or intensity of heat treatment (Ross et al., 1999).

Mostly, LAB bacteriocins interact synergistically with traditional and novel preservation methods (e.g., high pressure processing and pulsed electric fields) to inactivate target spoilage or pathogenic bacteria (Bennik et al., 1999; Kalchayanand et al., 1994; Steeg et

18 al., 1999). These combinations are effective because bacterial cells suffer more intensive

damage and may require more energy to repair damage after combination treatments,

resulting in energy exhaustion and cell death (Gálvez et al., 2007). Consequently, the

hygienic quality of food and food products is improved and shelf-life is extended (Hanlin

et al., 1993; Song and Richard, 1997; Vignolo et al., 2000).

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30 Class II Bacteriocins Producer Organism Class IIa (Pediocin-like bacteriocin) Pediocin PA1 Pediococcus acidilactici PAC-1.0 Pediocin AcH P. acidilactici H Leucocin A-UAL 187 Leuconostoc gelidum UAL 187 Mesentericin Y105 Leuc. mesenteroides Y105 Mesentericin 52B Leuc. mesenteroides FR52 Mesentericin B105 Leuc. mesenteroides Y105 Acidocin A Lactobacillus acidophilus TK9201 Bavaricin A Lb. bavaricus MI401 Curvacin A Lb. curvatus LTH1174 Sakacin A Lb. sakei LB706 Sakacin P Lb. sakei LTH673 Sakacin 674 Lb. sakei LB674 Carnobacteriocin BM1 Carnobacterium piscicola LB17B Carnobacteriocin B2 C. piscicola LV17B Divercin V41 C. divergens V41 Enterocin A Enterococcus faecium CTC492 Class IIb (two-peptide bacteriocins) Lactococcin M Lactococcus cremoris 9B4 Lactococcin G L. lactis LMG2081 Acidocin J1132 Lb. acidophilus JCM1132 Lactacin F Lb. johnsonii VPI11088 Plantaricin S Lb. plantarum LCPO10 Plantaricins EF Lb. plantarum C11 Plantaricins JK Lb. plantarum C11 Leucocin H Leuconostoc sp. MF215B Termophilin 13 Streptococcus thermophilus Spi 13 Class IIc (sec-dependent bacteriocins) Acidocin B Lb. acidophilus M46 Divergicin A C. divergens LV13 Enterocin P E. faecalis P13 Lactococcin 972 L. lactis IPLA972 Class IId (other bacteriocins) Lactococcins A and B L. cremoris 9B4 L. lactis WM4 Peptide A Lb. acidophilus LF221 Lactobin A Lb. amylovorous LMG P-13139 Divergicin 750 C. divergens 750 Carnobacteriocin A C. piscicola LV17A Leucocin B-TA33a Leuconostoc mesenteroides TA33a Enterocin B E. faecium T136 Enterocins L50 E. faecium L50 Enterocin Q E. faecium L50

Table 1.1 Overview of biochemically and genetically characterized Class II bacteriocins (Cintas et al., 2001)

Figure 1.1 Lanthionine syntheses (Cotter et al., 2005).

Figure 1.2 The structure of mature nisin A (Cheigh and Pyun, 2005). The substitution of Asn27 for His27 in nisin Z is indicated. Dha is didehydroalanine, Dhb is didehydrobutyrine, Ala-S-Ala is lanthionine and Abu-S-Ala is β-methyl-lanthionine.

IR *

Figure 1.3 Organization of nisin biosynthesis gene cluster (Kuipers et al., 1995). P* indicates regulated promoters and P indicates constitutive promoters. IR denotes an extensive inverted repeat sequence.

Figure 1.4 Model for nisin biosynthesis and regulation (Cheigh and Pyun, 2005).

35 CHAPTER 2

INVESTIGATION OF VIRULENCE FACTORS AND ANTIBIOTIC

RESISTANCE AMONG BACTERIOCIN-PRODUCING ENTEROCOCCI

ISOLATED FROM FOOD PRODUCTS

ABSTRACT

Enterococci are a controversial group of lactic acid bacteria. Some strains of

Enterococcus faecalis and E. faecium have been successfully used as dairy starter cultures or probiotics, but nosocomial infections associated with enterococci and prevalence of antibiotic resistance have been identified. Therefore, it is necessary to screen and evaluate virulence factors and antibiotic resistance among bacteriocin- producing enterococcal isolates from foods or food products.

Fourteen bacteriocin-producing enterococci with activity against food-borne

pathogenic bacteria were isolated from different foods. Each isolate was analyzed by polymerase chain reaction (PCR) for the presence of genes encoding known virulence factors: collagen-binding protein (ace), endocarditic antigen (efaA), hemolysin activator

(cylA), gelatinase (gelE), aggregation substances (agg), and extracellular surface protein

36 (esp). In addition, phenotypic screening of the activity of gelatinase and hemolysin on

THAG or TSA-blood agar and antibiotic resistance using broth dilution method was performed among fourteen enterococcal isolates. PCR results indicated that all isolates encoded some combination of virulence factors. The esp gene, encoding extracellular surface protein, was not detected in any of the isolates. Phenotype of gelatinase and hemolysin indicated the presence of a silent gelA gene in two isolates, OSY-3E4 and

OSY-16D2. All strains encoding cylA gene were positive for hemolysin activity on TSA- blood agar. Phenotype of antibiotic resistance indicated that all isolates were resistant to kanamycin (25 µg/ml). Three isolates, OSY-3E1, OSY-6F2, and OSY-B1, were also resistant to tetracycline (16 µg/ml). All isolates were sensitive to ampicillin, erythromycin, and chloramphenicol.

INTRODUCTION

Enterococci are a group of lactic acid bacteria (LAB) with proteolytic and lipolytic activity and citrate breakdown that contributes to the taste and flavor profile of some cheeses (Giraffa and Carminati, 1997; Manolopoulou et al., 2003). Some strains of enterococci have been successfully used as starter or adjunct cultures in cheeses and are also known as probiotics (Giraffa, 2003; Hugas et al., 2003; Lund et al., 2002; Mikes et al., 1995; Pollman et al., 1980). Bacteriocins produced by enterococci, generally referred to as enterocins, have been also reported to inhibit Gram-positive food-borne pathogens

(Aymerich et al., 1996; Franz et al., 1996; Laukova et al., 1993; Torri et al., 1994;

Vlaemynck et al., 1994). 37 However, the harmless status of enterococci used in foods is being challenged

(Giraffa, 2002). With the increasing prevalence of nosocomial infections, enterococci are being considered as emerging pathogens (Lewis and Zervos, 1990; Morrison et al., 1997).

Their pathogenesis is commonly related to their resistance to antibiotics and efficient gene transfer mechanism because enteroocci are easy to transform (Landman and Quale,

1997). Virulence cannot be explained by their antibiotic resistance alone and enterococcal virulence factors, such as collagen-binding protein and endocarditic antigen, have been identified in previous studies (Archimbaud et al., 2002; Eaton and Gasson 2001; Franz et al., 2001; Johnson, 1994; Mundy et al, 2000).

Eaton and Gasson (2001) identified the presence of known virulence factors in enterococcal isolates from medical and food samples. Food and dairy starter enterococci may contain virulence factors due to conjugation with clinical enterococcal isolates.

Therefore, evaluation of the presence of virulence factors and antibiotic resistance in enterococci from foods is necessary to determine the potential applications of bacteriocin-producing isolates.

The goals of the present study were to (1) screen and identify bacteriocin-producing bacteria from foods and food products, and (2) determine the prevalence of encoded virulence factors and quantify antibiotic resistance among food isolates.

MATERIALS AND METHODS

Screening foods for bacteriocin-producing lactic acid bacteria

38 Nineteen different foods, such as cheeses, sausages, fruits, fermented olives, raw

meat, purchased from local market (Columbus, OH), and raw cow’s milk (The Ohio State

University dairy farm, Columbus, OH), were selected for this study. Cheeses were made

of raw milk by small producers. Food were screened for bacteriocinogenic strains using a

soft agar overlay procedure (Lewus et al., 1991). Briefly, a 10-gram portion of food was

homogenized with 90 ml of 0.1% peptone water (Difco, BD Diagnostic Systems, Sparks,

MD), to obtain a 1:10 dilution (w/v). Serial dilutions were spread-plated onto Lactobacilli

DeMann, Rogosa, and Sharpe (MRS) (Difco, Sparks, MD) agar plates and incubated at

30ºC for 48 h. Plates with approximately 100 colonies were overlaid with 10 ml of MRS

soft agar (0.75%) seeded with 100 µl overnight culture of indicator strains. The overlaid

plates were incubated at 30oC for 24 h. Colonies surrounded by a clear inhibition zone were considered as potential bacteriocin producers. These colonies were isolated on MRS agar and examined for Gram reaction and cellular morphology. Stock cultures were prepared in 20% glycerol and maintained at –80oC.

Indicator strains and growth conditions

Food isolates were screened for antimicrobial production using Lactobacillus

plantarum ATCC 8014 or Lb. cellobiosus OSU 919 as indicators. Stock cultures of indicator strains were stored at -80oC in MRS broth supplemented with 20% glycerol.

Cultures were prepared for experiments by streaking onto MRS agar and incubation overnight at 30oC. A single colony from the incubated MRS agar plate was inoculated

into MRS broth and the tubes were incubated overnight at 30oC. The resulting culture was used to seed the soft agar overlay for bacteriocin activity.

Antimicrobial activity assay

Food isolates exhibiting antimicrobial properties were tested for the presence of

bacteriocin-like substances using the spot-on-lawn assay (Lewus et al., 1991). The cell-

free supernatant from overnight culture of each isolate was obtained by centrifugation

(8,000 x g for 10 min at 4oC) (Sorvall RC-5B, DuPont, Wilmington, Del.). An aliquot (10

µl) of cell-free supernatant was spotted onto MRS soft agar plate seeded with overnight

culture of Lb. cellobiosus OSU 919 or Lb. plantarum ATCC 8014. Plates were incubated at 30oC for 18 h and clear areas of inhibition were observed. To exclude the possibility that this inhibition is due to the presence of lytic bacteriophages, one piece of agar from the inhibition area was cut, homogenized with 0.1% peptone water, mixed with MRS soft agar seeded with overnight culture of Lb. plantarum ATCC 8014, poured into Petri dish, and tested for the presence of phage plaques (Lewus et al., 1991).

Inhibitory spectrum

The antimicrobial spectrum of supernatants was tested against several Gram-positive and Gram-negative bacterial strains, using the spot-on-lawn method, as previously described. The indicator strains used in this study were obtained from the culture collection of the Food Safety Laboratory at The Ohio State University (Columbus, OH) and are listed in Table 2.2. All lactic acid bacteria indicators were inoculated into 5 ml

MRS broth and incubated overnight at 30oC without shaking. Other Gram-positive bacteria and Gram-negative bacteria indicators were transferred to 5 ml of tryptic soy broth (TSB, BBL, Sparks, MD) and incubated overnight at 37oC without shaking. 10 µl

40 aliquots of cell-free supernatant (adjusted to pH 6.5-7.0 using 1M NaOH to exclude the

inhibitory effect of acid) of each food isolate were spotted onto soft agar seeded with

overnight cultures of indicators. The plates were then incubated overnight at 30oC for lactic acid bacteria indicators or at 37oC for all additional indicators and inhibitory activities were recorded. Fourteen isolates with broad-spectrum antimicrobial activity were chosen for further study.

Identification of isolated strains

Species identification of isolated strains was determined based on 16S ribosomal

RNA gene sequence analysis. Total DNA of each isolated strain was extracted from overnight culture grown in MRS broth at 30oC using a commercial DNA isolation kit

(DNeasy Tissue Kit; QIAgen Sciences Inc., Valencia, CA). The gene for 16S rRNA was amplified using universal oligonucleotide primers fD1 (5’-CCG AAT TCG TCG ACA

ACA GAG TTT GAT CCT GGC TCA G-3’), and rD1 (5’-CCC GGG ATC CAA GCT

TAA GGA GGT GAT CCA GCC-3’) (Weisburg et al., 1991). Polymerase chain reaction

(PCR) amplification was accomplished using Taq DNA polymerase kit (QIAGEN,

Valencia, CA). PCR conditions included an initial denaturation (3 min at 95oC), followed

by 30 cycles of denaturation (95oC for 1 min), annealing (52oC for 30 sec) and elongation

(72oC for 2 min), ending with a final extension step (72oC for 10 min). All PCR

amplifications were carried out in a Techgene DNA Thermal Cycler (geneAmp PCR

system 2400, Applied Biosystems, Foster City, CA). PCR products were examined on

0.8% agarose gel electrophoresis followed by ethidium bromide staining and visualization by ultraviolet light. The amplified product was purified using a commercial

41 DNA extraction kit (QIAquick gel extraction kit; QIAGEN, Valencia, CA), ligated to

pGEM-T Easy vector (Promega Corporation, Madison, WI), and transformed into

Escherichia coli DH5α competent cells via chemical transformation. The recombinant plasmid was harvested from 5 ml overnight culture in Luria-Bertani (LB) broth containing ampicillin (100µg/ml) using silica spin columns (QIAprep Spin Miniprep kit;

QIAGEN). In order to determine if the size of inserted DNA fragment was consistent with the expected (~1,500 kb), the recombinant plasmid was digested by Not I restriction enzyme (New England Biolabs, Ipswich, MA) and then examined on 0.8% agarose gel electrophoresis followed by ethidium bromide staining. In addition, the inserted DNA fragment was sequenced by the Plant and Microbe Genomics Facility (The Ohio State

University, Columbus, OH) (3730 DNA Analyzer; Applied Biosystems, Foster City, CA) using T7 terminator and SP6 promoter primers. The obtained gene sequence ( 1.5 kb)

was compared to known bacterial sequences in the NCBI GenBank

(http://www.ncbi.nlm.nih.gov/) using BLAST tool. The final phylotype identification was achieved only based on the highest-score queries with 97% minimum similarity

(Stackebrandt and Goebel, 1994).

Detection of potential virulence factors by PCR

Detection of genes encoding potential virulence factors in isolated bacteriocinogenic strains was performed by PCR amplification and subsequent agarose gel electrophoresis.

The virulence factor genes tested were ace (collagen-binding protein), efaA (endocarditic antigen), cylA (haemolysin activator), gelE (gelatinase), agg (aggregation substances), and esp (extracellular surface protein) (Creti et al., 2004; Eaton and Gasson 2001;

42 Shankar et al., 1999). The primer sequences for these potential virulence factors were chosen from previous studies. Primers and product size are listed in Table 2.1.

PCR amplification was performed using total bacterial DNA as template. PCR

amplification was performed using Taq DNA polymerase kit (QIAGEN, Valencia, CA).

Samples were amplified for ace, efaA, cylA, gelE, agg, and esp genes by denaturation at

95oC for 5 min, followed by 30 cycles of denaturation at 95oC for 1 min, annealing at

58oC for 1min, with the exception of gelE at 52oC and esp at 63oC (Shanker et al., 1999), elongation at 72oC for 1 min, and a final extension step at 72oC for 10 min. All PCR

amplifications were done in a Techgene DNA Thermal Cycler (Applied Biosystems).

PCR products were examined on 2.0% agarose gel via electrophoresis followed by

ethidium bromide staining. The PCR products from one isolate were sequenced after

purification (QIAquick Gel Extraction Kit, QIAGEN) for confirmation. Homologies of

the sequences were verified in the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) using

BLAST tool.

Production of gelatinase and hemolysin

Production of gelatinase was determined on Todd-Hewitt agar (THA) containing 3%

gelatin (Difco, Sparks, MD) per liter (THAG), as described by Coque et al. (1995).

Isolated colonies previously grown on MRS agar were streaked onto plates. After

incubation at 37oC for 24 h, colonies with opaque zones were considered gelatinase positive. To determine production of hemolysin, strains were streaked onto layered fresh horse blood (5%) in tryptic soy agar (TSA, Becton Dickinson, Sparks, MD) and

43 incubated at 37oC for 24 to 48 h. Clear zones around colonies on blood agar plates indicated hemolysin production.

Detection of minimum inhibitory concentrations (MICs) of various antibiotics

MICs were determined by the broth dilution method recommended by the National

Committee for Clinical Laboratory Standards (1997). Before testing, stock solutions of antibiotics were diluted either in distilled water (kanamycin, ampicillin, and) or in ethanol

(chloramphenicol and erythromycin in 95% ethanol) to working concentration.

Kanamycin, ampicillin, and tetracycline solution were microfiltered (0.2- µm-pore-size low-protein-binding filter; Millipore, Billerica, MA). MIC was determined as the lowest antibiotic concentration to prevent cell growth. Ampicillin, erythromycin, chloramphenicol, and kanamycin were purchased from Fisher Scientific (Pittsburgh, PA).

Tetracycline was purchased from Sigma-Alorich (St. Louis, MO).

RESULTS AND DISCUSSION

Isolation and identification of isolates with antimicrobial activity

Fifty-one strains producing potential antimicrobial agents against indicators (Lb. plantarum ATCC 8014 or Lb. cellobiosus OSU 919) were isolated from nineteen food samples including different types of cheeses, sausages, fermented vegetables, raw milk, and fresh fruit, using soft agar overlay method. These food isolates were all Gram- positive, non-spore-forming cocci.

44 Fourteen of these isolates produced antimicrobial activity against the food-borne

pathogens Listeria monocytogenes and Bacillus cereus. These isolates were designated as

OSY-3E1, OSY-3E2, OSY-3E3, OSY- 3E4, OSY-3E5, OSY-3E6, OSY-5B1, OSY-6F2,

OSY-6F3, OSY-15I2, OSY-16D1, OSY-16D2, OSY-B1, and OSY-RM1; these were chosen for further investigation. The antimicrobial spectra of these strains are shown in

Table 2.2. None of Gram-negative bacteria tested, including E. coli K12, E. coli P220,

Pseudomonas fluorescens, P. putida ATCC 49451, were inhibited by antimicrobial

agents produced by any food isolate tested (data not shown). In addition, the

antimicrobial properties were characterized as bacteriocin using spot-on-lawn method.

The probability that the antimicrobial properties are due to bacteriophage was excluded

by the absence of plaque-forming phage in the inhibition zone on indicator lawns.

Based on 16S rRNA gene sequence analysis, all fourteen isolates were identified as

E. faecalis. The identified enterococcal isolates and their origin are listed in Table 2.3.

Other studies have reported the high prevalence of E. faecalis in non-food systems and

this bacterium is a prevalent member of the natural microbiota of a variety of foods

(Campo et al., 2001; du Toit et al., 2000).

PCR screening for virulence factors

The fourteen bacteriocin-producing enterococcal isolates were screened for

virulence factors by PCR. Following amplification, the PCR products from isolate OSY-

3E1 were chosen to purify and sequence for confirmation. The obtained nucleotide

sequences demonstrated 99% similarity to efaA gene, 93% to agg gene, 98% to gelE gene, 98% to ace gene, 100% identity with cylA gene. Predicted amino acid sequence

45 was 99% similar to endocarditis specific antigen, 86% to aggregation substance, 97% to gelatinase, 88% to collagen adhesion precursor, and 100% identical with hemolysin activator.

All fourteen isolates were found to encode a different combination of virulence factors (Table 2.4). However, the esp gene, encoding extracellular surface protein, associated with colonization and persistence of E. faecalis in urinary tract infections due to its multiple-drug-resistance (Shankar et al., 2001), was not detected in any of the fourteen isolates.

Usually, virulence genes are plasmid-encoded and enterococci possess effective gene transfer mechanisms (Clewell, 1990; Simjee and Gill, 1997). Therefore, investigation and evaluation of the present virulence factors among food isolates are necessary, especially with regard to the introduction of novel and commercial cultures, due to the risk of acquiring virulence factors via conjugation with other bacteria in the food processing environment.

A virulence factor is an effector molecule that enhances the bacterial ability to cause disease in a host (Mundy et al., 2000). Although combinations of virulence factors appeared in food isolates, enteroocci need to express a combination of virulence traits including adhesion, translocation, and evasion of the immune system to be pathogenic

(Johnson, 1994). Further studies would be necessary to determine the in vivo pathogenicity of these isolates.

Production of gelatinase and hemolysin

46 Hemolysin activity was detected in isolates OSY-3E1, OSY-15I2 and OSY-16D2 on

TSA-blood agar. These strains were also identified genotypically as encoding the cylA

gene. Gelatinase activity was not detected in isolates OSY-3E4 and OSY-16D2 on

THAG agar, although they carried the gelE gene. Isolates OSY-3E2 and OSY-15I2

possessed the gelE gene and produced gelatinase activity on THAG agar. Other isolates

were negative for genotypic and phenotypic gelatinase and hemolysin activity.

The appearance of silent gelA gene in OSY-3E4 and OSY-16D2 indicates the low

levels or down regulation of this gene expression or an inactive gene product. Thus, the

presence of a silent gene in food strains suggests that these strains may be used safety in

food.

Antibiotic susceptibility

Antibiotic susceptibility of E. faecalis isolates is shown in Table 2.5. All isolates

were resistant to kanamycin (25 µg/ml). Isolates, such as OSY-3E1, OSY-6F2, and OSY-

B1, showed resistance to tetracycline (16 µg/ml). No antibiotic resistance to

chloramphenicol, ampicillin, and erythromycin was detected in any of the food isolates.

Evaluating the risk of antibiotic-resistant enterococci from foods or food products on

potential human pathogenicity is not straightforward (Franz et al., 2001). Antibiotics may

lead to selection of pathogenic enterococci prevalent in the hospital environment, especially strains resistant to glycopeptide antibiotics, such as vancomycin (Murray,

1990). Although antibiotic susceptibility tests showed that food isolates were resistant to some antibiotics, they did not show resistance to clinically relevant antibiotics, such as ampicillin. 47

In conclusion, virulence factors and antibiotic resistance were investigated and

assessed among bacteriocin-producing enterococci isolated from food products. Strains

encoding virulence factors did not necessarily express functional gene products.

Encoding virulence factors may be considered strain-specific among E. faecalis. All

strains were found to harbor multiple virulence factors. Strains tested were sensitive to

ampicillin, erythromycin, and chloramphenicol. Only three strains possessed resistance to

both kanamycin and tetracycline.

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Genes Sequences Product Reference

size

esp F: 5’-GGAATGACCGAGAACGATGGC-3’ 616 bp Shankar et

(extracellular surface protein) R: 5’-GCTTGATGTTGGCCTGCTTCCG-3’ al., 1999

ace F: 5’-GCCAATTGGGACAGACCCTC-3’ 688 bp Eaton and

(collagen-binding antigen) R: 5’-CGCCTTCTGTTCCTTCTTTGGC-3’ Gasson,

2001

efa A F: 5’-GACTCGGGGATTGATAGGC-3’ 688 bp Franz et

(endocarditic antigen) R: 5’-GCTGCTAAAGCTGCGCTTAC-3’ al., 2001

cyl A F: 5’-ACCCCGTATCATTGGTTT-3’ 405 bp Chow et

(hemolysin activator) R: 5’-ACGCATTGCTTTTCCATC-3’ al., 1993

gel E F: 5’-CCAGCCAACTATGGCGGAATC-3’ 529 bp Eaton and

(gelatinase) R: 5’-CCTGTCGCAAGATCGACTGTA-3’ Gasson,

2001

agg F: 5’-TTGCTAATGCTAGTCCACGACC-3’ 932 bp Chow et

(aggregation substance) R: 5’-GCGTCAACACTTGCATTGCCGA-3’ al., 1993

Table 2.1 PCR primers and product size for detection of virulence factors in enterococcal isolates

Indicator species a Inhibitory activityb

3E1 3E2 3E3 3E4 3E5 3E6 5B1 6F2 6F3 15I2 16D1 16D2 B1 RM1

Lactobacillus casei ATCC + - - - + - + + - - - - + +

7469

Lb. cellobiosus OSU 919 + + + + + + + + + + + + + +

Lb. bulgaricus OSU 135 + + + + + + + + + + + + + +

Lb. plantarum ATCC 8014 + + + + + + + + + + + + + +

Leuconostoc mesenteroides + - + + + + + + - + + + + -

ATCC 15935

Lactococcus lactis + + + + + + + + + + + + + +

ATCC 11454

Pediococcus acidilactici PO2 + + + + + + + + + + + + + +

Listeria monocytogenes + + + + + + + + + + + + + +

Scott A

Bacillus cereus ATCC 14579 + + + + + + + + + + + + + +

Staphylococcus aureus ------

OSU150 a Strains were obtained from the culture collection of the Food Safety Laboratory at The Ohio State University b (+-) inhibition; (-) no inhibition.

Table 2.2 Antimicrobial activities of supernatants from overnight culture of E. faecalis

isolates

53 Food Isolates Source(s)

Enterococcus faecalis OSY-3E1 Comté Cheese

E. faecalis OSY-3E2 Comté Cheese

E. faecalis OSY-3E3 Comté Cheese

E. faecalis OSY-3E4 Comté Cheese

E. faecalis OSY-3E5 Comté Cheese

E. faecalis OSY-3E6 Comté Cheese

E. faecalis OSY-5B1 Salamett Sausage

E. faecalis OSY-6F2 Prosciutto Diparma Sausage

E. faecalis OSY-6F3 Prosciutto Diparma Sausage

E. faecalis OSY-15I2 Dairy cheese

E. faecalis OSY-16D1 Fermented Olive

E. faecalis OSY-16D2 Fermented Olive

E. faecalis OSY-B1 Banana

E. faecalis OSY-RM1 Raw cow’s milk

Table 2.3 Identified enterococcal isolates and their sources.

54 Isolated strains Genotype Phenotype

OSY-3E1 ace+ efaA+ cylA+ agg+ cylA+ gelE-

OSY-3E2 gelE+ agg+ cylA- gelE+

OSY-3E3 ace+ efaA+ agg+ cylA- gelE-

OSY-3E4 ace+ efaA+ gelE+ agg+ cylA- gelE-

OSY-3E5 ace+ efaA+ agg+ cylA- gelE-

OSY-3E6 ace+ efaA+ agg+ cylA- gelE-

OSY-5B1 efaA+ agg+ cylA- gelE-

OSY-6F2 ace+ efaA+ agg+ cylA- gelE-

OSY-6F3 ace+ efaA+ agg+ cylA- gelE-

OSY-15I2 efaA+ cylA+ gelE+ agg+ cylA+ gelE+

OSY-16D1 ace+ efaA+ cylA- gelE-

OSY-16D2 ace+ efaA+ cylA+ gelE+ agg+ cylA+ gelE-

OSY-B1 ace+ efaA+ agg+ cylA- gelE-

OSY-RM1 ace+ efaA+ agg+ cylA- gelE-

Table 2.4 Occurrence of virulence factors from genotypic and phenotypic screening among E. faecalis isolates

55 Isolated Antibiotic Resistance (MIC µg/ml)

Strains Chloramphenicol Tetracycline Kanamycin Ampicillin Erythromycin

OSY-3E1 S R R S S

OSY-3E2 S S R S S

OSY-3E3 S S R S S

OSY-3E4 S S R S S

OSY-3E5 S S R S S

OSY-3E6 S S R S S

OSY-5B1 S S R S S

OSY-6F2 S R R S S

OSY-6F3 S S R S S

OSY-15I2 S S R S S

OSY-16D1 S S R S S

OSY-16D2 S S R S S

OSY-B1 S R R S S

OSY-RM1 S S R S S

Susceptibility (S) and resistance (R) were defined according to MIC breakpoints from the National Committee for Clinical Laboratory Standards (broth dilution method). MICs: chloramphenicol (S: ≤8 µg/ml; R: 32 ≥ µg/ml), tetracycline (S: ≤4 µg/ml; R: 16 ≥ µg/ml), kanamycin (S: ≤6 µg/ml; R: 25 ≥ µg/ml), ampicillin (S: ≤8 µg/ml; R: 16 ≥ µg/ml), erythromycin (S: ≤0.5 µg/ml; R: 8 ≥ µg/ml)

Table 2.5 Resistance of E. faecalis strains isolated from foods to selected antibiotics

56 CHAPTER 3

ENTEROCIN OSY-RM1, A BACTERIOCIN FROM Enterococcus faecalis:

ISOLATION AND CHARACTERIZATION OF THE PEPTIDE AND

STRUCTURAL GENE

ABSTRACT

Bacteriocins produced by lactic acid bacteria have received increasing attention due

to their potential use as natural preservatives and as natural substitute for chemical

preservatives. In this study, a bacteriocin, produced by Enterococcus faecalis OSY-RM1,

referred to as enterocin OSY-RM1 (Ent OSY-RM1), was isolated and characterized.

Results showed that Ent OSY-RM1 was heat stable, sensitive to proteinase K, and stable

over a wide pH range. Extracted Ent OSY-RM1 (44.7 nisin equivalent IU/ml) by

ammonium sulfate precipitation had a bactericidal effect on Listeria monocytogenes Scott

A and a bacteriostatic effect on Bacillus cereus ATCC 11778 in sterile milk. Although

Ent OSY-RM1 was not active against Gram-negative bacteria, it displayed synergistic inactivation of Escherichia coli K12 when combined with high pressure processing in phosphate buffer saline.

57 According to 16% tris-tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis, the molecular mass of Ent OSY-RM1 was estimated to be 4.0-4.5 kDa.

Polymerase chain reaction and direct sequence provided evidence for similarity between

Ent OSY-RM1 and the published enterocin EJ-97. Unlike enterocin EJ-97 that is encoded

on a plasmid, enterocin OSY-RM1 might be encoded on the chromosome because the

antimicrobial activity of plasmid-cured E. faecalis OSY-RM1 still retained.

INTRODUCTION

Bacteriocins produced by lactic acid bacteria (LAB) are investigated extensively due

to their antimicrobial activity against food-borne pathogens (Jack et al., 1995;

Klaenhammer, 1998; Nes et al., 1996). Nisin, for example, produced by several strains of

Lactococcus lactis, has been licensed for use as a food preservative due to its potent

bactericidal activity and non-toxicity to humans (Delves-Broughton 2005; Hurst, 1981).

Bacteriocin-producing LAB include lactococci, lactobacilli, pediococci,

leuconostoc, and enterococci; the latter are of particular relevance to the current

investigation. Enterococci can be found as members of the natural microbiota of various

foods such as milk, cheese, fermented sausages, vegetables and plant materials (Giraffa,

2002). Enterococci persists adverse environmental conditions, surviving at 60oC for 30 min, in the presence of 6.5% NaCl, or with alkaline treatment pH of 9.6 (Sherman, 1937).

Because of their proteolytic and lipolytic activity and citrate utilization, enterococci play an important role in the ripening and aroma development of some cheeses, especially

Mediterranean products (Copploa et al., 1990; Giraffa and Carminati, 1997;

58 Manolopoulou et al., 2003). Some strains of Enterococcus faecium and E. faecalis have

been successfully used as starter or adjunct cultures in cheeses and were used in

commercial probiotic preparations (Giraffa, 2003; Hugas et al., 2003; Lund et al., 2002;

Mikes et al., 1995; Pollman et al., 1980).

Some enterococci produce bacteriocins, generally referred to as enterocins.

Enterocins are reported to inhibit some food-borne pathogens, such as Staphylococcus

spp., Clostridium spp., Bacillus spp., and Listeria monocytogenes (Ennhar et al., 1998;

Franz et al., 1996; Galvez et al., 1998; Giraffa, 1995; Jennes et al., 2000). Most enterocins belong to class II bacteriocins, which are small, heat-stable, and unmodified peptides, with the exception of cytolysin (class I bacteriocin), a novel two-peptide lytic toxin and enterolysin A (class III bacteriocin), a heat-labile protein with a broad inhibitory spectrum (Gilmore et al., 1994; Nes and Holo, 2000; Nilsen et al., 2003). Due to the production of enterocins, enterococci have an advantage in competing with other bacteria sharing the same ecological niches (Eijsink et al., 2002).

The goals of the present study were to (1) characterize the enterocin produced by E.

faecalis OSY-RM1, an isolate from raw cow’s milk, (2) measure the sensitivity of

selected spoilage and food-borne pathogenic bacteria to enterocin OSY-RM1, and (3)

determine the location and sequence the structural gene of enterocin OSY-RM1.

MATERIALS AND METHODS

59 Bacterial strain and growth condition

A bacteriocin-producing bacterium, E. faecalis OSY-RM1 isolated from raw cow’s milk (as mentioned in Chapter 2), was used in this study due to its strong antimicrobial activity against the indicator Lactobacillus plantarum ATCC 8014. The bacteriocin produced was referred to as an enterocin, and thus abbreviated as Ent OSY-RM1. The stock culture was maintained in Lactobacilli DeMann, Rogosa, and Sharpe (MRS) broth

(Difco; BD Diagnostic Systems, Sparks, MD) and stored at -80oC supplemented with

20% glycerol. Before experiment, frozen stock culture was inoculated into 5 ml MRS broth and incubated overnight at 30oC without shaking. Antimicrobial activity of cell-free

supernatant from overnight culture of OSY-RM1, obtained by centrifugation at 8,000 x g

for 10 min (Sorvall RC-5B, DuPont, Wilmington, Del.), was measured against indicator

Lb. cellobiosus OSU 919 using spot-on-lawn method (Lewus et al., 1991) because Lb.

cellobiosus OSU 919 is more sensitive to Ent OSY-RM1 than Lb. plantarum ATCC

8014. Basically, 10 µl of cell-free supernatant was spotted onto MRS soft agar (0.75%)

plate seeded with overnight culture of Lb. cellobiosus OSU 919. Plates were incubated at

30oC for 18 h and clear inhibition zone were observed. Overnight cultures with high

activity were used in the study.

Indicator strains and growth conditions

Indicator strains, such as Lb. plantarum ATCC 8014, Lb. cellobiosus OSU 919, L. monocytogenes Scott A, Bacillus cereus ATCC 11778, and Escherichia coli K12, were obtained from the culture collection of the Food Safety Laboratory at The Ohio State

University (Columbus, OH) for testing in this study. Strains were stored in Lactobacilli

60 MRS broth (for LAB) or in tryptic soy broth (TSB, BBL, Sparks, MD) (for non LAB),

supplemented with 20% glycerol at -80oC. Stock cultures were prepared for experiments by streaking onto MRS agar or tryptic soy agar (TSA) and incubated overnight at 30oC, at

35oC or at 37oC. Before testing, a single colony from the incubated agar plate was inoculated into fresh broths and incubated overnight at 30oC, 35oC or 37oC

Determination of bacteriocin activity

The agar well-diffusion method was performed, as described by Benkerroum et al.

(1993), to determine antimicrobial activity of Ent OSY-RM1. MRS soft agar (15 ml) was seeded with 150 µl of overnight culture of Lb. plantarum ATCC 8014 and was poured into a Petri dish. Plates were held at 25°C for 10 min, and then holes (4-mm diameter) were cut from the indicator-seeded agar. Two-fold dilutions of cell-free supernatant of

OSY-RM1 in 0.1% peptone water (Difco; BD Diagnostic Systems, Sparks, MD) were prepared, and aliquots (50 µl) were placed in the agar wells. As a control, 50 µl of sterile

0.1% peptone water were used. The plates were incubated overnight at 30oC, and the zones of inhibition around the wells were measured and recorded. Bacteriocin titer was measured in arbitrary activity unit (AU), defined as the reciprocal of the highest dilution showing a clear inhibition zone of the indicator lawn, multiplied by a factor of 20 to obtain AU/ml.

Effects of enzymes and heat treatment

The effect of various enzymes on Ent OSY-RM1 activity was determined. Cell-free supernatant (1ml) from overnight culture of OSY-RM1 grown in MRS broth was treated

61 with the following enzymes (final concentration of 1 µg/ml) at 37oC for 2 h: α- chymotrypsin in 20 mM Tris-HCl, pH 8.0; pepsin in 0.002 N HCl; lipase in 0.1 M potassium phosphate, pH 6.0; papain in 0.05 M sodium phosphate acetate, pH 7.0; trypsin in 40 mM Tris-HCl; pH 8.2; protease in 20 mM Tris-HCl, pH 7.8; proteinase K and ficin in 20 mM sodium phosphate, pH 7. All enzymes were purchased from Sigma-Alorich (St.

Louis, MO). Untreated cell-free supernatant served as the positive control. The residual

bacteriocin activity was determined by spot-on-lawn method against Lb. cellobiosus OSU

919 (Lewus et al., 1991).

To evaluate the heat stability of the Ent OSY-RM1, cell-free supernatant (1ml) was treated at 100oC for 5, 10, 20 or 30 min. The remaining activity was then assayed by spot-on-lawn method against Lb. cellobiosus OSU 919.

Stability at different pH values

To determine the stability of Ent OSY-RM1 activity in wide pH range, three pH

values (pH 3.0, 7.0, and 9.0) were chosen. Briefly, cell-free supernatant from overnight

culture of OSY-RM1 were adjusted to tested pH values using either 1M HCl or 1M

NaOH. The adjusted supernatants were then incubated at room temperature (~25oC) for 1 h. After incubation, the tested supernatant was readjusted to neutral pH, and the antimicrobial activity against Lb. cellobiosus OSU 919 was tested using spot-on-lawn

method as described earlier. Unadjusted cell-free supernatant was used as the control.

Crude extraction of enterocin OSY-RM1

62 Overnight culture (5 ml) of OSY-RM1 was transferred to 1000 ml fresh MRS broth

and incubated at 30oC for 24 h. Stationary-phase cells were removed by centrifugation at

12,000 x g for 15 min at 4oC (Sorvall RC 5C Plus Superspeed® Centrifuge, Thermo

Fisher Scientific, Inc., Waltham, MA). Ammonium sulfate (Fisher Scientific Co.,

Pittsburgh, Pa.) was added into cell-free supernatant to reach 60% saturation and the mixture was held overnight at 4oC with gentle stirring. After centrifugation (12,000 x g for 30 min at 4oC), the pellet was suspended in 50 ml potassium phosphate buffer saline

(PBS, 50 mM, pH 6.5). The suspension was dialyzed at 4oC using a dialysis tube with a

1-kDa cut-off membrane (Spectra/Por® Biotech Cellulose Ester Dialysis Membranes,

Spectrum Laboratories, Inc., Rancho Dominguez, CA) against 1,000 ml potassium

phosphate buffer (100 mM, pH 7.0) for at least 18 h with four changes of buffer. After

dialysis, the remaining solution (~20 ml), designated as crude extract of Ent OSY-RM1

(CE OSY-RM1), was sterilized using a 0.2- µm-pore-size low-protein-binding filter

(Millipore Corp., Billerica, MA) and stored at -20oC until further study.

Quantification of the activity of CE OSY-RM1

The bioassay to determine the relative activity of CE OSY- RM1 was determined as described by Yousef and Carlstrom (2003), using commercial nisin as a standard.

Commercial nisin preparation (Nisaplin, Aplin and Barrett Ltd., Trowbridge, UK) was

dissolved in distilled water (targeted concentration was 1,000 IU/ml), adjusted to pH 2 with 1M HCl, and sterilized at 121oC for 15 min. Twofold serial dilutions of the solution of Nisaplin were made using sterile deionized water. Portions (5 µl) of diluted nisin with concentration of 100, 50, 12.5, 6.25, and 3.13 IU/ml, respectively, were spotted onto the 63 MRS soft agar seeded with overnight culture of Lb. cellobiosus OSU 919. CE OSY-RM1

(5 µl) was also spotted onto MRS soft agar. The spotted plate was incubated at 30oC for

24 h and examined for the presence of clear areas of inhibition. The diameter of the inhibition zone was measured and a dose-response plot was constructed to determine nisin equivalent unit of CE OSY-RM1.

Molecular weight estimation

To estimate the molecular weight of Ent OSY-RM1, CE OSY-RM1 was analyzed with 16% Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) (Ready Gel; Bio-Rad, Laboratories, Inc., Hercules, CA), using pre-stained low-

range molecular weight protein marker (Rainbow; BIO-RAD, Laboratories, Inc.,

Hercules, CA) as a size reference (Schagger and Jagow, 1987). Two identical aliquots (10

µl) of the CE OSY-RM1 were loaded on the gel, and these two sets of lanes were separated after electrophoresis. One half of the gel was used for molecular weight determination by fixing and staining with Coomassie Blue. The other half was assayed for antimicrobial activity by fixing in 20% (v/v) isopropanol and 10% (v/v) acetic acid, consecutively (30 min each), washed in distilled water, and then held in distilled water overnight at room temperature (~25oC) (Schagger and Jagow, 1987). The washed gel then was overlaid with MRS soft agar seeded with overnight culture of Lb. plantarum

ATCC 8014. The overlaid gel was incubated at 30oC for 18 h and inspected for the presence of inhibition area surrounding the protein band.

64 Antimicrobial properties of CE OSY-RM1 in ultra-high temperature (UHT)

sterilized milk

To determine the inhibitory activity of Ent OSY-RM1 against pathogenic bacteria in

food system, CE OSY-RM1 was tested against L. monocytogenes Scott A and B. cereus

ATCC 11778 in commercially sterilized milk (Parmalat® milk). CE OSY-RM1 (1ml)

was added to 9 ml of Parmalat® milk that was inoculated with overnight cultures of

pathogens (103 CFU/ml). The inoculated milk was incubated at 35oC for 24 h. In addition, inoculated Parmalat® milk without addition of CE OSY-RM1 was used as control. At selected time points, inoculated milks were serially diluted in 0.1% peptone water and plated on TSA to determine effect of CE OSY-RM1 on growth of pathogens in milk. Plates were incubated at 35oC for 24 to 48 h to enumerate the survivors (CFU/ml).

Combined effect of high pressure processing (HPP) and CE OSY-RM1 on

inactivation of Escherichia coli K12

Efficacy of CE OSY-RM1 and HPP combination against E.coli K12 was tested. E.

coli K12 cells (25 ml) at stationary phase (109 CFU/ml) were collected by centrifugation

(12,000 x g for 10 min at 4oC), washed twice with 25 ml of sterile phosphate buffer saline

(PBS, 12.5 mM, pH 7.2), and suspended in 25 ml of PBS. Cell suspensions (900 µl) were mixed with either PBS (100 µl) or CE OSY-RM1 (100 µl), aseptically transferred to a sterile polyethylene bags (Fisher Scientific Co.), and then heat-sealed. Sample bags were pressurized at 500 MPa and 25°C ± 2°C for 1 min in a high-pressure processor (Quintus

QFP-6, Flow Pressure Systems, Kent, Wash.) containing 1:1 (v/v) glycol/water pressure

transmitting fluid (Houghto-Safe 620 TY, Houghton International Inc., Valley Forge, Pa). 65 In addition, samples containing PBS (100 µl) or CE OSY-RM1 (100 µl) were used as

controls and were held on ice during HPP. After HPP, all samples (control and treated)

were serially diluted in 0.1% peptone water and plated on TSA. The plates were

incubated at 37oC for 24 to 48 h to enumerate the survivors (CFU/ml). This experiment

was repeated independently three times.

Identification of the structural genes of enterocin OSY-RM1

The structural gene of Ent OSY-RM1 was identified through Polymerase Chain

Reaction (PCR) amplification and direct sequencing. Total DNA of OSY-RM1 was

extracted from overnight culture grown in MRS broth at 30oC using a commercial DNA isolation kit (DNeasy Tissue Kit; QIAgen Sciences Inc., Valencia, CA). Purified total

DNA was used as a DNA template for PCR amplification. The genomic DNA was screened for eight published structural genes (entA, entB, entP, entAS-48, entL50A/B,

bac31, entEJ-97, ent1071AB) which encode these bacteriocins: enterocin A, enterocin B,

enterocin P, enterocin AS-48, enterocin L50A/B, bacteriocin 31, enterocin EJ-97, and

enterocin 1071AB, respectively. Specific primers used are listed in Table 3.1.

PCR amplification reactions were carried out using Taq DNA polymerase kit

(QIAGEN, Valencia, CA). For entA, entB, and entP, amplification for PCR included

initial denaturation (5 min at 95oC), followed by 30 cycles of denaturation (95oC for 1

min), annealing (56oC for 1min), and elongation (72oC for 1 min), ending with a final

extension step (72oC for 10 min). For entL50A/B, bac31, entAS-48, entEJ-97,

ent1071AB, the annealing temperature and cycle number were changed to 60oC for 35 cycles, 42oC for 20 cycles, 52oC for 30 cycles, 55oC for 30 cycles, and 50oC for 25

66 cycles, respectively. New primer set, namely EntEJ-97-F2 and EntEJ-97-R2,

corresponding to nucleotide positions 1581-1601 upstream, and positions 1695–1715

downstream, of the enterocin EJ-97 structural gene (Sanchez-Hidalgo et al., 2003), was

designed. The annealing temperature and cycle number for PCR amplification using

newly designed primer set were 54oC and 30 cycles, respectively.

All PCR amplifications were performed in a Techgene DNA Thermal Cycler

(geneAmp PCR system 2400, Applied Biosystems, Foster City, CA). After amplification,

PCR products were examined on 2.0% agarose gel electrophoresis followed by ethidium bromide staining. The gels were run at 100 V for 45 min, using the 100 base pair (bp)

DNA ladder (Gibco BRL, Gaithersburg, MD) as the molecular weight standard. The amplified product was purified using a commercial DNA extraction kit (QIAquick gel extraction kit; QIAGEN) and then sequenced by the Plant Microbe Genomics Facility

(The Ohio State University, Columbus, OH) (3730 DNA Analyzer; Applied Biosystems,

Foster City, CA). The obtained nucleotide sequence was compared with those in the

NCBI GenBank (http://www.ncbi.nlm.nih.gov/) using BLAST (Altschul et al., 1997).

Curing OSY-RM1 plasmids

To determine the location of Ent OSY-RM1, E. faecalis OSY-RM1 was cured of

any potential plasmids using acridine orange and the antimicrobial activity of plasmid-

cured OSY-RM1 was tested using the well diffusion method, as described earlier (Barker

and Hardman, 1978). Overnight culture of OSY-RM1 (109 CFU/ml, 50 µl) was

transferred to 5 ml of MRS broth containing acridine orange (AO; 20 µg/ml). The

mixture was incubated overnight at 30°C under dark condition without shaking. Aliquots

67 (100 µl) of the 10-5 dilution were spread-plated onto several MRS agar plates and the

plates were incubated at 30°C until appearance of colonies (~24 h). The plates were then

overlaid with MRS soft agar seeded with overnight culture of Lb. plantarum ATCC 8014, and incubated overnight at 30oC. Colonies of AO-treated OSY-RM1, with or without

clearing zones, were carefully 3-phase streaked on MRS agar, and incubated overnight at

30oC. Colonies of AO-treated OSY-RM1 were examined microscopically and confirmed to contain cocci (target), not rods (indicator). Additionally, AO-untreated culture (wild type) was tested as a control. Cell-free supernatants of AO-treated and untreated OSY-

RM1 were tested for the production of antimicrobial agents using the agar well diffusion method.

Plasmid DNAs of AO-treated and non-treated OSY-RM1 were extracted using a commercial kit (QIAprep Spin Miniprep kit, QIAGEN) as described by the manufacturer.

Before extracting plasmid DNA, the cell pellet (prepared by centrifugation at 8,000 x g for 5 min from 1 ml of overnight culture) was incubated at 37°C for 1.5 h in 180 µl enzymatic lysis buffer (containing 20 mM Tris.Cl, pH 8.0; 2 mM sodium EDTA; 1.2%

Triton® X-100; and 20 mg/ml lysozyme). After extraction, plasmid DNA from AO- treated and non-treated OSY-RM1 was examined on 0.8% agarose gel electrophoresis

followed by ethidium bromide staining. The gels were run at 100 V for 45 min, using the

1,000 base pair (kb) DNA ladder (Gibco BRL, Gaithersburg, MD) as the molecular

weight standard.

RESULTS AND DISCUSSION

68 Properties of the enterocin OSY-RM1

The antimicrobial activity of Ent OSY-RM1 against Lb. plantarum ATCC 8014 was

detected in culture supernatant, revealing a strong antimicrobial titer of 1,280 AU/ml. In

addition, its activity was examined for sensitivity to heat and various enzyme treatments

(Table 3.2). After treatment at 100oC for 10 min, the antimicrobial activity of Ent OSY-

RM1 was retained, indicating relative heat stability. But the activity of Ent OSY-RM1 was completely abolished after heat treatment at 100oC for 20 min. In addition, Ent OSY-

RM1 was stable over a wide pH range. Unlike nisin, the solubility and activity of which decreases with the increasing pH value (Delves-Broughton, 2005), Ent OSY-RM1 still retained its activity after incubation at room temperature for 1 h in a pH range from 3.0 to

9.0. Ent OSY-RM1 was sensitive to proteinase K and α−chymotrypsin, indicating its proteinaceous nature, but its activity was not altered by pepsin, lipase, papain, protease and ficin and partially lost by trypsin. Resistance to lipase suggests that Ent OSY-RM1 does not require a lipid moiety for activity. These results indicated that Ent OSY-RM1 could be a potentially useful additive in pasteurized, acidic or alkaline foods or food products.

Relative enterocin OSY-RM1 activity value

To quantify CE OSY-RM1, the relative bacterioicn activity value was determined using commercial nisin as a reference standard. After incubation, the diameters of circular clear zones, indicating growth inhibition of indicator (Lb. cellobiosus OSU 919) were measured. Then a dose-response plot was constructed, revealing the correlation between nisin concentration and the diameter of inhibition zone (Figure 3.1). The

69 diameter of inhibition zone, resulting from the antimicrobial activity of CE OSY-RM1,

was 16 mm, and was matched with a similar point on the standard dose-response plot to

determine the equivalent concentration of CE OSY-RM1 as 44.7 IU/ml.

Estimation of molecular weight of enterocin OSY-RM1

The molecular weight of Ent OSY-RM1 was analyzed by 16% Tris-tricine SDS-

PAGE. After incubation, an inhibition area around a band appeared (Figure 3.2, Lane C),

suggesting that the molecular weight of Ent OSY-RM1 was approximately 4.0-4.5 kDa.

However, the molecular weight reported here is only an estimated value, as size determination by SDS-PAGE is approximate.

Inactivation of food-borne pathogens

The antimicrobial activity of CE OSY-RM1 against food-borne pathogens L. monocytogenes Scott A and B. cereus ATCC 11778 in Parmalat® milk is shown in

Figure 3.3 A and 3.3 B, respectively. Results showed that the population of L. monocytogenes Scott A decreased below detection limit (101 CFU/ml) after 1 h

treatment, suggesting that Ent OSY-RM1 has a bactericidal mode of action against this pathogen (Figure 3.3 A). This result is similar that reported by García et al. (2004), who demonstrated bactericidal activity of Ent EJ-97 (30 IU/ml) against L. monocytogenes with

no viable cells detected after incubation for 24 h at 37oC in brain heat infusion broth.

CE OSY-RM1 inhibited the growth of B. cereus ATCC 11778 (Figure 3.3 B), with no considerable growth of this pathogen throughout the incubation period. This finding suggests that Ent OSY-RM1 has a bacteriostatic mode of action against B. cereus.

70 Synergistic effect of HPP and CE OSY-RM1

To inactivate Gram-negative bacteria, combination treatment of high pressure

processing (HPP) and CE OSY-RM1 against E. coli K12 was performed (Figure 3.4). E.

coli K12 controls with or without CE OSY-RM1 treatment were not different, indicating

Ent OSY-RM1 alone had no antimicrobial activity against this Gram-negative bacterium, likely due to the barrier properties of outer membrane (Breukink and Kruijff, 2006).

Although the porins, the one of membrane transport proteins of Gram-negative bacteria, allow hydrophilic molecules below 600 Da to freely diffuse, Ent OSY-RM1 is too large

(4.0-4.5 kDa) to transverse the outer membrane (Klaenhammer, 1993; Stiles and

Hastings, 1991). Therefore, Ent OSY-RM1 cannot reach the cytoplasmic membrane which is the target attacked by most bacteriocins of LAB (Cintas et al., 2001).

CE OSY-RM1 in combination with HPP caused significant inactivation of E. coli

K12, leading to an average population decrease of 5.5 log, compared with control

(without any treatment), and of 3.0 log decrease caused by HPP alone, indicating the

antimicrobial synergy between HPP and Ent OSY-RM1. Because the outer membrane of

Gram-negative bacteria is thought to be damaged due to the denaturation of outer

membrane protein after HPP, Ent OSY-RM1 may access the cytoplasmic membrane,

resulting in the lethality of cells (Ritz, 2000).

Identification of enterocin OSY-RM1 structural gene

Total DNA extracted from overnight culture of OSY-RM1 was subjected to PCR

amplification to determine the presence of structural genes (entA, entB, entP, entEJ-97,

ent1071AB, entL50, entAS-48 and bac31) encoding for previously described enterocins.

71 A PCR product was obtained from the primer set enterocin EJ-97. However, the PCR

product resulting from amplification using the enterocin EJ-97 primer set (EntEJ-97-F1

and EntEJ-97-R1) could not be sequenced effectively. PCR amplification was repeated using newly designed primers (EntEJ-97-F2 and EntEJ-97-R2). A DNA fragment of about 150 bp was obtained and was purified and sequenced (Figure 3.5). The obtained nucleotide sequence showed 98% similarity to the gene encoding for published enterocin

EJ-97, and the predicted amino acid sequence was 95% similar to the mature enterocin

EJ-97 protein (Figure 3.6).

Location of enterocin OSY-RM1

In order to determine the origin of Ent OSY-RM1 genetic code, plasmids of E.

faecalis OSY-RM1 were cured and the antimicrobial activities of wild type and plasmid-

cured strains of E. faecalis OSY-RM1 were tested using agar-well diffusion method

(Figure 3.7). Results indicated that AO-treated OSY-RM1 (plasmid-cured) retained

antimicrobial activity and no plasmid were detected on agarose gel following

electrophoresis, compared with wild type OSY-RM1, in which its plasmids were detected

on agarose gel as showing three bands after electrophoresis. This finding suggests that

Ent OSY-RM1 is most likely encoded on the chromosome of E. faecalis OSY-RM1.

There was a report of the structural gene of enterocin A, produced by E. faecium, being

located on the bacterial chromosome (Aymerrich et al., 1996), however, to our

knowledge, this is the first report that a bacteriocin produced by E. faecalis is

chromosomally encoded.

72 Although the nucleotide sequences between Ent OSY-RM1 and Ent EJ-97 showed

similarity, some of the properties were clearly different (Table 3.3). The molecular

weights of Ent OSY-RM1 and Ent EJ-97 were similar, 4.0-4.5 kDa and 4.8 kDa,

respectively, based on SDS-PAGE analysis (Gálvez et al., 1998). Ent EJ-97 was more

heat stable than Ent OSY-RM1 with the activity of Ent EJ-97 retaining after heat

treatment at 100oC for 30 min. Importantly, unlike Ent EJ-97 (Sanchez-Hidalgo et al.,

2003), the structural gene of which was encoded on a plasmid, the structural gene of Ent

OSY-RM1 might be encoded on the chromosome.

In conclusion, an enterocin, produced by the food isolate E. faecalis OSY-RM1, was

further characterized. Extracted Ent OSY-RM1 by ammonium sulfate precipitation

showed a bactericidal effect against L. monocytogenes Scott A and a bacteriostatic effect

against B. cereus ATCC 11778 in Parmalat® milk. Gram-negative bacterium E. coli K12 also can be inactivated by the combination treatment of high pressure processing and Ent

OSY-RM1. In addition, obtained nucleotide sequences from PCR products, predicted amino acid sequences, and some properties of Ent OSY-RM1 are different from published enterocin EJ-97. Unlike enterocin EJ-97 that is encoded on a plasmid, Ent

OSY-RM1 is likely chromosomally encoded.

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77 Eijsink, V. G. H., L. Axelsson, D. B. Diep, L. S. Havarstein, H. Holo, and I. F. Nes. 2002. Production of class II bacteriocin by lactic acid bacteria; an example of biological warfare and communication. Antonie van Leeuwenhoek. 81: 639-654.

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78 Enterocins Primers Amino acid Classification Reference size Enterocin AS-48 (entAS-48) 67 Class II Galvez et al. F: 5’-GAGGAGTITCATGITTAAAGA-3’ 1994 R: 5’-CATATTGTTAAATTACCAAGCAA-3’ Enterocin L50 A/B (ent L50A/B) Ent L50A: 44 Class II Cintas et al., F: 5’-TGGGAGAATCGCAAAATTAG-3’ Ent L50B: 36 2000 R: 5’-ATTGCCCATCCTTCTCCAAT-3’ Enterocin EJ-97 (entEJ-97) 48 Class II This study F2: 5’-AAGCGATGATTAAGAAGTTT-3’ R2: 5’-ACGACCGTATTGTTGTTTAT-3’ Enterocin EJ-97 (entEJ-97) 48 Class II Gálvez et al., F1: 5’-AGTCGAAGAGAAATTGG-3’ 1998 R1: 5’-AGGGGAATTTGAACAGA-3’ Enterocin A (entA) 47 Class IIa Aymerrich et F: 5’-AAATATTATGGAGTGTAT-3’ al., 1996 R: 5’-GCACTTCCCTGGAATTGCTC-3’ Enterocin 1071AB (ent 1071AB) Ent 1071A: 39 Class IIb Balla et al., F: 5’-ATGAAGCAATATAAAGTATTG-3’ Ent 1071B: 35 2000 R: 5’-ATACATTCTTCCACTTATTTTT-3’ Bacteriocin 31 (bac31) 43 Class IIc Tomita et al., F: 5’- TATTACGGAAATGGTTTATATTGT-3’ 1996 R: 5’-TCTAGGAGCCCAAGGGCC-3’ Enterocin P (entP) 44 Class IIc Cintas et al., F: 5’-TATGGTAATGGTGTTTATTGTAAT-3’ 1997 R: 5’-ATGTCCCATACCTGCCAAAC-3’ Enterocin B (entB) 53 Class IId Casaus et al., F: 5’-GAAAATGATCACAGACCTA -3’ 1997 R: 5’-GTTGCATTTAGAGTATACATTG-3’

Table 3.1 Primers used in the PCR reactions for attempted amplification of Enterococcus faecalis OSY-RM1 bacteriocin

79 Treatment Conditions Residual activitya of Ent OSY-RM1

Temperature (100oC) 5 min +

10 min +

20 min -

30 min -

Enzymes a-chymotrypsin -

Pepsin +

Lipase +

Papain +

Trypsin -/+

Protease +

Proteinase K -

Ficin +

Acidic pH 3.0 +

Neutral pH 7.0 +

Alkaline pH 9.0 +

a (+) activity retained; (-/+) activity lost partially; (-) activity lost.

Table 3.2 Susceptibility of enterocin OSY-RM1 to heat, enzyme, and wide pH value range treatments

Properties Enterocin OSY-RM1 Enterocin EJ-97a

Molecular weight (Based on SDS-PAGE) 4.0-4.5 kDa 4.8 kDa

Heat stability (100oC for 30 min) Total loss of activity Partial loss of

activity

Sensitivity to proteases trypsin Partial loss of activity Total loss of activity

Antimicrobial activity against No inhibitory effect Inhibitory effect Staphylococcus aureus Location of enterocin structural gene Chromosome Plasmid

a Reported by Galvez et al. (1998)

Table 3.3 Different properties between enterocin OSY-RM1 and enterocin EJ-97

25 ) m m ( 20 ne o z

o 15 i t bi hi

in 10 of r e t

e 5 m a i D 0 00.511.522.5 commercial nisin concentration (Log IU/ml)

Figure 3.1 Dose-response plot of the concentration of commercial nisin and determination of nisin-equivalent units of CE OSY-RM1 (Lactobacillus cellobiosus OSU 919 served as the indicator organism for spot-on-lawn assay).

Figure 3.2 Analysis of peptide extracted from Enterococcus faecalis OSY-RM1 culture by 16% Tris-Tricine SDS-PAGE. Lane A: Molecular weight marker, coomassie blue stained Lane B: Crude extract of enterocin OSY-RM1, coomassie blue stained Lane C: Inhibition area caused by crude extract of enterocin OSY-RM1, on the Lactobacillus plantarum overlaid gel

l 9 m /

U 8 F C

g 7

Lo 6 s e

n 5 e g

o 4

cyt No CE OSY -RM1 o 3 n

o CE OSY -RM1

m 2 a i

er 1 st i

L 0 0 2 4 6 8 1012141618202224 A Time (hours)

l 8 m /

U 7 F 6 g C

Lo 5 s u

e 4 r 3 s ce No CE OSY -RM1 u l l

ci 2 CE OSY -RM1 a B 1

0 0 2 4 6 8 1012141618202224 B Time (hours)

Figure 3.3 Changes in the populations of Listeria monocytogenes Scott A (A) and Bacillus cereus ATCC 11778 (B) in Parmalat® milk treated with enterocin OSY-RM1 crude extract (44.7 IU added /ml of milk) and incubated at 35°C for 24 h. Error bars represent standard error, n=2.

Figure 3.4 Survivor of Escherichia coli K12 (log CFU/ml) treated with CE OSY-RM1 (44.7 IU added /ml) and/or HPP (500 MPa, 25oC ± 2oC for 1 min). Error bars represent standard error, n=3.

Figure 3.5 PCR amplification of enterocin EJ-97 structural gene using total DNA of Enterococcus faecalis OSY-RM1 as template. Lane A: 100bp DNA ladder Lane B: E. faecalis OSY-RM1

Figure 3.6 Comparison of the nucleotide sequences of structural gene of enterocin OSY- RM1 and enterocin EJ-97, including the predicted amino acid sequences. Sequence differences are marked by red letters.

Figure 3.7 Relation between plasmid curing by acridine orange (AO) and persistence of antimicrobial activity in Enterococcus faecalis OSY-RM1 culture. (a) stained 0.8% agarose gel showing plasmid of non-treated (lane B) and AO-treated (lane C) OSY-RM1 cultures; (b) inhibition areas produced by cell-free supernatants from non-treated (D) and AO-treated (E) OSY-RM1 cultures. Lane A contains 1 kb DNA ladder.

88 CHAPTER 4

CHARACTERIZATION OF A BACTRERIOCIN PRODUCED BY Enterococcus

faecalis OSY-3E1 ISOLATED FROM COMTĖ CHEESE

ABSTRACT

A bacteriocin, produced by Enterococcus faecalis OSY-3E1, referred to as enterocin

OSY-3E1 (Ent OSY-3E1), was characterized and identified by polymerase chain reaction

(PCR) and direct sequencing. Ent OSY-3E1 retained its antimicrobial activity after treatment at 100oC for 20 min. In addition, Ent OSY-3E1 was sensitive to proteases enzyme and stable over a wide pH range (pH 3.0 to pH 9.0). Using PCR amplification

and sequencing, Ent OSY-3E1 was found to be similar to published enterocin AS-48. In

spite of undesirable properties commonly associated with E. faecalis, Ent OSY-3E1

(25,600 AU/ml), obtained from modified MRS broth containing 5% glucose, was effective against the spoilage bacterium, Leuconostoc mesenteroides ATCC 14935, in sterile milk.

89 INTRODUCTION

Bacteriocins are bacterially produced peptides or proteins that are active against closed related bacteria (Jack et al., 1995). Recently, bacteriocins produced by lactic acid bacteria (LAB) have attracted increasing interest due to their potential for use as biopreservatives in the food industry to eliminate food-borne pathogenic bacteria, such as

Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens and

Clostridium botulinum (Abee et al., 1995; Jack et al., 1995; Klaenhammer, 1988; Nes et al., 1996; Stiles, 1996).

Enterococcus is one of LAB genera and species of this genus are natural inhabitants of the gastrointestinal tract of humans and other animals. They are also present in vegetables, plant material and foods (Giraffa 2002). Some strains of enterococci are used as cheeses starter cultures and even as animal and human probiotics (Coppola et al.,

1990; Lund et al., 2002). Bacteriocins produced by enterococci are generally referred to as enterocins and several enterocins have been characterized (Aymerich et al., 1996;

Casaus et al., 1997; Cintas et al., 1997, 1998, 2000; Du Toit et al., 2000; Ennahar et al.,

1998, 2001; Galvez et al., 1989, 1998; Herranz et al., 1999, 2001; Marekova et al., 2003;

Ohmomo et al., 2000; Sabia et al., 2002, 2004).

Results from Chapter 2 showed E. faecalis OSY-3E1 produced antimicrobial agent(s) against several LAB (e. g., Lactobacillus casei, Lb. cellobiosus, Lb. bulgaricus,

Lb. plantarum, Leuconostoc mesenteroides, Lactococcus lactis, and Pediococcus acidilactici) and food-borne pathogens (Listeria monocytogenes and Bacillus cereus).

The goals of the present study were to (1) characterize the enterocin produced by E.

90 faecalis OSY-3E1 isolated from comtė cheese, (2) optimize the production of Ent OSY-

3E1, and (3) measure the sensitivity of selected spoilage bacterium, Leuc. mesenteroides,

to Ent OSY-3E1 in sterile milk.

MATERIALS AND METHODS

Bacterial strain and growth condition

A bacteriocin-producing bacterium, E. faecalis OSY-3E1 isolated from comté cheese (Chapter 2), was used in this study due to its strong antimicrobial activity against the indicator, Lactobacillus cellobiosus OSU 919. The bacteriocin produced was referred

to as an enterocin OSY-3E1 (Ent OSY-3E1). The stock culture of E. faecalis OSY-3E1

was maintained in Lactobacilli DeMann, Rogosa, and Sharpe (MRS) broth (Difco; BD

Diagnostic Systems, Sparks, MD) and stored at -80oC supplemented with 20% glycerol.

Before experiment, the frozen stock culture was inoculated into 5 ml MRS broth and incubated overnight at 30oC. Antimicrobial activity of cell-free supernatant from overnight culture of OSY-3E1, obtained by centrifugation at 8,000 x g for 10 min

(Sorvall RC-5B, DuPont, Wilmington, Del.), was measured against Lb. cellobiosus OSU

919, a sensitive indicator, using spot-on-lawn method (Lewus et al., 1991). Basically, 10

µl cell-free supernatant was spotted onto MRS soft agar (0.75%) plate seeded with overnight culture of Lb. cellobiosus OSU 919. Plates were incubated at 30oC for 18 h and clear inhibition zone were observed. Overnight cultures with high activity were tested in this study.

91 Indicator strains and growth conditions

Ent OSY-3E1 sensitive strains, Lb. cellobiosus OSU 919 and Leuc. mesenteroides

ATCC 14935, were obtained from the culture collection of the Food Safety Laboratory at

The Ohio State University (Columbus, OH). Strains were stored in Lactobacilli MRS

broth supplemented with 20% glycerol at -80oC. Stock cultures were prepared for

experiments by streaking onto MRS agar and incubated overnight at 30oC. Before testing,

a single colony from the incubated agar plate was inoculated into fresh MRS broths and

grown to stationary phase by incubation overnight at 30oC.

Effects of enzymes and heat treatment

Susceptibility of Ent OSY-3E1 to various enzymes and heat was tested as described by Lewus et al. (1991) with modifications. Cell-free supernatant (1 ml) from overnight culture of OSY-3E1 grown in MRS broth was incubated at 37oC for 1 h with the

following enzymes: proteinase K, trypsin, and pepsin at a final concentration of 1 µg/ml

in phosphate buffer (50 mM, pH 7.0). All enzymes were purchased from Sigma-Alorich

(St. Louis, MO). Untreated cell-free supernatant served as the positive control. The residual bacteriocin activity was determined by spot-on-lawn method against Lb. cellobiosus OSU 919 (Lewus et al., 1991).

To evaluate the heat stability of the Ent OSY-3E1, cell-free supernatant (1 ml) was

treated at 100oC for 5, 10, 20 and 30 min. The remaining activity was then assayed by spot-on-lawn method against Lb. cellobiosus OSU 919.

Stability in a wide pH range

92 To determine the stability of Ent OSY-3E1 activity in wide pH range, three pH

values (pH 3.0, 7.0, and 9.0) were tested. Briefly, cell-free supernatant from overnight

culture of OSY-3E1 were adjusted to tested pH values using either 1M HCl or 1M

NaOH. The adjusted supernatants were then incubated at room temperature (~25oC) for 1 h. After incubation, the tested supernatant was readjusted to neutral pH, and the activity against Lb. cellobiosus OSU 919 was tested using spot-on-lawn method as described earlier. Unadjusted cell-free supernatant served as a positive control.

Identification of the structural genes of enterocin OSY-3E1

Total DNA of OSY-3E1 was extracted from overnight culture grown in MRS broth at 30oC using a commercial DNA isolation kit (DNeasy Tissue Kit; QIAgen Sciences

Inc., Valencia, CA). Purified total DNA was used as a DNA template for polymerase

chain reaction (PCR) amplification. The genomic DNA was screened against eight

published structural genes (entA, entB, entP, entAS-48, entL50A/B, bac31, entEJ-97,

ent1071AB) of bacteriocins (enterocin A, enterocin B, enterocin P, enterocin AS-48,

enterocin L50A/B, bacteriocin 31, enterocin EJ-97, and enterocin 1071AB, respectively)

using the specific primers listed in Table 4.1.

PCR amplification reactions were carried out using Taq DNA polymerase kit

(QIAGEN, Valencia, CA). For entA, entB, and entP, amplification for PCR included

initial denaturation (5 min at 95oC), followed by 30 cycles of denaturation (95oC for 1

min), annealing (56oC for 1min), and elongation (72oC for 1 min), ending with a final

extension step (72oC for 10 min). For entL50A/B, bac31, entAS-48, entEJ-97,

ent1071AB, the annealing temperature and cycle number were changed to 60oC for 35

93 cycles, 42oC for 20 cycles, 52oC for 30 cycles, 55oC for 30 cycles, and 50oC for 25

cycles, respectively. PCR products were examined on 2.0% agarose gel electrophoresis

followed by ethidium bromide staining. The gels were run at 100 V for 45 min, using the

100 base pair (bp) DNA ladder (Gibco BRL, Gaithersburg, MD) as the molecular weight

standard. The amplified product was purified using a commercial DNA extraction kit

(QIAquick gel extraction kit; QIAGEN) and then sequenced by the Plant Microbe

Genomics Facility (The Ohio State University, Columbus, OH) (3730 DNA Analyzer;

Applied Biosystems, Foster City, CA). The obtained nucleotide sequence was compared with nucleotide sequences in the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) using

BLAST (Altschul et al., 1997).

Optimizing production of enterocin OSY-3E1

Overnight culture of E. faecalis OSY-3E1 (109 CFU/ml, 50 µl) was transferred to

three tubes of MRS broth (5 ml) containing 1% glucose, 5% glucose, or 10% glucose

(w/v), respectively, and incubated at 30oC overnight. Non-modified MRS was used as control. After incubation, the relative Ent OSY-3E1 activity from cell-free supernatant was determined using Lb. cellobiosus OSU 919 as the indicator for the spot-on-lawn method, as described earlier. Bacteriocin activity value was measured in arbitrary activity unit (AU), which is defined as the reciprocal of the highest dilution showing a clear inhibition zone of the indicator lawn, multiplied by a factor of 100 to obtain AU/ml. In addition, the pH values of each supernatant were measured (pH meter 430, Corning,

Lowell, MA).

94 Efficacy of enterocin OSY-3E1 against Leuconostoc mesenteroides in milk

The inhibitory activity of Ent OSY-3E1 from modified MRS broth was tested

against Leuc. mesenteroides ATCC 14935 in commercially sterile milk (Parmalat®

milk). Cell-free supernatant of OSY-3E1 from modified MRS containing 5% glucose was

sterile filtrated (0.2-µ m-pore-size low-protein-binding filter; Millipore, Billerica, MA)

and 100 µl of filtrate was added to 900 µl of sterile milk containing 104 CFU/ml Leuc. mesenteroides ATCC 14935. The inoculated milk was incubated at 16oC for 5 days.

Inoculated milk without Ent OSY-3E1 served as the control. At selected time points, samples of inoculated milks were serially diluted in 0.1% peptone water and plated on

MRS agar, and the plates were incubated at 30oC for 24 to 48 h to enumerate the survivors (CFU/ml).

RESULTS AND DISCUSSION

Properties of the enterocin OSY-3E1

The antimicrobial activity of Ent OSY-3E1 was examined for sensitivity to heat treatment and various enzymes (Table 4.2). Enzyme sensitivity assay demonstrated that the antimicrobial activity of Ent OSY-3E1 was completely eliminated by treatment with proteinase K, pepsin and trypsin, indicating its proteinaceous nature. Although the inhibitory activity of Ent OSY-3E1 was relatively heat stable, its activity was lost when incubated at 100oC for 30 min. The inhibitory activity of Ent OSY-3E1 was retained after

exposure to pH 9.0, and lost partially at pH 3.0. These results offer several desirable

characteristics that make Ent OSY-3E1 suitable for food preservation. 95 Identification of enterocin OSY-3E1 structural gene

Total DNA extracted from overnight culture of OSY-3E1 was subjected to PCR amplification to determine the presence of structural genes (entA, entB, entP, entEJ-97, ent1071AB, entL50, entAS-48 and bac31) encoding for previously described enterocins.

After PCR amplification and agarose gel electrophoresis, a DNA fragment was obtained when using the primer set for enterocin AS-48. This fragment (~250 bp) was purified and sequenced (Figure 4.1).

The obtained nucleotide sequence showed 97.5% similarity to the gene encoding for enterocin AS-48, and the predicted amino acid sequence was 96.7% similar to the mature enterocin AS-48 protein (Figure 4.2). The amino acid residues, glycine (Gly), glycine and glutamic acid (Glu), found in the mature enterocin AS-48 at the position 13, 22 and 49, respectively (Gonzalez et al., 2000), were replaced by the predicted amino acids, histidine

(His), lysine (Lys) and glutamine (Gln), respectively, for Ent OSY-3E1. Unlike enterocin

AS-48 that has a broad inhibitory activity spectrum against not only Gram-positive bacteria but also Gram-negative bacteria (i.e. Escherichia coli and Shigella sonnei)

(Galvez et al., 1989), Gram-negative bacteria tested in this study were not inhibited by

Ent OSY-3E1 (data not shown). Although the relationship between the structure of enterocins and the inhibitory activity spectrum is not clearly understand, the globular molecular structure of AS-48 probably determines its broad antimicrobial activity by stimulating pore formation in target cell membranes (Maqueda et al., 2004).

96 Optimizing production of enterocin OSY-3E1

To enhance the production of Ent OSY-3E1, modified MRS broth containing

different concentrations of glucose was used to cultivate E. faecalis OSY-3E1. After incubation, the supernatants from modified and non-modified MRS broth were examined the antimicrobial activity against Lb. cellobiosus OSU 919, and the pH value of each

supernatant was also tested. Results were shown in Table 4.3. The highest relative Ent

OSY-3E1 activity value (25,600 AU/ml) was obtained from the modified MRS broth

containing 5% glucose. The pH values of cell-free supernatants did not vary significantly

after incubation.

Demonstration of antimicrobial activities of enterocin OSY-3E1 in food

Ent OSY-3E1 from modified MRS broth (5% glucose) was effective against

Leuconostoc mesenteroides ATCC 14935 in Parmalat® milk (Figure 4.3). The initial

load of Leuc. mesenteroides ATCC 14935 was 104 CFU/ml, but the population decreased during the storage period and no viable cells (detection limit 101 CFU/ml) were detected after a 4-day treatment. These findings indicate Ent OSY-3E1 has a bactericidal mode of action against this spoilage bacterium.

According to studies by Chenoll et al. (2007) and Garcia-Gimeno et al. (2005),

Leuc.mesenteroides is a main species responsible for meat product spoilage, mostly due

to gas production, slime formation, or pH drop. Although the antimicrobial activity of Ent

OSY-3E1 against Leuc. mesenteroides in meat was not tested in this study, the result

indicates that Ent OSY-3E1 could be a potential preservative to prevent the spoilage

caused by this bacterium.

97 In conclusion, an enterocin produced by the food isolate, E. faecalis OSY-3E1, was

characterized. Nucleotide sequences comparison showed that enterocin OSY-3E1 was

similar to the published sequence of enterocin AS-48. Unlike enterocin AS-48 that has

antimicrobial activity against Gram-negative bacteria, Ent OSY-3E1 did not possess

similar activity. Ent OSY-3E1 with high antimicrobial activity was obtained from

modified MRS broth containing 5% glucose and had a bactericidal effect on the spoilage

bacterium, Leuc. mesenteroides ATCC 14935, in sterile milk.

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102

Enterocins Primers Amino acid Classification Reference size Enterocin AS-48 (entAS-48) 67 Class II Galvez et al. F: 5’-GAGGAGTITCATGITTAAAGA-3’ 1994 R: 5’-CATATTGTTAAATTACCAAGCAA-3’ Enterocin L50 A/B (ent L50A/B) Ent L50A: 44 Class II Cintas et al., F: 5’-TGGGAGAATCGCAAAATTAG-3’ Ent L50B: 36 2000 R: 5’-ATTGCCCATCCTTCTCCAAT-3’ Enterocin EJ-97 (entEJ-97) 48 Class II Gálvez et al., F1: 5’-AGTCGAAGAGAAATTGG-3’ 1998 R1: 5’-AGGGGAATTTGAACAGA-3’ Enterocin A (entA) 47 Class IIa Aymerrich et F: 5’-AAATATTATGGAGTGTAT-3’ al., 1996 R: 5’-GCACTTCCCTGGAATTGCTC-3’ Enterocin 1071AB (ent 1071AB) Ent 1071A: 39 Class IIb Balla et al., F: 5’-ATGAAGCAATATAAAGTATTG-3’ Ent 1071B: 35 2000 R: 5’-ATACATTCTTCCACTTATTTTT-3’ Bacteriocin 31 (bac31) 43 Class IIc Tomita et al., F: 5’- TATTACGGAAATGGTTTATATTGT-3’ 1996 R: 5’-TCTAGGAGCCCAAGGGCC-3’ Enterocin P (entP) 44 Class IIc Cintas et al., F: 5’-TATGGTAATGGTGTTTATTGTAAT-3’ 1997 R: 5’-ATGTCCCATACCTGCCAAAC-3’ Enterocin B (entB) 53 Class IId Casaus et al., F: 5’-GAAAATGATCACAGACCTA -3’ 1997 R: 5’-GTTGCATTTAGAGTATACATTG-3’

Table 4.1 Primers used in the PCR reactions for attempted amplification of Enterococcus faecalis OSY-3E1 bacteriocin

103

Treatment Conditions Residual Activitya of Ent OSY-3E1

Temperature 5 min +

(100oC) 10 min +

20 min +

30 min -

Enzymes Pepsin -

Trypsin -

Proteinase K -

Acidic pH 3.0 -/+

Neutral pH 7.0 +

Alkaline pH 9.0 +

a (+) activity retained; (-/+) activity lost partially; (-) activity lost.

Table 4.2 Susceptibility of enterocin OSY-3E1 to heat, enzyme, and wide pH value range treatments

104

Medium Activity value (AU/ml) pH values of cell-free

supernatant

MRS 6,400 4.73

+1% glucose 6,400 4.69

+5% glucose 25,600 4.78

+10% glucose 3,200 4.81

Table 4.3 Antimicrobial activity and pH value of supernatants from MRS broth supplemented with various concentration of glucose after inoculation with OSY-3E1 and incubation overnight

105

Figure 4.1 PCR amplification of enterocin AS-48 structural gene using total DNA of Enterococcus faecalis OSY-3E1 as template. Lane A: 1000 bp DNA ladder Lane B: E. faecalis OSY-3E1

106

Figure 4.2 Comparison of the nucleotide sequences of structural gene of enterocin OSY- 3E1 and enterocin AS-48, including the predicted amino acid sequences. Sequence differences are marked by red letters.

107

9 s de

i 8 o r

e 7 l nt m e / 6 s U e F 5 m c g C No Ent OSY-3E1 o

t 4 Lo

os 3 Ent OSY-3E1 on 2 uc

Le 1 0 012345 Time (days)

Figure 4.3 The change in population of Leuconostoc mesenteroides ATCC 14935 in Parmalat® milk treated with enterocin OSY-3E1 (25,600AU added /ml of milk) and incubated at 16°C for 5 days. Error bars represent standard error, n=2.

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