Enterococcus Faecalis, but Other Species Also Can Be Recovered

Antibiotic resistant enterococci from food and clinical samples: microbiological characterization, molecular typing and genetic relation of strains

Inauguraldissertation der Philosophisch-naturwissenschaftlichen Fakultät der Universität Bern

vorgelegt von

Stefanie Petra Templer

von Zürich

Leiter der Arbeit:

Dr. Andreas Baumgartner Bundesamt für Gesundheit

Antibiotic resistant enterococci from food and clinical samples: microbiological characterization, molecular typing and genetic relation of strains

Inauguraldissertation der Philosophisch-naturwissenschaftlichen Fakultät der Universität Bern

vorgelegt von

Stefanie Petra Templer

von Zürich

Leiter der Arbeit:

Dr. Andreas Baumgartner Bundesamt für Gesundheit

Von der Philosophisch-naturwissenschaftlichen Fakultät angenommen.

Bern, 19. Juni 2006 Der Dekan Prof. Dr. P. Messerli Table of Contents

Table of Contents

Introduction ...... 2 1. Enterococci: a general overview ...... 2 1.1 Historical perspective and genus definition...... 2 2. Human and nonhuman reservoirs of enterococci ...... 3 2.1 Host specificity of enterococci...... 3 2.2 Distribution of enterococcal species in humans ...... 4 2.3 Importance of animal reservoirs according to gene transfer to human enterococci... 5 2.4 Enterococci in Foods...... 5 3. Enterococcal Virulence ...... 7 3.1 Secreted Factors...... 7 3.2 Enterococcal Adhesins...... 8 4. Acquired Antibiotic Resistances in Enterococci ...... 9 4.1 β-lactam Resistance ...... 10 4.2 Glycopeptide Resistance...... 10 4.3 Macrolide Resistance...... 11 4.4 Chloramphenicol Resistance...... 11 4.5 Tetracycline Resistance ...... 12 5. Conjugation and Genetic Exchange in Enterococci ...... 12 5.1 Plasmids...... 12 5.2 Transposons ...... 13 6. Enterococcal Disease and Treatment ...... 13 6.1 Clinical Disease...... 13 6.2 Treatment with antimicrobial agents...... 14 Reference List Introduction ...... 15 Aim of the Work ...... 21 Publications...... 22 Summary and Discussion ...... 68 Acknowledgments...... 71 Curriculum Vitae ...... 72

i Introduction

INTRODUCTION

1. Enterococci: a general overview

The enterococci are a complex and diverse group of bacteria in respect of their interactions with humans. Some enterococcal strains can be used in the production of fermented foods or as probiotics. At the same time, however, enterococci have been associated with a number of human and animal infections. Several virulence factors have been described and the number of vancomycin-resistant enterococci is increasing. However, enterococci occur ubiquitous and are prevailed in nearly everything we humans come into contact with. They are commonly found in the alimentary tracts of humans and other animals, in the soil and water, and in the foods.

1.1 Historical perspective and genus definition

In 1899 Thiercelin et al. [62] described bacteria seen in pairs and short chains in human feces and called them “streptococcus of fecal origin”. Their publication is probably the first report on enterococci In the following years several researchers made discoveries of similar bacteria isolated from a cesspool or a case of acute endocarditis. In 1906, The name Streptococcus faecalis was used by Andrews and Horder [4] to identify an organism of fecal origin that clotted milk. Orla-Jensen [47] described a second organism of this group, Streptococcus faecium, which differed from the fermentation patterns of S. faecalis. In the following 30 years several other species came to the so called group “enterococcal streptococci” such as Streptococcus durans which was very similar to S. faecium but had less fermentation activity. In 1970, Kalina proposed that a genus for the enterococcal streptococci be established and suggested that, based on cellular arrangement and phenotypic characteristics, S. faecalis and S. faecium and their subspecies of these two taxons be named Enterococcus [38]. It took another 14 years, based on the publication of Schleifer and Klipper-Balz [53], until the genus Enterococcus has been generally accepted. Enterococci are gram-positive cocci that occur singly, in pairs, or as short chains. They are facultative anaerobes with an optimum growth temperature of 37°C and a growth range from 10 to 45°C. Growth also occurs in the presence of 6.5% NaCl, at pH 9.6, and enterococci survive heating at 60°C for 30 min. To date, 28 species have been added to the genus Enterococcus on the basis of phylogenetic evidence strengthened by 16S rRNA DNA sequencing and/or DNA – DNA hybridization studies. Characteristically, enterococci inhabit harsh environments like the intestinal tract of humans and animals. Growth under such hostile conditions demands metabolic flexibility. In

2 Introduction this respect, enterococci are capable of much more than simple sugar fermentation to produce lactic acid. These organisms readily catabolize a spectrum of energy sources including diverse carbohydrates, glycerol, lactate, malate, citrate, the diamino acids arginine and agmatibe, and many α-keto acids [35].

2. Human and nonhuman reservoirs of enterococci

Enterococci are a natural part of the intestinal flora in most mammals and birds. For almost a century, enterococci are used as indicators of fecal contamination of water and food for human consumption. The emergence of multiple antimicrobial-resistant strains worldwide, led to the awareness of enterococci being an important agent of human disease. Six years ago, an interdisciplinary federal team of researchers in Switzerland worked out a situation analysis with regard to antibiotic resistant microorganisms in human and veterinary medicine and in food [10]. They reasoned that the spread of antibiotic resistant microorganisms is in a first place retraced back to human medicine and the hospital environment, followed by the veterinary medicine. It was recognized that foods were important for the transfer of antibiotic resistance, but only in a third place.

2.1 Host specificity of enterococci

Enterococci are ubiquitous and can therefore be found, aside the intestinal flora of mammals and birds, in soil, on plants, and in water. In water they are generally considered as fecal contaminants and belong mainly to the species Enterococcus faecium and Enterococcus faecalis, but other species also can be recovered. Some of the enterococcal species are typically associated with a certain environment or select animal species, which points to a moderate host specificity. Several enterococcal species, although identifiable in a number of hosts, can be divided into host-specific ecovars on the basis of phenotypic or genotypic characteristics. Phenotypically, E. faecium strains from poultry origin are frequently able to ferment raffinose, whereas this characteristic generally is not present in E. faecium strains from other origins [20]. Ahmed et al. recently published the results of a biochemical, phenotypic characterization of 4057 enterococcal isolates from surface water. Based on the biochemical phenotypes (BPTs), they traced the source of faecal contamination in a local creek. Furthermore, they were able to distinguish the enterococcal isolates between human and animal source as well as between animal species [2].

3 Introduction

2.2 Distribution of enterococcal species in humans

The healthy human body is colonized by microbial communities that, together, constitute the “normal microflora.” These communities can be detected in the upper respiratory tract, oral cavity, distal intestinal tract, and vagina and on the skin. Each of these regions of the body is biochemically and histologically distinct. The prevailing physocochemical factors in each of the body regions provide conditions that select for appropriately adapted microbes. Enterococci are minority members of the bacterial community inhabiting the large bowel of adult humans. Molecular analysis has shown that these bacteria make up no more than 1% of the intestinal microflora of an adult [55]. The medical importance of the enterococci far outweighs their relative numbers in the intestinal tract, and Enterococcus species now rank among the leading causes of nosocomial infections of humans. For example, it is estimated that, in the United States, 800’000 cases each year of enterococcal infection are causing about $500 million of health care costs [23]. In Switzerland it is difficult to say anything about the costs caused, because there exists no reporting system for human infections due to enterococci. Among the 17 species of enterococci that have been described, Enterococcus faecalis and Enterococcus faecium appear to be the most commonly detected in human feces [24]. The prevalence and population sizes of these three species recorded during a study of 62 humans consuming a “western diet” are given in Table 1. Enterococcus avium has been detected occasionally in human feces [24]. E. faecalis accounts for most of the enterococcal infections of humans usually representing 80 to 90% of clinical isolates. E. faecium is detected much less frequently (less than 10%) but is nevertheless of significance because of a high incidence of resistance to multiple antibacterial agents. Our results for enterococcal isolates from three university hospitals in Switzerland showed the same distribution according to species identification (see results of Publicationin this thesis).

Table 1 Populations of enterococcal species in human fecesa

Mean log10 CFU/g Range log 10 CFU/g Prevalence (detected Species (dry wt) (dry wt) in % of subjects) E. faecalis 7.4 3.6 – 10.9 82 E. faecium 8.0 3.5 – 10.9 36 E. durans 7.5 4.7 – 12.7 11 aSixty-two subjects. See Finegold et al. [24]

4 Introduction

2.3 Importance of animal reservoirs according to gene transfer to human enterococci

The therapeutic use of antimicrobial agents in clinical medicine and animal husbandry has been one of the most significant medical achievements in the 20th century. In this context, enterococci inhabiting nonhuman reservoirs appear to play an important role in the acquisition and dissemination of antibiotic resistance determinants. The most effective way to limit the spread of antimicrobial resistance, and thereby extend the usefulness of antimicrobials is through their restricted use. Avoparcin, as an example, was used as a growth-promoting feed additive in several European countries. Several studies showed an association between the use of avoparcin and the occurrence of VanA-type glycopeptide-resistant enterococci (GRE) in farm animals [1,40]. Therefore, all European Union countries banned avoparcin as a growth promoter. In recent study Sorum et al. published a survey of GRE in poultry three to eight years after the ban on avoparcin and could show that the prevalence of faecal GRE in poultry declined significantly [59].

2.4 Enterococci in Foods

Enterococci are used in the fermentation of foods for the production of certain cheeses and other fermented milk products [5,26,28]. They are also present as contaminants on raw meat, milk, and milk products. Ordinarily, E. faecium and E. faecalis are the most frequently encountered species, even though the prevalence varies greatly between different countries and different types of products [5,9,31,32,42]. Comparable to what is found in animals, a high genetic diversity has been demonstrated among enterococcal isolates from food [42]. Enterococci are very tolerant to extremes in temperature, salinity, and pH and are among the most thermotolerant of the non-sporulating bacteria [26]. E. faecium strains have been able to survive heating to 65°C for 20 min, 71°C for 10 min, and 80°C for 3 min [39]. Thus, enterococci may survive some types of food processing, and enterococci have been implicated in food spoilage of processed cooked meat [26]. The Ordinance on Hygiene of the Swiss Food legislation decrees no microbiological criteria for enterococci in foods. It regulates only the occurrence of enterococci in drinking water [16].

2.4.1 Enterococci as Probiotics

The definition of a probiotic has been evolving with the increasing understanding of the mechanisms by which they influence human health [18]. The term was first introduced to describe substances produced by one microorganism (protozoa) that stimulate the growth of other microorganisms [41]. Nowadays, the definition most commonly used is that of Fuller [30],

5 Introduction i.e. “probiotics are live microbial feed supplements, which beneficially affect the host animal by improving its intestinal microbial balance”. Over the years, the definition was expanded to include food and non-food uses as well as the use of mono and mixed cultures. Hence, a probiotic can be described as a preparation of or a product containing viable, defined microorganisms in sufficient numbers, which alters the microbiota in a compartment of the host and that exerts beneficial health effects in this host [18]. Probiotic strains are applied in foods such as yogurt, fermented and non-fermented milks, infant formulas, and pharmaceutical preparations. A group of requirements have been identified for a microorganism to be defined as an effective probiotic [52]. These include the ability to a) adhere to cells; b) exclude or reduce pathogenic adherence; c) persist and multiply; d) produce acids, hydrogen peroxide, and bacteriocins antagonistic to pathogen growth; e) be safe, noninvasive, non-carcinogenic, and nonpathogenic; and f) coaggregate to form a normal balanced flora.

In Switzerland, milk products which are described in the Food Legislation can be supplemented with probiotic bacteria without an authorization procedure. However, the added probiotic microorganisms must fulfill minimal requirements which is 106 CFU/g of living cells. Other probiotic foods, such as drinks or capsules, have to be authorized. In this the added strains must be specified and fulfill the minimal requirement valid for probiotic milk products. Furthermore health claims must be based on adequate scientific studies. Unfortunately, there are no legal requirements with regard to transferable antibiotic resistance or virulence factors [16,17,19]. The use of enterococci as probiotics remains a controversial issue. While the probiotic benefits of some strains are well established, the emergence and the increased association of enterococci with human disease and multiple antibiotic resistances (see below) have raised concern regarding their use as probiotics. The fear that antimicrobial resistance genes or genes encoding virulence factors can be transferred to other bacteria in the gastrointestinal tract contributes to this controversy [28].

2.4.2 Enterococci as starter culture in cheese

The presence of enterococci in high numbers in many different types of cheeses, their potential contribution to the organoleptic properties of fermented food products, and their ability to produce bacteriocins (enterococins) are important characteristics to be considered in food processing and manufacturing. During the last few years, the reports about enterococci

6 Introduction proposed to be used as starter cultures or co-cultures have considerably increased. The aim of these studies was mainly to elucidate the role that enterococci play in cheese ripening. However, as enterococci have been recognized in recent years as major nosocomial pathogens, one should carefully consider the potential virulence factors of this group of microorganisms before use.

3. Enterococcal Virulence

A virulence factor is an effector molecule that enhances the ability of a microorganism to cause disease beyond that intrinsic to the species background [44]. Typical examples are aggregation substance, gelatinase, and extracellular surface protein (see below).

3.1 Secreted Factors

3.1.1 The Cytolysin

The cytolysin is a novel, two-peptide lytic toxin expressed by some strains of Enterococcus faecalis. It is toxic in animal models of enterococcal infection, and associated with acutely terminal outcome in human infection. The cytolysin can be encoded by large pheromone- responsive plasmids, or on the chromosome within the pathogenicity island. It is produced by a complex process that involves the products of eight genes. The cytolysin toxin, maturation and regulatory genes are organized into two divergent transcripts: a structural transcript and a regulatory transcript. The active cytolysin subunits are synthesized ribosomally as non- identical peptides, post-translationally modified, then secreted and activated. The cytolysin operon within the E. faecalis pathogenicity island is associated with other virulence determinants, including aggregation substance and enterococcal surface protein, Esp (see Figure 1).

3.1.2 Gelatinase

Gelatinase (Gel) is an extracellular metallo-endopeptidase involved in the hydrolysis of gelatin, collagen, haemoglobin, and other bioactive peptides [60]. The association between an enterococcal protease and virulence was first suggested in 1975 by Gold et al., who found that a gelatin-liquefying, human, oral E. faecalis isolate induced caries formation in germ-free rats, while non-proteolytic strains did not. Singh et al. [58] demonstrated that Gel, which is commonly produced by nosocomial, fecal and clinical enterooccal isolates is a virulence factor of enterococci, at least for peritonitis in mice. The presence of Gel production among food E. faecalis strains is high [22,27]. However, Eaton and Gasson [22] demonstrated that even

7 Introduction when the gel gene is present, a negative phenotype can be found. None of the E. faecium strains involved in both studies produced Gel.

3.2 Enterococcal Adhesins

3.2.1 Aggregation substance

Aggregation substance (Agg) is a pheromone-inducible surface protein of E. faecalis, which promotes aggregate formation during bacterial conjugation [14]. As an important component of the bacterial pheromone-responsive genetic exchange system, Agg mediates efficient enterococcal donor-recipient contact to facilitate plasmid transfer [14]. This trait may contribute to the pathogenesis of enterococcal infection through different mechanisms. The cells that express this trait form large aggregates in vivo. However, it is unknown how this phenomenon influences phagocytosis and subsequent damage of the vital functions of the organism. Also, Agg may bind on and present its cognate ligand to the surface of the organism, possibly resulting in superantigen activity. Finally, Agg increases the hydrophobicity of the enterococcal surface, which may induce localization of cholesterol to phagosomes, and prevent or delay fusion with lysosomal vesicles [44]. Studies on edocarditis showed synergism between cytolysin and Agg. Up to now, Agg is exclusively found in E. faecalis strains; however its incidence among food isolates seems to be high [22,27].

Enterococci Aggregation Aggregation Quorum + substance substance sensing Target Cell induced mediated autoinduction clumping adherence of cytolysin

Figure 1. Model for the synergy of the cytolysin and aggregation substance [35]

Chow et al. [11] noted that the expression of both cytolysin and aggregation substance resulted in a eightfold increase in lethality relative to the expression of either factor alone. It is likely that these traits work in concert: aggregation substance functions to mediate clumping or adherence to eukaryotic target cells, increasing the local density of bacteria to the level necessary to achieve a quorum. Sufficiently high local concentrations of the cytolysin inducer,

CylLS, may then occur, inducing high-level cytolysin expression (see Figure 1)

8 Introduction

3.2.2 Extracellular surface protein (Esp)

Extracellular surface protein (Esp) was first described in a clinical E. faecalis isolate by Shankar et al [56]. Studies on the distribution of this surface protein revealed a significant enrichment in infection-derived E. faecalis isolates [57]. Esp is thought to play a role in adhesion and evasion of the immune response of the host. The incidence of Esp in food isolates differs for E. faecium and E. faecalis. While it is hardly found in E. faecium, the incidence in E. faecalis is higher than expected. Up to now, no explanation could be found for this phenomenon [22,27].

4. Acquired Antibiotic Resistances in Enterococci

Enterococci are intrinsically resistant to a broad range of antimicrobial agents, and this has always limited the choice of therapeutic agents against these organisms. Because enterococci have increased in prevalence worldwide as pathogens in nosocomial infections, antimicrobials have been used in greater frequency in hospitals. Some of these antimicrobial agents have

DNA cell Transcription membrane Replication

mRNA

DNA Translation cell wall Tetracycline, Macrolide β-Laktame Chloramphenicol Glykopeptide Protein

Figure 2 Mechanism of action of different antimicrobial agents

also been used widely as growth promoters in animal husbandry. Enterococci have developed increased resistance to each of these agents either by the acquisition of antibiotic resistance genes on plasmids or transposons from other organisms or by spontaneous mutations that give the enterococci an enhanced level of resistance.

9 Introduction

4.1 β-lactam Resistance

β-lactam antibiotics, such as penicillin or ampicillin, act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls. The peptidoglycan layer is important for cell wall structural integrity, especially in Gram-positive organisms. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by transpeptidases known as penicillin binding proteins (PBPs). Inhibition of PBPs may also lead to the activation of autolytic enzymes in the bacterial cell wall (see Figure 2). The mode of β-lactam resistance is due to enzymatic hydrolysis of the β-lactam ring. If the bacteria produce β-lactamase or penicillinase, these enzymes will break the β-lactam ring of the antibiotic, rendering the antibiotic ineffective. The genes encoding these enzymes may be inherently present on the bacterial chromosome or may be acquired via plasmid transfer, and β-lactamase gene expression may be induced by exposure to β-lactams. The production of a β-lactamase by a bacterium does not necessarily rule out all treatment options with β-lactam antibiotics. In some instances, these agents may be co-administered with a β-lactamase inhibitor.

4.2 Glycopeptide Resistance

The glycopeptide antibiotics vancomycin and teicoplanin are used to treat serious infections due to resistant gram-positive organisms. They had been used in the clinical setting for over 30 years before reports of resistance appeared. In 1986, the first vancomycin-resistant E. faecium isolates were isolated in France from patients with leukemia and in England from patients with renal failure [64]. Since then, resistant strains have disseminated widely across the globe. In 1998, over 20% of enterococcal isolates in the United States were resistant to vancomycin [29]. Most reports of vancomycin-resistant enterococci (VRE) come from intensive care units, oncology wards, outpatient dialysis units, and long-term care facilities. Once they have colonized a patient, VRE often persist in the gastrointestinal tract and may be spread horizontally to other patients.

4.2.1 Mechanism of Action of Glycopeptides

Gylcopeptides act on gram-positive organisms by inhibiting cell wall biosynthesis. Gram- negative organisms are inherently resistant to glycopeptides because their outer membrane blocks the glycopeptides from reaching their targets in the cell wall (see Figure 2). Glycopeptides do not act on the cell wall biosynthesis enzymes like penicillins do, but on the substrates of these enzymes, the peptidoglycan pentapeptide precursors. They prevent the cross-linking of these precursors, which leads to a loss in cell wall structural integrity followed by cell death [65].

10 Introduction

4.2.2 Vancomycin resistance

Glycopeptide-resistant enterococci are phenotypically and genotypically heterogeneous. Of the six different VRE-phenotypes known to date [34], VanA and VanB are of the highest clinical importance as they are most frequently observed in two predominant enterococcal species, E. faecalis and E. faecium [46]. VanA-type strains display high-level inducible resistance to both vancomycin and teicoplanin, following the acquisition of transposon Tn1546 or closely related mobile genetic elements. VanB-type strains display variable levels of inducible resistance to vancomycin only [6,7].

4.3 Macrolide Resistance

The mechanism of action of the macrolides, such as erythromycin, is the inhibition of bacterial protein synthesis by binding reversibly to the subunit 50S of the bacterial ribosome, thereby inhibiting translocation of peptidyl-tRNA. This action is mainly bacteriostatic, but can also be bactericidal at high concentrations in the gut. Macrolides tend to accumulate within leukocytes, and are therefore actually transported into the site of infection (see Figure 2). Three different mechanisms of erythromycin resistance have been described: - Target modification, mediated by a rRNA erm(A,B,C) methylase that alters a site in 23S rRNA common to the binding of macrolides; - Drug inactivation, by enzymes (EreA and EreB) that hydrolyze the lactone ring of the macrocyclic nucleus and phosphotransferases that inactivate macrolides [50,61]; - Efflux systems, encoded by mef(A/E) gene, an efflux protein that pumps macrolide antibiotics out of the cell, or the msr(A) gene, which confers resistance via an ATP-binding transporter protein [61].

4.4 Chloramphenicol Resistance

Chloramphenicol stops bacterial growth by inhibiting the enzyme peptidyl transferase, which inhibits ribosomal activity and protein synthesis by preventing the binding of amino acyl-tRNA to the A site on the 50S subunit [63]. Resistance to chloramphenicol is conferred by the cat gene. This gene codes for an enzyme called "chloramphenicol acetyltransferase" which inactivates chloramphenicol by covalently linking one or two acetyl groups, derived from acetyl-S-coenzyme A, to the hydroxyl groups on the chloramphenicol molecule. The acetylation prevents chloramphenicol from binding to the ribosome.

11 Introduction

4.5 Tetracycline Resistance

Tetracycline resistance is present in at least 60 to 65% of enterococcal clinical isolates even though these antibiotics are not routinely used to treat infections [21,33,37]. Tetracyclines inhibit protein synthesis by interfering with the binding of aminoacyl-tRNA to the ribosome (see Figure 2). There are two major mechanisms of tetracycline resistance in enterococci: - Active efflux of the drug across the cell membrane, mediated e.g. by the tet(L) gene located on a conjugative plasmid or on the chromosome, encoding a large protein with 14 transmembrane domains [3]; - Ribosomal protection, mediated e.g. by the tet(M) or the tet(S) gene, encoding proteins that confer resistance by binding of the resistant proteins to the ribosomes and subsequent alteration of the ribosomal conformation to prevent binding of tetracyclines. The tet(M) gene is usually carried by Tn916 or related conjugative transposons, but it can also be found on conjugative plasmids [3,21,33].

5. Conjugation and Genetic Exchange in Enterococci

5.1 Plasmids

Three classes of plasmids are known to be capable of replication in the enterococci: the rolling circle replicating (RCR) plasmids, the Inc18 plasmids, and the pheromone-responsive plasmids. The RCR and Inc18 plasmids are capable of replication in a broad range of gram- positive bacteria, and some RCR plasmids are also capable of replication in gram-negative species. The replication of pheromone-responsive plasmids appears to be restricted to the enterococci, although plasmids with clearly related replicons are present in a variety of gram- positive organisms. The gene exchange among enterococci of plasmids coding for antibiotic resistances and virulence determinants has been evaluated in vivo and ex vivo [15].

12 Introduction

5.2 Transposons

A large number of individual transposons and several transposon classes have been described in enterococci. Enterococcal transposons generally fall into one of three classes: Tn3-family transposons, composite transposons, and conjugative transposons. The first two classes are widespread throughout the bacterial domain and their transposition mechanisms have been well described in E. coli and other gram-negative bacteria [35]. Conjugative transposons were initially discovered in E. faecalis and are now known to be widespread in gram-positive bacteria [13].

5.2.1 Conjugative Transposon Tn916-Tn1545 family

Tn916, which confers resistance to tetracycline, belongs to the Tn916-Tn1545 family of conjugative transposons [8]. Transposition of Tn916 and other conjugative transposons is a three-step process involving excision from the donor chromosome and formation of a covalently closed circular intermediate, conjugation by way of single-stranded transfer to the recipient cell , and nonspecific integration into the recipient chromosome. Multiple copies of Tn916 can occur on the same chromosome [54].

6. Enterococcal Disease and Treatment

During the past several decades, enterococci have emerged as important nosocomial pathogens [43,48]. This fact is attributed primarily to the high degree of antibiotic resistance that is exhibited by most enterococci. Of particular concern has been the rapid spread of enterococci with resistance to vancomycin (VRE) [51]. The species responsible for most infections in the community, long-term care, and hospital setting is E. faecalis, which is most likely to be susceptible to vancomycin and penicillins [36,49]. Enterococcus faecalis with high level resistance to gentamicin and other aminoglycosides emerged in the United States in the 1980s, creating therapeutic problems for patients with serious infections, such as endocarditis [66]. In Switzerland, there are no statistically solid data about the emergence of enterococci in the nosocomial environment because there is no nation-wide surveillance system.

6.1 Clinical Disease

The genus Enterococcus is associated with a variety of different clinical infections. Frequently, other bacterial species are isolated from the same sites of infection. In those cases, it is often not clear whether disease manifestation is a result of growth and invasion of tissues by enterococci or whether these relatively avirulent organisms are merely playing a minor role in

13 Introduction the infection [35]. However, enterococci have been implicated as an important cause of endocarditis, bacteremia, and infections of the urinary tract, central nervous system and intra- abdominal and pelvic infections [25].

6.2 Treatment with antimicrobial agents

Enterococci are intrinsically resistant to many antimicrobial agents, including cephalosporins, clindamycin, and penicillinase-resistant penicillins [46]. They have low-level intrinsic resistance to aminoglycosides and they are, when compared with most streptococci, are relatively resistant to penicillins as well. Piperacillin and the carbapenems show a good activity against enterococci but have no advantage over ampicillin. Plamid-mediated beta-lactamase production by some strains of E. faecalis and E. faecium has led to further problems with treatment of serious enterococcal infections [45]. In addition to vancomycin and high-level aminoglycoside resistance, most strains of VRE also have chromosomally mediated resistance to penicillins. In the treatment of enterococcal infections, several factors have to be taken into consideration. It is important to know whether the causative strain is susceptible to beta-lactams, aminoglycosides, and glycopeptides or whether it is resistant to various combinations of these antimicrobial classes. An important point to know is also whether the infection involves the patient’s heart valves. Finally, it is to consider whether the infection is monomicrobial, as it is the case with most urinary tract infections, or polymicrobial as it is true of most wound infections and abscesses [35].

Enterococci are associated with a variety of different clinical syndromes, including bacteremias, endocarditis, and urinary tract infections. The emergence of resistance has made clinicians keenly aware of this organism, previously considered a nonpathogen except in certain circumstances. Molecular methods have helped delineate the epidemiology of VRE and demonstrate nosocomial acquisition and transmission among patients. Treatment of serious enterococcal disease requires a synergistic combination of a cell wall agent and an aminoglcoside. The few antimicrobial agents that are available to treat VRE are often ineffective or poorly tolerated, making treatment of a serious infection challenging. Given the limitations of antimicrobial therapy, removal of infected foci, such as intravenous catheters, and drainage of abscesses are important adjunctive measures.

14 Reference List Introduction

REFERENCE LIST INTRODUCTION

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15 Reference List Introduction

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15. Cocconcelli, P.S., Cattivelli, D. & Gazzola, S. Gene transfer of vancomycin and tetracycline resistances among Enterococcus faecalis during cheese and sausage fermentations. Int. J. Food Microbiol. 88, 315-323 (2003).

16. Eidgenössisches Departement des Innern. Hygieneverordnung des EDI vom 23. November 2005 (HyV). (SR 817.024.1), 26-32. 2005. Switzerland.

17. Eidgenössisches Departement des Innern. Verordnung des EDI über Lebensmittel tierischer Herkunft vom 23. November 2005. (SR 817.024.1), 26-32. 2005. Switzerland.

18. De Vuyst, L., Avonts, L. & Makras, E. Functional Foods, Ageing and Degenerative Disease. Remacle, C. & Reusens, B. (eds.), pp. 416-482 (Woodhead Publishing Ltd., Cambridge, United Kingdom,2004).

19. Schweizerischer Bundesrat. Lebensmittel- und Gebrauchsgegenständeverordnung (LGV) vom 23. November 2005. (SR 817.024.1), 26-32. 2005. Switzerland.

20. Devriese, L.A., Pot, B. & Collins, M.D. Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J. Appl. Bacteriol. 75, 399-408 (1993).

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22. Eaton, T.J. & Gasson, M.J. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl. Environ. Microbiol. 67, 1628-1635 (2001).

23. Edwards, D.D. Enterococci attract attention of concerned microbiologists. ASM News 66, 540-545. 2000.

24. Finegold, S.M., Sutter, V.L. & Mathisen, G.E. Human intestinal microflora in health and disease. Hentges, D.J. (ed.), pp. 3-31 (Academic Press, New York, N.Y.,1983).

25. Foulquie Moreno, M.R., Sarantinopoulos, P., Tsakalidou, E. & De Vuyst, L. The role and application of enterococci in food and health. Int. J. Food Microbiol. 106, 1-24 (2006).

26. Franz, C.M., Holzapfel, W.H. & Stiles, M.E. Enterococci at the crossroads of food safety? Int. J. Food Microbiol. 47, 1-24 (1999).

27. Franz, C.M. et al. Incidence of virulence factors and antibiotic resistance among Enterococci isolated from food. Appl. Environ. Microbiol. 67, 4385-4389 (2001).

28. Franz, C.M., Stiles, M.E., Schleifer, K.H. & Holzapfel, W.H. Enterococci in foods - a conundrum for food safety. Int. J. Food Microbiol. 88, 105-122 (2003).

29. Fridkin, S.K. & Gaynes, R.P. Antimicrobial resistance in intensive care units. Clin. Chest Med. 20, 303-16, viii (1999).

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31. Gelsomino, R. et al. Antibiotic resistance and virulence traits of enterococci isolated from Baylough, an Irish artisanal cheese. J. Food Prot. 67, 1948-1952 (2004).

32. Gelsomino, R., Vancanneyt, M., Condon, S., Swings, J. & Cogan, T.M. Enterococcal diversity in the environment of an Irish Cheddar-type cheesemaking factory. Int. J. Food Microbiol. 71, 177-188 (2001).

33. Gevers, D., Danielsen, M., Huys, G. & Swings, J. Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage. Appl. Environ. Microbiol. 69, 1270-1275 (2003).

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34. Gholizadeh, Y. & Courvalin, P. Acquired and intrinsic glycopeptide resistance in enterococci. Int. J. Antimicrob. Agents 16 Suppl 1, p.11-17 (2000).

35. Gilmore, M. et al. The Enterococci (Pathogenesis, Melocular Biology, and Antibiotic Resistance). (First Edition ASM Press, Washington, DC 2002).

36. Graninger, W. & Ragette, R. Nosocomial bacteremia due to Enterococcus faecalis without endocarditis. Clin. Infect. Dis. 15, 49-57 (1992).

37. Huys, G., D'Haene, K., Collard, J.M. & Swings, J. Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food. Appl. Environ. Microbiol. 70, 1555-1562 (2004).

38. Kalina, A.P. The taxonomy and nomenclature of enterococci. Int. J. Syst. Bacteriol. 20, 185-189 (1970).

39. Kearns, A.M., Freeman, R. & Lightfoot, N.F. Nosocomial enterococci: resistance to heat and sodium hypochlorite. J. Hosp. Infect. 30, 193-199 (1995).

40. Kruse, H., Johansen, B.K., Rorvik, L.M. & Schaller, G. The use of avoparcin as a growth promoter and the occurrence of vancomycin-resistant Enterococcus species in Norwegian poultry and swine production. Microb. Drug Resist. 5, 135-139 (1999).

41. Lilly, D.M. & Stillwell, R.H. Probiotics: growth-promoting factors produced by microorganisms. Science 147, 747-748 (1965).

42. Mannu, L. & Paba, A. Genetic diversity of lactococci and enterococci isolated from home-made Pecorino Sardo ewes' milk cheese. J. Appl. Microbiol. 92, 55-62 (2002).

43. Moellering, R.C., Jr. Emergence of Enterococcus as a significant pathogen. Clin. Infect. Dis. 14, 1173-1176 (1992).

44. Mundy, L.M., Sahm, D.F. & Gilmore, M. Relationships between enterococcal virulence and antimicrobial resistance. Clin. Microbiol. Rev. 13, 513-522 (2000).

45. Murray, B.E. Beta-lactamase-producing enterococci. Antimicrob. Agents Chemother. 36, 2355-2359 (1992).

46. Murray, B.E. Diversity among multidrug-resistant enterococci. Emerg. Infect. Dis. 4, 37- 47 (1998).

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47. Orla-Hensen, S. The lactic acid bacteria. Mem. Acad. Roy. Sci. Danemark. Sect. Sci. Ser. 2 5, 81-197 (1919).

48. Papanicolaou, G.A. et al. Nosocomial infections with vancomycin-resistant Enterococcus faecium in liver transplant recipients: risk factors for acquisition and mortality. Clin. Infect. Dis. 23, 760-766 (1996).

49. Patterson, J.E. et al. An analysis of 110 serious enterococcal infections. Epidemiology, antibiotic susceptibility, and outcome. Medicine (Baltimore) 74, 191-200 (1995).

50. Portillo, A. et al. Macrolide resistance genes in Enterococcus spp. Antimicrob. Agents Chemother. 44, 967-971 (2000).

51. Robredo, B., Singh, K.V., Baquero, F., Murray, B.E. & Torres, C. Vancomycin-resistant enterococci isolated from animals and food. Int. J. Food Microbiol. 54, 197-204 (2000).

52. Salminen, S., Isolauri, E. & Salminen, E. Clinical uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenges. Antonie Van Leeuwenhoek 70, 347-358 (1996).

53. Schleifer, K.H. & Klipper-Balz, R. Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov. Int. J. Syst. Bacteriol. 34, 31-34 (2006).

54. Scott, J.R. & Churchward, G.G. Conjugative transposition. Annu. Rev. Microbiol. 49, 367-397 (1995).

55. Sghir, A. et al. Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization. Appl. Environ. Microbiol. 66, 2263-2266 (2000).

56. Shankar, N., Coburn, P., Pillar, C., Haas, W. & Gilmore, M. Enterococcal cytolysin: activities and association with other virulence traits in a pathogenicity island. Int. J. Med. Microbiol. 293, 609-618 (2004).

57. Shankar, V., Baghdayan, A.S., Huycke, M.M., Lindahl, G. & Gilmore, M.S. Infection- derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein. Infect. Immun. 67, 193-200 (1999).

58. Singh, K.V., Qin, X., Weinstock, G.M. & Murray, B.E. Generation and testing of mutants of Enterococcus faecalis in a mouse peritonitis model. J. Infect. Dis. 178, 1416-1420 (1998).

19 Reference List Introduction

59. Sorum, M. et al. Prevalence, persistence, and molecular characterization of glycopeptide-resistant enterococci in Norwegian poultry and poultry farmers 3 to 8 years after the ban on avoparcin. Appl. Environ. Microbiol. 72, 516-521 (2006).

60. Su, Y.A. et al. Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp. liquefaciens. Infect. Immun. 59, 415-420 (1991).

61. Sutcliffe, J., Grebe, T., Tait-Kamradt, A. & Wondrack, L. Detection of erythromycin- resistant determinants by PCR. Antimicrob. Agents Chemother. 40, 2562-2566 (1996).

62. Thiercelin, E. Sur un diplocoque saprophyte de lîntestin susceptible de devenir pathogene. C. R. Soc. Biol. 5, 269-271 (1899).

63. Trieu-Cuot, P. et al. Study of heterogeneity of chloramphenicol acetyltransferase (CAT) genes in streptococci and enterococci by polymerase chain reaction: characterization of a new CAT determinant. Antimicrob. Agents Chemother. 37, 2593-2598 (1993).

64. Uttley, A.H., Collins, C.H., Naidoo, J. & George, R.C. Vancomycin-resistant enterococci. Lancet 1, 57-58 (1988).

65. Walsh, C.T., Fisher, S.L., Park, I.S., Prahalad, M. & Wu, Z. Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story. Chem. Biol. 3, 21-28 (1996).

66. Zervos, M.J. et al. Nosocomial infection by gentamicin-resistant Streptococcus faecalis. An epidemiologic study. Ann. Intern. Med. 106, 687-691 (1987).

20 Publications I-III

AIM OF THE WORK

Persistence of particular antibiotic resistant enterococcal strains in food

Previous studies showed that enterococci can be found in a variety of ready-to-eat products in Switzerland (see reference 3 and 5 of publication). Furthermore, in a high amount of enterococcal isolates from raw-milk cheese a triple resistance against chloramphenicol (Chl), tetracycline (Tet) and erythromycin (Ery) could be demonstrated. The aim of this study was to determine the diversity of enterococci in two types of artisan raw-milk cheese (Schabziger and Appenzeller) and to investigate whether particular strains with triple resistance against Chl, Tet and Ery persist in a production system.

Enterococcal clinical isolates from three hospitals in Switzerland compared to food isolates from Appenzeller and Schabziger raw milk cheese

The prevalence of different species of clinical enterococcal isolates, their antibiotic susceptibility and virulence factors was studied. Enterococci were recovered from three different hospitals in Switzerland. In order to understand the extent of similarity between strains from clinical sources and such from foods, we compared the antimicrobial resistance, virulence factors and pulsed field gel electrophoresis (PFGE) profiles of these strains.

Horizontal transfer of genes for tetracycline and erythromycin resistance among E. faecalis strains in vanilla cream

Horizontal transfer of antibiotic resistance genes between bacteria and the following accumulation of resistance in strains, gains a serious problem for the treatment of enterococcal infections. The food chain is a possible vector for the transfer of antibiotic resistance genes among bacteria. We investigated the possibility of genetic exchange between enterococcal strains in a food model with enterococcal counts, which normally occur in certain cheeses.

21 Publications I-III

PUBLICATIONS

Publication I

Enterococci from Appenzeller and Schabziger raw milk cheese: antibiotic resistance, virulence factors and persistence of particular strains in the products

Submitted 12 April 2006 to the Journal of Food Protection

Conference Contribution I

Enterococci from Appenzeller and Schabziger raw milk cheese: antibiotic resistance, virulence factors and persistence of particular strains in the products. (Poster)

Proceedings of the 65th annual assembly of SSM, Lausanne (Switzerland), 7th-8th March 2006

Publication II

Enterococcal clinical isolates from three hospitals in Switzerland: antibiotic resistance patterns, virulence determinants, and genotypic characterization by PFGE.

Submitted in May 2006 to the Journal of Clinical Microbiology

Conference Contribution II

Enterococcal clinical isolates: antibiotic resistance patterns and genotypic characterization by PFGE. (Poster)

Proceedings of the 2nd International ASM-FEMS Conference on Enterococci. Helsingor (Denmark), 28th-31st August 2005

Publication III

Horizontal gene transfer of tetracycline and erythromycin resistances among Enterococcus faecalis in vanilla cream.

Submitted in May 2006 to Applied and Environmental Microbiology

22 Publication I

Publication I

Enterococci from Appenzeller and Schabziger raw milk cheese: antibiotic resistance, virulence factors and persistence of particular strains in the products

S. P. TEMPLER1, A. BAUMGARTNER1* 1Section of Microbiology and Biotechnology, Swiss Federal Office of Public Health, Schwarzenburgstrasse 165, CH-3097 Liebefeld, Switzerland * Tel: +41 31 322 95 82, Fax: +41 31 322 95 74, E-mail: [email protected]

Submitted 12 April 2006 to the Journal of Food Protection

23 Publication I

Enterococci from Appenzeller and Schabziger raw milk cheese: antibiotic resistance, virulence factors and persistence of particular strains in the products

Submitted 12 April 2006 to the Journal of Food Protection

Abstract

Enterococci are natural residents of human and animal intestinal tracts and grow to high levels in a variety of cheeses. The aim of this study was to determine the diversity of enterococci in two types of artisanal raw-milk cheese (Schabziger and Appenzeller) and to investigate whether particular strains with triple resistance against chloramphenicol (Chl), tetracycline (Tet) and erythromycin (Ery) persist in the production system. Out of forty-six cheese samples, a total of 312 Enterococcus strains were isolated over a five-months-period on selective agar plates containing either Chl, Tet or Ery. Enterococcus faecalis was the predominant species (80.7%), followed by Enterococcus faecium (5.1%) and Enterococcus durans (11.7%). According to the phenotypic resistance patterns, a selection of 150 strains was analysed with PCR for the presence of genes encoding resistance to Ery (ereA, ereB, mphA, ermA, ermB, ermC, mrsA/mrsB, mefA/mefE), and Tet (tetM, tetL). Since virulence factors have been linked to the pathogenicity of enterococci, the strain selection was also tested for the presence of the following virulence factors: Agg, GelE, Cyl, Esp, EfaAfs, EfaAfm, Cpd, Cob and Ccf. All tested strains contained at least two of the nine virulence genes taken into analysis. Pulsed-field gel electrophoresis (PFGE) patterns of the isolates showed a limited persistence of several strains over a period of one to two months in Schabziger and more than two months in Appenzeller. Finally, the enterococcal flora in the two types of cheeses seems to be rather unrelated. Within 150 strains out of 25 different cheese samples (11 Appenzeller and 14 Schabziger), 41 PFGE-patterns could be identified and only one of these was found in enterococci from both types of cheese

24 Publication I

Introduction

Enterococci are found in a variety of cheeses made from raw or pasteurized milk from cows [3,13]. For a long time, the presence of these bacteria in foods was associated with faecal contamination, but nowadays they are considered as normal part of the food micro flora [19]. Enterococci seem to improve the flavor development and cheese quality [13]. Because of antilisterial activity based on bacteriocin production, they are also used in food preservation [13,14,16]. In spite of the beneficial activities of enterococci, they have become important over the past decade as one of the most frequently encountered nosocomial pathogens and appear to have increasing resistance to antimicrobials [20,22,23]. An even greater threat is the transfer of resistance to vancomycin or other antimicrobials within and over the species boarder [9-11,20]. But the antibiotic resistance alone cannot explain enterococcal pathogenicity. To cause infection, enterococci must be able to colonize the host tissue, to resist host specific and unspecific defense mechanisms and to cause pathological changes. Thus they have to be virulent, and virulence factors including cytolysin (CylABM), enterococcal surface protein (Esp), aggregation substance (Agg), and gelatinase (GelE) should be taken into consideration too [12,13,22,25]. The cytolysin CylABM causes ruptures of a variety of membranes, including those of bacterial cells, erythrocytes, and other mammalian cells [29]. The Esp factor [30] produces a cell wall-associated protein that is involved in the immune evasion. It is possible, that esp and cylABM genes are associated on a pathogenicity island [25]. The aggregation substance (Agg) is a surface-localized protein encoded by pheromone-responsive, self-transmissible plasmids that mediates binding of donor bacterial cells with plasmid-free recipients, allowing efficient conjugal transfer in a liquid environment [9]. Another adhesin-like virulence factor is the E. faecalis and E. faecium antigen A (EfaAfs and EfaAfm, respectively) that is expressed in the serum [21]. Gelatinase is an extracellular metalloendopeptidase that hydrolyzes gelatin, collagen, hemoglobin, and other bioactive compounds [31]. The sex pheromones (Cpd, Cob, Ccf) are thought to be involved in eliciting an inflammatory response. These pheromones are chemotactic for human leukocytes and induce production and secretion of lysosomal enzymes and thereby facilitate conjugation [8]. Previous studies showed that enterococci can be found in a variety of ready-to-eat products in Switzerland [3,5]. Further, in a high amount of enterococcal isolates from raw- milk cheese a triple resistance against Chl, Tet and Ery could be demonstrated. The aim of this study was to determine the diversity of enterococci in two types of artisan raw-milk cheese (Schabziger and Appenzeller) and to investigate whether particular strains with triple resistance against chloramphenicol (Chl), tetracycline (Tet) and erythromycin (Ery) persist in the production system.

25 Publication I

Appenzeller is a hard type cheese, produced from raw cow milk. Milk is inoculated with a starter culture containing lactic acid bacteria. The milk coagulation is started with addition of rennet. Cheese is normally ripened for about 3 to 4 months. Schabziger cheese (also known as Sapsago) is a creamery, hard, fat free, pungent, grating cheese. It is spiced with fenugreek. During the ripening process the cheese is grated to avoid rind-forming. Schabziger cheese is produced in only one production plant in Switzerland and is therefore an ideal model system to show persistence of particular strains of enterococci.

Materials and Methods

Cheese sampling and isolation of enterococci

Over a period of five months, a cheese sample of every new batch production of Schabziger cheese (21 samples) and Appenzeller cheese (26 samples) were analyzed. The samples were diluted 1:10 in 0.85% (w/v) NaCl + 0.01% (w/v) trypton casein-peptone, pH 7.00 and homogenized for 2 min in a laboratory blender (Stomacher 400, Seward, PBI International, Milano, Italy) and dilution series were plated on Kanamycin Aesculin Azide Agar Base (KAA) (OXOID Ltd., Hampshire, England) to determine the number of enterococci per g of product. To determine the number of chloramphenicol, erythromycin and tetracycline resistant enterococci per g of product, dilution series of the cheese samples were also plated on KAA containing either 20µg/ml of chloramphenicol (Chl), 10µg/ml of erythromycin (Ery), or 10µg/ml of tetracycline (Tet). Plates were incubated at 37°C for 48h. Up to ten colonies were randomly selected from each dilution series containing antibiotics of a cheese sample. The colonies were purified twice on Brain Heart Infusion Agar (BHIA) (OXOID Ltd., Hampshire, England). The strain labeling was done as follows: the cheese type was abbreviated with “SCH” for “Schabziger” and “APP” for “Appenzeller respectively. This abbreviation was followed by a consecutively numbering and the hyphenated cheese sample-number (example: SCH003-1; indicating the third enterococcal isolate coming from the Schabziger cheese sample No. 1).

Phenotypic characterization

All 216 isolates from Schabziger and 100 isolates from Appenzeller were characterized using the Gram stain method. Gram positive and coccal-shaped isolates were further characterized with API 20 Strep (Biomérieux, France).

26 Publication I

Phenotypic and genotypic assessment of antibiotic susceptibility

Strains were screened for phenotypic resistance to 12 antibiotics using the disc diffusion method and Mueller-Hinton agar (Oxoid, Basingstoke, UK). The following antibiotic discs (Becton, Dickinson and Company, Le Pont de Claix, France) were used: penicillin (10 µg) ampicillin (10 µg), amoxicillin/clavulanic acid (30 µg), chloramphenicol (30 µg), tetracycline (10 µg), erythromycin (15 µg), vancomycin (30 µg), teicoplanin (30 µg), imipenem (10 µg), ciprofloxacin (5 µg), nitrofurantoin (300 µg), and rifampicin (5 µg). Inhibition zones were interpreted following the guideline tables of the NCCLS [24]. Total genomic DNA was prepared using a protocol based on the method of Pospiech et al. [26]. PCR assays were performed using pairs of previously reported primers [1,32,33,36] for the detection of resistance genes: erythromycin drug inactivation (ereA, ereB, mphA), methylation mechanism (ermA, ermB, ermC), and efflux systems (mrsA/mrsB, mefA/mefE); tetracycline resistance by ribosomal protection mechanism (tetM) and an efflux system (tetL).

Phenotypic and genotypic assessment of virulence traits

PCR assays were performed using pairs of previously reported primers [12] for the detection of the following genes: agg, gelE, cylM, cylB, cylA, esp, efaAfs, efaAfm, cpd, cob, and ccf.

PCR assay

For all detection assays, a common PCR core mix (total volume 50 µl) was used consorting of 1 × PCR buffer (Promega, Madison, WI), 200 µM concentrations of deoxynucleoside triphosphates (Promega, Madison, WI), 1 U of Taq DNA Polymerase (Promega, Madison,

WI), 4 mM MgCl2, and 20 pmol of the corresponding primers (Thermo Electron GmbH, Ulm, Germany). A 50 ng portion of intact total DNA was used as PCR template.

PFGE typing

The chromosomal DNA was prepared as previously described [4] by growing the cells on BHI agar for 48 h at 37°C. Cells were suspended in 10 mM Tris / 100 mM EDTA, pH 8.0 (TE-

100 buffer) and consequently the suspension’s OD600 was adjusted to 2.5. This suspension was mixed with an equal volume of 1.5 % SeaKem Gold Agarose (Cambrex Bio Science Rockland, Rockland, ME) in 10 mM Tris / 1 mM EDTA, pH 8.0 (TE-buffer) to pour plugs. The solidified agar plugs were transferred in 1.5 ml TE-100 buffer with 25 mg/ml lysozyme (Sigma-Aldrich, Buchs, Switzerland) and rotated over night at 10 rpm (Hybaid Mini 10, MWG- Biotech, Ebersber, Germany) at 37°C. After washing the plugs twice in TE-100 buffer for 15 min, 1.5 ml 0.5 M EDTA / 1%N-Lauroylsarcosine (pH 8.0) and 2 mg/ml proteinase K (Roche,

27 Publication I

Basel, Switzerland) was added and incubated overnight at 50°C under rotation. Subsequently, the plugs were washed five times in TE-buffer for one hour under rotation. Subtyping of strains was performed by using pulsed-field gel electrophoresis (PFGE) with Sma-digested (Fermentas Inc., Maryland, USA) chromosomal DNA. PFGE was performed with a 1.0% agarose gel by using a CHEF-DR III apparatus (BioRad Laboratories, Hercules, California, USA) in 0.5× Tris-borate-EDTA buffer at 12°C at 6 V/cm with an angle of 120°. A linearly ramped switching time from 1 to 16 s was applied for 16.5 h.

Results

Enterococcal populations in Schabziger and Appenzeller cheese

Twenty-three of 26 tested Appenzeller and 17 of the 21 tested Schabziger cheeses were positive for enterococci. The counts varied from 3.2 x 104 to 1.6 x 106 CFUg-1 in Schabziger cheese and from 2.0 x 102 to 2.5 x 106 CFU per g in Appenzeller cheese respectively, which is in accordance with published data [3,17]. The screening for ChlR, TetR or EryR enterococcal strains resulted in 100 isolates from Appenzeller and 216 from Schabziger respectively. It was noticeable that Schabziger more frequently harboured resistant enterococci than Appenzeller (Table 1).

Table 1 Enterococci isolated from 21 Schabziger and 26 Appenzeller recovered from KAA agar plates containing either Chl, Tet, or Ery

Samples Analyzed Positive for with ChlR strains with TetR strains with EryR strains enterococci Appenzeller 26 23 (88.5%) 4 (15.4%) 11 (42.3%) 6 (23.1%) Schabziger 21 17 (81.0%) 9 (42.9%) 14 (66.7%) 13 (62.0%)

All tested isolates were Gram positive and coccal shaped. 270 of the 312 isolates were shown to be E. faecalis by biochemical genus and species identification, followed by 37 E. durans, and 16 E. faecium isolates. Eight isolates could only be characterized as enterococci on the genus level.

Antibiotic susceptibility: disc test, E-test and PCR

One of the most important characteristic for the evaluation of enterococci is their susceptibility to different antibiotics. All 312 enterococcal isolates were tested with 12 different antibiotics using the disc method. According to the zone diameter interpretive

28 Publication I standards for Enterococcus spp., all the tested strains were sensitive to vancomycin (Van). This finding was confirmed with the E-test. The disc test method showed 94 isolates (30%) resistant against Chl, 157 (50%) against Ery and 212 (68%) against Tet. Arranging the 312 isolated enterococci according to their phenotypic resistance resulted in 16 different patterns (Table 2).

Table 2 Enterococci from Appenzeller and Schabziger cheese with resistance to either Chl, Ery or Tet: resistance patterns by testing with 11other antimicrobial substances (penicillin (Pen), ampicillin (Amp), amoxicillin (Amo), chloramphenicol (Chl), tetracyclin (Tet), erythromycin (Ery), vancomycin (Van), teicoplanin (Tec), imipenem (Imi), ciprofloxacin (Cip), nitrofurantoin (Nit), and rifampicin (Rif)).

Resistance E. faecalis E. durans E. faecium n = 253 n = 39 n = 20 Tet 57 (22.5%) 4 (10.3%) 1 (5.0%) Ery Tet 55 (21.7%) 2 (5.1%) 4 (20.0%) Chl Tet 5 (2.0%) 0 (0%) 0 (0%) Ery Nit 0 (0%) 2 (5.1%) 0 (0%) Rif Tet 21 (8.0%) 0 (0%) 0 (0%) Chl Ery Tet 74 (29.2%) 0 (0%) 0 (0%) Chl Rif Tet 17 (6.7%) 0 (0%) 0 (0%) Ery Nit Tet 1 (0.4%) 29 (74.4%) 2 (0%) Ery Rif Tet 1 (0.4%) 0 (0%) 0 (0%) Cip Rif Tet 1 (0.4%) 0 (0%) 0 (0%) Nit Rif Tet 0 (0%) 0 (0%) 3 (15.0%) Ery Pen Tet 0 (0%) 0 (0%) 1 (5.0%) Ery Nit Pen Tet 0 (0%) 2 (5.1%) 0 (0%) Chl Ery Rif Tet 21 (8.0%) 0 (0%) 0 (0%) Cip Nit Rif Tet 0 (0%) 0 (0%) 8 (40.0%) Cip Ery Imi Pen Tet 0 (0%) 0 (0%) 1 (5.0%)

For PFGE typing, analysis of virulence factors and resistance genes, a selection was done as follows: from each cheese sample at most two isolates representing one of the sixteen resistance patterns were chosen, resulting in a selection containing 150 isolates out of 25 different cheese samples (11 Appenzeller and 14 Schabziger). Within this selection 146 were resistant against Tet and 92 against Ery. Further characterization of the resistance mechanisms resulted in 102 Tetr isolates showing both tetM and tetL genes together. Only six isolates had the tetL resistance alone and the remaining 38 isolates were positive for the tetM resistance alone. Most of the Eryr isolates showed their resistance being based on the

29 Publication I methylation mehanism of ermB. Eleven ermC, mph and msrA/msrB gene could be amplified with PCR. Within the 150 isolates 114 were positive for the transposon integrase gene (int) of the Tn916-Tn1545 family.

Strain typing with PFGE

PFGE was performed on 150 selected isolates and all additional 50 isolates from the resistance profiles “Chl Tet Ery” and “Chl Tet Ery Rif” . The resulting 200 PFGE band patterns were clustered and compared visually and with the Bionumerics software.

Figure 1 Three E. faecalis strains from one Schabziger sample showing the same resistance profile (ChlR, EryR, TetR) and different PFGE-patterns. Lane 1, SCH086-23; lane 2, SCH089-23; lane 3, SCH092-23; lane 4, MidRange PFG Marker I (New England BioLabs, USA) 1 2 3 4 Kb - 242.5 - 197.0 - 145.5

- 97.0

- 48.5 - 33.5 - 15

Thirty-four different PFGE patterns were found among the 200 isolates tested. With one exception, there were no strains with common patterns in Appenzeller and Schabziger cheese. All tested Schabziger samples showed at least two different PFGE patterns. Six Schabziger samples harbored strains that showed the same PFGE pattern but had different resistance profiles, whereas seven showed the same profile within one pattern. Four Schabziger samples were each dominated by a particular single clone. Only four Appenzeller samples showed heterogeneous PFGE patterns, whereof two samples harbored strains with a single resistance profile. In six cheese samples (2 Appenzeller, 4 Schabziger) several strains with a triple resistance (ChlR, EryR, TetR) showed completely different PFGE patterns (Figure 1). As previously shown [3], enterococci with the resistance profile “Chl Ery Tet” are commonly found in Swiss raw milk cheese. In order to show whether enterococci with this combination of resistance persist in cheese samples produced over a certain period of time, the resulting PFGE patterns were clustered. Consequently, five E. faecalis clones

30 Publication I from an Appenzeller and four Schabziger samples, could be found throughout one to three months of the whole screening period. (Figure 2).

Figure 2 Five enterococcal clones repeatedly isolated throughout one to three months from Appenzeller and Schabziger cheese (x: samples taken)

xxxxxxxxxxxxxxxxx

Nov 02 Dec 02 Jan 03 Feb 03 Mar 03 Apr 03 May 03 June 03

Schabziger SCH055-20 E.faecalis Schabziger SCH156-35 E.faecalis Schabziger SCH086-23 E.faecalis Schabziger SCH116-29 E.faecalis Appenzeller APP025-9 E.faecalis

PCR analysis for virulence factors

The prevailing species in Appenzeller and Schabziger cheese is E. faecalis, which is known to harbor multiple virulence factors [12]. The presence of virulence factors (Figure 3) in enterococci isolated from Appenzeller and Schabziger varied between 2 and 10.

Figure 3 Incidence of virulence factors among E. faecalis stains isolated from Appenzeller and Schabziger cheese.

ccf 98

cob 72

cpd 84.7

efaAfm 37.3 s r

o efaAfs 88 t c a esp 64 e f enc l cylA 6 u r i V cylB 6

cylM 3.3

gelE 76

agg 53.3

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 [%]

The sex pheromone genes (cpd, cob, ccf) were present in 72 – 98% of the strains tested. Further, no tested enterococcal strain possessed all 11 virulence factors. The gelE gene was

31 Publication I detected with 76% of the isolates, while cylM and esp genes occurred with the frequency of 6% and 64% respectively. The presence of cylAB factors only (no cytolysin production) occurred in 4% of the isolates.

Discussion

In this study two types of raw-milk cheese were sampled for enterococci within a period of five months. Both phenotypic and genotypic analyses were used for identification and further characterization of the isolates. Enterococcal counts in Schabziger and Appenzeller cheese were as expected. The higher level of enterococci in Schabziger can be explained with the shorter ripening period and the lower salt content of the cheese. Preliminary experiments have shown, that the enterococcal counts of young (ripening at least 3 months, lower salt content) compared with older (ripening at least 4 months, higher salt content) Appenzeller cheese (data not shown) are higher. E. faecalis was the most frequently isolated species which is in accordance with similar observations reported by other European researchers. Andrighetto et al [2] stated that most of the strains isolated in Italian cheeses were identified as E. faecalis, likewise E. faecalis dominated in the milk and Irish Cheddar-type cheese as reported by Gelsomino et al. [15] and did so in Slovenian Tolmic cheese [6]. The level of enterococci resistant to Chl, Tet or Ery was higher than expected. Resistance to tetracyclin was widespread in enterococci isolated from Schabziger and Appenzeller, which is in accordance to findings of previous studies [18]. Chloramphenicol and erythromycin resistance occurred at a lower level, but could often be found in combination with tetracycline resistance. In the last years, this multiresistance was described by several researchers [3,18,28,35]. In order to assess the potential risk associated with the presence and its possible ability of conjugative exchange of this multiresistance in the cheese investigated in this study, detailed analyses of the potential mobility of the detected resistances are required. The tetM gene was found in all but 11 Tetr E. faecalis isolates and 110 out of 146 isolates showed the efflux system tetL. A similar situation was found during a major survey of 229 enterococcal isolates collected in 10 hospitals in France, where tetM and tetL were the dominant Tetr determinants [7]. In many enterococci and streptococci of clinical or food origin, drug resistance genes occur more frequently on conjugative transposons than on plasmids. Also in this study, 78% of the tetM containing isolates (106 of 136 isolates) were positive by PCR for the integrase gene int, indicating that they contain a member of the broad-host-range Tn916-Tn1545 conjugative transposon family (Table 2). Further characterization of the 87 Eryr isolates resulted in 83 containing erm(B) gene, which is considered to be the most widespread macrolide resistance gene among enterococci from food animals or foods and from clinical isolates [34]. Furthermore, 69 of these erm(B) containing strains were also positive for a Tn916-Tn1545 element. Erm(B) genes in

32 Publication I enterococci can also occur on other mobile elements, such as conjugative multiresistance plasmids [35] or members of the Tn917 family [27]. The findings of PFGE typing showed, that enterococcal isolates with a very similar PFGE profile can have completely different resistance patterns. Furthermore, identical resistance pattern could be found in completely different PFGE profile. These results support the idea of possible conjugative gene exchange during cheese production. Such a gene transfer was previously shown by Cocconcelli and coworkers [10]. Five E. faecalis PFGE profiles could be found throughout one to three months. Since, there exists only one plant for the Schabziger production in Switzerland, it is possible, that these particular strains could persist in the manufacturing plant over a limited period. During this period conjugative gene transfer could have occurred, which could explain, that these isolates had in some cases different resistance patterns. None of the five strains was found along the entire screening period of five months. It is possible, that the cleaning concept of the manufacturing plant includes procedures which broadly eliminate microorganisms in the producing facility. With a next production batch, new strains can establish in the production system until they are eliminated again. As for Appenzeller cheese, the situation is probably not similar since there are several production plants. Nearly all strains included in this study contained one or more virulence genes that have previously been found in human isolates [12]. Over 75% of all tested isolates carried the gelE gene, although phenotypic GelE activity could not be detected. Similar results were obtained from hemolysis testing. Collectively, these results seem to indicate that the investigated strains contain silent gelE and cylABM genes. According to Eaton and Gasson [12], there are several environmental or temporal factors that may account for the apparent lack of phenotypic expression of enterococcal virulence genes. Similar to GelE, the presence of the cylABM gene is not always linked to the phenotypic expression of hemolysin activity. This phenomenon may be due to low-level gene expression or to the presence of an inactive gene product. Fifty-three percent of the strains contained the agg gene responsible for the clumping factor aggregation substance. This virulence factor was in most cases associated with the presence of pheromone determinants which is in accordance with findings published by Eaton and Gasson [12]. The incidence of Esp-positive enterococci was in the same range (64%) as previously published [12]. Since pheromone determinants are thought to facilitate conjugation, their high level (72 – 98% of all 150 isolates) could indicate possible gene exchange during cheese production. The results of the current study indicate that the enterococci present in Schabziger and Appenzeller can harbor various antibiotic resistance and virulence traits. All virulence traits found in the strains examined have also been found in human clinical isolates. However, these results as such do not allow conclusions with regard to safety of the cheese

33 Publication I itself. Complete risk assessment would require further research on the potential source and the in vitro and in situ transferability of these resistance and virulence properties.

Acknowledgments

This work was granted by the Swiss Federal Office of Public Health (SFOPH).

References

1. Aminov, R.I., Garrigues-Jeanjean, N. & Mackie, R.I. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. Appl. Environ. Microbiol. 67, 22-32 (2001).

2. Andrighetto, C. et al. Phenotypic and genetic diversity of enterococci isolated from Italian cheeses. J. Dairy Res. 68, 303-316 (2001).

3. Baumgartner, A., Kueffer, M. & Rohner, P. Occurrence and Antibiotic Resistance of Enterococci in Various Ready-to-eat Foods. Archiv für Lebensmittelhygiene 52, 16-19 (2004).

4. Baumgartner, A., Küffer, M. & Grand, M. Quantitative analysis and molecular typing with pulsed-field gel electrophoresis of two probiotic Lactobacillus strains from sour milk products available on the Swiss food market. Mitt. Lebensm. Hyg. 94, 452-460 (2003).

5. Baumgartner, A., Schmid, H., Steffen, C., Piguet, A.F. & Hartmann, D.W. Bakterielle Antibiotikaresistenz in den Bereichen Humanmedizin, Veterinärmedizin und Lebensmittel. Eine Situationsanalyse der "Koordinationsgruppe antibiotikaresistente Mikroorganismen". Koordinationsgruppe antibiotikaresistente Mikroorganismen. 1- 147. 15-6-1999. Bundesamt für Gesundheit, Infodienst.

6. Canzek, M.A., Rogelj, I. & Perko,B. Enterococci from Tolminc cheese: population structure, antibiotic susceptibility and incidence of virulence determinants. Int. J. Food Microbiol. 102, 239-244 (2005).

7. Charpentier, E., Gerbaud, G. & Courvalin, P. Presence of the Listeria tetracycline resistance gene tet(S) in Enterococcus faecalis. Antimicrob. Agents Chemother. 38, 2330-2335 (1994).

8. Clewell, D.B., An, F.Y., Flannagan, S.E., Antiporta, M. & Dunny, G.M. Enterococcal sex pheromone precursors are part of signal sequences for surface lipoproteins inverted question markletter. Mol. Microbiol. 35, 246-247 (2000).

9. Clewell, D.B., Francia, M.V., Flannagan, S.E. & An, F.Y. Enterococcal plasmid transfer: sex pheromones, transfer origins, relaxases, and the Staphylococcus aureus issue. Plasmid 48, 193-201 (2002).

34 Publication I

10. Cocconcelli, P.S., Cattivelli, D. & Gazzola, S. Gene transfer of vancomycin and tetracycline resistances among Enterococcus faecalis during cheese and sausage fermentations. Int. J. Food Microbiol. 88, 315-323 (2003).

11. Dzidic, S. & Bedekovic, V. Horizontal gene transfer-emerging multidrug resistance in hospital bacteria. Acta Pharmacol. Sin. 24, 519-526 (2003).

12. Eaton, T.J. & Gasson, M.J. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl. Environ. Microbiol. 67, 1628-1635 (2001).

13. Franz, C.M., Holzapfel, W.H. & Stiles, M.E. Enterococci at the crossroads of food safety? Int. J. Food Microbiol. 47, 1-24 (1999).

14. Garcia, M.T. et al. Inhibition of Listeria monocytogenes by enterocin EJ97 produced by Enterococcus faecalis EJ97. Int. J. Food Microbiol. 90, 161-170 (2004).

15. Gelsomino, R., Vancanneyt, M., Condon, S., Swings, J. & Cogan, T.M. Enterococcal diversity in the environment of an Irish Cheddar-type cheesemaking factory. Int. J. Food Microbiol. 71, 177-188 (2001).

16. Giraffa, G. Enterococci from foods. FEMS Microbiol. Rev. 26, 163-171 (2002).

17. Giraffa, G. Functionality of enterococci in dairy products. Int. J. Food Microbiol. 88, 215-222 (2003).

18. Huys, G., D'Haene, K., Collard, J.M. & Swings, J. Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food. Appl. Environ. Microbiol. 70, 1555-1562 (2004).

19. Klein, G. Taxonomy, ecology and antibiotic resistance of enterococci from food and the gastro-intestinal tract. Int. J. Food Microbiol. 88, 123-131 (2003).

20. Kruse, H. & Sorum, H. Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments. Appl. Environ. Microbiol. 60, 4015-4021 (1994).

21. Lowe, A.M., Lambert, P.A. & Smith, A.W. Cloning of an Enterococcus faecalis endocarditis antigen: homology with adhesins from some oral streptococci. Infect. Immun. 63, 703-706 (1995).

22. Mundy, L.M., Sahm, D.F. & Gilmore, M. Relationships between enterococcal virulence and antimicrobial resistance. Clin. Microbiol. Rev. 13, 513-522 (2000).

23. Murray, B.E. Diversity among multidrug-resistant enterococci. Emerg. Infect. Dis. 4, 37-47 (1998).

24. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. NCCLS, Villanova, Pa. (1999).

35 Publication I

25. Pillar, C.M. & Gilmore, M.S. Enterococcal virulence--pathogenicity island of E. Faecalis. Front Biosci. 9, 2335-2346 (2004).

26. Pospiech, A. & Neumann, B. A versatile quick-prep of genomic DNA from gram- positive bacteria. Trends Genet. 11, 217-218 (1995).

27. Rollins, L.D., Lee, L.N. & Le Blanc, D.J. Evidence for a disseminated erythromycin resistance determinant mediated by Tn917-like sequences among group D streptococci isolated from pigs, chickens, and humans. Antimicrob. Agents Chemother. 27, 439-444 (1985).

28. Schwarz, F.V., Perreten, V. & Teuber, M. Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25. Plasmid 46, 170- 187 (2001).

29. Shankar, N., Coburn, P., Pillar, C., Haas, W. & Gilmore, M. Enterococcal cytolysin: activities and association with other virulence traits in a pathogenicity island. Int. J. Med. Microbiol. 293, 609-618 (2004).

30. Shankar, V., Baghdayan, A.S., Huycke, M.M., Lindahl, G. & Gilmore, M.S. Infection- derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein. Infect. Immun. 67, 193-200 (1999).

31. Su, Y.A. et al. Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp. liquefaciens. Infect. Immun. 59, 415-420 (1991).

32. Sutcliffe, J., Grebe, T., Tait-Kamradt, A. & Wondrack, L. Detection of erythromycin- resistant determinants by PCR. Antimicrob. Agents Chemother. 40, 2562-2566 (1996).

33. Sutcliffe, J., Tait-Kamradt, A. & Wondrack, L. Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system. Antimicrob. Agents Chemother. 40, 1817-1824 (1996).

34. Teuber, M. Spread of antibiotic resistance with food-borne pathogens. Cell Mol. Life Sci. 56, 755-763 (1999).

35. Teuber, M., Schwarz, F. & Perreten, V. Molecular structure and evolution of the conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw-fermented sausage. Int. J. Food Microbiol. 88, 325-329 (2003).

36. Trieu-Cuot, P. et al. Study of heterogeneity of chloramphenicol acetyltransferase (CAT) genes in streptococci and enterococci by polymerase chain reaction: characterization of a new CAT determinant. Antimicrob. Agents Chemother. 37, 2593- 2598 (1993).

36 Conference Contribution I

Conference Contribution I

Enterococci from Appenzeller and Schabziger raw milk cheese: antibiotic resistance, virulence factors and persistence of particular strains in the products.

S. P. TEMPLER,1 P. ROHNER2, A. BAUMGARTNER1 1Section of Microbiology and Biotechnology, Swiss Federal Office of Public Health, Schwarzenburgstrasse 165, CH-3097 Liebefeld, Switzerland 2Laboratory of Clinical Microbiology, University Hospital Geneva, CH-1205 Geneva, Switzerland

Proceedings of the 65th annual assembly of SSM, Lausanne (Switzerland), 7th-8th March 2006

37 Enterococci from Appenzeller and Schabziger Raw Milk Bundesamt für Cheese: Antibiotic Resistance, Virulence Factors and b Gesundheit Office fédéral Persistence of particular Strains in the Products de la santé publique Ufficio federale delle sanità pubblica Stefanie P. Templer1,2, Andreas Baumgartner1 Swiss Federal Office 1Section of Microbiology and Biotechnology, Swiss Federal Office of Public Health, 3003 Bern, of Public Health Switzerland; 2Institute of Cell Biology, University of Bern, 3012 Bern, Switzerland

Abstract Pearson correlation (Opt:1.50%) [0.0%-100.0%] Enterococci are natural residents of human and animal intestinal tracts and PFGE-SmaI resistance source strain 80 100 C TE ERA grow to high levels in a variety of artisanal cheeses. The aim of this study Schabziger. SCH116-29 was to determine the diversity of enterococci in two types of artisanal raw- clinical. isolate GE029 milk cheese (Schabziger and Appenzeller) and whether a strain showing clinical. isolate LU026 I triple resistance against chloramphenicol (C), tetracycline (TE) and clinical. isolate GE027 erythromycin (E) persists in cheese samples. Out of forty-six cheese clinical. isolate LU025 clinical. isolate GE004 samples, a total of 312 Enterococcus strains were isolated over a five- clinical. isolate GE055 II months-period. Enterococcus faecalis was the predominant isolated species Schabziger. SCH137-35 (80.7%); other Enterococcus species identified were Enterococcus faecium Schabziger. SCH138-35 (5.1%) Enterococcus durans (11.7%). According to the phenotypic Schabziger. SCH043-17 Schabziger. SCH022-14 resistance patterns a selection of 155 strains was made to determine Schabziger. SCH023-14 resistance genes against E and TE. The occurrence of conjugative Schabziger. SCH010-11 III transposons of the Tn916-Tn1545 was determined by detecting the clinical. isolate LU020 integrase (int) gene. All selected strains contained at least two of the clinical. isolate LU002 clinical. isolate LU003 IV virulence genes tested. The Pulsed-field gel electrophoresis (PFGE) Appenzeller. APP063-34 patterns of the isolates showed a limited persistence over a period of one to clinical. isolate LU022 two months in Schabziger and more than two months in Appenzeller. clinical. isolate AG003 Compared with clinical isolates (previous studies), some food samples Appenzeller. APP004-5 Appenzeller. APP025-9 V showed the same PFGE patterns. Appenzeller. APP003-5 Schabziger. SCH208-44 Methods Schabziger. SCH209-44 Schabziger. SCH210-44 Isolation of Enterococci from Appenzeller and Schabziger (raw milk cheese) Fig.3 Comparison of E. faecalis isolates: 11 clinical isolates from 3 different hospitals and 14 food Biochemical characterization by API 20 Strep, biomérieux, France isolates from 9 different cheese samples. Cases I – V indicate multiple-strain genetic clusters. Screening for resistance determinants by the disc diffusion method and PCR-based detection methods Screening for virulence determinants by PCR-based detection methods Genotypic characterization by Pulsed-field gel electrophoresis (PFGE) cytolysin antigen A sex pheromones

Results and Discussion virulence efa efa agg gel E cylA cylB cylM esp cpd cob ccf Enterococcal food isolates (n=312) from raw-milk cheese were collected over a five- factor Afs Afm months-period. 21 Schabziger and 26 Appenzeller samples were analyzed. % of Enterococcal count varied from 3.2 x 104 to 1.6 x 106 CFUg-1 in Schabziger and from 56 54 14 14 8 57 86 46 78 52 92 occurrence 2.0 x 102 to 2.5 x 106 per g in Appenzeller respectively.

270 Enterococcus faecalis, 37 E. durans, 16 E. faecium and 8 non-further classified Tab.1 Incidence of virulence factors among E. faecalis stains isolated from Appenzeller and Schabziger Enterococcus spp. strains could be isolated from the cheese samples. This distribution is cheese. in accordance with similar observations reported by European researchers. Isolates were characterized by antibiotic resistance determination (disc diffusion, E- test and PCR) Antibiotics tested were penicillin (P), ampicillin (AM), amoxicillin/ Chromosomal SmaI restriction patterns from 155 cheese isolates were determined and PFGE types were analyzed with Bionumerics software for Windows, version 4.5 clavulanic acid (AMC), chloramphenicol (C), tetracycline (TE), erythromycin (E), (Applied Maths) vancomycin (VA), teicoplanin (TEC), imipenem (IPM), ciprofloxacin (CIP), nitrofurantoin (FM), rifampicin (RA). No resistance against VA or TEC could be detected Five clones could repeatedly been isolated throughout several weeks during the (see Fig. 1A) screening (see Fig. 2). A high percentage of triple resistant (C, TE, E) strains could be detected which A comparison of the PFGE patterns of clinical isolates of a previous study (data not suggests a persistence of a resistance plasmid coding for the resistance determinants shown) showed high similarities to the patterns from four food isolates. This could against C, E, and TE. Further conjugation experiments with plasmid free strains will be support the hypothesis of spreading strains along the food chain (see Fig. 3). performed. Determination of resistance mechanisms of TE and E resistant strains by PCR: Nearly all of the TE resistant strains had the ribosomal protection gene tet M. The methylation mechanism erm B was predominant in the E resistant strains (see Fig. 1B). Determination of virulence factors of 155 selected strains (see Tab. 1). All virulence traits found in the strains examined have also been found in human clinical isolates. However, these results as such do not allow conclusions with regard to safety of the Nov 02 Dec 02 Jan 03 Feb 03 Mar 03 Apr 03 May 03 June 03 cheese itself. Schabziger SCH055-20 E.faecalis Schabziger SCH156-35 E.faecalis Schabziger SCH086-23 E.faecalis 80 Schabziger SCH116-29 E.faecalis 70 Appenzeller APP025-9 E.faecalis 60 Schabziger Fig.2 Five enterococcal clones repeatedly isolated throughout one to three months from Appenzeller and 50 Appenzeller Schabziger cheese.

[%] 40 30 20 References 10 0 A Eaton, T. J. and M. J. Gasson. Molecular screening of Enterococcus virulence P AM AMC C TE E VA TEC IPM CIP FM RA determinants and potential for genetic exchange between food and medical isolates. Appl.Environ.Microbiol. 67:1628-1635 (2001). methylation ribosomal Dicuonzo,G. et al. Antibiotic resistance and genotypic characterization by PFGE of mechanism protection 100 clinical and environmental isolates of enterococci. FEMS Microbiol. Lett. 201, 205-211 conjugative 80 drug efflux transposon (2001). 60 inactivation system Ery resistance

[%] efflux 40 system Tet resistance Tenover,F.C. et al. Interpreting chromosomal DNA restriction patterns produced by 20 Integrase pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, 0 B C S L 2233-2239 (1995). re A re B h A mA mB m int e e p r r r tet M tet tet m e e e A/msrBfA/mefE sr e m m Fig.1 Antibiotic-resistant profiles among food isolates; A: phenotypic determination by disc diffusion Acknowledgements method, B: genotypic determination by PCR. This work was granted by the Swiss Federal Office of Public Health (SFOPH). Publication II

Publication II

Enterococcal clinical isolates from three hospitals in Switzerland: antibiotic resistance patterns, virulence determinants, PFGE-typing and relation to food isolates.

S. P. TEMPLER,1 P. ROHNER2, A. BAUMGARTNER1* 1Section of Microbiology and Biotechnology, Swiss Federal Office of Public Health, Schwarzenburgstrasse 165, CH-3097 Liebefeld, Switzerland * Tel: +41 31 322 95 82, Fax: +41 31 322 95 74, E-mail: [email protected] 2Laboratory of Clinical Microbiology, University Hospital Geneva, CH-1205 Geneva, Switzerland

Submitted in May 2006 to the Journal of Clinical Microbiology

39 Publication II

Enterococcal clinical isolates from three hospitals in Switzerland: antibiotic resistance patterns, virulence determinants, PFGE-typing and relation to food isolates.

Submitted 12 April 2006 to the Journal of Clinical Microbiology

Abstract

Clinical enterococcal isolates (n=114) from three different hospitals of the German and French part of Switzerland were characterized with testing for antibiotic resistance, pulsed field gel electrophoresis (PFGE) and the occurrence of virulence factors in a one-year-study (January 2003 to February 2004). Enterococcus faecalis was the predominant species (75.4%), followed by Enterococcus faecium (11.4%) and Enterococcus gallinarum (2.6%). Phenotypic determination of antibiotic resistance resulted in 18% of 114 isolates showing a triple-resistance against chloramphenicol (Chl), tetracycline (Tet), erythromycin (Ery), and 16 isolates (14%) exhibiting a multi-resistance (minimally four resistances). Four (3.5%) isolates showed vancomycin (Van) resistance. All isolates were analysed with PCR for the presence of genes encoding resistance to Ery (ereA, ereB, mphA, ermA, ermB, ermC, mrsA/mrsB, mefA/mefE), Tet (tetM, tetL), and Van (vanA, vanB, vanC, vanD, vanE, vanG). Since virulence factors have been linked to the pathogenicity of enterococci, the strain selection was also tested for the presence of the following virulence factors: agg, gelE, cyl, esp, efaAfs, efaAfm, cpd, cob and ccf. All tested strains contained at least two of the nine virulence genes taken into analysis. Clustering of the PFGE-profiles showed two dominating groups of 9 (8%) respectively 7 (6%) strains with highly similar genotypes. These strains originated from all three hospitals included in the present study. Seventy isolates (61%) occurred as unique, patient-specific clones. Several PFGE-types were associated with shared features in their antibiotic resistance patterns, indicating clonal spread between and within wards. A comparison of the PFGE-types of the clinical isolates with food isolates from

40 Publication II raw milk cheese resulted in genetically strongly related multiple-strain clusters consisting of genetic strongly related clinical and food isolates.

Introduction

Enterococci are part of the natural gut microflora in mammals. They are also found in a variety of food products, including milk and cheese [2,5,13-15,20,29]. In many of these cheeses enterococci play a key role in the maturation and to develop final taste [3,12]. For many years enterococci have been considered as harmless to humans. However, in the last decade, they have become important nosocomial pathogens and are often responsible for infections such as urinary tract infections, endocarditis or bacteremia [17]. The species responsible for most of the infections in the community, long-term care, and hospital settings is Enterococcus faecalis, which is most likely to be susceptible to vancomycin and penicillins [16,19,24]. Enterococcus faecium, intrinsically more often resistant than E. faecalis, accounts for approximately 10% of enterococcal infections overall, but in recent years a disproportionate number of nosocomially acquired infections arose [23,24]. Because of the increasing incidence of antibiotic resistant enterococci, the treatment of these infections has become increasingly difficult, especially in the case of vancomycin resistant strains. Antibiotic resistance alone cannot explain enterococcal pathogenicity. To cause infection, enterococci must be able to colonize the host tissue, to resist host specific and unspecific defense mechanisms and to cause pathological changes. Thus, they have to be virulent, and virulence factors including cytolysin (CylABM), enterococcal surface protein (Esp), aggreagation substance (Agg), and gelatinase (GelE) should be taken into consideration too [10,12,21,26]. The cytolysin causes ruptures of a variety of membranes, including those of bacterial cells, erythrocytes, and other mammalian cells [30]. The Esp factor produces a cell wall-associated protein that is involved in the immune evasion [31]. It is possible, that esp and cylABM genes are associated on a pathogenicity island [26]. The aggregation substance (Agg) is a surface-localized protein encoded by pheromone- responsive, self-transmissible plasmids that mediates binding of donor bacterial cells with plasmid-free recipients, allowing efficient conjugal transfer in a liquid environment [8]. Another adhesin-like virulence factor is the E. faecalis and E. faecium antigen A (EfaAfs and EfaAfm, respectively) that is expressed in the serum [18]. Gelatinase (GelE) is an extracellular metalloendopeptidase that hydrolyzes gelatin, collagen, hemoglobin, and other bioactive compounds [33]. The sex pheromones (Cpd, Cob, Ccf) are thought to be involved in eliciting an inflammatory response. These pheromones are chemotactic for human leukocytes and induce production and secretion of lysosomal enzymes and thereby facilitate conjugation [7].

41 Publication II

Most of the human clinical enterococcal isolates belong to species that normally colonize humans [32]. It has been also shown that the same resistance genes were found in bacteria isolated from unpasteurized cheese and in bacteria isolated from human patients [37]. Moreover, a recent study showed that both the milk and cheese samples and the human fecal samples of the personnel involved in cheese making contained the same Enterococcus species and, more importantly, the same three dominant clones [14]. These observations support the hypothesis that enterococci carried on food are either colonizing humans or exchanging antibiotic resistance genes with bacteria that colonize humans. However, there is still no demonstrated correlation between ingestion of food products containing enterococci and infections. In this study we determined the prevalence of different species of clinical enterococcal isolates, their antibiotic susceptibility and virulence factors. The isolates were recovered from three different hospitals in Switzerland. In order to understand the extent of similarity among enterococci from clinical sources and enterococcal strains obtained in a previous study in the context of foods, we compared the antimicrobial resistance, virulence factors and pulsed field gel electrophoresis (PFGE) profiles of these strains.

Materials and Methods

Clinical isolates

Hundred-and-fourteen isolates of enterococci isolated from patients of different wards of three university hospitals in Switzerland during a one-year period (2003) were included in the present study.

Food isolates

Strains from food were isolated in a previous study. Over a period of five months, a cheese sample of every new batch production of Schabziger cheese (21 samples) and Appenzeller cheese (26 samples) were analyzed. To determine the number of chloramphenicol, erythromycin and tetracycline resistant enterococci per g of product, dilution series of the cheese samples were plated on KAA containing either 20µg/ml of chloramphenicol (Chl), 10µg/ml of erythromycin (Ery), or 10µg/ml of tetracycline (Tet). Up to ten colonies were randomly selected from each dilution series containing antibiotics of a cheese sample. The strain labeling was done as follows: the cheese type was abbreviated with “SCH” for “Schabziger” and “APP” for “Appenzeller respectively. This abbreviation was followed by a consecutively numbering and the hyphenated cheese sample-number (example: SCH003-1; indicating the third enterococcal isolate coming from the Schabziger cheese sample No. 1).

42 Publication II

Identification

All 114 isolates from the three different hospitals were Gram stained and Gram positive and coccal-shaped isolates were further characterized with API 20 Strep (Biomérieux, France).

Phenotypic assessment of antibiotic susceptibility

Genotypic assessment of antibiotic susceptibility and detection of int genes

All isolates showing against Van, Tet, Chl or Ery were further characterized with PCR. The occurrence of conjugative transposons of the Tn916-Tn1545 family was determined in all clinical isolates with primers Int-FW and Int-RV targeting the transposon integrase (int) gene (Doherty et al. 2000, Gevers et al. 2003). Total genomic DNA was prepared using a protocol based on the method of Pospiech et al. [27]. PCR assays were performed using pairs of previously reported primers [1,4,9,11,25,34,35,39] for the detection of resistance genes: erythromycin drug inactivation (ere(A), ere(B), mphA), methylation mechanism (erm(A), erm(B), erm(C)), and efflux systems (mrsA/mrsB, mefA/mefE); chloramphenicol drug inactivation (cat); tetracycline resistance by ribosomal protection mechanism (tet(M)) and an efflux system (tet(L)); and resistance to vancomycin (van(A), van(B), van(C), van(D), van(E), van(G)).

Phenotypic and genotypic assessment of virulence traits

PCR assays were performed using pairs of previously reported primers for the detection of the following genes: agg, gelE, cylM, cylB, cylA, esp, efaAfs, efaAfm, cpd, cob, and ccf [10].

PCR assay

For all detection assays, a common PCR core mix (total volume 20 µl) was used consorting of 1 x PCR buffer (Promega, Madison, WI), 200 mM concentrations of deoxynucleoside triphosphates (Promega, Madison, WI), 1 U of Taq DNA Polymerase (Promega, Madison,

43 Publication II

WI), 4 mM MgCl2, and 20 pmol of the corresponding primers (Thermo Electron GmbH, Ulm, Germany). A 50 ng portion of intact total DNA was used as PCR template.

PFGE typing

The chromosomal DNA was prepared as previously described (4) by growing the strains to be typed on BHI agar for 48 h at 37°C. Cells were suspended in 10 mM Tris / 100 mM EDTA, pH 8.0 (TE-100 buffer) and consequently, the suspension’s OD600 was adjusted to 2.5. This suspension was mixed with an equal volume of 1.5 % SeaKem Gold Agarose (Cambrex Bio Science Rockland, Rockland, ME) in 10 mM Tris / 1 mM EDTA, pH 8.0 (TE-buffer) to pour plugs. The solidified agar plugs were transferred in 1.5 ml TE-100 buffer with 25 mg/ml lysozyme (Sigma-Aldrich, Buchs, Switzerland) and rotated over night at 10 rpm (Hybaid Mini 10, MWG-Biotech, Ebersber, Germany) at 37°C. After washing the plugs twice in TE-100 buffer for 15 min, 1.5 ml 0.5 M EDTA / 1%N-Lauroylsarcosine (pH 8.0) and 2 mg/ml proteinase K (Roche, Basel, Switzerland) was added and incubated overnight at 50°C under rotation. Subsequently, the plugs were washed five times in TE-buffer for one hour under rotation at 15 rpm on a rotamix (Heto Lab Equipment A/S, Denmark). Subtyping of strains was performed by using pulsed-field gel electrophoresis (PFGE) with SmaI-digested (Fermentas Inc., Maryland, USA) chromosomal DNA. PFGE was performed with a 1.0% agarose gel by using a CHEF-DR III apparatus (BioRad Laboratories, Hercules, California, USA) in 0.5 Tris-borate-EDTA buffer at 12°C at 6 V/cm with an angle of 120°. A linearly ramped switching time from 1 to 16 s was applied for 16.5 h.

Cluster analysis

PFGE types were analyzed with Bionumerics software for Windows, version 4.0 (Applied Maths). The DNA banding patterns were normalized with MidRange PFG Marker I (New England Biolabs, USA). Comparison of the banding patterns was performed by Pearson correlation. A tolerance of 1.5% in band position was applied during comparison of the DNA patterns.

Results

Enterococcal species distribution

The clinical isolates were isolated from blood cultures (75%), from miscellaneous sites, including catheter tips, peritoneal fluid and urine (13%) and from surgical and non-surgical wound swabs (12%). Over 50% of all isolates were recovered from patients older than 65 years. Among the 114 isolates of enterococci, the most prevalent species found was

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E. faecalis at a frequency of 87 isolates (76.3%), followed by 20 (17.5%) E. faecalis, four (3.5%) E. gallinarum, two (1.8%) E. avium isolates and one (0.9%) E. durans isolate.

Antibiotic susceptibility: disc test, E-test and PCR

All 114 enterococcal isolates were tested with 12 antibiotics using the disc method. According to the zone diameter interpretive standards for Enterococcus spp. [22], five of the tested strains were resistant to vancomycin (Van). Confirmation with the E-test resulted in MICs for Van of 4µg/mL for all five isolates. From the 114 isolates, 11 E. faecalis, one E. gallinarum and one E. avium strains were susceptible to all tested substances. Arranging the remaining 101 isolated enterococci according to their phenotypic resistance resulted in 35 different patterns. Eight E. faecium harbored six and more resistances whereas only one E. faecalis did (Table 1). Within the 114 clinical isolates 61 were resistant against Tet and 39 against Ery. Further characterization of the resistance mechanisms resulted in 32 Tetr isolates showing both tet(M) and tet(L) genes together. Only one isolate had the tet(L) resistance alone and the remaining 29 isolates were positive for the tet(M) resistance alone. Most of the Eryr isolates showed their resistance being based on the methylation mehanism of erm(B). No erm(C), mph and msrA/msrB gene could be amplified with PCR. Within the 114 isolates 45 were positive for the transposon integrase gene (int) of the Tn916-Tn1545 family (Table 2). The five clinical isolates from two different hospitals showing a moderate phenotypic resistance against vancomycin, could be characterized as van(B) and van(C1) genotype.

Table 2 Resistance mechanisms of 61 Tet resistant and 39 Ery resistant clinical enterococcal isolates. Mechanisms tested for Ery resistant isolates: drug inactivation ere(A), ere(B) and mph, methylation mechanism erm(A), erm(B) and erm(C), efflux system msrA/msrB and mefA/mefE. Mechnaisms tested for Tet resistant isolates: ribosomal protection protein tet(M), efflux system tet(L). All isolates are also tested for the presence of the transposon integrase gene int.

Occurrence of resistance mechanism

Species ere(A) ere(B) erm(A) erm(B) mefA/mefE tet(M) te(L) int

E. faecalis (n = 50) 6 (12%) 3 (6%) 1 (2%) 21 (42%) 0 (0%) 46 (92%) 24 (48%) 20 (40%)

E. facium (n = 14) 1 (7%) 1 (7%) 2 (14%) 6 (43%) 2 (14%) 10 (71%) 8 (57%) 8 (57%)

E. gallinarum (n = 2) 1 (50%) 0 (0%) 1 (50%) 0 (0%) 0 (0%) 1 (50%) 1 (50%) 2 (100%)

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Table 1 Antibiotic-resistant profiles of 114 clinical enterococcal isolates tested with 12 antimicrobial agents no. of Antibiotica E. faecalis E. faecium E. gallinarum E. avium E. durans resistances Resistance n = 87 n = 20 n = 4 n = 2 n = 1 1 Chl 1 (1.2%) - - - - Ery 1 (1.2%) - - - - Imi - 1 (5.0%) - - - Rif 21 (24.4%) 1 (5.0%) 1 (25%) - 1 (100%) Tet 8 (9.3%) 1 (5.0%) - - - 2 Cip, Imi - 1 (5.0%) - - - Cip, Van 1 (1.2%) - - - - Chl, Rif 1 (1.2%) - - - - Ery, Tet 1 (1.2%) - - - - Pen, Tet - 1 (5.0%) - - - Ery, Nit - 1 (5.0%) - - - Rif, Tet 12 (14.0%) 1 (5.0%) - - - Rif, Van - 1 (5.0%) - - - 3 Cip, Ery, Rif 1 (1.2%) - - - - Cip, Ery, Tet 1 (1.2%) - - - - Cip, Chl, Rif 1 (1.2%) - - - - Cip, Rif, Van 1 (1.2%) - - - - Cip, Pen, Tet 1 (1.2%) - - - - Chl, Ery, Tet 8 (9.3%) - - 1 (50.0%) - Ery, Rif, Tet 2 (2.4%) - - - - Pen, Rif, Tet 2 (2.4%) - - - - 4 Cip, Ery, Pen, Tet - 1 (5.0%) - - - Cip, Imi, Nit, Pen 1 (1.2%) - - - - Chl, Ery, Rif, Tet 8 (9.3%) - - - - Chl, Ery, Tet, Van 1 (1.2%) - - - - Cip, Nit, Rif, Tet - - 8 (40.0%) Cip, Ery, Imi, Pen, 5 - - 1 (5.0%) Tet Cip, Chl, Ery, Rif, 1 (1.2%) - - - - Tet Chl, Ery, Rif, Tet, 1 (1.2%) - - - - Van Amo, Amp, Pen, 6 - 1 (5.0%) - - - Rif, Tec, Tet Amp, Cip, Ery, - - 1 (25%) - - Imi, Pen, Tet Amo, Amp, Ery, - 1 (5.0%) 1 (25%) - - Imi, Pen, Rif Amo, Amp, Cip, 7 - 1 (5.0%) - - - Imi, Pen, Rif, Tet Amo, Amp, Cip, 8 Ery, Imi, Pen, Rif, 1 (1.2%) 4 (20%) - - - Tet Amo, Amp, Cip, 9 Ery, Imi, Nit, Pen, - 1 (5.0%) - - - Rif, Tet a According to Clinical and Laboratory Standards Institut (formerly NCCLS) definitions. Pen, penicillin; Amp, ampicillin; Amo, amoxicillin; Chl, chloramphenicol; Tet, tetracycline; Ery erythromycin; Van, vancomycin; Tec, teicoplanin; Imi, imipenem; Cip, ciprofloxacin; Nit, nitrofurantoin; Rif, rifampicin

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Distribution of virulence determinants

PCR amplification of the enterococcal total DNA revealed distinct trends in the occurrence of virulence determinants in the clinical isolates. A summary of the pattern of virulence gene incidence in E. faecalis and E. faecium strains is presented in Table 3. E. faecalis strains harbored significantly more virulence determinant than do E. faecium strains. All of the E. faecalis strains tested possessed multiple virulence determinants (between 5 and 11). Two E. faecalis strains showed all of the virulence genes. At least two oft the sex pheromone determinants occurred in all of the strains tested. We could observe a trend to association of agg and esp virulence determinants with the presence of pheromone determinants. A significantly different pattern was seen in the E. faecium strains. Only one strain possesed the cylMBA determinants and only four strains had the agg determinants. Therefore it was not possible to conclude anything about association of agg with the pheromone determinants.

Table 3 Frequency of virulence determinants agg, gelE, cylM, cylB, cylA, esp, efaAfs, efaAfm, cpd, cob and ccf among clinical isolates of E. faecalis and E. faecium

Frequency of virulence determinants

Species agg gelE cylABM esp efaAfs efaAfm cpd cob ccf

E. faecalis 39 (45%) 56 (64%) 11 (13%) 34 (39%) 62 (71%) 55 (63%) 74 (85%) 67 (77%) 71 (82%) (n=87) E. faecium 4 (20%) 8 (40%) 1 (5%) 9 (45%) 7 (35%) 8 (40%) 7 (35%) 5 (25%) 11 (55%) (n=20)

Strain typing with PFGE

Chromosomal SmaI restriction patterns were determined to genotype all of the 114 clinical isolates by PFGE to explore clonal relatedness of human strains from different hospitals. Wide genotypic variability was found among the 87 clinical isolates of E. faecalis. Fourty- three different PFGE-types, and 58 subtypes among E. faecalis strains were identified. Within the PFGE-types, 14 multiple-strain genetic clusters could be defined. Seven of these clusters consisted of isolates from two hospitals and one cluster consisted of strains from all three hospitals. Genotyping of the 20 E. faecium strains resulted in 12 pulsotypes with 18 subtypes We compared the PFGE-patterns of all clinical E. faecalis strains with food isolates from two different Swiss raw milk cheeses, Appenzeller and Schabziger from previous experiments (manuscript in preparation). The comparison resulted in 5 multiple-strain clusters which consisted of genetic strongly related clinical and food isolates (Figure 1).

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Figure 1 Dendrogram of E. faecalis isolates, based on PFGE-profiles: 11 clinical isolates from 3 different hospitals and 14 food isolates from 9 different cheese samples. Cases I – V indicate multiple-strain genetic clusters. (Pearson correlation (Opt:1.50%) [0.0%-100.0%])

a PFGE-SmaI resistance source strain 80 100 Chl Tet Ery Rif Schabziger SCH116-29 clinical isolate GE029 I clinical isolate LU026 clinical isolate GE027 clinical isolate LU025 clinical isolate GE004 clinical isolate GE055 II Schabziger SCH137-35 Schabziger SCH138-35 Schabziger SCH043-17 Schabziger SCH022-14 Schabziger SCH023-14 III Schabziger SCH010-11 clinical isolate LU020 clinical isolate LU002 clinical isolate LU003 IV Appenzeller APP063-34 clinical isolate LU022 clinical isolate AG003 Appenzeller APP004-5 Appenzeller APP025-9 V Appenzeller APP003-5 Schabziger SCH208-44 Schabziger SCH209-44 Schabziger SCH210-44 a Abbreviations in strain name: SCH, Schabziger raw milk cheese; APP, Appenzeller raw milk cheese; GE, University Hospital of Geneva; LU, Cantonal Hospital of Lucerne; AG Cantonal Hospital of Aarau

Discussion

In this study 114 clinical isolates were recovered from three different hospitals in Switzerland. Both phenotypic and genotypic analyses were used for identification and further characterization of the isolates. E. faecalis was the predominant species followed by E. faecium which is in accordance to the literature. The highest frequencies of resistance were recorded with Chl, Ery, Rif or Tet. Resistance to tetracyclin was detected in over 55% of the isolates, which is in accordance to findings of previous studies [17]. Chloramphenicol and erythromycin resistance occurred at a lower level, but could often be found in combination with tetracycline resistance. In the last years, this multiresistance was described by several

48 Publication II authors (3,17,27,33). As shown in table 3, the tet(M) gene was found in all but four Tetr E. faecalis isolates and 24 out of 50 isolates showed the efflux system tet(L). A similar situation was found during a major survey of 229 enterococcal isolates collected in 10 hospitals in France, where tet(M) and tet(L) were the dominant Tetr determinants [6]. The five Vanr isoltates further defined as van(B,C1) resistance, indicate, that also in Switzerland resistance against Van occurs. However, resistance of VanB and VanC1 type is only moderate and does not suggest a trend of emergence. To assess the potential risk associated with the presence these resistances in the gut of humans, analyses of the potential mobility of the detected resistances would be required. In many enterococci and streptococci of clinical or food origin, drug resistance genes occur more frequently on conjugative transposons than on plasmids. Also in this study, over 50% of the tet(M) containing isolates (30 of 57 isolates) were positive by PCR for the integrase element int, indicating that they contain a member of the broad-host-range Tn916- Tn1545 conjugative transposon family (Table 2). Further characterization of the 39 Eryr isolates resulted in 27 containing erm(B) gene, which is considered to be the most widespread macrolide resistance gene among enterococci from food animals or foods and from clinical isolates [36]. Furthermore, all of these erm(B) containing strains were also positive for a Tn916-Tn1545 element. Erm(B) genes in enterococci can also occur on other mobile elements, such as conjugative multiresistance plasmids [38] or members of the Tn917 family [28]. Nearly all strains included in this study contained one or more virulence genes that have previously been found in human isolates [10]. Over 50% of all tested isolates carried the gelE gene, although phenotypic GelE activity could not be detected. Similar results were obtained from hemolysis testing. Together, these results seem to indicate that the investigated strains contain silent gelE and cylABM genes. According to Eaton and Gasson [10], there are several environmental or temporal factors that may account for the apparent lack of phenotypic expression of enterococcal virulence genes. Similar to gelE, the presence of the cylABM gene is not always linked to the phenotypic expression of hemolysin activity. This phenomenon may be due to low-level gene expression or to the presence of an inactive gene product. Fourty-five percent of the E. faecalis strains and 20% of the E. faecium strains respectively contained the agg gene responsible for the clumping factor aggregation substance. This virulence factor was in most cases associated with the presence of pheromone determinants which is in accordance with findings published by Eaton and Gasson [10]. The incidence of esp-positive enterococci was in the same range (39% in E. faecalis and 45% in E. faecium) as previously published (Eaton et al. 2001). Because pheromone determinants are thought to facilitate conjugation, their high level (71 – 91% of all isolates) could indicate possible gene exchange, which should be further analyzed.

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PFGE-typing showed that enterococcal isolates with a very similar PFGE-profile can have completely different resistance patterns. Furthermore, identical resistance pattern could be found in completely different PFGE-profiles. Such phenotypic differences can be caused by conjugative elements that exhibit no or only slight difference in the PFGE-profiles. The wide genotypic variability of clinical isolates of enterococci in Switzerland showed that infections are mostly of sporadic nature. However, the finding of 14 multiple-strain genetic clusters suggests small clinical outbreaks. To our knowledge, this is the first report of a comparison of PFGE-patterns of clinical and food isolates showing such strong relatedness between clinical and food isolates. To understand the spread of genetically related clusters of strains from different settings, further epidemiological investigations would be required.

Acknowledgments

We gratefully acknowledge Dr. I. Heinzer, cantonal hospital Aarau, Switzerland, and Mrs. T. Rutz, cantonal hospital Lucerne, Switzerland for providing clinical isolates. This work was granted by the Swiss Federal Office of Public Health (SFOPH).

References

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53 Conference Contribution II

Conference Contribution II

Enterococcal clinical isolates: antibiotic resistance patterns and genotypic characterization by PFGE.

S. P. TEMPLER,1 T. SEEBECK2, A. BAUMGARTNER1 1Section of Microbiology and Biotechnology, Swiss Federal Office of Public Health, Schwarzenburgstrasse 165, CH-3097 Liebefeld, Switzerland 2Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland

Proceedings of the 2nd International ASM-FEMS Conference on Enterococci. Helsingor (Denmark), 28th-31st August 2005 Submitted in May 2006 to the Journal of Clinical Microbiology

54 Enterococcal clinical isolates: antibiotic resistance patterns and genotypic characterization by PFGE. Stefanie P. Templer1,3, Peter Rohner2, Thomas Seebeck3, Andreas Baumgartner1 1 Section of Microbiology and Biotechnology, Swiss Federal Office of Public Health, 3003 Bern, Switzerland; 2 Laboratory of Clinical Microbiology, University Hospital Geneva, 1205 Geneva, Switzerland; 3 Institute of Cell Biology, University of Bern, 3012 Bern, Switzerland

Swiss Federal Office b of Public Health

Pearson correlation (Opt:1.00%) [0.0%-100.0%] Abstract PFGE-SmaI PFGE-SmaI Antibiogramme kb Clinical enterococcal isolates (n=114) from three different hospitals of the 300.00 250.00 200.00 140.00 120.00 100.00 80.00 60.00 30.00 15.00 10.00 6.00 4.00 80 100 German and French speaking part of Switzerland were characterized by P C-30 TE-30 E-15 VA-30 RA-5 antibiotic resistance determination and pulsed field gel electrophoresis GE034 E.faecalis (PFGE) in a one-year-study (January 2003 to February 2004). 18% of 114 GE055 E.faecalis isolates showed a triple-resistance against chloramphenicol, tetracyclin, LU025 E.faecalis erythromycin, and 16 (14%) exhibited a multi-resistance (minimally four GE004 E.faecalis GE053 E.faecalis resistances). I GE054 E.faecalis Clustering of the PFGE-profiles showed two dominating groups of 9 (8%) GE027 E.faecalis respectively 7 (6%) strains with highly similar genotypes. These strains LU026 E.faecalis originated from all three hospitals included in the present study. The GE029 E.faecalis majority of isolates, 70 (61%) occurred as unique, patient-specific clones. LU018 E.faecalis Several PFGE-types were associated with shared features in their antibiotic GE045 E.faecium resistance patterns, indicating clonal spread between and within wards. AG010 E.faecalis GE006 E.faecalis II Methods GE010 E.faecalis GE039 E.faecalis Isolation of clinical isolates from patients of three different hospitals GE035 E.faecalis Biochemical characterization by API 20 Strep, biomérieux, France LU016 E.faecalis Screening for resistance determinants by the disc diffusion method, GE002 E.faecalis III E-test and PCR-based detection methods Genotypic characterization by Pulsed field gel electrophoresis (PFGE) LU022 E.faecalis AG003 E.faecalis IV LU024 E.faecalis Results LU014 E.faecalis LU032 E.faecalis Clinical enterococcal isolates (n=114) 100 V AG011 E.faecalis from inpatents were recovered from three 90 different hospitals in Switzerland during a Fig.5 Genetic relatedness of E.faecalis isolates from Aarau (AG), Lucerne (LU), and Geneva (GE), based 80 one-year-period. on the PFGE banding patterns of the isolates. 70 86 Enterococcus faecalis, 11 E. 60 faecium, 3 E. gallinarum, 2 E. avium, 10 Chromosomal SmaI restriction patterns from 114 clinical isolates were determined and non-further classified Enterococcus spp. 50 PFGE types were analyzed with Bionumerics software for Windows, version 4.5 [%] strains (see Fig.1) included in this one 40 (Applied Maths) year study (January 2003 to February 30 Clustering showed five groups containing strains of at least two different hospitals 2004), 53% of all isolates came from 20 (see Fig. 5). Two groups (I + II) were dominating patiets older than 65 (see Fig 2) 10 61% of all isolates did not correspond to one of these groups, occurring as unique, Distribution by site of isolation – 75% 0 patient-specific clones. bloodstream, 12% surgical and non- alis ium c c Several PFGE types were associated with shared features in their antibiotic surgical wound swabs, 13% E. spp E.avium resistance patterns E.fae E.fae miscellaneous sites, including catheter E.gallinarum tips, peritoneal fluid and urine (see Fig. 3) Fig.1 Identification of the 114 species of enterococci isolated from human Isolates were characterized by Discussion antibiotic resistance determination (disc 9% diffusion, E-test and PCR) Antibiotics 2% The high percentage of triple resistant strains suggests a persistence of a resistance age 0-5 plasmid coding for the resistance determinants against C, E, and TE. tested were penicillin (P), ampicillin (AM), age 6-25 amoxicillin/clavulanic acid (AMC), age 26-64 Preliminary experiments suggest that identical resistance patterns within different chloramphenicol (C), tetracycline age 65+ PFGE-profiles are a result of horizontal transfer of DNA sequences encoding for (TE), erythromycin (E), vancomycin (VA), 53% 37% antibiotic resistance. Conjugation experiments in different in vitro or in vivo systems are imipenem (IPM), ciprofloxacin (CIP), planned to obtain information about conjugation frequencies. nitrofurantoin (FM), rifampicin (RA) (see Fig.2 Distribution of the 114 isolates by The clustered PFGE groups included strains of all three hospitals. With further Fig. 4). patients age experiments, the hypothesis will be tested whether resistant strains are spread along the

14 isolates showed no resistance 12% food chain (or vice versa). against one of the tested antibiotics. 13% A comparison of the PFGE patterns of the clinical isolates showed high similarities to 4 isolates showed a vancomycin the patterns from four food isolates of a previous study (data not shown). This could resistance according to the disc diffusion support the hypothesis of spreading strains along the food chain. method which could be confirmed neither blood stream 75% by E-test nor by PCR. miscellaneous sites surgical and non-surgical wound swabs 21 isolates showed a triple resistance References against C, E, and TE, and 16 exhibited a Fig.3 Distribution of the 114 isolates by multi resistance (minimally 4 resistances) site of isolation van den Bogaard,A.E. et al. Antibiotic resistance of faecal enterococci in poultry, poultry farmers and poultry slaughterers. J. Antimicrob. Chemother. 49, 497-505 (2002). Dicuonzo,G. et al. Antibiotic resistance and genotypic characterization by PFGE of 70 clinical and environmental isolates of enterococci. FEMS Microbiol. Lett. 201, 205-211 60 (2001). 50 Tenover,F.C. et al. Interpreting chromosomal DNA restriction patterns produced by 40 pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, [%] 30 2233-2239 (1995). 20 10 Acknowledgements 0 PAMAMC C TE E VA IPM CIP FM RA We gratefully acknowledge Dr. I. Heinzer, cantonal hospital Aarau, Switzerland, and Fig.4 Antibiotic-resistant profiles among clinical isolates Mrs. T. Rutz, cantonal hospital Lucerne, Switzerland for providing clinical isolates. Publication III

Publication III

Horizontal transfer of genes for tetracycline and erythromycin resistances among Enterococcus faecalis in vanilla cream. S. P. TEMPLER1, A. BAUMGARTNER1* 1Section of Microbiology and Biotechnology, Swiss Federal Office of Public Health, Schwarzenburgstrasse 165, CH-3097 Liebefeld, Switzerland * Tel: +41 31 322 95 82, Fax: +41 31 322 95 74, E-mail: [email protected]

Submitted in May 2006 to Applied and Environmental Microbiology

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Horizontal transfer of genes for tetracycline and erythromycin resistance among Enterococcus faecalis in vanilla cream

Submitted in May 2006 to Applied and Environmental Microbiology

Abstract

This study assessed the frequency of transfer of mobile genetic elements coding for virulence determinants and antibiotic resistance factors, from food associated enterococci into the plasmid free recipient strain E. faecalis JH2-2. The transfer of two mechanisms of tetracycline resistance (tetM and tetL), several erythromycin resistance mechanisms (erythromycin drug inactivation (ereA, ereB), methylation mechanism (ermA, ermB, ermC), and efflux systems (mrsA/mrsB, mefA/mefE)) and the aggregation substance (agg) and cell- wall associated adhesin (esp) as virulence factors could be shown in vanilla cream inoculated with donor and recipient strains at a level similar to those found in raw milk cheese. The frequencies varied between 2.8 x 10-4 and 9.8 x 10-7 transconjugants/donors. Furthermore, we analyzed the transposition of members of the Tn916-Tn1545 family. At refrigeration temperatures of 4°C, no transconjugants occurred after 48 h. This study showed that even in the absence of selective pressure, mobile genetic elements carrying antibiotic resistance and virulence determinants can be transferred at high frequency among E. faecalis strains in a food model system.

Introduction

Enterococci are found in a variety of cheeses made from raw or pasteurized milk from cows [4,14]. For a long time, the presence of these bacteria in foods was associated with faecal contamination, but nowadays they are considered as normal part of the food micro flora [19]. Enterococci seem to improve the flavor development and cheese quality [14]. Because of antilisterial activity based on bacteriocin production, they are also used in food preservation

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[14,15,18]. It is a major interest of food producers to guarantee the safety of technologically used bacterial cultures that can be consumed as living cells and in large quantities. Therefore, it is important to use well defined cultures, which should be free of factors harmful to humans. In this context, transmissible resistance to antimicrobial agents is an aspect which should also be taken into consideration although there are not yet legal requirements decreed in this field. Horizontal transfer of antibiotic resistance genes between bacteria has attracted much attention, and numerous investigations of gene transfer in environmental settings have been reported [20,24]. One concern is the dissemination and accumulation of resistance genes in the environment, which might be enhanced by use of antimicrobial agents and constitutes a risk for spread of resistance to pathogenic bacteria [33]. Since the first report of conjugative transposons in 1981 [13], increasing attention has been given to these self- transmissible discrete DNA elements and their role in spreading antibiotic resistance between bacteria [28]. Presence of members of the Tn916-Tn1545 family can be identified by detecting an integrase (int) gene in the total DNA of an enterococcal strain. These transposons are known to replicate in a wide range of clinically important gram-positive and gram-negative species [7]. Transposition of conjugative transposons is a three-step process involving (i) excision from the donor chromosome and formation of a covalently closed circular intermediate, (ii) conjugation by way of single-stranded transfer to the recipient cell, and (iii) nonspecific integrations into the recipient chromosome [27]. Multiple copies of transposons can exist on the same chromosome [23]. However, the ratio of transfer is low compared to that of conjugative transfer of plasmids [3]. Three classes of plasmids are known to be capable of replication in the enterococci: the rolling circle replicating (RCR) plasmids, the Inc18 plasmids, and the pheromone-responsive plasmids. The gene exchange among enterococci of plasmids coding for antibiotic resistances and virulence determinants has been evaluated in vivo and ex vivo [8]. The high frequencies of conjugal transfer are related to the presence of aggregation substances (agg), which mediate the binding between enterococcal cells [10] These bacterial adhesions, coded by conjugative plasmids in Enterococcus faecalis, has been studied in detail and its role in the high frequency of conjugal transfer of sex pheromone plasmids has been demonstrated. Sex pheromones are also thought to be involved in eliciting an inflammatory response [6,12]. The purpose of the present study was to asses the frequency of gene transfer of tetracycline and erythromycin resistances, virulence determinants and of members of the Tn916-Tn1545 family among E. faecalis strains in vanilla cream, a model system for other food stuffs with homogenous matrices such as soft cheese.

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Materials and Methods

Bacterial strains and growth media

The rifampicin- and fusidic acid-resistant strain E. faecalis JH2-2 [31] was used as the recipient strain. The three donor strains were isolated from food in a previous study. Over a period of five months, a cheese sample of every new batch production of Schabziger cheese (21 samples) and Appenzeller cheese (26 samples) were analyzed. Brain heart infusion broth (Oxoid, Hampshire, England) was used for all broth cultures. Chromocult Enterococi-Agar (Merck, Darmstadt, Germany) was used for selective plating of enterococci. Recipients plus transconjugants, donors, and transconjugant enterococci were selected on Chromocult Enterococci-Agar plates containing either fusidic acid and tetracycline (Tet) or fusidic acid and erythromycin (Ery). All plates were incubated aerobically at 37°C for two days. Antibiotics (Sigma) were used at the following concentrations: fusidic acid, 100 µg/ml; tetracycline, 5 µg/ml; erythromycin 10 µg/ml.

Filter matings

Filter matings were done on the basis of the protocol described by Perreten and coworkers [25]. In short, 1ml of a culture of the donor or recipient strain grown overnight in brain heart infusion (BHI) broth at 37°C was added to 5 ml of fresh BHI broth and further incubated for 4h. Equal volumes (1ml) of donor and recipient cultures were mixed and filtered through a sterile membrane filter with a pore size of 0.45 µm (MF-Millipore membrane filter HAQP 2500; Millipore, Bedford, Mass.) contained in a Swinnex filter holder (SX00 02500; Millipore). Subsequently, filters were gently rinsed once with 2 ml of a sterile peptone-physiological saline (PPS) solution (8.5g of NaCl per liter, 1g of neutralized bacteriological peptone (Oxoid) per liter). Filters were incubated on BHI agar for 24h at 37°C, and after mating cells were washed from the filter with 2ml of PPS. Finally, serial dilutions in PPS of the mixed suspension and of the donor and recipient strains were plated on BHI agar supplemented with 5 µg/ml of Tet or 10 µg/ml of Ery respectively and 100 µg/ml of fusidic acid. The Tet- susceptible JH2-2 recipient strain and the potential donor strains are resistant and susceptible, respectively, to the latter two antibiotics at the indicated concentrations. Following incubation at 37°C for 48 h, plates were checked for the absence (donor and recipient plates) or presence (mating mixture plate) of growth.

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Mating experiments in vanilla cream

To perform mating experiments in 50 ml portions of vanilla cream (Coop, Basel, Switzerland) were cooked following the manufacturer’s instructions and inoculated with 2 x 106 cells g-1 donor and recipient strains. These are enterococcal counts as they can be found in several raw milk products. After mixing, the inoculated vanilla cream portions were incubated at 37°C and 4°C for two days. Selection of transconjugants was achieved on Chromocult enterococci-agar (Merck) added with fusidic acid and Tet or Ery respectively. Antibiotics were used at the concentration reported above.

Sampling from food

At specific times, 250 µl of inoculated vanilla cream was spread onto the appropriate selective Chromocult enterococci-agar plates. Transconjugants were selected using the antibiotic concentration as described above.

Genotypic assessment of resistance genes, virulence factors and integrase

Total genomic DNA was prepared using a protocol based on the method of Pospiech et al. [26]. PCR assays were performed using pairs of previously reported primers [1,29,30,32] for the detection of resistance genes: erythromycin drug inactivation (ereA, ereB, mphA), methylation mechanism (ermA, ermB, ermC), and efflux systems (mrsA/mrsB, mefA/mefE); tetracycline resistance by ribosomal protection mechanism (tetM) and an efflux system (tetL). PCR assays were performed using pairs of previously reported primers [11] for the detection of the following genes: agg, esp, cpd, cob, and ccf. The occurrence of conjugative transposons of the Tn916-Tn1545 family was determined in all clinical isolates with primers Int-FW and Int-RV targeting the transposon integrase (int) gene [9,17]. For all detection assays, a common PCR core mix (total volume 20 µl) was used consorting of 1 × PCR buffer (Promega, Madison, WI), 200 µM concentrations of deoxynucleoside triphosphates (Promega, Madison, WI), 1 U of Taq DNA Polymerase (Promega, Madison, WI), 4 mM MgCl2, and 20 pmol of the corresponding primers (Thermo Electron GmbH, Ulm, Germany). A 25 ng portion of intact total DNA was used as PCR template.

Results and Discussion

60 Publication III transferred between Gram-negative and Gram-positive bacterial cells. This leads to an accumulation of resistance genes in a variety of bacteria important in the human and veterinary medicine and can cause serious problems in the treatment of such infections. A possible way of transferring the resistance genes is in the food chain, either during production or processing of a product or during storage. In this context, the gene transfer among the natural microflora of the product and the technologically needed starter strains can play a major role.

Table 1 Summary of PCR results for E. facaelis isolates from conjugation experiments in vanilla cream (all transconjugants contained the sex pheromones cpd, cob and ccf, not shown in table) Strain Species Genotype

Recipient JH2-2 (plasmid free) E. faecalis fus+ cpd+ cob+ ccf+

Donors APP003 E. faecalis tetM+ tetL+ int+ ereA+ ermB+ agg+ esp+ cpd+ cob+ ccf+ APP063 E. faecalis tetM+ int+ ermB+ agg+ esp+ cpd+ cob+ ccf+ APP091 E. faecalis tetM+ tetL+ int+ ermB+ mefA/E+ agg+ cpd+ cob+ ccf+ SCH092 E. faecalis tetM+ tetL+ int+ ermA+ ermB+ ermC+ msrA/B+ mefA/E+ agg+ cpd+ cob+ ccf+ SCH138 E. faecalis tetM+ tetL+ int+ ermB+ agg+ esp+ cpd+ ccf+ SCH212 E. faecalis tetM+ tetL+ int+ ereA+ ereB+ ermA+ ermB+ ermC+ cpd+ cob+ ccf+ Transconjugants JHA003T1 E. faecalis fus+ tetM+ tetL+ int+ ereA+ ermB+ agg+ esp+ JHA003T3 E. faecalis fus+ tetM+ tetL+ int+ ereA+ ermB+ agg+ esp+ JHA003E1 E. faecalis fus+ tetM+ tetL+ int+ ereA+ ermB+ agg+ esp+ JHA003E2 E. faecalis fus+ tetM+ tetL+ int+ ereA+ ermB+ agg+ esp+ JHA003E3 E. faecalis fus+ tetM+ tetL+ int+ ereA+ ermB+ agg+ esp+ JHA091T1 E. faecalis fus+ tetM+ tetL+ int+ ermB+ mefA/E+ agg+ JHA091E1 E. faecalis fus+ tetM+ tetL+ int+ ermB+ agg+ JHS092T1 E. faecalis fus+ tetM+ tetL+ int+ ermB+ mefA/E+ agg+ JHS092T2 E. faecalis fus+ tetL+ int+ ermB+ agg+ JHS212T1 E. faecalis fus+ tetM+ tetL+ int+ ereA+ ereB+ ermB+ ermC+ JHS212T2 E. faecalis fus+ tetM+ tetL+ int+ ereA+ ereB+ ermA+ ermB+ ermC+ JHS212E1 E. faecalis fus+ tetM+ tetL+ int+ ereB+ ermB+ JHS212E2 E. faecalis fus+ tetM+ ereA+ ermB+ ermC+ JHS212E3 E. faecalis fus+ tetM+ tetL+ int+ ereA+ ermB+

The role of the food chain as a possible source of antibiotic resistant enterococci has been proposed, and recently strains harboring glycopeptide resistance were detected in various foods in Europe [2,5,16,21,22]. In cheese, which generally contains relevant amounts of

61 Publication III viable enterococci, multiple antibiotic resistant strains have been detected, and a self transmissible transposon, the tetracycline resistance TnFO1, has been characterized [25].

This study assessed the frequency of transfer of mobile genetic elements coding for virulence determinants and antibiotic resistance factors, from food associated enterococci into the plasmid free recipient strain E. faecalis JH2-2. Six enterococcal food isolates were screened for their potential of transferring Tet and Ery resistances and virulence factors to a plasmid free recipient strain (E. faecalis JH2-2). In preliminary experiments the donor and recipient strains were further characterized. PCR assays for the detection of resistance genes in donor strains were done, resulting in all donor strains harboring the methylation mechanism ermB, a ribosomal protection mechanism (tetM) and an efflux system (tetL). The presence of a transposon of the Tn916-Tn1545 family could be confirmed in all donor strains (see Table 1). In order to assess the presence of aggregation substance (agg), cell wall- associated protein (esp) and sex pheromones (cpd, cob and ccf) PCR was done with previously described primers [11]. All donor strains and the recipient strain were positive for sex pheromones. E. faecalis SCH138 harbored only two pheromone genes (cpd, ccf) whereas all other strains harbored all three genes. With the exception of E. faecalis SCH212 all donor strains were positive for agg, and the recipient strain was negative. Esp, a cell wall- associated protein involved in immune evasion could be detected in E. faecalis APP003, APP063 and SCH138, whereas he recipient strain was negative for this factor (see Table 1).

Table 2 Transfer frequencies of Ery and Tet resistances in filter mating experiments and in the model system vanilla cream of food isolates. Transfer frequency in filter Transfer frequency in vanilla mating (transconjugants/donors) cream (transconjugants/donors)

Donor strain Conjugation at 37°C (24h) Conjugation at 37°C (48h)

E. faecalis APP003 2.8 x 10-4 7.8 x 10-6 E. faecalis APP063 9.8 x 10-7 No conjugation E. faecalis APP091 1.7 x 10-5 4.0 x 10-6 E. faecalis SCH092 9.8 x 10-7 2.7 x 10-6 E. faecalis SCH138 8.0 x 10-7 No conjugation E. faecalis SCH212 2.4 x 10-6 6.0 x 10-7

Filter mating experiments demonstrated that the Tet and Ery resistance could be transferred only from four of the six isolates tested. The resistance transfer was only the phenotypically analyzed. Transferability of virulence determinants was not tested in filter matings. The frequencies varied between 2.8 x 10-4 and 9.8 x 10-7 transconjugants/donors (see Table 2).

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Food model experiments were designed to assess in standardized conditions the gene transfer frequency in vanilla cream, which is very similar to several milk products such as soft cheese with respect to matrix and enterococcal counts. A consumable portion of vanilla cream (50 ml) was prepared and inoculated with donor and recipient strains resulting in enterococcal counts of 2 x 106 CFU/g, as detectable in raw milk cheese such as Swiss Appenzeller or Schabziger cheese. Two different conjugation temperatures (4°C and 37°C) were used. At 4°C no donor strain gained transconjugants after 48h of incubation. When the temperature of incubation was maintained at 37°C E. faecalis APP003 reached the highest level of transfer frequency which correlates with the frequencies detected in the filter mating experiments (see Table 2). The two E. faecalis strains APP063 and SCH138 showed the lowest transfer frequencies in the filter mating and in vanilla cream it was not possible to detect any transconjugants of these donor strains after 48h of conjugation at 37°C. In all strains showing transconjugants the transfer frequency in vanilla cream was always lower than the one detected in filter mating. These results suggests, that the conjugation frequency is dependent on the incubation temperature and time. The analysis of 14 transconjugants resulted in transfer of agg, esp and ermB, in all transconjugants possible. One donor strain was not able to transfer either tetM, tetL or int. The transfer of the Ery resistance by an efflux system could not be detected for msrA/B (see Table 1 and 3).

Table 3 Transferability of virulence factors (agg, esp), erythromycin drug inactivation (ere(A), ere(B)), methylation mechanism (erm(A), erm(B), erm(C)), and efflux systems (mrsA/mrsB, mefA/mefE), tetracycline resistance by ribosomal protection mechanism (tet(M)) and an efflux system (tet(L)) and of conjugative transposons of the Tn916-Tn1545 family. Ratio of positive transconjugants and transconjugants coming from donor strains harboring the corresponding gene

Virulence factors EryR mechanisms TetR mechanisms Integrase

agg esp ermA ermB ermC ereA ereB msrA/B mefA/E tetL tetM int

9/9 5/5 1/7 14/14 3/7 9/10 3/5 0/2 2/4 13/14 13/14 13/14

The experiments performed demonstrated that further analysis on the dependence of incubation temperature and time during conjugation are needed. This is of importance in the view of particular products harboring high viable enterococcal counts. Conjugation conditions have to be adapted to longer storage times and specific temperatures. Furthermore, the results of Cocconcelli et al. [8] showed that gene transfer can occur during ripening of fermented cheese and sausages. These findings and those of the current study suggest

63 Publication III further experiments to complete the data which are needed to perform a risk assessment with regard to enterococci resistant to antimicrobial agents in foods.

Acknowledgments

This work was granted by the Swiss Federal Office of Public Health (SFOPH).

References

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9. Doherty, N., Trzcinski, K., Pickerill, P., Zawadzki, P. & Dowson, C.G. Genetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 44, 2979-2984 (2000).

10. Dunny, G.M. & Clewell, D.B. Transmissible toxin (hemolysin) plasmid in Streptococcus faecalis and its mobilization of a noninfectious drug resistance plasmid. J. Bacteriol. 124, 784-790 (1975).

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13. Franke, A.E. & Clewell, D.B. Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid. J. Bacteriol. 145, 494-502 (1981).

14. Franz, C.M., Holzapfel, W.H. & Stiles, M.E. Enterococci at the crossroads of food safety? Int. J. Food Microbiol. 47, 1-24 (1999).

15. Garcia, M.T. et al. Inhibition of Listeria monocytogenes by enterocin EJ97 produced by Enterococcus faecalis EJ97. Int. J. Food Microbiol. 90, 161-170 (2004).

16. Gelsomino, R. et al. Antibiotic resistance and virulence traits of enterococci isolated from Baylough, an Irish artisanal cheese. J. Food Prot. 67, 1948-1952 (2004).

17. Gevers, D., Danielsen, M., Huys, G. & Swings, J. Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage. Appl. Environ. Microbiol. 69, 1270-1275 (2003).

18. Giraffa, G. Enterococci from foods. FEMS Microbiol. Rev. 26, 163-171 (2002).

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21. Mannu, L. & Paba, A. Genetic diversity of lactococci and enterococci isolated from home-made Pecorino Sardo ewes' milk cheese. J. Appl. Microbiol. 92, 55-62 (2002).

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25. Perreten, V., Kolloeffel, B. & Teuber, M. Conjugal transfer of the Tn916-like transposon TnF01 from Enterococcus faecalis isolated from cheese to other gram- positive bacteria. Syst. Appl. Microbiol. 20, 27-38 (1997).

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67 Summary and Discussion

SUMMARY AND DISCUSSION

Enterococci are part of the natural gut microflora in mammals. They are also found in a variety of ready-to-eat foods, including milk products such as cheese. In many types of cheese, enterococci play a key role in the maturation and developing of final taste. For many years, enterococci have been considered as harmless to humans. However, they were recognized as important nosocomial pathogens causing urinary tract infections, endocarditis or bacteremia. Because of the increasing incidence of antibiotic resistant enterococci, the treatment of these infections has become increasingly difficult, especially in the case of vancomycin resistant strains. This situation led the Swiss Federal Office of Public Health in 1999 to launche an interdisciplinary study to critically analyze the topic of bacterial antibiotic resistance in human and veterinary medicine and in the field of food stuffs. At the same time, the Swiss National Science Foundation started with a national research program on resistance to antimicrobial agents. In this context, it was recorded, that foods may be a vector for the spread of antibiotic resistances. Of particular interest are enterococci because they naturally and frequently occur in various food products and because they easily transfer resistance genes between strains and even over the species boarder.

The starting point of this thesis was a study on antibiotic resistant enterococci in Swiss ready-to-eat foods such as cheeses types, salads and vegetables. This study showed that enterococcal strains with specific antibiotic resistance profiles could repeatedly be isolated. One of the major aims of this thesis was to determine the diversity of enterococci in two types of artisanal raw-milk cheese (Schabziger and Appenzeller) and to investigate whether particular strains with triple resistance against chloramphenicol (Chl), tetracycline (Tet) and erythromycin (Ery) persist in the production system. Out of forty-six cheese samples, a total of 312 Enterococcus strains were isolated over a five-months-period on selective agar plates containing either Chl, Tet or Ery. Enterococcus faecalis was the predominant species (80.7%), followed by Enterococcus faecium (5.1%) and Enterococcus durans (11.7%). According to the phenotypic resistance patterns, a selection of 150 strains was analysed with PCR for the presence of genes encoding resistance to Ery (ereA, ereB, mphA, ermA, ermB, ermC, mrsA/mrsB, mefA/mefE), and Tet (tetM, tetL). Since virulence factors have been linked to the pathogenicity of enterococci, the strain selection was also tested for the presence of the following virulence factors: Agg, GelE, Cyl, Esp, EfaAfs, EfaAfm, Cpd, Cob and Ccf. All tested strains contained at least two of the nine virulence genes taken into analysis. Pulsed-field gel electrophoresis (PFGE) patterns of the isolates showed a limited persistence of several strains over a period of one to two months in Schabziger and more

68 Summary and Discussion than two months in Appenzeller. Finally, the enterococcal flora in the two types of cheeses seems to be rather unrelated. Within 150 strains out of 25 different cheese samples (11 Appenzeller and 14 Schabziger), 41 PFGE-patterns could be identified and only one of these was found in enterococci from both types of cheese. However, these results as such do not allow conclusions with regard to safety of the cheese itself. Complete risk assessment would require further research on the potential source and the in vitro and in situ transferability of these resistance and virulence properties. The limited persistence shown in a specific cheese type has to be further analyzed to determine whether the insertion of antibiotic resistant enterococci in the food chain can be reduced.

We determined the prevalence of different species of clinical enterococcal isolates, their antibiotic susceptibility and virulence factors. The isolates were recovered from three different University hospitals in Switzerland. In order to understand the extent of similarity among enterococci from clinical sources and enterococcal strains from foods, we compared the antimicrobial resistance, virulence factors and pulsed field gel electrophoresis (PFGE) profiles of these strains. Enterococcus faecalis was the predominant species (75.4%), followed by Enterococcus faecium (11.4%) and Enterococcus gallinarum (2.6%). Phenotypic determination of antibiotic resistance resulted in 18% of 114 isolates showing a triple- resistance against chloramphenicol (Chl), tetracycline (Tet), erythromycin (Ery), and 16 isolates (14%) exhibiting a multi-resistance (minimally four resistances). Four (3.5%) isolates showed vancomycin (Van) resistance. All isolates were analysed with PCR for the presence of genes encoding resistance to Ery, Tet and Van. The strain selection was also tested for the presence of the following virulence factors: agg, gelE, cyl, esp, efaAfs, efaAfm, cpd, cob and ccf. All tested strains contained at least two of the nine virulence genes taken into analysis. Clustering of the PFGE-profiles showed two dominating groups of 9 (8%) respectively 7 (6%) strains with highly similar genotypes. Seventy isolates (61%) occurred as unique, patient-specific clones. Several PFGE-types were associated with shared features in their antibiotic resistance patterns, indicating clonal spread between and within wards. A comparison of the PFGE-types of the clinical isolates with food isolates from raw milk cheese resulted in genetically strongly related multiple-strain clusters consisting of genetic strongly related clinical and food isolates. To our knowledge, this is the first report of a comparison of PFGE-patterns of clinical and food isolates showing such strong relatedness between clinical and food isolates. To understand the spread of genetically related clusters of strains from different settings, further epidemiological investigations would be required.

69 Summary and Discussion

The frequent detection of resistance to antimicrobial agents among enterococci could be related to the efficient transfer mechanisms of resistance genes associated with these organisms. Enterococci are known to harbor transferable genetic elements, conjugative plasmids and transposons, which have an unusually broad range and can even be transferred between Gram-negative and Gram-positive bacterial cells. This study assessed the frequency of transfer of mobile genetic elements coding for virulence determinants and antibiotic resistance factors, from food associated enterococci into the plasmid free recipient strain E. faecalis JH2-2. With vanilla cream, a food model was designed which is, with respect to the homogenous matrix and Enterococcal counts, very similar to several milk products such as cheese. The transfer of two mechanisms of tetracycline resistance, several erythromycin resistance mechanisms and the aggregation substance (agg) and cell-wall associated adhesin (esp) as virulence factors could be shown in vanilla cream inoculated with donor and recipient strains at a level similar to those found in raw milk cheese. The frequencies varied between 2.8 x 10-4 and 9.8 x 10-7 transconjugants/donors. Furthermore, we analyzed the transposition of members of the Tn916-Tn1545 family. At refrigeration temperatures of 4°C, no transconjugants occurred after 48 h. This study showed that even in the absence of selective pressure, mobile genetic elements carrying antibiotic resistance and virulence determinants can be transferred at high frequency among E. faecalis strains in a food model system. The findings of this experiments should possibly be confirmed with animal models to examine whether transconjugants colonize the gut or not.

For risk assessments in the filed of bacteria resistant to antimicrobial agents in food stuffs, the present thesis contributes with concrete findings and it identifies various aspects where further research work is needed. If possible, the future outcome of such basic scientific work should be risk management measures to reduce the spread of antibiotic resistant bacteria with foods and the transfer of resistance genes in foods.

70 Acknowledgments

ACKNOWLEDGMENTS

I gratefully acknowledge Dr. A. Baumgartner for giving me the opportunity to do my PhD in the Swiss Federal Office of Public Health. Specially, I thank him for his help and great support during my thesis. A special thank goes to Prof. Dr. Thomas Seebeck for his support and the constructive comments during our meetings. I would also like to express my gratitude to Dr. Peter Rohner for his support and helpful advice during the analysis of the clinical samples.

A special and sincere thank to all the present and former members of our group. Without their help this thesis would not have been possible. In particular I am thankful to Marianne Küffer for introducing me to the subject of PFGE analysis, to Dr. R. Fretz for sharing with me his experience in publishing and direct contribution to this work.

And finally, I wish to thank my parents, my sister and Jürg for their motivation, support, appreciation and love.

71 Curriculum Vitae

CURRICULUM VITAE

PERSONAL DATA

Name: Stefanie P. Templer Birth date: 23th March 1977 Adress: Blümlisalpstrasse 18, CH-3076 Worb EDUCATION AND QUALIFICATIONS From 2002 Doctoral dissertation in microbiology/molecular biology (PhD) at the Institute of Cell Biology in Bern and at the Swiss Federal Office of Public Health in Bern, Switzerland 1997 – 2002 Studies in Biology at the University of Bern, Switzerland; MSc thesis (“The phosphodiesterase 3 (TbPDE3) and the cyclic nucleotide binding protein 1J11 of Trypanosoma brucei”) at the Institute of Cell Biology in Bern. 1993 – 1997 Gymnasium in Bern, Switzerland; economy/law Matura diploma (A-levels)

PROFESSIONAL EXPERIENCE From 2000 Biosafety Officer and scientific consultant for the microbiology laboratory at the Haco AG, Gümligen, Switzerland 2005 Technical instructor for non-scientific personnel of the production plant Haco Asia Pacific, Kualalumpur, Malaysia in laboratory practice 2004 Consultant in generating the outline of the microbiology laboratory at the new production plant Haco Asia Pacific, Kualalumpur, Malaysia 2002 March - August 2002: six months traineeship in marine microbiology at the Max-Planck- Institute in Bremen, Germany

ACHIEVEMENTS Conference Contributions 2005 Templer S., Rohner P., Seebeck T., Baumgartner A. (2005) “Enterococcal clinical isolates: antibiotic resistance patterns and genotypic characterization by PFGE.” Proceedings of the 2nd International ASM-FEMS Conference on Enterococci. Helsingor (Denmark), 28th-31st August 2005 2006 Templer S., Seebeck T., Baumgartner A. (2006) “Enterococci from Appenzeller and Schabziger raw milk cheese: antibiotic resistance, virulence factors and persistence of particular strains in the products.” Proceedings of the 65th annual assembly of SSM, Lausanne (Switzerland), 7th-8th March 2006

ADDITIONAL SKILLS Languages German Native language English Cambridge Certificate in Advanced English (CAE) B-level; Cambridge Certificate in Proficiency English (CPE) in preparation French Good working knowledge Italian Basic knowledge

Computer Skills MS Office Good working knowledge Bionumerics (Applied Maths) Good working knowledge (integral databasing and analysis of biodata)