Human Listeriosis – Grouping of Human Listeria Monocytogenes Isolates with Pfge and Asci Restriction Enzyme

Department of Food Hygiene and Environmental Health Faculty of Veterinary Medicine University of Helsinki, Finland

HUMAN LISTERIOSIS – GROUPING OF HUMAN LISTERIA MONOCYTOGENES ISOLATES WITH PFGE AND ASCI RESTRICTION ENZYME

GLORIA LOPEZ VALLADARES

ACADEMIC DISSERTATION To be presented, with permission of the Faculty of Veterinary Medicine, University of Helsinki, for public examination in Auditorium B4, room 107, ”Metsätieteiden talo”, Viikki campus, Helsinki, on May 24, 2019 at 12 noon.

Helsinki 2019

Supervising Professor Miia Lindström Professor Department of Food Hygiene and Environmental Health Faculty of Veterinary Medicine University of Helsinki Helsinki, Finland

Supervisors Professor Wilhelm Tham School of Hospitality, Culinary Arts and Meal Science University of Örebro Örebro, Sweden

Professor Marie-Louise Danielsson-Tham School of Hospitality, Culinary Arts and Meal Science University of Örebro Örebro, Sweden

Professor Hannu Korkeala Department of Food Hygiene and Environmental Health Faculty of Veterinary Medicine University of Helsinki Helsinki, Finland

Reviewed by Professor Aivars Bērzinš Faculty of Veterinary Medicine Latvian University of Agriculture Jelgava, Latvia

Professor Kathie Grant Public Health England London, UK

Opponent Professor Carl Påhlson Department of Medical Sciences, Infectious Medicine Akademiska sjukhuset Uppsala University Uppsala, Sweden

ISBN 978-951-51-5090-5 (paperback) ISBN 978-951-51-5091-2 (PDF) Helsinki University Print Helsinki 2019

For his invisible qualities are clearly seen from the world’s creation onward, because they are perceived by the things made, even his eternal power and Godship, so that they are inexcusable.

(to the Romans 1:20)

To my family

ABSTRACT

Isolates of Listeria monocytogenes (N=932) collected from human cases of invasive listeriosis in Sweden between 1958 and 2010 were serotyped and characterised with pulsed-field gel electrophoresis (PFGE) and AscI restriction enzyme. The genotype diversity of L. monocytogenes isolates was investigated and related to genotypic results from epidemiological information on human infection, in order to detect possible clustering of L. monocytogenes genotypes over time, season, location, age, or gender (Paper I). From 1972 to 1995, serovar 4b was the predominant serovar; however, in 1996, serovar 1/2a became the major serovar among human listeriosis cases in Sweden. Based on the number and distribution of all bands in the profile, 63 PFGE types belonging to serovars 1/2b, 3b and 4b and 119 PFGE types belonging to serovars 1/2a and 1/2c were identified (Paper I). The PFGE types were further assembled into PFGE groups, based on the number and distribution of small bands below 145.5 kb (Papers II and III). As the genomic region of small bands is genetically more conservative than in large bands, the distribution of small bands establishes relatedness of strains and defines genetic markers for both lineages. Cold-smoked salmon (Salmo salar) and gravad salmon packed under modified atmosphere or vacuum from three manufacturers were purchased in Sweden and Germany in 2005 and the occurrence and levels of L. monocytogenes were analysed (Paper IV): 56 products were analysed and eleven harboured L. monocytogenes. From the positive samples, 56 isolates were analysed with AscI, and 11 isolates were further analysed with ApaI: five AscI PFGE types were identified, four belonging to serovar 1/2a and one to 4b. Forty-three (n=43: 76.8%) isolates shared serovar 1/2a and 13 (23.2%) shared serovar 4b and all AscI types were identified among human clinical strains in Sweden. Moreover, three gravad salmon samples harboured two PFGE types each from different lineages, serovar 1/2a and serovar 4b. Although, in most of the products, the level of L. monocytogenes was less than 100 cfu/g, the highest level was 1500 cfu/g. The occurrence of L. monocytogenes was 12.9% in gravad salmon, encountered in three manufacturers (A, B, C) and 28% in cold-smoked salmon only from manufacturer A. Although the level of L. monocytogenes in RTE fish products is generally low, these products, should be considered possible sources of listeriosis in Sweden. A patient may harbour more than one L. monocytogenes PFGE type that can be determined through PFGE and AscI restriction enzyme. However, to avoid misleading conclusions, several L. monocytogenes colonies should be isolated and characterised from different sites from the same patient or mother-baby pairs (Paper V).

ACKNOWLEDGEMENTS

The project and the research started at the section of Microbial Food Safety, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden, and continued at the School of Hospitality, Culinary Arts and Meal Science, Örebro University, Örebro, Sweden, and finished at the Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.

I wish to express sincere gratitude to everyone who has in different ways contributed and encouraged me to complete this project over the years, especially:

Prof. Miia Lindström, my supervising professor for critical reading of the thesis, for the corrections, comments and suggestions for improving the work, and for giving me the opportunity to complete this project. Without your support this work would possibly never have been completed;

Prof. Wilhelm Tham, supervisor, for allowing me to be part of your Listeria research group, for introducing me to the fascinating world of Listeria, for sharing your vast scientific knowledge in microbiology, molecular biology and food hygiene, for contribution to the writing of the scientific papers and the thesis, for inspiring me to start this project, for all the interesting discussions on listeriosis and other topics, for continuous support and encouragement both in good times and in the challenging times;

Prof. Marie-Louise Danielsson-Tham, supervisor, for continuing encouragement and support throughout the project, for all the interesting lectures on food hygiene, for the valuable suggestions and comments made on the scientific papers and the thesis, for all funny moments and generous help you have given me over the years. I am thankful to you and to Wilhelm for the hospitality, genuine friendship and for taking care of me as part of your family;

Prof. Hannu Korkeala, supervisor, for support and encouragement, for all help and assistance, for solid knowledge in bacteriology, particularly in Listeria monocytogenes, for the valuable comments on the thesis and effective collaboration;

Prof. Håkan Ringberg, Birgitta Andersson and Ingela Tjernberg, co-authors (Paper I) for collecting and sending L. monocytogenes isolates from human cases in Skåne;

Enevold Falsen, ex-curator at the Culture Collection University of Gothenburg, Sweden, for collecting and sending L. monocytogenes isolates from human cases in Gothenburg;

Prof. Birgitta Henriques-Normark and Christina Johansson, co-authors (Paper I) from the Public Health Agency of Sweden for providing isolates from domestic human cases;

Sofia Ivarsson, co-author (Paper I) and Margareta Löfdahl from the Public Health Agency of Sweden for all the epidemiological information regarding human listeriosis cases and L. monocytogenes isolates;

Inoka Peiris, co-author (Paper IV) for valuable contributions and the fun moments at the Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden;

Seved Helmersson, co-author (Papers I, IV, and V) for guiding me in laboratory work at the Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden;

Prof. Richard Goering, co-author (Papers II and III) from the Department of Medical Microbiology and Immunology, Creighton University Medical Center, School of Medicine, Omaha, California, USA, for solid knowledge in PFGE, for the valuable comments on the papers and excellent collaboration;

Susanne Thisted Lambertz, colleague, for excellent laboratory assistance in PCR, PFGE and BioNumerics programme for L. monocytogenes according to EFSA, for interesting and useful discussions at the National Food Administration (SLV), Uppsala, Sweden;

The staff and Hans Lindmark, head of the Division of Microbiology, Development and Research Department, the National Food Administration, Uppsala, Sweden, for support, friendship and enjoyable coffee breaks;

Ingela Hedenström, for guidance in practical molecular techniques, for the good advice concerning genotyping and other aspects of molecular biology, for excellent cooperation, for having a warm heart, for being so kind and helpful and for your great support at the Public Health Agency of Sweden;

Eva, Kristina, Annelie, Lena, Linda, Jonas, Görel, Cecilia, Britta, Tara, Juan Carlos and Prof. Lars Engstrand, head of the Department of Bacteriology at the Public Health Agency of Sweden for excellent assistance, for friendship and all our laughs and joyful moments;

Tobias Nygren and Stefan Wennström, heads of the School of Hospitality, Culinary Arts and Meal Science, for giving me the opportunity to continue the research work at Örebro University, even though the staff represented other professional groups, for unforgettable meal experiences, interesting meetings and events;

Hans Lundholm for all IT help, for the technical assistance, for interesting discussions, and friendship at the School of Hospitality, Culinary Arts and Meal Science;

Laila, Åsa, Annelie, Christina, Annika, Birgitta, Marie, Johan, Ulf, Lars, Inger, Ute, Bente, Jesper, Asgeir, Carl Jan, the staff of School of Hospitality, Culinary Arts and Meal Science, for assistance and friendship, for interesting conversations and discussions on varied matters during my stay in Grythyttan and for making me feel welcome and comfortable over these years;

Göran Ternebrandt and Carola Tedenbring for providing excellent library service and prompt technical assistance at the School of Hospitality, Culinary Arts and Meal Science;

Susan Pajuluoma for valuable lessons on scientific English and for linguistic improvements of all scientific papers and the thesis;

Anna-Lisa Wattrang for having an enormous warm heart, for being generous, for financial support and for taking care of Peruvian children in Lima, Peru, over the years;

The Stiftelsen Grythytte Stipendiefond, Sweden, for financial support;

The Research Foundation of Ivar and Elsa Sandberg, Uppsala, Sweden, for financial support;

The Foundation of Stadsveterinär Billström, Uppsala, Sweden, for financial support;

Anas Al-Makhzoomi, Master student, for promoting and encouraging me to write the Doctoral thesis, for unfailing enthusiasm and friendship at the Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden;

Esteban Rivera, colleague, for your recommendation to Prof. Wilhelm Tham, for valuable help and advice in my first job as a veterinarian at the research laboratory, for support and constructive discussion at the National Veterinary Institute (SVA), Uppsala, Sweden;

Prof. Tomas Hökfelt from Karolinska Institutet, Stockholm, Sweden, for support and encouragement, for your trust and for seeing much more in me than many others are able to see;

All my other wonderful relatives, friends and colleagues, for support and encouragement, for making life more enjoyable and much easier;

My parents for giving me the life that every child deserves and being such wonderful parents, for helping me to be a stronger and a better person, for raising me to believe in God Almighty my creator, who has throughout all my life always been my source of inspiration, wisdom, insight, knowledge and understanding. Let the praise, the glory, the thanksgiving, the honour, the power, and the strength be to God forever and ever. I love you and you will forever be in my mind and in my heart;

My sisters and their families and my brother with whom I have shared knowledge, frustrations, disappointments and laughs, for encouragement and support, for believing in my ideas and me as a person and for always being there for me. I will always love you.

CONTENTS

Abstract………………………………………………………………………………..4

Acknowledgements……………………………………………………………………5

List of papers……………………………………………………………………...... 10

Abbreviations……………………………………………………………………….. 11

1 Introduction………………………………………………………………..... 13

2 Review of the literature……………………………………………………... 15 2.1 History……………………………………………………………………………………. 15 2.2 Taxonomy………………………………………………………………………..……….. 15 2.3 Morphology………………………………………………………………………………. 16 2.4 Growth and biochemical properties…………………………………………………….....16 2.5 Habitat……………………………………………………………………………………. 17 2.6 Phenotypic typing methods………………………………………………………………..17 2.6.1 Conventional serotyping………………………………………………………………….. 18 2.6.2 Molecular serotyping……………………………………………………………………... 18 2.7 The genome of Listeria monocytogenes………………...………………………………... 19 2.7.1 Extrachromosomal DNA in L. monocytogenes genome……...………………………….. 20 2.7.2 Virulence factors…………………………………………………………………………..20 2.7.3 Phylogeny of L. monocytogenes…………...……………………………………………... 21 2.7.4 PFGE fragments………………………………………………………………………….. 24 2.8 Listeriosis in humans……………………………………………………………………... 24 2.8.1 Human listeriosis in Europe…………………………………………………………….... 26 2.8.2 Human listeriosis in Sweden……………………………………………………………... 33 2.9 Contaminated food as source of listeriosis……………………………………………….. 36 2.9.1 Outbreaks of listeriosis and presence of L. monocytogenes in food……………………... 37 2.9.2 Contamination in cheeses…………………………………………….…………………... 41 2.9.3 Contamination in fish……………………………………………………………………...46

3 Aims of the thesis…………………………………………………………… 54

4 Materials and methods………………………………………………………. 55

4.1 Bacterial isolates…………………………………………………………………………. 55 4.1.1 Human isolates………………………………………………………………………….... 55 4.1.2 Food isolates……………………………………………………………………………… 55 4.2 Isolation and detection methods for L. monocytogenes…………………………………...55 4.3 Conventional serotyping of L. monocytogenes……………………………………………56

4.4 Molecular typing by pulsed-field gel electrophoresis (PFGE) of L. monocytogenes…...... 56 4.4.1 Preparation of gel plugs…………………………………………………………………... 56 4.4.2 Restriction digestion of DNA…………………………………………………………….. 57 4.4.3 Electrophoresis………………………………………………………………………….... 57 4.4.4 Analysis of PFGE profiles………………………………………………………………. .. 58 4.5 In silico analyses……………………………………………………………………...... 58

5 Results and discussion……………………………………………………... .. 59

5.1 Serovars of L. monocytogenes…………………………………………………………... .. 59 5.1.1 Human L. monocytogenes isolates from 1958–1971 in Sweden………………………... .. 59 5.1.2 Human L. monocytogenes isolates from 1972–1995 in Sweden………………………... .. 59 5.1.3 Human L. monocytogenes isolates from 1996–2010 in Sweden……………………...... 59 5.1.4 The shift from serovar 4b to serovar 1/2a in human listeriosis…………………………. .. 60 5.2 Seasonal variation……………………………………………………………………….. .. 62 5.3 Geographical variation………………………………………………………………….....63 5.4 Age and gender………………………………………………………………………….. .. 65 5.5 Pulsed-field gel electrophoresis (PFGE) types and PFGE groups………………………...66 5.6 Relationship between PFGE group, PFGE type and serovar……………………………...75 5.7 PFGE and L. monocytogenes genetic variants………………………………………….....76

6 Conclusions………………………………………………………………..... 80

7 References…………………………………………………………………... 82

LIST OF PAPERS

The thesis is based on the following papers, which will be referred to in the text by Roman numerals:

I. Lopez-Valladares, G., Tham, W., Parihar, V.S., Helmersson, S., Andersson, B., Ivarsson, S., Johansson, C., Ringberg, H., Tjernberg, I., Henriques-Normark, B., Danielsson-Tham, M.-L. (2014). Human isolates of Listeria monocytogenes in Sweden during half a century (1958-2010). Epidemiology and Infection 20, 1–10, PMID: 24480252 II. Lopez-Valladares, G., Danielsson-Tham, M.-L., Goering, R., Tham, W. (2015). Division of human Listeria monocytogenes Pulsed-Field Gel Electrophoresis (PFGE) types belonging to lineage I (serovar 4b, 1/2b and 3b) into PFGE groups. Foodborne Pathogens and Disease 12, 447– 453 III. Lopez-Valladares, G., Danielsson-Tham, M.-L., Goering, R., Tham, W. (2017). Lineage II (serovar 1/2a and 1/2c) human Listeria monocytogenes Pulsed-Field Gel Electrophoresis types divided into PFGE groups using the band patterns below 145.5 kb. Foodborne Pathogens and Disease 14, 8–16 IV. Peiris, P. I., Lopez-Valladares, G., Parihar, V.S., Helmersson, S., Barbuddhe, A., Tham, W., Danielsson-Tham, M.-L. (2009). Gravad (Gravlax) and cold-smoked salmon, still a potential source of listeriosis. Journal of Foodservice, 20, 15–20. V. Tham, W., Lopez-Valladares, G., Helmersson, S., Österlund, A., Danielsson-Tham, M-L. (2007). More than one variant of Listeria monocytogenes isolated from each of two human cases of invasive listeriosis. Epidemiology and Infection 135, 854–856.

ABBREVIATIONS

BHI brain heart infusion bp base pair BSA bovine serum albumin Cas CRISPR associated CC clonal complex cfu colony forming units CNS central nervous system CRISPR clustered regularly interspaced short palindromic repeats CSF cerebrospinal fluid DNA deoxyribonucleic acid EC epidemic clone ECDC European Centre for Disease Prevention and control EDTA ethylenediaminetetraacetic acid EFSA European Food Safety Authority ESP N-lauroylsarcosin, EDTA, pronase ET electrophoretic type ETEC enterotoxigenic Escherichia coli EU European Union EURL European Union reference laboratory G-C guanine and cytosine ISOPOL International Symposium on Problems of Listeriosis kb kilobase L. Listeria LPSN list of prokaryotic names with standing in nomenclature MEE multilocus enzyme electrophoresis MLGT multilocus genotyping MLST multilocus sequence typing MLVA multiple-locus variable-number tandem-repeat analysis MvLST multi-virulence-locus sequence typing NaCl sodium chloride NMKL Nordic committee on food analysis NRLs National Reference Laboratories PALCAM polymyxin acriflavin lithium chloride ceftazidime aesculin mannitol agar PCR polymerase chain reaction PEFA 4-(2-Aminoethyl)-benzenesulfonyl fluoride, hydrochloride PFGE pulsed-field gel electrophoresis PI-PLC phosphoinositide phospholipase C RFLP restriction fragment length polymorphism RTE ready-to-eat SNP single nucleotide polymorphism SOP standard operating procedures ST sequence typing SLV the National Food Administration SVA National Veterinary Institute

TESSy the European Surveillance System TBE Trisbas, Boric acid, EDTA buffer TE Tris-HCl, EDTA buffer TN Tris-HCl, NaCl VTEC verotoxin-producing Escherichia coli WGS whole genome sequencing

1 INTRODUCTION

Listeriosis is commonly a zoonotic food-borne disease mostly transmitted through ready-to-eat (RTE) foods contaminated with Listeria monocytogenes, such as gravad/smoked fishes, soft cheeses and deli meat products (Novelli et al., 2017). The new generation of refrigerated RTE foods, that pose most concern for listeriosis, are products normally eaten without further bactericidal treatment, with no inhibitory organic acids or lack of high counts of competing microorganisms, and with extended shelf life (Luber et al., 2011). The disease listeriosis can lead to clinical manifestations, such as gastroenteritis, septicaemia, encephalitis, meningitis, and abortion (McLauchlin et al., 2004). Listeriosis is one of the food-borne infections with a high fatality rate: in 2015, fatality was 12.3% of 2200 cases in EU (EFSA, 2016). L. monocytogenes is psychrotrophic and can grow without oxygen; therefore, food products/dishes stored in vacuum and in modified atmospheres with extended shelf life provide the opportunity for L. monocytogenes to multiply to large numbers towards the end of the shelf life without having to compete with most other food-borne microorganisms (Lado and Yousef, 2007). The prevalence of L. monocytogenes in RTE foods is generally low, and seldom exceeds the EU safety limit (< 100 cfu/g) on the day of production. At retail levels, non-compliance has been highest in fishery products and soft/semi-soft cheeses (EFSA, 2016).

In 2015, 2206 cases of invasive listeriosis were reported from 28 member states in the EU, and the incidence of listeriosis in 2014 and in 2015 was 4.6 cases per million habitants (EFSA, 2016). Elderly, pregnant women, neonates and immuno-compromised individuals are particularly susceptible to L. monocytogenes, although, the number of cases among the elderly population (> 64 years) increased from 56% in 2008 to 64% in 2013 (EFSA, 2016).

According to the World Health Organisation, food hygiene comprises the conditions and measures necessary to ensure the safety of food from production to consumption. In times of decreasing budgets, there is little room for preventive food hygiene. Inadequate cleaning procedures, lack of interest or knowledge, and complex equipment also increase the risk of foodborne infections. In addition, personal hygiene could be improved, such as hand cleaning, among food workers (Lindqvist et al., 2000; Çakıroğlu & Uçar, 2008). Changes in lifestyle, social attitudes, and eating habits increase the opportunity for transmission of pathogenic microorganisms through contaminated foods (de Oliveira et al., 2010). Since the consumption of contaminated food was identified as the main vehicle for Listeria infection (causing large outbreaks in Europe and USA), both the disease and L. monocytogenes have become a concern for food-processing manufacturers and public health authorities globally, and a health hazard and economic problem for the food industry (Giovannacci et al., 1999; Stasiewicz et al., 2015).

Molecular typing used to classify and compare food borne bacterial pathogens and with epidemiological investigations enable effective control and preventative measures to be implemented. Molecular typing methods are used in human listeriosis surveillance, for tracing the source of outbreaks, and for assessing the genetic diversity (generated by mutations, recombination or gene transfer) and relationships among L. monocytogenes isolates from one source or between various sources. Pulsed-field gel electrophoresis (PFGE) with restriction enzymes AscI and ApaI has been the gold standard molecular method for characterising L. monocytogenes since the middle of 1990s (Brosch et al., 1996; Kérouanton et al., 1998;

Cantinelli et al., 2013; Michelon et al., 2015; Kramarenko, et al., 2016; Camargo, et al., 2016). Various typing methods that analyse nucleotides within specific genes have been compared with PFGE including multiple-locus variable-number tandem-repeat analysis (MLVA), multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MvLST). Although, PFGE typing is labour-intensive, it probes the entire genome and is reproducible, whereas, MLST only analyses seven housekeeping genes. MvLST only analyses six to eight virulence genes, and MLVA analyses multiple tandem repeat sequences (Miya et al., 2008; Cantinelli et al., 2013; Lunestad et al., 2013). In a comparison of results from PFGE, MLST and MvLST among L. monocytogenes clonal groups, Cantinelli et al. (2013) observe MvLST is no more discriminative than MLST, and PFGE has a higher discriminatory power than MLST.

PFGE characterisation of L. monocytogenes isolates from human patients and foods has contributed to the identification of human listeriosis outbreaks in Sweden since 1994 (Ericsson et al., 1997; Danielsson-Tham et al., 2004; Thisted Lambertz et al., 2013). In several epidemiological investigations, a large number of PFGE types with identical DNA restriction fragments profiles have been identified among humans, animals, foods, food-processing environments, and other sources. Despite these diversifications, researchers conclude there are few prevalent L. monocytogenes clonal groups distributed worldwide (den Bakker et al., 2010; Chenal-Francisque et al., 2011; Cantinelli et al., 2013; Haase et al., 2014; Chen et al., 2016a).

Various PFGE types have been involved in large national and multinational outbreaks and these types, called epidemic clones (EC), have been defined based on MEE, ribotyping and PFGE, and later by MvLST. Some widespread epidemic clones such as ECI and ECII, have been included in major groups through MLST and are called Clonal Complex (CC), e.g., ECI was included in CC1 and ECII in CC6 (Cantinelli et al., 2013). Most CC of L. monocytogenes have persisted over decades and only a small number of new CC have been identified in recent years (Haase et al., 2014).

However, whole genome sequencing (WGS) analysis of the virulence determinants and genetic diversity (prophage, plasmids, SNP and insertion/delition mutations) in the bacterial genome provides a greater level of discrimination for investigating L. monocytogenes isolates to establish epidemiologic links during surveillance, outbreak investigations, and for source tracking in Sweden and within the EU and other countries (Gilmour et al., 2010; Jackson et al., 2016; Kwong et al., 2016). Predictions of conventional typing results (MLST, MvLST, PFGE, serotyping) are even possible in silico from WGS data (in silico typing) (Bikandi et al., 2004; Chen et al., 2016a; Kwong et al., 2016).

The current thesis compiles information on listeriosis and characteristics (phenotypical and molecular) of L. monocytogenes isolates from humans and foods in Sweden and in several countries, especially within the EU. Furthermore, as all AscI PFGE types identified during the study period could be further assembled into PFGE groups based on small restriction fragments below 145.5 kb, a new procedure for improving the identification of a L. monocytogenes isolate is proposed.

2 REVIEW OF THE LITERATURE

2.1 History

Gustav Hülphers, a veterinarian at the Veterinary Institute of Stockholm, Sweden, first identified the bacterium Listeria monocytogenes in laboratory rabbits in 1910. In 2004, the original Swedish paper was translated into English and was published in the proceedings of “ISOPOL XV, International Symposium on Problems of Listeriosis”, Uppsala, Sweden. Hülphers described carefully the morphology, the cultural characteristics, the biochemical properties, the growth limits of the bacterium and the pathological-anatomical changes in rabbits: he called the new bacterium Bacillus hepatis (Hülphers, 1911 and 2004; Nyfeldt, 1940; Gray & Killinger, 1966). In 1924, researchers from United Kingdom observed a similar bacterium, isolated from laboratory rabbits and guinea pigs at the University of Cambrige and called it Bacterium monocytogenes (Murray et al., 1926). A researcher from South Africa (Pirie, 1927), found this pathogen in gerbils (Iatera lobenquiae), known as desert or African jumping mouse, and called it Listerella hepatolytica in honour of Lord Lister; thereafter, Pirie called the organism Listerella monocytogenes. In 1929, the first human case of listeriosis was reported by Nyfeldt in Denmark (Nyfeldt, 1929), where the microorganism was obtained from blood. However, in 1919, an unknown bacterium of diphtheroid type was isolated from cerebrospinal fluid (CSF) of a soldier in France and after 20 years, the organism was identified as Listeria monocytogenes (McLauchlin et al., 1986). In Australia, cases of meningitis due to diphtheroids, possibly Listeria, were reported by Atkinson in 1917. In 1935, a case of meningo-encephalitis was reported in an adult, and in 1936, four cases of listeriosis in three newborn infants and an adult in USA were reported. Milk was suggested as a possible source of infection due to a similar organism being found in cows with encephalitis in 1934–1935 (Burn, 1935 and 1936). A similar organism was recovered from the brain of sheep by Gill in 1931 (Nyfeldt, 1940). Pirie changed the name of the bacterium to Listeria monocytogenes in 1940, as the name Listerella was used for other species (Pirie, 1940). One of the eminent researchers who contributed the most to the study of L. monocytogenes since the beginning of the 1950s was Heinz P. R. Seeliger from East Germany. Since 1955, he has published more than 500 papers (scientific papers, popular science papers and textbooks), mostly about Listeria (Miller et al., 1990). Seeliger considered the theory of food infectious disease of substantial importance, as Listeria could be proved to cause epidemics (Seeliger, 1955). Due to his intensive work, he observed the first listeriosis outbreak reported in the world (1949–1957 in Halle, Germany) and suspected unpasteurised milk, sour milk, whipped cream and cottage cheese were the sources of infection in the outbreak. For this reason, Seeliger speculated contaminated food could be the route of infection by L. monocytogenes in humans, i.e. the food-borne route (Seeliger, 1961).

2.2 Taxonomy

L. monocytogenes belongs to the phylum: Firmicutes, class: Bacilli, order: Bacillales, family: Listeriaceae, and the genus Listeria. The genus Listeria comprises seventeen species and six subspecies: L. monocytogenes, L. grayi subsp. grayi, L.grayi subsp. murrayi, L. innocua, L. seeligeri, L. welshimeri, L. ivanovii subsp. ivanovii, L. ivanovii subsp. londoniensis, L. marthii, L. rocourtiae, L. weihenstephanensis, L. fleischmannii subsp. fleischmannii, L. fleischmannii

15 subsp. coloradonensis, L. riparia, L. grandensis, L. floridensis, L. cornellensis, L. aquatica, L. newyorkensis, L. booriae (Weller et al., 2015; Orsi & Wiedmann, 2016; LPSN, 2017). Six species belonging to Listeria sensu strictu (L. monocytogenes, L. marthii, L. innocua, L. welshimeri, L. ivanovii, L. seeligeri) are ubiquitous and commonly found in diverse environments, according to phenotypic characteristics such as motility and growth at 4°C. The remaining 11 species belong to Listeria sensu lato. There are proposals (Orsi & Wiedmann, 2016) for dividing and reclassifying the genus Listeria (17 species) into four genera: genus Listeria (including the six Listeria sensu strictu), Murraya (L. grayi), Mesolisteria (L. fleischmannii, L. floridensis and L. aquatica), and Paenilisteria (the remaining 7 species). The last three genera are not pathogenic; among those, only L. grayi is motile and positive for the Voges-Proskauer test, and only L. floridensis is unable to reduce nitrate (Weller et al., 2015; Orsi & Wiedmann, 2016).

2.3 Morphology

Morphologically, Listeria spp. are rod-shaped with rounded ends, vary in size between 0.4 and 0.5 µm in diameter and 1 and 2 µm in length, and do not form spores or capsules. Characteristically, young cultures are Gram-positive but may become Gram-negative as they mature. Listeria colonies grow on nutrient agar after 24 hours at 20–25°C and are round, 0.5–1.5 mm in diameter, translucent, low convex with a glistening and smooth surface (S-forms). After 3–7 days of incubation, the colonies are 3–5 mm in diameter with rough surface (R-forms). All species belonging to Listeria sensu strictu and L. grayi are motile due to peritrichous flagella especially when grown in liquid culture at 10° – 25°C (Seeliger & Jones; 1986, Farber & Peterkin, 1991; Weller et al., 2015).

2.4 Growth and biochemical properties

The growth range for Listeria bacteria extends from pH 4.4 to pH 9.5 at temperatures between 0.4°C and 52°C, in food with up to 14% NaCl and at a water activity (aw) as low as 0.92. The metabolism is aerobic and facultative anaerobic (Seeliger & Jones, 1986; Farber & Peterkin, 1991; Swaminathan et al., 2007; Velge & Roche, 2010; Carpentier et al., 2011; Iannetti et al., 2016; Wemmenhove et al., 2016). Only L. monocytogenes, L. ivanovii, L. seeligeri and a few L. innocua strains are positive for haemolysis (Orsi & Wiedmann, 2016). All species are able to produce the antioxidant enzyme catalase, hydrolyse esculin and produce acid from N- acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, D-fructose, and D-mannose. However, they are not able to reduce nitrite and produce acid from D-arabinose, D-adonitol, methyl β-D-xylopyranoside, raffinose, glycogen, L-fucose, potassium gluconate, potassium 2- ketogluconate and potassium 5-ketogluconate (Weller et al., 2015). The biochemical characteristics of some of the Listeria species are presented in Table 1.

Table 1. Biochemical properties of some Listeria species (Seeliger & Jones, 1986; Leclercq et al., 2010; Weller et al., 2015; Orsi & Wiedmann, 2016) 49.3074.,6 :<9:0<>40= $! " ## $ "! " ! J3,0796A=4= "4><,>0<0/?.>498 !9>464>A 96 <,-4>96 +A69=0 %3,789=0 I!0>3A6 26?.9=4/0 %4-9=0 6?.9=0 :39=:3,>0 ',2,>9=0 )920=$<9=5,?0< PI-PLC, phosphoinositide phospholipase C. - = negative reaction. + = positive reaction

2.5 Habitat

Listeria spp. are widely distributed in the environment and can be isolated from a variety of sources such as soil, surface, rivers, canal waters, sewage, decaying vegetation (Beuchat, 1996) and from the surfaces of equipment, floors and wall of the food processing plants (Franco-Abuín, et al., 1996; Unnerstad et al., 1996). Listeria spp. have also been isolated from faeces of domestic and wild animals, birds, tics, larvae, ensilage, processed foods (ready-to-eat food, food requiring cooking or reheating) and different kinds of raw foods such as vegetables, fruits, milk, cheese, meat, fish and crustaceans (Gray, 1963; Loncarevic et al., 1996a; Loncarevic et al. 1996b).

2.6 Phenotypic typing methods

Serotyping is the standard subtyping method for analysing phenotypic characteristics of Listeria and is widely used in epidemiological surveillance of human and food isolates. Furthermore, the method has the advantage that results can be compared between different laboratories. Although conventional serotyping by agglutination is traditionallly used, molecular serotyping based on specific genes has been developed (Seeliger & Höhne, 1979; Bannerman, 1995; Borucki & Call, 2003; Doumith et al., 2004; Nightingale et al., 2007; Kérouanton et al., 2009).

2.6.1 Conventional serotyping

Serological analysis of somatic and flagellar antigens is the common technique for characterisation of different genus Listeria isolates. There are 15 somatic (I–XV) and 5 flagellar antigenic factors (A–E). The species L. monocytogenes is divided based on 13 somatic antigenic factors (O-factor) into four serogroups (1/2, 3, 4, 7). Serogroups 1/2 and 3 are further divided into 6 serovars (1/2a, 1/2b, 1/2c, 3a, 3b, 3c), based on four flagellar antigenic factors (H-factor). Although, the remaining serogroups 4 and 7 share the same flagellar factors (ABC), the somatic antigenic factors further divide them in serovars: 4a, 4ab, 4b, 4c, 4d, 4e and 7. Thus, the species L. monocytogenes are divided into 13 serovars (Table 2) (Seeliger & Höhne, 1979).

2.6.2 Molecular serotyping

Polymerase chain reaction (PCR) serotyping is a molecular method based on the amplification of specific regions in the bacterial genome. Two PCR assays with seven different marker genes are used. These PCR assays are based on the detection of serogroup specific regions leading to five molecular serogroups IIa, IIb, IIc, IVa and IVb. The first PCR assay detects the presence of six genes: prfA (specific for L. monocytogenes), prs (specific for the genus Listeria), orf 2819 (specific for molecular serogroup IIb and IVb), orf 2110 (specific for molecular serogroup IVb), lmo 0737 and lmo 1118 (specific for molecular serogroups IIa and IIc). The second PCR detects the flaA gene specific for molecular serogroups IIa and IVa (Table 2). However, the combination of sigma factor sigB gene, which is involved in the stress response regulation of L. monocytogenes (Severino et al., 2007), with multiplex PCR increases discrimination of atypical serovar 4b. Unfortunately, PCR serotyping is unable to distinguish all serovars, as with conventional serotyping. However, PCR serotyping distinguishes the most common serovars involved in human listeriosis (Borucki & Call, 2003; Doumith et al., 2004; Nightingale et al., 2007; Kérouanton et al., 2009).

Table 2. Comparision of conventional and molecular serotyping !960.?6,<=0<92<9?: , . - ), )- &0<9@,< ,, .. -- ,. -,-/0 $% $% &0<9@,< , . - , - #1,.>9< ))+ )) 1,.>9< &0<9@,< , . - . ,- #1,.>9< ) )++ )++ )) )))++ 1,.>9< &0<9@,< 8/ 8/ 8/ / #1,.>9< 8/ 8/ ++ 8/ ))) 1,.>9< 8/ 8/ 8/ &0<9@,< 8/ 8/ 8/ 8/ 0 #1,.>9< 8/ 8/ 8/ 8/ )))+ 1,.>9< 8/ 8/ 8/ 8/ - = gene is absent. nd= no data

2.7 The genome of Listeria monocytogenes

The genome of L. monocytogenes is a circular chromosome with a size varying between 2.7 and 3.0 Mb (Hain et al., 2007; Bécavin et al., 2014). The nitrogenous bases of a DNA molecule are bound by hydrogen bonds and are either Guanine or Cytosine (G-C) and Adenine or Thymine (A-T). The G-C pair is bound by three bonds, whereas, A-T pairs are connected by two bonds. The G-C base pairs are more stable under higher temperatures than A-T base pairs, although the G-C content does not play a role in the stability of DNA. The percentage of these base pairs may refer to whole genome or a specific fragment of DNA or RNA, i.e. a fragment of the genetic material that is part of a single gene, gene clusters, or even a non-coding region. The genomic G- C content of L. monocytogenes is low, between 36–39% (Brosch et al., 1991; Yakovchuk et al., 2006; Hain et al., 2007; Zheng & Wu, 2010; Bécavin et al., 2014). In Listeria PFGE, the restriction enzymes with recognition sequences containing G and C nucleotides are used to avoid multiple bands in bacterial PFGE profiles. Among the different L. monocytogenes serovars, the genomes are highly syntenic: display a similar size, G-C content, percentage of protein coding DNA and average length of protein coding genes (Kuenne et al., 2013), where surface proteins display the highest number of single nucleotide polymorphisms (SNP) (Hain et al., 2012).

The species L. monocytogenes have a highly conserved but open pan-genome permiting limited integration of foreign chromosomal DNA (called mobile genetic elements), such as bacteriophages, plasmids, transposons, genomic islands and insertion sequences, which contribute to genome diversity through mutation, recombination and duplication (Nelson et al., 2004; den Bakker et al., 2010; Kuenne et al., 2013). L. monocytogenes genomes contain some accessory genes located in a highly variable chromosomal region or hyper variable hotspots (Kuenne et al., 2013).

2.7.1 Extrachromosomal DNA in L. monocytogenes genome

The presence of extrachromosomal DNA is common in L. monocytogenes genome. The Listeria genomes harbour at least one prophage or part of bacteriophage genomes. The species L. monocytogenes has a cryptic prophage region in a single locus called monocin lma locus, but the gene content in the lma operon vary in different genomes of L. monocytogenes (Hain et al., 2012). Comparative analysis of genome sequences belonging to serovars 4b or 1/2a strains reveal 4b strains contain fewer prophages than 1/2a strains, and are inserted into different chromosomal loci adjacent to tRNA genes (Hain et al., 2006 and 2012; Kuenne et al., 2013). The plasmids of genus Listeria are related to plasmids in Bacillus, Enterococcus and Streptococcus and contain a large number of mobile genetic elements (Kuenne et al., 2010). Although, the L. monocytogenes genomes rarely harbour plasmids, several plasmid genes are frequently involved in heavy metal resistance (cadmium and arsenite resistance operons), benzalkonium chloride resistance, oxidative stress response and multidrug efflux (Lebrun et al., 1992; Hadorn et al., 1993; McLauchlin, 1996c; Harvey & Gilmour, 2001; Elhanafi et al., 2010; Kuenne et al., 2010). The presence of plasmids in L. monocytogenes serovar 4b isolates is lower than in serogroup 1/2 isolates (Lebrun et al., 1992; Peterkin et al., 1992). The L. monocytogenes genome contains CRISPR (clustered regularly interspaced short palindromic repeats) and protein coding genes Cas (CRISPR associated), which is an adaptive immune system to protect the bacteria against bacteriophages. Prophages and plasmid content in the bacteria can be associated with acquisition or loss events occurring during human infection, in processing plants, environment or during passage of isolates in a medium containing acriflavine, a plasmid curing agent (Margolles et al., 1998b; Orsi et al., 2008c; Chen et al., 2016b; Chen et al., 2017a). Loss or gain of prophages in the bacterial genome appears to occur promptly and frequently (Orsi et al., 2008a). The variation in genome size can be attributed to the complete or partial absence of prophages and plasmid in the genome. The diversification of prophages in the species L. monocytogenes signifies a fundamental mechanism for short-term genome development (Orsi et al., 2008c).

2.7.2 Virulence factors

L. monocytogenes is able to invade, survive and proliferate within phagocytes (macrophages) and in different tissues of several eukaryotic, non-phagocytic cells such as epithelial, endothelial, neurons and hepatocytes. Specific virulence factors provide this ability and are crucial for causing an infection in the host (Mackaness, 1962; Chakraborty et al., 2000; Vázquez-Boland et al., 2001a). The bacteria induce their own uptake into non-phagocytic cells. During the adhesion

20 and internalisation process of the bacterium into the cell, several genes such as inlA, inlB, inlC are involved and these are regulated by the transcriptional activator PrfA. The L. monocytogenes internalin genes (inlA, inlB and inlC) encode the surface proteins internalin A, B, C (InlA, InlB and InlC), which are associated with the attachment and invasion of the host cells. The gene for invasion-associated protein (iap) encodes the extracellular protein p60, a hydrolase essential for bacterial metabolism. The surface proteins Ami (an autolysin) and Lap contribute to the adhesion of L. monocytogenes to eukaryotic cells.

Six virulence factors (prfA, plcA, hly, mpI, actA and plcB) are involved in the intracellular parasitic life cycle of L. monocytogenes. In Listeria, these virulence genes, located in a central virulence gene cluster physically linked to a 9 kb chromosomal island, are organised into discrete genetic units known as pathogenicity islands (PAIs) or LIPI-1. The positive regulatory factor A (prfA) codes for the PrfA protein, a thermo-regulated transcriptional activator that activates the expression of all six genes in the cluster. In Listeria, virulence genes have maximal expression at 37°C (body temperature) but are weak below 30°C. Two different phospholipase C are involved in L. monocytogenes invasion and spread to host. The first bacterial gene phospholipase C (plcA) encodes the enzyme phosphatidylinositol-specific phospholipase C (PlcA), also known as PI- PLC, and participates in the rupture of the phagocytic vacuole, the primary phagosome of cells. The haemolysin gene (hly) encodes the haemolytic toxin protein called listeriolysin O (Hly or LLO) in L. monocytogenes, where its cytolytic activity is maximised at pH 5.5. The haemolytic toxin in combination with PlcA lyses the primary phagosome.

After the bacterium escapes into the cytoplasm, it multiplies intracellular. In this step, the gene actin A (actA) produces the surface protein actin A (ActA) responsible for motility and cell-to- cell spread. The bacterium is covered with a layer of actin filaments that subsequently rearrange into a tail to provide motility. Through movement, the organism makes contact with the plasma membrane and induces pseudopod-like protrusions that reach the adjacent cell: the organism is ingested by a double plasma membrane. The second bacterial gene phospholipase C (plcB) produces the enzyme phosphatidylcholine-specific phospholipase C (PlcB), a zinc-metallo- enzyme. In the adjacent cell, the PlcB enzyme in combination with Hly lyses the two-membrane phagocytic vacuole, the secondary phagosome, which surrounds the bacterium during transfer; thus, a new intracellular parasitic life cycle reinitiates. The gene mpI encodes the metalloprotease enzyme (MpI), a zinc-dependent metalloprotease that processes extracellular PlcB enzyme to its mature form and contributes to dissemination of the bacterium from cell to cell (Chakraborty et al., 2000; Vásquez-Boland et al., 2001a; Vásquez-Boland et al., 2001b; Alvarez & Agaisse, 2016).

2.7.3 Phylogeny of L. monocytogenes

Epidemiological studies use methods for determining the genetic diversity and relationships among L. monocytogenes isolates recovered from different sources and geographic locations. Two primary phylogenetic divisions within the species L. monocytogenes were first identified at the end of 1980s by multilocus enzyme electrophoresis (MEE). Among electrophoretic types (ET), division I consists of serovars 1/2b, 3b, 4a, 4b and division II serovars 1/2a, 1/2c (Bibb et al., 1989; Piffaretti et al., 1989). In the 1990s, new molecular subtyping methods consistently

21 divided L. monocytogenes into two corresponding groups (divisions I and II). Nucleotide sequence of the listeriolysin gene (hly) coincides with flagellar antigens and groups the isolates into sequence type I (1/2b, 4b) and sequence type II (1/2a, 1/2c) (Rasmussen et al., 1991). MEE and ribotyping analysis divided serovars 1/2b, 3b, 4b, 4ab and serovars 1/2a, 1/2c, 3a into two subgroups (Graves et al., 1994). PFGE analysis with restriction enzyme AscI divided isolates into two genomic divisions (division I: serovars 1/2a, 1/2c, 3a, 3c and division II: serovars 1/2b, 3b, 4b, 4d, 4e) that correlate with the flagellar (H) antigen. Furthermore, restriction enzyme ApaI recognises two clusters in each genomic division i.e. cluster IA (serovars 1/2c, 3c), cluster IB (serovars 1/2a, 3a), cluster IIA (serovars 1/2b, 3b) and cluster IIB (serovars 4b, 4d, 4e) (Brosch et al., 1994).

Rasmussen et al. (1995) analysed several L. monocytogenes isolates from different sources and countries and distinguished three evolutionary lines through nucleotide sequences of flaA (flagellin), iap (invasive associated protein), hly (listeriolysin O) and 23S rRNA genes. The grouping correlated with the serovars: the first sequence type included serovars 1/2b, 4b, the second sequence type 1/2a, 1/2c, 3 and the third serovar 4a. In addition, Wiedmann et al. (1997) define three distinct genetic lineages, these combine ribotype patterns and PCR-restriction fragment length polymorphism (RFLP) types of three virulence genes (hly, actA, inlA). All three lineages are designated in accordance with Rasmussen et al. (1995), e.g. sequence type 1 corresponds to lineage I, sequence type 2 corresponds to lineage II, and sequence type 3 corresponds to lineage III.

Nadon et al., (2001) describe the relationships between L. monocytogenes serotypes, ribotypes and genetic lineages. Lineage I contains serotypes 1/2b, 3b, 3c, 4b, lineage II contains serotypes 1/2a, 1/2c, 3a and lineage III contains serotypes 4a, 4c. Thereafter, the designation “lineage” has been used by other researchers and remains unchanged. Multilocus sequence typing (MLST: developed by Salcedo et al., 2003) for L. monocytogenes uses 7–9 housekeeping genes and divides the species into two genetic divisions, according to Brosch et al. (1994). In the 2000s, lineage III was divided into multiple distinct subgroups by sequence analyses of one or more bacterial genes. Some published studies use RFLP, nucleotide sequences of sigB encoding the stress response sigma factor sigB, prfA encoding a virulence regulator, MLST and PCR/Southern hybridisation (Tran & Kathariou, 2002; Moorhead et al., 2003; Meinersmann et al., 2004; Ward et al., 2004; Liu et al., 2006a). Sequence analyses of sigB and actA identify three subgroups in lineage III (IIIA, IIIB and IIIC) that include serovars 4a, atypical 4b, and 4c. However, no association between subgroups and serovars has been found and “these three subgroups may represent separate evolutionary lineages” (Roberts et al., 2006). With a sequence of five genes (cheA, phoP, lmo0693, flaR and lmo2537), Orsi et al. (2008b) observed L. monocytogenes is divided into four clusters, with the fourth cluster containing the lineage III subgroup IIIb, which forms an “independent cluster”. With the use of multilocus genotyping (MLGT), Ward et al. (2008) identified lineage III subgroup IIIB as a fourth lineage of L. monocytogenes (lineage IV) which contains serotypes 4a, atypical 4b and 4c (Table 3). Lineage I appears to be almost clonal whereas, considerable horizontal gene transfer or recombination occurrs in other lineages. The lineages most frequently involved in human listeriosis are lineage I (serovars 1/2b and 4b) and lineage II (serovars 1/2a and 1/2c), whereas, lineages III and IV are usually isolated from animals, food and the enviroment (Meinersmann et al., 2004; Ward et al., 2004; Liu et al., 2006b; Orsi et al., 2008a, b; Orsi et al., 2011).

Table 3. Identification of Listeria monocytogenes phylogenic lineages.

%010<08.0 !0>39/ 480,20 480,20 480,20 480,20) &0<9@,< &0<9@,< &0<9@,< &0<9@,< $411,<0>>4! ! 4@4=498 4@4=498 8/ 8/ -,- , . 4--! ! 6?=>0< 6?=>0< 8/ 8/ --- , %,=7?==08! 080=0;?08.0 &0;?08.0>A:0 &0;?08.0>A:0 8/ 8/ 9<$ -- , . <,@0=! %4-9>A:482 %4-9>A:0J %4-9>A:0I 8/ 8/ ! ---,- , ., <9=.3! $ 4@4=498 4@4=498 8/ 8/ ---/ , ., 0 . %,=7?==08! 080=0;?08.0 &0;?08.0>A:0 &0;?08.0>A:0 &0;?08.0>A:0 8/ $ &<%" -- , . , *40/7,88! %4-9>A:482 480,20 480,20 480,20 8/ % $$! -- , . , ",/98! %4-9>A:482 480,20 480,20 480,20 8/ ---. , ., ,. &,6.0/9! ! &'G 4@4=498 4@4=498 8/ 8/ 39?=0500:4822080= -- , !99<30,/! 080=0;?08.0 480,20 480,20 480,20 8/ &"$ -- , . ,.,8/ ,>A:4.,6-. !0480<=7,88 ! &' 480,20 480,20 480,20 8/ ! ---0 , ., ,-. *,=! 080=0;?08.0 8/ 8/ &?-2<9?:= 8/ ! 4?! $%,8/&9?>30<8 -- , . &?-2<9?:= 8/ - -69>> #<=4! 080=0;?08.0 - , &?-2<9?:= 8/ - ,8/ *,A:4.,6 ,,>A:4.,6 - -,8/. nd = no data

2.7.4 PFGE fragments

Pulsed-field gel electrophoresis (PFGE) is able to estimate the whole genome size of a microorganism, i.e. the number of base pairs contained in the genomic DNA, through the measurement of restriction fragments (Hielm et al., 1998; Alduina & Pisciotta, 2015). These fragments are generated by restriction enzymes that cleave the DNA at different cleaveage sites (Wilhelm et al., 2003). As the genomic G-C content of L. monocytogenes is low, restriction enzymes with recognition sequences containing G and C nucleotides are used to avoid generating multiple bands. Suitable rare cutting restriction enzymes for L. monocytogenes PFGE analyses are AscI and ApaI. Usually, a PFGE type (pulsotype or pulsovar) is established based on the number and distribution of all fragments within a DNA restriction profile. AscI PFGE profiles of L. monocytogenes have two well-defined regions above and below the size of 145.5 kb. Several L. monocytogenes AscI PFGE profiles from isolates identified in outbreaks, and even among isolates from different sources, time and location, display PFGE types that differ in the region above 145.5 kb, but are identical in the region below 145.5 kb (Brosch et al., 1994; Cantinelli et al., 2013). The large restriction fragments usually harbour prophages and the insertion or deletion of these prophages change the number and distribution of these fragments in the upper region, generating new variants or AscI PFGE profiles of L. monocytogenes strains (Stasiewicz et al., 2015; Chen et al., 2017b; Li et al., 2017). Among highly related L. monocytogenes strains the AscI PFGE profiles differ by only two fragments in the upper region and are similar in the lower region (Kathariou et al., 2006; Gilmour et al., 2010; Chen et al., 2016b; Kvistholm Jensen et al., 2016). These observations suggest AscI restriction fragments below 145.5 are more conserved than larger fragments, supporting the utility of smaller fragments in grouping L. monocytogenes. Furthermore, there is a correlation between L. monocytogenes PFGE types and serovars in both lineages I and II. However, a few L. monocytogenes isolates with indistinguishable PFGE profiles displaying different serovars (1/2a and 3a or 1/2b and 3b) are reported (Brosch et al., 1994; Nadon et al., 2001; Lukinmaa et al., 2003; Revazishvili et al., 2004; Gianfranceschi et al., 2009).

PFGE in silico Analysis in silico ‘‘is conducted via computer simulations with models closely reflecting the real world’’ (Apache Software Foundation, 2016). By selecting a specific strain of L. monocytogenes from the available whole genome sequences (http://insilico.ehu.es), it is possible to make a theoretical cleavage (in silico) by choosing a required restriction enzyme, e.g., AscI. Bikandi et al., (2004) analysed in silico with PFGE and restriction enzyme AscI some sequenced L. monocytogenes strains belonging to lineage II.

2.8 Listeriosis in humans

Although listeriosis is considered a rare disease in humans veterinarians have long observed listeriosis in animals. Increased knowledge of human listeriosis, improved bacteriological diagnoses, and an increasing number of bacteriological routine tests may partly explain the increase in the number of diagnosed cases of listeriosis (Larsson, 1960; Sepp & Roy, 1963; Gerdin, 1981; McLauchlin, 1996b). In addition, changes in food habits may contribute to the increase in human listeriosis cases (Allerberger & Wagner, 2010; de Oliveira et al., 2010).

Modern food technology, i.e. the use of vacuum packaging, has led to increased opportunities for L. monocytogenes to multiply in RTE foods (Bērzinš et al., 2007). Morever, the susceptible human population is ever increasing, people are living longer and more people have underlying health conditions which themselves or the treatment of them increases susceptibility to listeriosis infection (Allerberger & Wagner, 2010; Todd & Notermans, 2011).

The Listeria species pathogenic to humans and animals are L. monocytogenes, L. ivanovii and L. seeligeri, although, L. monocytogenes is associated with the vast majority of cases of listeria infection. Listeriosis in humans manifests primarily as septicaemia, meningitis, encephalitis, gastrointestinal infection and abortion. Other less common manifestations are endocarditis, pericarditis, myocarditis, arteritis, pneumonia, sinusitis, conjunctivitis, ophthalmitis, otitis, joint infection and skin infection (Seeliger & Jones, 1986; Radostits et al., 1994; Vázquez-Boland et al., 2001b). Incubation times range from one day to three months (Linnan et al., 1988). Goulet et al., (2013) report four different median incubation periods related to clinical manifestations: 24 hours (range 6–24 h) for gastrointestinal forms; 2 days (range 1–12 days) for bacteraemia; 9 days (range 1–14 days) for central nervous system (CNS) cases; and, 27.5 days (range 17–67 days) for pregnancy-related cases.

The disease occurs sporadically and in outbreaks involving few or substantial numbers of affected individuals. However, most human cases are sporadic and thus, it is difficult to trace the implicated source. Although the incidence of listeriosis is lower than for other bacterial diseases, the fatality rate is between 20% and 40% in humans (Hof et al., 1994; McLauchlin, 1996a; Magalhães et al., 2015). Fatality rate is highest in susceptible populations with underlying immunosuppressive conditions, such as pregnant women, neonates and the elderly (Schlech et al., 1983; Linnan et al., 1988; McLauchlin et al., 1991; Büla et al., 1995).

Clinically healthy and convalescent humans can be carriers of L. monocytogenes. Positive samples have been obtained from faeces of slaughterhouse and laboratory workers and even from office workers that have no contact with listerial material. L. monocytogenes has been isolated from 1.2% individuals in Denmark, 2% in West Germany, 12 to 77% in The Netherlands, 5.4% in France and 0.6% in England. Pharyngeal and vaginal carriage is also reported (Bojsen-Møller, 1964; Kampelmacher & van Noorle Jansen, 1972; Carbonnelle et al., 1978; Kampelmacher & van Noorle Jansen, 1979; McLauchlin et al., 1986). Domestic ruminants can shed the organism in faeces or milk and the percentage of carriers among cattle is e.g. 52% in Denmark, 6.7% in Finland, and 6% in Sweden (Skovgaard & Morgen, 1988; Husu et al., 1990; Unnerstad et al., 2000). In the pathogenesis of listeriosis, certain factors play an important role, such as age (neonates and elderly) and deficient immune status due to underlying illness (Nieman & Lorber, 1980; McLauchlin, 1990a and 1990b). In vitro, the pathogenic species of Listeria are susceptible to many common antibiotics such as penicillin, ampicillin, tetracycline and erythromycin. However, treatment in vivo is complicated because the bacteria grow and multiply intracellular, where antibiotics have to penetrate through the cell walls (Bannister, 1987; Nichterlein & Hof, 1991). Although ampicillin and gentamicin are still the drugs of choice for treating listeriosis, aminoglycosides, such as gentamicin, may be harmful to patients with renal failure (Mitjà et al., 2009).

2.8.1 Human listeriosis in Europe

Several countries report an increasing tendency of human listeriosis since the 1990s. In the European Union, the incidence of listeriosis increased 30% from 2013 to 2014 (Mammina et al., 2013; Iannetti et al., 2016).

United Kingdom Between 1967 and 1982, there were less than 100 human cases of listeriosis annually in England, Wales and Northern Ireland. Since the 1960s, the Public health laboratory service of London, UK, has monitored human listeriosis (McLauchlin et al., 1991). From 1967 to 1985, 786 cases were reported in Britain and of 722 viable L. monocytogenes human isolates, 248 (34%) were associated with pregnancy, with a 36% fatality rate. Among 474 (66%) non-pregnant cases, 58% were male and 42% female. The fatality rate was higher among non-pregnant cases (44%), with the highest fatality rate being among patients over 60 years old with underlying diseases (54%). Among all 722 isolates, 423 (59%) isolates shared serovar 4b, 130 (18%) shared serovar 1/2a, 99 (14%) shared serovar 1/2b, and 29 (4%) shared serovar 1/2c (McLauchlin, 1987, 1990a and 1990b). In France, during the same period, overall fatality rate was 34% among pregnancy- associated cases and 47% among non-pregnant cases (Humbert et al., 1977a & 1977b).

In Britain between 1976 and 1979, a L. monocytogenes serovar 4b strain belonging to a particular phage type, called “Liverpool type”, was isolated from several human patients, which meant a probable common source outbreak (McLauchlin et al., 1986). In 1985, in England, Wales and Northern Ireland, the number of reported human cases was 149, and the cases increased considerably between 1987 and 1989 to more than 250 cases annually (McLauchlin et al., 1991). At the end of 1987, the Department of Health in London, UK, issued a hazard warning for contaminated soft Swiss cheeses after the Swiss outbreak of 1983–1987. In February 1989, the government reiterated the warning on soft cheeses to vulnerable groups, and in July 1989, issued a warning on the comsumption of paté (McLauchlin et al., 1988 and 1991). The majority of L. monocytogenes human isolates from 1987 belonged to serovar 4b (71%) and 42 people died among 130 non-pregnant cases (McLauchlin et al., 1988).

Between 1967 and 1988 in Scotland, 220 cases of listeriosis were reported, with feto-maternal cases frequently involved (198 cases). Among the feto-maternal cases, the rate increased from 38 (1977–1981) to 72 cases (1987–1988). L. monocytogenes serovar 4b was predominant, especially in the last two years (63%). The incidence in Scotland subsequently increased from 0.5 cases per million habitants in 1967–1971 to 0.6 (1972–1976), 1.5 (1977–1981), 2.2 (1982–1986), and 7.0 cases per million habitants (1987–1988) (Campbell, 1990). During the period 1983 to 1994 in England, Wales and Northern Ireland, 1844 cases of listeriosis were reported, of which 655 (36%) of cases were associated with pregnancy. Among 1620 available L. monocytogenes human isolates from this period, serovar 4b accounted for 39% to 72% of cases annually, and at least 57% among pregnancy-associated cases. Among non-pregnancy associated cases, serovar 4b accounted for 47% to 64% of cases; however, the serovar became less common in 1993 (34%) and 1994 (24%). Instead, serovar 1/2a became more dominant in 1993 (37%) and 1994 (43%) (McLauchlin & Newton, 1995). Most common cause of outbreaks in UK in recent years has been due to consumption of prepared sandwiches served to patients at hospitals (Little et al., 2008; Shetty et al., 2009; Coetzee et al., 2011; Little et al., 2012).

France In France, the average incidence of listeriosis between 1948 and 1958 was 1.1 cases per year, whereas, between 1958 and 1970, it was 40.5 cases per year. Subsequently, the number of cases increased considerably between 1970 and 1975 to 165 cases annually. In Western France before 1975, the reported cases of listeriosis in humans did not exceed 15 cases per year, but there were 113 cases reported in 1975 and 54 cases in 1976. This increase was possibly due to the first outbreak observed in France. The increase was located in Anjou and involved 128 neonates and 39 adults. However, the source of infection was not determined. Most L. monocytogenes isolates belonged to serovar 4b (98%), whereas, serovar 1/2a was only 2%. Previously, serovar 4b was observed in high proportions, between 63% and 100% of L. monocytogenes clinical isolates, which indicates this serovar was predominant in France (Carbonnelle et al., 1978). In 1984, the incidence was 11.3 cases per million of population with 630 listeriosis cases being reported, of which 56% was associated with pregnancy and 44% with non-pregnancy cases. In 1986, human listeriosis cases increased to 807 (14.7 cases/million) and in 1987 decreased to 661cases (11.9 cases/million). The proportion of pregnancy and non-pregnancy cases in these two years was similar. However, in 1988, the incidence decreased to 11 cases/million (Goulet et al., 1990) and among 615 reported cases, the rate of pregnancy-associated cases was lower (43%) than non- pregnancy cases (57%). In 1987 in France, 384 L. monocytogenes isolates were characterised from 366 human cases. Serovar 4b was dominant (63%), followed by serovar 1/2a (22%) and 1/2b (13%): 8% of human serovar 4b isolates shared lysovar with food isolates. However, in 1988 among humans, serovar 4b isolates decreased to less than 50% (Espaze et al., 1989).

A further, French outbreak occurred in 1989 and involved 14 individuals. However, the source of infection was not determined (Sutherland et al., 2003). In the 1990s, six nationwide outbreaks were reported in France. The largest outbreak occurred in 1992 and involved 279 individuals, the fatality rate was 34% and most cases were associated with pregnancy (60%). The epidemic strain serovar 4b (pulsovar 2/1/3, related to the Swiss, Californian and Danish epidemic strain) was identified in 247 of 279 human isolates (89%), and 154 different food products (Goulet et al., 1993; Jacquet et al., 1995; de Valk et al., 2000). According to French National Reference Center (NRC), the incidence of human listeriosis in France decreased from 6.3 in 1987 to 4.1 cases per million in 1997. However, two hospital laboratory surveys reported the incidence was 16.7 cases per million in 1987 and 14.9 cases per million in 1988 (Goulet et al., 2001). In 1999, listeriosis surveillance was improved through new methods and the incidence of listeriosis in France decreased from 4.5 cases per million in 1999 to 3.5 cases per million in 2005, although in 2006, incidence increased to 4.7 cases per million, which was not due to any known outbreak (Goulet et al., 2008).

Germany In Germany, 20 cases were described in 1945 and up to 1958, nearly a thousand cases had been diagnosed (Linell et al., 1959). Between 1950 and 1956 in West Germany, 303 human cases of listeriosis were reported and in 1950 to1975, 2000 cases were reported (Carbonnelle et al., 1978). At the beginning of 1960s in Germany, 80% of listeriosis cases were associated with pregnancy, however, from 1969 to 1985, the percentage decreased to 54% (McLauchlin, 1990b). In the early 1980s, the incidence of listeriosis cases per million habitants was highest in East Germany (3.36), France (3.27), Denmark (2.27), and Switzerland (2.05). The incidence was

27 lower in The Netherlands (1.69), Sweden (1.48), West Germany (0.96), USA (0.66), and Canada 0.62 (McLauchlin et al., 1986).

In Germany, listeriosis has been a mandatory notifiable disease since 2001, and in this year, 217 cases were reported; an incidence of 2.6 cases per million population. In 2005, 510 cases were reported, with an incidence of 6.2 per million, but no outbreak was observed. Between 2001 and 2005, 1519 cases were reported and among all these cases, 15% were associated with pregnancy, with the most cases occurring in patients aged > 60 years (76%). Among 80 analysed isolates, 39 isolates shared serovar 1/2a, 38 shared serovar 4b and 3 shared serovar 1/2b (Koch & Stark, 2006). In 2009–2010, a multinational listeriosis outbreak occurred in Germany, Austria and the Czech Republic due to consumption of a red-smear cheese (Quargel) produced in Austria. The age of patients ranged between 57–89 years and most patients were male (76%). PFGE identified two different serovar 1/2a PFGE types in 14 and 20 human cases each (Fretz et al., 2010; Rychli et al., 2014). Between November 2012 and November 2015, 1765 human listeriosis cases were notified in Germany. The characterisation of 793 isolates identified a common source outbreak, where 66 patient isolates shared an identical PFGE pattern with AscI and ApaI enzymes, sharing serovar 1/2a and ST8 (Ruppitsch et al., 2015).

The Netherlands Between 1957 and 1976 in The Netherlands, 769 L. monocytogenes isolates were collected from human patients and clinically healthy individuals. The percentage of serovar 4b isolates (n=343) was 44.6%, whereas, the percentage of serogroup 1 (n=319) isolates was 41.8% (Kampelmacher & van Noorle Jansen, 1979). According to Kampelmacher (1962), human listeriosis in The Netherlands occurred among individuals who worked on farms or regularly had contact with animals. The annual incidence of listeriosis in 1981–1990 was 1.2 per million inhabitants, whereas, in 1991–1995, it was 0.7 per million. The decrease could be due to “effective microbiological monitoring of food sources, especially of soft cheeses” (Kampelmacher, 1962). In the period of 1976–1995, 207 isolates from listeria meningitis cases were collected; most of the patients were male (61%). Among 204 isolates, serovar 4b constituted 65% and 30% were 1/2a, 1/2b, or 1/2c (Aouaj et al., 2002). Between 1995 and 2003, 283 patients were hospitalised with listeriosis, an incidence of 2.0 cases per million inhabitants annually: of all cases, 149 (53%) were men, 40% were adults between 65–79 years old, and 24 cases (8.5%) were associated with pregnancy. Among 159 clinical isolates from 1999–2003, serovar 4b was predominant (44%), followed by serovar 1/2a (36%) and 1/2b (16%). It was recommended that pregnant women should avoid soft cheeses, raw milk and smoked salmon (Doorduyn et al., 2006).

Switzerland A large listeriosis outbreak involving 122 individuals occurred in Switzerland between 1983 and 1987, due to raw milk cheese: the fatality rate was 28%, and 53% of the cases were associated with pregnancy (Bille & Glauser, 1988; Büla et al., 1995). In a retrospective study (Boerlin et al., 1996), 47 L. monocytogenes isolates sharing ET1 type serovar 4b isolated from humans, cheese and the processing plants from this Swiss outbreak were compared with isolates from 145 humans and 117 animals (collected between 1988 and 1993) and 8 animal isolates from 1986 that shared ET1 type serovar 4b. Among L. monocytogenes isolates collected from the post epidemic period, 28% belonged to humans and 37% to animals. In total, 144 isolates (45 human

28 isolates and 44 animal isolates post-epidemic and 47 human and 8 animal isolates from the epidemic period) belonging to ET1 type were further analysed by PFGE with ApaI and SmaI. Through combining both enzymes, 29 subtypes were obtained, where five were predominant (I– V). Among 144 isolates, PFGE subtypes II and IV were identified in human isolates, and subtypes I, II, IV and XX in cheese isolates were associated with the Swiss outbreak. In addition, the four subtypes encountered in these cheeses were identified among animal isolates and three of them were dominant. Moreover, subtypes I and II were the main subtypes identified among human (5%) and animal (8%) isolates collected in the post-epidemic period (Boerlin et al., 1996). In Switzerland between 2011 and 2013, 93 L. monocytogenes isolates were collected from human individuals. Serovar 1/2a isolates were predominant (58 isolates), whereas, serovar 4b was less dominant (28 isolates), followed by serovar 1/2b (5 isolates) and 1/2c (2 isolates). PFGE yielded 70 different AscI types. The incidence over the last ten-year period fluctuated between 5.3 and 9.7 cases per million in Switzerland (Althaus et al., 2014).

Spain In Valencia, between 1978 and 1988, 14 human cases of listeriosis were diagnosed, where L. monocytogenes serogroup 1 was predominant, whereas, in 1989, 9 cases were reported with serogroup 4 being predominant. This increase was possibly due to the first outbreak observed in Spain. Six isolates were characterised and shared serovar 4b; phage typing identified two types. Three of the isolates were identical to a listeriosis outbreak strain in ovines occurring in the province of Leon in 1990 (Vázquez-Boland et al., 1991). On the island of Gran Canaria, only three cases of human listeriosis were reported between 1983 and 1987, whereas, five cases were reported during 1988–1991, and 19 cases in 1992. A common source outbreak was suspected due to this increase. Between December 1991 and May 1993 in Las Palmas, Gran Canaria, 24 cases of listeriosis were detected: 12 (50%) were pregnant-associated. Among 24 isolates, 21 (87.5%) shared serovar 4b and 2 (8.3%) serovar 1/2a. Of the 12 characterised isolates, seven had identical MEE and PFGE patterns. The majority of patients usually consumed fresh cheeses and RTE meat products (Elcuaz et al., 1996). In a Spanish study, 51 human isolates recovered from different regions of Spain between 1989 and 1998 were characterised: serovar 4b was dominant with 38 isolates (74.5%), whereas, only 12 (23.5%) isolates shared serovar 1/2a. PFGE identified 29 ApaI pulsotypes, and two profiles were common with 11 and 8 isolates (Vela et al., 2001). Subsequently, the incidence of listeriosis in Spain increased from 5.6 cases per million (2001 to 2007) to 9.3 cases per million in 2012, which is the second highest in the EU. An analysis of 17 human L. monocytogenes isolates, collected between 2006 and 2014, the majority from the region of Leon, Spain, found 58.8% of the isolates belonged to lineages I and III and 41.2% to lineage II. PFGE detected 14 AscI pulsotypes and MLST 11 STs. One ST, identified in 2012, was responsible for two outbreaks occurring in Northern Spain in 2013–2014 (Ariza-Miguel et al., 2015).

Italy According to the Listeriosis Surveillance System in Italy, among human isolates in 2002 and 2003, serovar 1/2a was the most common (44%) followed by serovar 1/2b (32%) and serovar 4b (20%) (Gianfranceschi et al., 2006). In a comparison of L. monocytogenes human isolates collected in 1990–1999 and 2002–2005, results revealed serovar 1/2a has increased, whereas, serovar 4b has decreased. In the second period (2002 to 2005), four different serovars were found among 57 human isolates: 1/2a (51%), 1/2b (16%), 1/2c (7%), and 4b (25%); and, PFGE

29 identified 23 AscI pulsotypes (Gianfranceschi et al., 2009). Listeriosis in humans in Italy has been notifiable since 1993. In 2004 to 2006, the incidence of listeriosis was lower than in other European countries, 0.8 cases per million annually. However, during 2002 and 2003, an incidence of 1.3 cases per million was reported. In 2005 and 2006, the incidence was 1.4 per million in Lombardy and 4.5 per million in Tuscany (Mammina et al., 2009b).

Between 1996 and 2007 in Lombardy and Tuscany regions of Italy, 54 human clinical isolates were collected. Overall, 25 (46.3%) L. monocytogenes human isolates shared serovar 1/2a, 23 (42.6%) isolates shared serovar 4b, and 6 (11.1%) isolates shared serovar 1/2b. PFGE identified 31 AscI pulsotypes, of which 19 pulsotypes were each represented by only one isolate (Mammina et al., 2009a). Lombardy region accounted for 55% of listeriosis cases in Italy during 2006–2010, although, only 16% of the population lives in this region. The regional reference laboratory of Lombardy collected 134 L. monocytogenes isolates from 132 human cases in 2006– 2010; 15 cases were associated with pregnancy and among 118 non-pregnant adults, 63 (53.4%) were females and 55 (46.6) males. From human isolates, 70 (52.2%) shared serovar 1/2a, 52 (38.8%) isolates shared serovar 4b, 9 (6.7%) isolates shared serovar 1/2b, and 3 (2.2%) shared serovar 1/2c. PFGE recognised 73 AscI pulsotypes, and MLST identified 22 isolates belonging to ST1 and ECI among serovar 4b. The high prevalence of listeriosis in Lombardy is possibly due to improved surveillance systems and a higher level of contamination in food products such as some soft-ripened cheeses (Mammina et al., 2013).

Portugal In Portugal, the incidence of listeriosis is underestimated, as the disease was not notifiable until 2014 and no active surveillance currently exists (Magalhães et al., 2015). However, incidence increased from 1.4 cases per million inhabitants in 2003 to 2.3 in 2007. In an analysis of 95 L. monocytogenes human isolates collected between 1994 and 2007, 14.8% were associated with pregnancy. PCR group IVb was predominant and accounted for 71.6% of cases, whereas, group IIb accounted for 17.9% and group IIa 10.5%. The combination of AscI and ApaI enzymes yielded 58 PFGE types including 18 pulsotypes, represented by 2 or more isolates. PCR group IVb yielded 14 pulsotypes, group IIb 3 pulsotypes and group IIa only one pulsotype. All PFGE pulsotypes were compared with the French human pulsotypes database and seven pulsotypes were indistinguishable from French pulsotypes: two pulsotypes were involved in the French pig tongue in aspic outbreak in 1992 (Almeida et al., 2006 and 2010). In 2008–2012, 203 cases of listeriosis in Portugal were reported and the incidence ranged from 2.0 to 7.0 cases per million annually. Among all cases, 9.5% were associated with pregnancy and 46.4% of patients were over 65 years. Of the 203 isolates, the majority shared molecular serogroup IVb (77.8%), followed by serogroups IIb (14.7%) and IIa (7.4%): 91 PFGE types were identified with AscI and ApaI enzymes. In the period 1994–2007, 26 of these types had already been isolated (Magalhães et al., 2014). Between 2009 and 2012, an outbreak in Portugal involving 30 individuals occurred due to consumption of pasteurised milk cheese that harboured L. monocytogenes serogroup IVb. The fatality rate was 36.7%. Only two cases were associated with pregnancy and 13 (43%) cases were older than 65 years (Magalhães et al., 2015).

Denmark In Scandinavia, the first human case of listeriosis was reported in 1929 in Denmark by Nyfeldt, who subsequently isolated L. monocytogenes from 13 humans up to 1940 (Bojsen-Møller, 1972). Between 1981 and 1987, L. monocytogenes was isolated from 138 individuals, and between 1958 and 1974, the annual incidence of listeriosis in Denmark was 2.3 per million, with 3.3 cases per million in 1981, and 1.5 cases in 1984. Subsequently, in 1985, the incidence increased to 4.3 cases, and increased again in 1986 to 8.4: in 1987, it was 5.3 cases per million. An outbreak occurred in 1985–1987 involving 35 individuals, where all 35 isolates shared serovar 4b and had identical phage type. Among this group, 15 cases were associated with pregnancy, 15 adults presented meningo-encephalitis and the mortality rate was 20%. The mean age among outbreak patients was 47 years, whereas among non-outbreak patients mean age was 62 years. The source of infection was not identified (Samuelsson et al., 1990; Nørrung & Skovgaard, 1993).

In a Danish study, 69 L. monocytogenes isolates were characterised from human cases and 30 isolates from meat products and slaughterhouse samples, collected in 1989–1990. Of these, 67% shared serogroup 4 and 33% belonged to serogroup 1. Among the 69 human isolates, one type was dominant, with 28 isolates belonging to serogroup 4, which were indistinguishable with three different typing methods (ribotyping, REA and MEE). The dominant type was not found among meat food and environmental isolates, but two types belonging to serogroup 4 and 1 from meat products isolates were identical to other human isolates with all three typing methods (Nørrung & Gerner-Smidt, 1993). Another study (Jensen et al., 1994) identified a second Danish outbreak, which occurred in 1989–1990 and involved 26 patients, where the vehicle of transmission was probably blue mould cheese.

The incidence of listeriosis in Denmark increased markedly between 2002 and 2012 and became one of the highest incidences reported worldwide, from 5.0 cases per million inhabitants in 2002–2003 to 9.0 cases in 2012, with a peak of 18.0 cases in 2009. Between 2002 and 2012, 570 cases were reported, where 52% were females and 4% were associated with pregnancy. Among non-pregnant cases, the median age was 71 years and overall fatality rate was 26%. Most isolates, 334 (58%) belonged to lineage II and serovar 1/2a was dominant, whereas, 235 (42%) isolates belonged to lineage I, where serogroup 4 accounted for 193 (34%). The combined AscI and ApaI profiles yielded 178 pulsotypes, with 116 of them being identified in only one isolate each. One pulsotype was predominant with 82 isolates, followed by two pulsotypes with 40 isolates each. These pulsotypes, accounting for 122 cases, were closely related and multilocus sequence typing (MLST) revealed that both belonged to CC8. Furthermore, these two pulsotypes have been isolated in other countries i.e. Sweden, Finland and Norway (Kvistholm Jensen et al., 2016).

Norway The first case of human listeriosis in Norway was reported in 1952. The fatal case was a 54-year- old farmer who became ill after he had cleaned the sheephouse. Two months later, a sheep died from listerial encephalitis (Bernander et al., 1983). Before 1959, three human meningitis cases due to L. monocytogenes were reported (Bassøe et al., 1959). In Norway, human listeriosis has been a notifiable disease since 1977 (Rosef et al., 2012) and the first documented food-borne outbreak of invasive listeriosis occurred in Trøndelag during summer 1992, which involved six patients. Five samples of vacuum-packed RTE meat products from one manufacturer collected

31 from patients’ refrigerators harboured L. monocytogenes and three samples taken from the processing plant were contaminated. The isolates from patients, meat products from the refrigerator, and the plant shared similar serovar and ET (Hellsnes et al., 1992).

During the period 1977 and 2003, 289 cases of listeriosis were reported in Norway. The annual incidence of listeriosis ranged from 0.2 to 4.9 cases per million inhabitants during this period: male and female cases were represented equally and 39 (13%) cases were associated with pregnancy. The fatality rate was 45% in 1977–1987 and 41% in 1990–2000. Between 1977 and 2000, most listeriosis cases (70.7%) were from individuals aged 60 years or older. Within this same period, two outbreaks due to consumption of meat products harbouring L. monocytogenes occurred. The first in 1992, involved eight cases, and the second in 2000, involved four cases. In 2005, a third outbreak occurred at a hospital canteen, where three individuals ingested contaminated food (Antal et al., 2007). In a retrospective study, 137 L. monocytogenes human isolates collected during 1992–2005 and 188 L. monocytogenes isolates collected during 1989– 2002 from fish (n=90), poultry (n=26), meat (n=43), processing plants environments (n=29) were characterised by ribotyping. Most human isolates, 76 (55.6%) belonged to lineage II, whereas, 57 (41.6%) isolates belonged to lineage I. Among food and environmental isolates, lineage II was dominant. Of the 37 identified ribotypes, 22 ribotypes were found among human isolates and 33 ribotypes in remaining isolates. The two most common ribotypes were found among all sources and accounted for 58 (18%) and 54 (17%). The rate of listeriosis, during 1994 to 2003, fluctuated between 15 and 30 cases annually in Norway. However, in 2007, 50 cases were reported and this high rate was due to an outbreak involving 17 patients from a hospital who consumed soft cheese contaminated with L. monocytogenes (Rosef et al., 2012).

Finland In Finland, human listeriosis has been a notifiable disease since 1994, although, L. monocytogenes isolates have been submitted to the Finish National Health Institute since the 1980s. Between 1990 and 2001, 20 to 50 human listeriosis cases were reported annually in Finland. During this period, 314 L. monocytogenes isolates (25 isolates linked with a butter- borne outbreak: Lyytikäinen et al., 2001; Maijala et al., 2001) were collected and most isolates were from male patients (57%), with 10.2% from pregnant-associated cases. Serovar 1/2a was predominant (53%), then, serovars 4b (27%), 3a (11%), 1/2c (6%) and 1/2b (3%). In 1990, serovar 4b was more dominant than serovar 1/2a (61%), but in 2000–2001, serovar 1/2a exceeded 60%. Among all isolates, 81 AscI PFGE types, including 11 PFGE types represented by five or more isolates, were detected. One PFGE type serovar 1/2a was predominant with 37 isolates (45.7%). This type was involved in an outbreak due to vacuum-packed cold-smoked rainbow trout in 1997 (Miettinen et al., 1999b). One PFGE type serovar 1/2c (5%) was only found among male L. monocytogenes isolates (Lukinmaa et al., 2003). In 2010, an outbreak of listeriosis caused by a specific AscI type, previously isolated in fish products, was investigated and in this year, 71 cases of human listeriosis were reported: the incidence was 13 cases/million, a pregnancy association of 7% and a fatality rate of 23%. Of 69 available L. monocytogenes isolates, serovar 1/2a was predominant (72%), with (23%) being serovar 4b. AscI PFGE type yielded 35 types and 13 (19%) isolates shared one PFGE type (Lm96). This type, identified in several European countries during 2010 and previous years, is commonly isolated from human and foodstuffs (Nakari et al., 2014).

2.8.2 Human listeriosis in Sweden

In Sweden, veterinarians had primary knowledge about listeriosis and reported the disease in a large number of species (Lilleengen, 1942; Wramby, 1944; Nilsson & Karlsson, 1959; Andersson, 1961; Nilsson & Söderlind, 1974; Tham et al., 1989). It was not until the 1950s that cases of listeriosis in humans were diagnosed in Sweden, especially among pregnant women and newborns (Ekelund et al., 1962). In May 1958, Linell et al. presented what appears to be the first confirmed human listeriosis case, a 7-day-old child with granulomatosis infantiseptica and meningitis, at a meeting of the Pathology Association in Malmö (Linell et al., 1959). A second report of human listeriosis was a fatal case of meningitis in an adult man (Brandt & Berg, 1959). Thereafter, during the 1960s, several cases of listeriosis in humans were diagnosed in different counties in Sweden; mainly perinatal listeriosis cases (Anonymous, 1960; Bergman et al., 1960; Larsson, 1960; Ekelund et al., 1962; Brorson et al., 1968; Söderling, 1974; Larsson et al., 1978a; Larsson et al., 1978c; Niklasson et al., 1978; Larsson et al., 1979a). Since 1960, human listeriosis has been a notifiable disease in Sweden, and both clinicians and microbiological laboratories are required to report listeriosis cases to the County Medical Office and to the Public Health Agency, according to the Swedish Communicable Disease Act. Until 1999, the reporting system from clinicians and laboratories were analysed separately. As laboratory reports were voluntary and contained poor information from patients, only reports from clinicians were included in zoonosis reports (Romanus, 1984; SVA, 2000 and 2009). The Swedish Zoonosis Council, which was created in 1997 to oversee the zoonosis situation, was represented by the National Board of Health and Welfare, Swedish Institute for Infectious Disease control, National Food Administration, Swedish Board of Agriculture, National Veterinary Institute, and the Swedish Association of Local Authorities (SVA, 1999).

Between 1958 and 1974, 110 cases of human listeriosis were reported in Sweden: 46 cases (42%) were associated with pregnancy, and in two cases involving twins, and resulted in eight abortions and three stillborn. Among 37 neonatal cases born alive, 17 died (46%). Among 64 (58%) non-pregnant cases reported, 34 were male and 30 females. The fatality rate in this group was 31.3% (20 cases). The majority of cases occurred in adults aged between 40 and 70 years, with the highest fatality rate being among patients aged 50 to 70 years old with underlying diseases, such as leukaemia and alcoholism. The diagnosis was meningoencephalitis in 52 cases (16 fatalities) and septicaemia in 10 cases (4 fatalities). Incidence of human listeriosis was high in 1959 (16 cases) and 1960 (13 cases), but decreased each year between 1961 and 1971; the incidence was between 2 and 6 cases per year. In 1974, the incidence increased to 16 cases, and was similar to 1959. Among pregnancy-associated and non-pregnant cases, serogroup 1 and serovar 4b were equally distributed, but neither clinical diagnosis nor mortality was related to serogroups (Larsson et al., 1978a; Larsson et al., 1979a; Larsson et al., 1979b). In 1975, two listeriosis outbreaks of nosocomial infection occurred in a hospital (two infants were infected in the first outbreak and five infants in the second), and was possibly due to a thermometer not being disinfected between patients (Larsson et al., 1978b).

From 1975 to 1983, between 19 and 34 human cases were reported annually. Of 128 cases reported in 1979–1983, clinicians reported 62 cases and laboratories reported 66 cases. The highest listeriosis incidence was among patients over 60 years old (56%), with 9 (7%) cases being neonatal, and 38% of the cases had underlying diseases. During this period, the incidence

33 rate of listeriosis was three cases per million inhabitants (Romanus, 1984), with most cases being sporadic, although small outbreaks are reported (Larsson et al., 1978a; Romanus, 1984).

In 1983, Bernander et al., (1983) reported a probable outbreak of invasive listeriosis occurring in Västerås in the county of Västmanland during July to September 1981. Five cases were involved, two pregnant women and three males: the age of the patients varied between 28 and 73 years. Four of the patients lived in the same residential area and the fifth’s parents lived in the area. An animal field with goats and sheep was located near the area, sometimes the animals got loose and moved within the residential area, and some families with children had rabbits that could borrow. L. monocytogenes serovar 4b was isolated from clinical samples and from samples of wool and straw. One sheep had a recent infection of mastitis and a milk sample harboured L. monocytogenes, serovar 4b. With the results from serotyping and epidemiological analysis, and that all cases occurred at the same time and same space, raised suspicion about a common route of infection.

During the 1990s, the reported number of cases of listeriosis was stable (between 25 to 35 cases annually): physicians reported 271 cases and the incidence was similar to the 1980s, with the highest incidence occurring among the elderly over 75 years (SVA, 1999).

The first documented sporadic case of foodborne listeriosis in Sweden (a woman suffering from meningitis) occurred in 1993 due to consumption of medwurst (Loncarevic et al., 1997). L. monocytogenes was isolated from food products in the patient’s refrigerator: vacuum-packed sliced pork brawn, sliced cooked medwurst, and berliner wurst of the same brand. At least three PFGE types were recovered, and one (in medwurst) was identical to L. monocytogenes PFGE type of the patient. This PFGE type was also isolated from the same products sold in the local retail store. The first documented outbreak of invasive listeriosis in Sweden occurred in the county of Värmland between 1994 and 1995 and involved nine patients. All patients had consumed gravad, cold- or hot-smoked rainbow trout/salmon vacuum-packed RTE products in the previous six months. Three different PFGE types all sharing serovar 4b were involved in the outbreak. The AscI/ApaI PFGE types were identified in both patients and samples from the same producer: gravad rainbow trout, cold-smoked rainbow trout and residues found in the packing machine. The fourth AscI/ApaI PFGE type was isolated from cold-smoked rainbow trout processed by another producer (Ericsson et al., 1997).

In 1998, two sporadic cases of listeriosis were reported in Sundsvall, Västernorrland County. A hard cheese collected from one of the patient’s refrigerator harboured < 100 cfu of L. monocytogenes, and Chinese cabbage samples from the second patient were heavily contaminated with L. monocytogenes (200–1000 cfu per gram). All isolates, both from patients and food, shared identical AscI/ApaI PFGE type and belonged to serovar 1/2a (Tham et al., 1998).

Since 2000, the total number of reported cases, both from clinicians and laboratories were included and surveillance was based on passive case findings. However, an increase in reported cases was observed during the 2000s with a peak in 2001 (67 cases, and an incidence of 8.0 cases per million) and 2009 (73 cases). Between 1983 and 2012, the incidence of listeriosis increased by 2.5% annually, especially during the 2000s, and became one of the highest incidences

34 reported worldwide, from 4.0 cases per million inhabitants in 2002 to 9.6 cases in 2013. The number of pregnant-associated cases remained low, between 1–2 cases annually with a peak in 2001 (5 cases), 2007 (5 cases), 2010 (7 cases), and 2014 (5 cases). Among non-pregnant cases, the elderly (over 70 years old) were overrepresented. Between 2003 and 2015, the northern region had the highest incidence of listeriosis (i.a. Jämtland and Västernorrland counties), whereas, the three largest counties (Stockholm, Skåne and Västra Götaland) reported most of the cases. Fatality rate was high and stable, one-third of patients died within three months. As this group usually constitutes elderly with underlying diseases, the effect of listeriosis is complicated to estimate. Most cases were of domestic origin (SVA, 1999–2015).

In 2001, a large gastrointestinal outbreak of listeriosis in Hälsingland, Gävleborg County, linked to fresh cheese made of raw goat and cow milk on a summer farm caused febrile gastroenteritis in at least 120 people. Besides L. monocytogenes, some cheeses were contaminated with VTEC, ETEC and staphylococci. The isolation of L. monocytogenes was the most consistent finding from both patients and cheese samples, which were heavily contaminated with this pathogen, some cheese samples harboured over 10 million cfu per gram of L. monocytogenes. All isolates from clinical cases, cheeses, dairy animals, environment and equipment shared identical AscI/ApaI PFGE type and belonged to serovar 1/2a (Carrique-Mas et al., 2003; Danielsson-Tham et al., 2004). This is the first substanteated reported outbreak of listeriosis caused by raw milk cheese, where the epidemic strain was cultured from the milk of a dairy animal. The milk was used for fresh cheese manufactured on the farm. A goat with subclinical mastitis excreted 18,100 cfu L. monocytogenes per ml milk.

Since 2011, the typing routines of L. monocytogenes isolates from human cases have changed. The Public Health Agency of Sweden has used molecular serotyping and this new method has identified four molecular serogroups among all viable isolates. Serogroup IIa were most common (59% – 83%), followed by serogroup IVb (8% – 25%), serogroup IIc (2% – 16%) and serogroup IIb (3% – 6%) (SVA, 2011–2015; Personal communication: Hedenström, 2017).

Two invasive listeriosis outbreaks occurred between 2013 and 2015. In the first outbreak, 27 clinical cases were reported between May 2013 and April 2015 (7 cases 2013, 17 cases 2014 and 3 cases 2015) from nine counties, mainly in the Götaland and Svealand regions of Sweden. The majority of cases were reported from Skåne (10 cases) and Stockholm (6 cases) counties. The mean age of the 27 patients was 79 years (40 to > 90 years) and 67% were male. An epidemiological investigation revealed the source of infection as gravad and smoked fish products produced by one manufacturer. The clinical and food isolates were characterised by molecular serotyping, PFGE with two restriction enzymes AscI and ApaI and MLST. The causative strain in this outbreak belonged to serovar 1/2a, AscI PFGE type 0194 (EFSA nomenclature) and ST19. The second outbreak occurred from October 2013 to October 2014, and 16 counties were involved. The majority of cases were from Skåne (16 cases) and Västra Götaland (9 cases). During this period, 51 patients were affected (61% were female), including four maternal cases: there were seven fatalities. The consumption of contaminated cold cuts was suspected to be the source of infection and this outbreak was dominated exclusively by AscI PFGE type 0039 belonging to serovar 1/2a and ST7. The National Food Agency of Sweden analysed 70 meat product samples from three different manufacturers and found different PFGE types: five samples from the same manufacturer harboured the same AscI PFGE type 0039 as the

35 clinical samples (Public Health Agency of Sweden reports from 2015 and 2016; SLV report, 2016; Dahl et al., 2017; Personal communication: Hedenström, 2017).

Since 2007, the incidence of human invasive listeriosis in Sweden has varied between 5.9 (2011) and 12.8 (2014) cases per million inhabitants: in 2015, there were 9.0 cases per million (Public Health Agency of Sweden, 2015). Although, contaminated food products are the main source of human listeriosis in Sweden, no official coordinated system exists for the surveillance of L. monocytogenes in food. Sampling is performed by national and local authorities, mostly at production units and retail levels. Sampling performed by the industry is not usually reported to the authorities (SVA, 2016).

As human listeriosis has increased in Sweden, five authorities jointly developed a five-year strategy in 2013 and 2014 for working with L. monocytogenes. These governmental authorities included the National Board of Health and Welfare, National Food Administration, Public Health Agency of Sweden, National Veterinary Institute, and the Swedish Board of Agriculture. The strategy document outlines certain measures these authorities consider particularly important (National Board of Health and Welfare, 2013). The Listeria research group at Campus Grythyttan, Örebro University, who reviewed the initial version of the strategy document, consider the measures listed in the strategy document are important. However, they recommend long-term basic research aimed at increasing understanding of L. monocytogenes and listeriosis should be included, and that characterisation of L. monocytogenes isolates from cases of human listeriosis should be obligatory.

In 2014, L. monocytogenes was included in the microbiological surveillance programme at the Public Health Agency of Sweden and included the typing of all L. monocytogenes isolates from human cases due to the last two outbreaks and the high incidence of listeriosis. In 2015, the Public Health Agency implemented a new molecular method for characterisation of clinical L. monocytogenes isolates: whole genome sequencing (WGS) (SVA, 2014 and 2015).

2.9 Contaminated food as source of listeriosis

Since first being identified, the disease listeriosis has been considered an occupational infection among veterinarians, farmers and butchers, or related to regular contact with animals (Sepp & Roy, 1963; Kampelmacher & van Noorle Jansen, 1979; Tham et al., 1989). This disease usually affected single cases as the person was infected while assisting bovine or ovine partus or during slaughter. The possible relationship between human listeriosis and animal products was discussed during the Giessen symposium in West Germany on listeriosis in 1957, although, this was based on a description of a few cases with suspected source of infection. In the second symposium on listeric infection in 1962 in Montana, USA, researchers continued to deliberate about potential sources of human listerial infection (e.g. raw milk, egg and egg products, meat and meat products). Researchers also realised hygiene in kitchens and food-producing plants could pose a problem, and how food products with Listeria contaminates hands, utensils and kitchen tables. Concerns were also expressed that fish, in particular trout, could be fed raw minced meat contaminated with Listeria and that humans could be infected by consuming this fish (Kampelmacher, 1962). During a possible outbreak of listeriosis in Uppsala, Sweden, in the

36 beginning of 1960s, bacteriological investigations were undertaken on e.g. eggs and poultry but no Listeria were detected (Ekelund et al., 1962): this investigation was two decades before the food-borne route for L. monocytogenes was confirmed.

2.9.1 Outbreaks of listeriosis and presence of L. monocytogenes in food

One of the first suspected human listeriosis outbreaks reported was in Halle, East Germany, between 1949 and 1957. The suspected sources of infection were raw milk, sour milk, cream and cottage cheese; approximately 100 individuals were implicated and the human L. monocytogenes isolates belonged to serogroup 1/2. Moreover, in 1954, an outbreak involving 26 individuals occurred in Jena, Germany, and one year later, an outbreak in 41 neonatals was reported from Prague, Czechoslovakia. The human L. monocytogenes isolates shared serogroup 1/2. In both outbreaks, the vehicle of transmission was not identified. In 1956, a correlation between the consumption of pork meat on a Soviet kolchoz and 19 human cases of listeriosis was suspected. In Bremen, Germany, two outbreaks occurred: in 1960–1961 (involving 81 individuals) and in 1963 (involving 20 individuals). In 1966, an outbreak of neonatal listeriosis occurred in Halle, East Germany, where 279 were affected and raw milk was suspected to be the source of infection; the human L. monocytogenes isolates belonged to serovar 1/2a. During the 1970s, three outbreaks were reported in the USA: the first in 1975 involving 6 neonatals, the second in 1976 among 20 non-pregnant individuals possibly due to raw salad; and, the third in 1979, also involving 20 individuals, where raw vegetables and cheese were suspected to be the sources of infection. The human L. monocytogenes isolates in the three American outbreaks belonged to serovar 4b (Kampelmacher, 1962; Ho et al., 1986; McLauchlin et al., 1986; Sutherland et al., 2003).

Nevertheless, awareness that contaminated food could be the vehicle of infection by L. monocytogenes in humans was raised when Schlech et al. (1983) reported an outbreak occurring in Canada in 1981 that was caused by coleslaw salad. Seven non-pregnant individuals and 34 neonatals were implicated; the isolates belonged to serovar 1/2b (Schlech et al., 1983). Thereafter, in the USA a further two outbreaks were reported, in 1983 in Massachusetts (49 cases), due to pasteurised milk, and 1985 in California (142 cases), due to soft cheeses; all isolates belonged to serovar 4b (Fleming et al., 1985; Linnan et al., 1988). Subsequently, several outbreaks due to contaminated foodstuffs have been reported in many countries. A wide variety of foods especially ready-to-eat foods with long shelf life have been identified as the vehicle for transmission of Listeria.

Since the 1970s, the Public Health Service of the UK has investigated the presence of L. monocytogenes in food and the environment. From 60,000 food items, 4% of RTE foods and 19% of food requiring cooking or reheating harboured L. monocytogenes (McLauchlin, 1996b). In the UK, an outbreak occurred between 1987 and 1989, probably due to imported paté. Two serovar 4b subtypes were identified in humans and these subtypes corresponded to 96% of all isolates collected from a single manufacturer. However, a smaller amount of both subtypes had been isolated before and after the outbreak, and from other foodstuffs and manufacturers. In 1989, the English government advised vulnerable people against eating soft cheeses and paté (McLauchlin et al., 1991). In 1991, pregnant women were again adviced to avoid infection from

37 food (McLauchlin, 1996b). A survey of food products between 1991 and 1992 revealed a significant decrease in L. monocytogenes contaminated soft cheeses and paté (one sample of 251 soft cheeses and 1 of 40 paté samples). However, other raw or minced meat products such as poultry samples (21 of 32), beef samples (9 of 26), lamb samples (8 of 20), pork samples (9 of 32), sausages samples (8 of 23) and raw fish samples (3 of 24) were more frequently contaminated with L. monocytogenes (MacGowan et al., 1994). Most outbreaks in recent years have been due to contaminated sandwiches served in hospitals to vulnerable groups (Little et al., 2008; Shetty et al., 2009; Coetzee et al., 2011; Little et al., 2012).

In the French outbreak in 1992, the vehicle of infection was RTE products: 11,343 L. monocytogenes food isolates were collected and 220 food products harboured the endemic phage type. The epidemic strain serovar 4b, pulsovar 2/1/3 was detected in 154 different food products: pork tongue in aspic (n=112), other meat products (n=19), cheeses (n=12), diverse foods (n=11); and, even from food-processing environments (n=17) and animals (n=4) (Goulet et al., 1993; Jacquet et al., 1995). In 1992, a control measure previously only related to cheese production (section 2.9.2) was applied to all meat-processing manufacturers and expanded to all foodstuffs the year after (Goulet et al., 2001). In 1993, contaminated rillettes harbouring L. monocytogenes serovar 4b caused an outbreak in France involving 39 individuals (Goulet et al., 1998; de Valk et al., 2000).

In France two large national samplings of RTE products (1993–1994, 5,809 samples: 1995– 1996, 6,147 samples) analysed the presence of L. monocytogenes. In the first national study (1993–1994), L. monocytogenes was isolated from meat products (12.6% of 1533), seafood or fish products (10.4% of 841), prepared salads (5.2% of 1740), and dairy products (5.4% of 1695). The proportion of RTE foods contaminated with ≥ 100 cfu L. monocytogenes per g was 1.3% in 1993–1994 and 0.8% during 1995–1996. In the second national study in 1995–1996, the number of contaminated foods was slightly lower (Goulet et al., 2001). During 1995–1996, PFGE type “a1” serovar 1/2a (187 isolates) was predominant among 287 L. monocytogenes isolates collected from five French pork slaughter houses combined with cutting plants. In total, 17 PFGE types with ApaI enzyme were identified and two genotypes (with 186 and 16 isolates) were isolated from four of these plants. After sanitisation, some genotypes were still isolated from two plants (Giovannacci et al., 1999).

During 2000 and 2001 in France, 398 L. monocytogenes clinical isolates and 271 food isolates were collected, but only 179 (45%) of the human isolates and 270 food isolates were characterised: seafood (79 isolates), dairy products (21), pork products (126) and other products (44). Among the food isolates, PCR serogroup IIa was predominant and mostly found in seafood isolates (89%), dairy products (57%) and pork products (39%). PCR serogroup IVb was also present in these products (5% in seafood, 14% in dairy products and 16% in pork products). In contrast, PCR serogroup IVb was predominant among human isolates (44%); IIa was less common (30%). Among human isolates, PFGE yielded 35 AscI PFGE types. Isolates from seafood (28 AscI PFGE types) and dairy products (9 AscI PFGE types) were the most diverse, whereas, isolates among pork products yielded 10 types, of which two types dominated that differed by one band below the size of 384.5 kb (Hong et al., 2007).

In 2006, the occurrence of L. monocytogenes was reported in 13 French pork-meat processing plants (Thévenot et al., 2006). Among 1028 isolates, four of the most common serovars that cause infection in humans were identified: serovar 1/2a (49.9%), serovar 1/2c (20.4%), serovar 1/2b (12%) and serovar 4b (8%). Furthermore, 83 ApaI PFGE types were identified and several were widespread in all 13 plants. The bacterium was probably introduced into the processing plants via contaminated raw meat (Thévenot et al., 2005 and 2006). In France, the bacterium is mostly found in dairy products (frequently in soft cheeses), cooked pork meats (especially RTE dishes, such as potted meat and pork tongue in aspic), poultry, meats, smoked fish and seafood (de Valk et al., 2001).

In Italy, two large national samplings of food products, environment, and human samples were taken in 1990–1999 and 2002–2005. Among L. monocytogenes food isolates serovar 1/2a was similar in both periods, whereas, among human isolates, serovar 4b decreased and serovar 1/2a increased. In the second period (2002–2005), serovar 1/2a was prevalent in humans (51%), food (39%) and environmental (51%) isolates, whereas, the prevalence of serovar 4b was 25% (humans), 10% (food) and 22% (environment). In the second period, serovar 1/2c was dominant in meat products (32%), especially Italian salami (69%), however, serovar 1/2a was frequently found in this product (23%). Around 80% of cheese isolates were from Gorgonzola cheese (blue- veined cheese) and among those isolates, serovar 1/2a was dominant (31%). Furthermore, in the second period, 85 AscI pulsotypes were identified among 558 food isolates. One of these pulsotypes was predominant in cheese, and was identical to 13 (22.8%) human isolates from the second period, comprising 57 human isolates (Gianfranceschi et al., 2009). In 1997, a large outbreak of febrile gastroenteritis occurred in Italy and was associated with corn contaminated with L. monocytogenes. All reported cases (1566 students and staff from two primary schools) had eaten salad with corn and tuna at the school’s cafeteria. L. monocytogenes serovar 4b with similar DNA profile were identified in 123 (among 141) feacal samples and one (among 40) blood sample (Aureli et al., 2000).

In 2005–2008, the occurrence of L. monocytogenes was investigated in an Iberian pork- processing plant and slaughterhouse in Spain, which exported pork meat products to the USA. Isolates (n=541) were collected from raw and dry-cured products, carcasses, the environment, and equipment. Four of the most common serovars were found among the isolates: serovar 1/2a was dominant (68%), followed by serovar 1/2b (21%). Furthermore, in combined AscI and ApaI profiles, 29 pulsotypes were identified; 15 pulsotypes belonged to lineage II and 14 pulsotypes to lineage I. Among 349 (64%) raw products isolates, serovar 1/2a accounted for 74% of isolates and 25 pulsotypes were identified. However, only three pulsotypes were dominant (73%) and found in all types of samples (Ortiz et al., 2010).

In Finland, 295 L. monocytogenes isolates were characterised from diverse food products from 41 producers in nine countries that were sampled between 1988 and 1999. Among the food isolates, 241 were from 28 Finnish producers and 54 food isolates were from Poland (19 isolates), Hungary (16 isolates), Estonia (7 isolates), France (5 isolates), Denmark (3 isolates), Norway (2 isolates), Belgium (1 isolate) and Spain (1 isolate): the isolates were from meat products (100), poultry (40), dairy (14), fish (76), vegetables (39), and other products (26). Six serovars sharing lineage I (1/2b, 4b), lineage II (1/2a, 1/2c, 3a) and lineage III (4c) were detected. AscI pulsotype yielded 61 types and ApaI pulsotype yielded 47 types, and a

39 combination of profiles of both enzymes yielded 66 pulsotypes. Of these, 10 pulsotypes were found in various food products. Furthermore, 17 pulsotypes were found in food products from two or more producers from several countries and isolated in different years e.g. in fish products (Autio et al., 2002).

Between 1990 and 1997, the presence of L. monocytogenes at an ice cream plant in Finland was investigated: 1225 samples were collected from unpasteurised raw material and ice cream and 1320 samples from the processing environment and equipment. L. monocytogenes was isolated from 71 samples (2.8%). Two different L. monocytogenes serovars were identified among 41 isolates, serovar 1/2b was predominant with 37 isolates (90.2%) and serovar 4b had four isolates (9.8%). All serovar 4b isolates were from environmental/equipment samples and shared one PFGE type identified by three restriction enzymes; whereas, serovar 1/2b isolates were found at all sites, except raw materials and distributed into 11 PFGE types. Serovar 1/2b PFGE types mostly differed by one or two fragments and one predominant PFGE type accounted for 26 isolates (63%). Only two serovar 1/2b PFGE types were found in ice cream (Miettinen et al., 1999a).

L. monocytogenes isolates from food products (n= 486) collected between 1997 and 1999 originated from 17 Finnish producers: five fish plants (206 isolates), eight meat plants (139 isolates), three dairy plants (38 isolates) and one poultry plant (103 isolates). Only 121 isolates were characterised: the most common serogroup was 1/2 (90%), with 4b only constituting 3.3%. PFGE with AscI yielded 42 types, PFGE with SmaI yielded 24 types and a combination with two enzymes yielded 46 types. Of these, 24 types were each represented by only one isolate. One PFGE type was isolated from three different plants: fish, meat and poultry but L. monocytogenes serovar 4b was only found in two plants (Aarnisalo et al., 2003).

In Denmark in 1988, the prevalence of L. monocytogenes was 26% in minced beef and 47% in raw poultry (Skovgaard, 1988). In another Danish study (Skovgaard & Morgen, 1988) 67% of minced beef samples harboured Listeria spp. and 28% L. monocytogenes. From neck-skin samples of 17 carcasses of poultry, 94% harboured Listeria spp. and 47% L. monocytogenes. Serogroups 1 and 4 were common among the samples (Skovgaard & Morgen, 1988). In 1989– 1990, 17 isolates were recovered from various sausages and minced raw meat manufactured by five Danish producers. In addition, 13 isolates from meat and the environment of a slaughterhouse were characterised. In meat products, 89% shared serogroup 4 and 11% serogroup 1; all isolates from the slaughterhouse belonged to serogroup 1 (Nørrung & Gerner- Smidt, 1993). In 2000, the occurrence of L. monocytogenes in a Danish turkey processing plant and turkey products was reported. L. monocytogenes was presented in 53 of 204 samples from the processing line and 12 of 101 samples from turkey products. Two common ApaI PFGE types were found in both the plant and the products and identified in 9 and 1 human cases among 41 human isolates collected during and after the study period (Ojeniyi et al., 2000).

The first documented case of food-borne listeriosis occurred in Sweden in 1993, due to the consumption of medwurst (Loncarevic et al., 1997). Another meat-borne listeriosis outbreak occurred from October 2013 to October 2014 and included 49 patients. The National Food Agency of Sweden analysed 70 meat product samples from three different manufacturers and

40 found different PFGE types. Five of these samples harboured identical AscI PFGE type encountered in clinical samples (Public Health Agency of Sweden reports from 2015 & 2016).

In the USA, a multi-state listeriosis outbreak, linked to the consumption of cantaloupe melon, occurred in 2012: 146 cases were involved and the fatality rate was 20.5%. PFGE typing with AscI and ApaI restriction enzymes identified two closely related types serovar 1/2a and two closely related types serovar 1/2b in both cantaloupes and patients (Laksanalamai et al., 2012; Lomonaco et al., 2013).

The European Union Reference Laboratory for L. monocytogenes (EURL for Lm) coordinates a European network of 35 National Reference Laboratories (NRLs) that are responsible for typing food isolates. The PFGE standard operating procedure (EURL SOP) has been used by EURL since 2007. To facilitate the exchange of PFGE profiles between laboratories, EURL recommends the NRLs to use EURL SOP, and for this reason, the database EURL Lm DB was created. At the same time, the European Centre for Disease Prevention and control (ECDC) created the European Surveillance System (TESSy) to exchange PFGE profiles and epidemiological information from clinical isolates (Felix et al., 2013; Michelon et al., 2015).

As L. monocytogenes is capable of persisting for long periods in processing plants producing a wide variety of RTE foods and is resistent to heat, cold, salt and acidity, the food-processing environment has been the main source of contamination in the final product. Listeria has a large impact on global food industries, because the economic losses incurred every year are extremely high (Camargo et al., 2016).

2.9.2 Contamination in cheeses

Cheeses are very complicated microcosms. The raw product, i.e. the milk, is produced in a contaminated environment, not least with faecal impurities. Cheese is manufactured at the optimum environment for growth of several pathogenic bacteria. Furthermore, this dairy product constitutes an excellent culture medium for pathogenic as well as non-pathogenic microorganisms. This is especially true for soft cheeses with their high moisture contents and water activity which offer favourable conditions for bacterial survival and growth. The cheese may become contaminated as a result of unsanitary conditions caused by food-contact surfaces, dirty hands, airborne organisms or packaging material. Cheese, particularly if prepared from untreated milk, has been associated with a wide range of infections.

In 1974, a study (Sipka et al., 1974) in Yugoslavia on the survival of L. monocytogenes in white brined cheese made of unpasteurised cow’s milk lead to the recommended use of only pasteurised milk in the manufacture of fresh and ripened cheeses. However, in 1987 the first documented sporadic case of listerial meningitis due to heavily contaminated French pasteurised soft country cheese was reported in Britain. The implicated L. monocytogenes strain, serovar 4b, was found in the package consumed by the patient and was indistinguishable from the patient’s isolate with phagetyping. A further six cheeses from the same manufacturer were analysed without finding L. monocytogenes (Bannister, 1987). A second reported sporadic case in 1988 was associated with the consumption of vacuum-packed fresh goat cheese (Anari) from an

English manufacturer (X). Forty cheese samples from this producer were obtained from retail outlets and the processing plant during 11 months and 57% of the samples were contaminated with L. monocytogenes. All 73 isolates collected belonged to serovar 4b and were indistinguishable by phagetyping from the patient’s isolates. During 1987 and 1988 in Britain, 533 clinical isolates (one per case) were collected, of which 378 (71%) belonged to serovar 4b. From 277 isolates that were further analysed, seven were indistinguishable by sero- and phagetyping from isolates found in the English manufacturer X (McLauchlin et al., 1990c). Routine surveillance in the UK revealed the contamination of soft cheeses with L. monocytogenes decreased from 10% in 1987 to 1% in 1995 and even the cfu of L. monocytogenes per g declined (McLauchlin, 1996b). In 1997–1998, seven human cases infected by identical strains found in cheeses produced in England were identified (McLauchlin et al., 2004).

In 1986 in France, the first control measure was established for cheese-processing manufacturers that sold cheeses to the USA. In 1988, after the Swiss cheese outbreak in 1983–1987, French authorities expanded the control to cover all cheese-processing plants (Goulet et al., 2001). During the 1990s, three outbreaks occurred in France due to consumption of soft cheeses contaminated with L. monocytogenes serovar 4b. The first documented outbreak was associated with soft raw milk cheese, Brie de Meaux, in 1995 and involved 36 patients. The French NRC isolated the epidemic strain from four samples of Brie de Meaux among 2500 food isolates (Goulet et al., 1995; Vaillant et al., 1998; de Valk et al., 2000; de Buyser, et al., 2001). The second outbreak was in 1997 due to consumption of the soft raw milk cheese Pont l’Évêque, Livarot that infected 14 people (Jacquet et al., 1998; de Valk et al., 2000; de Buyser, et al., 2001). In a case-control study in France during 1997 (de Valk et al., 1998), 49% of enrolled sporadic cases of listeriosis could be attributed to eating soft cheese and that “Soft cheese may account for a substantial proportion of sporadic listeriosis” (de Valk et al., 1998). The third documented outbreak in 1999 reported three listeriosis cases due to the consumption of the soft raw milk cheese Epoisses (de Valk et al., 2000).

French investigators suggested a substantial part of the decrease in L. monocytogenes in RTE French food, including cheese, during the 1990s was related to control measures implemented at the food production level. Milking hygiene improved and cows with L. monocytogenes mastitis were not used for milk production. In addition, several precautionary measures during raw milk cheese preparation were taken (de Valk et al., 2000; de Buyser et al., 2001; Sanaa et al., 2004). As a consequence of fewer contaminated ready-to-eat products, the number of listeriosis cases in France decreased by 68% from 1987 to 1997: the number of listeriosis cases per million population and year in France decreased from 5.2 in 1995 to 3.8 in 1996 (Jacquet et al., 1998). “Because of the importance in the rural economy of food made from raw material of animal origin, especially in France, a great deal of effort has been placed on hygiene during production, transformation and distribution of cheese” (Sanaa et al., 2004).

In Switzerland during 1983–1987, a Swiss soft cheese, Vacherin Mont d’Or, caused 122 cases of listeriosis (Bille & Glauser, 1988; Büla et al., 1995). More than 200 soft cheeses of different brands and types, both domestic and imported, were analysed for the presence of L. monocytogenes. Among the soft cheeses analysed, 8 to 10% harboured L. monocytogenes. However, the only cheese isolates that matched the two particular epidemic human strains of L.

42 monocytogenes serovar 4b with two different phagetypes, were found in Vacherin Mont d’Or soft cheese (Bille, 1988; Bille & Glauser, 1988). Vacherin Mont d’Or was also sold in Sweden during the same period, however, currently Vacherin Mont d’Or is made of pasteurised milk (Personal communication: Tham, 2017). In 1986 in Switzerland, 9 of 70 soft cheese samples (7 smear and 2 white mould cheeses) harboured L. monocytogenes and 7 of 9 isolates belonged to serovar 4b. Four of the nine cheeses were made from pasteurised and five from raw milk (Breer, 1986). In 2005, a small cheese outbreak involving 10 individuals was detected in Norwest, Switzerland. A local manufacturer produced the implicated soft cheese, known as tomme cheese: all cheese and butter samples that were analysed of this brand harboured L. monocytogenes serovar 1/2a. Of two PFGE types identified by AscI and ApaI enzymes, one type was predominant among human and cheese isolates (Bille et al., 2006).

Between 1992 and 1994, 32 French unpasteurised soft cheeses, 32 French pasteurised soft cheeses, 20 unpasteurised and 2 pasteurised German cheeses were purchased on the German market. The presence of L. monocytogenes was higher (47%) in French unpausterised cheeses, but was lower (9% each) in both French pasteurised and German cheeses (Eppert et al., 1995). In 1999, German researchers analysed 374 cheese samples produced in six European countries from raw milk and pasteurised milk. The results confirmed samples from four countries were contaminated with L. monocytogenes (6.4%), including France (5 of 150), Germany (11 of 120), Italy (4 of 23), and Austria (1 of 10); from Switzerland 1 of 22 samples contained L. innocua, and from Denmark, no Listeria species were isolated (0 of 4). The incidence of L. monocytogenes was highest (7.6%) in semi-soft cheeses, followed by soft cheeses (6.3%) and hard cheeses (4.4%). Furthermore, 8% of contaminated cheeses were manufactured from pasteurised milk and 4.8% from raw milk. The high incidence of listeriosis in Europe was associated with red smear cheeses. In 2000, German authorities recalled 80 tons of soft and semi- soft red smear cheeses due to the presence of L. monocytogenes (Rudolf and Scherer, 2001).

In 2006–2007, a large listeriosis outbreak (189 cases) occurred in Germany due to the consumption of acid curd soft cheese made from pasteurised milk and ripened with a red smear. The implicated cheeses were produced by one manufacturer but sold under distinct brand names. Eleven cases were associated with pregnancy, among the adults, 62% were male, and the overall fatality rate was 14%. The L. monocytogenes isolates shared an identical AscI PFGE type serovar 4b (Koch et al., 2010). In Austria between 1997 and 2000, the occurrence of L. monocytogenes in 181 small-scale Austrian cheese-processing plants was investigated: 182 L. monocytogenes isolates were recovered from 50 manufacturers (mostly from smear cheese and cheese) and 14 plants were heavily contaminated. Among 177 isolates, 88 (49.7%) shared serovar 1/2b, 47 (26.6%) isolates shared serovar 1/2a, 39 (22%) serovar 4b and 3 (1.7%) serovar 1/2c. Twenty-six manufacturers were contaminated with serovar 1/2a, 18 with serovar 1/2b, and 21 with serovar 4b. PFGE typing with AscI and SmaI enzymes identified 76 pulsotypes among 182 isolates. One AscI pulsotype serovar 4b was identified in eight manufacturers. However, among 41 human isolates from 1997–2002, no pulsotype could be associated with cheese manufacturers (Wagner et al., 2006).

In 2009–2010, a multinational outbreak occurred in Germany, Austria and the Czech Republic, where two different L. monocytogenes clones serovar 1/2a caused 34 cases of listeriosis. The source of infection was a red-smear cheese (Quargel) produced in Austria. These cheeses were

43 also distributed to Poland and Slovakia, where about 50 and 60 tons of cheese were recalled (Fretz et al., 2010; Schoder et al., 2012; Rychli et al., 2014). In 2015, an Austrian study reported 108 cheeses purchased through the Internet from seven European countries, only 19 cheeses (17.6%) were properly labelled and conformed to European requirements, and L. monocytogenes was detected in two cheeses (1.9%). A semi-hard cheese from The Netherlands yielded 9.5 x 103 cfu/g at the end of expiry date and a French soft cheese < 10 cfu/g. According to EFSA, L. monocytogenes prevalence among 3452 soft and semi-soft cheeses analysed in the European Union was 0.47% at the end of expiry date (Schoder et al., 2015).

In a Belgian study, L. monocytogenes isolates recovered from cheeses and human cases during 1990–1992 were characterised. The esterase typing revealed that among cheese isolates esterase type I B serovar 1/2a was dominant (44.2%), followed by esterase types II B and C serovar 4b (10.6%), whereas, among human isolates esterase types II B and C serovar 4b were dominant (40%) followed by esterase type I B serovar 1/2a (4.2%) (Gilot & Andre, 1995). In 1997, a fatal case of listeriosis associated with the consumption of French Camembert cheese was reported in Belgium: the isolates from both patient and cheese belonged to serovar 1/2a (Gilot et al., 1997).

The first reported case of listeriosis due to the consumption of cheese in Italy was in 2003. Indistinguishable L. monocytogenes PFGE pulsotype, serovar 1/2b isolates, were recovered from the patient’s blood sample and a Gorgonzola cheese made of pasteurised milk (Italian blue- veined cheese) that the patient had in the refrigerator. Cheeses from the same brand purchased in food stores and sampled from the production plant harboured 100 to 1200 cfu/g of identical pulsotype. Moreover, three previous studies conducted in Italy and USA, revealed 5–9% of Italian blue-veined cheese samples were contaminated with L. monocytogenes (Gianfranceschi et al., 2006). Between 2002 and 2005 in Italy, 94 L. monocytogenes cheese isolates were collected, mostly from Gorgonzola cheese (blue-veined cheese), and 25 isolates from cheese-processing plants. Among the cheese isolates, serovar 1/2a was dominant (67 isolates) followed by 1/2b (20 isolates), 1/2c (3 isolates) and 4b (2 isolates). Serovar 1/2a was also dominant in the environment (92%), whereas, serovar 4b was only found in 8% of samples. One pulsotype was predominant both in cheese (44) and environmental (17) isolates (Gianfranceschi et al., 2009).

Between 2011 and 2012 in Italy, 2205 cooked meat products and 1292 soft and semi-soft cheese products were collected from 12 manufacturers. Among 901 meat samples and 894 cheese samples, 1.7% of meat samples and 1.9% of cheese samples harboured L. monocytogenes. Of 894 meat samples and 398 cheese samples analysed at the end of their shelf life, 2.1% of meat samples and 1% of cheese samples harboured L. monocytogenes. One of four meat product types (Spalla cotta) was most frequently contaminated (16%), and among eight types of cheese products, three harboured L. monocytogenes: Taleggio (14.5%), Gorgonzola (4.2%) and Brie (2.3%). Of all 85 isolates characterised, serovar 1/2a was predominant (84.7%), serovar 1/2b, 7% and serovar 4b and 1/2c, 3.5% each. PFGE with AscI and ApaI yielded 38 pulsotypes, with four pulsotypes accounting for 41.2% of all 85 isolates; these were associated with meat products or soft and semi-soft cheese (Iannetti et al., 2016).

In Spain during 1992–1993, six types of regional cheeses from Asturias manufactured with pasteurised cow, ewe and goat milk were collected. In total, 101 samples of short-ripened cheeses were collected from five farms (home-produced) and one factory (manufactured) and

44 analysed. Nine samples of four homemade cheese types harboured L. monocytogenes, with one cheese type having the highest incidence of positive samples (7 samples). Thirty L. monocytogenes isolates from positive samples were further characterised: 10 isolates shared serovar 1/2a, nine isolates shared serovar 4b and 1/2b each. PFGE with ApaI and SmaI enzymes yielded two PFGE types of serovar 4b, three types of serovar 1/2a and two types of 1/2b (Margolles et al., 1996; Margolles et al., 1998a; Margolles et al., 1998b). In Galicia (Northwest of Spain), the prevalence of L. monocytogenes in a dairy processing plant was studied during one year: 376 samples from i.a. the environment, raw milk, cheese, untreated water, and butter were collected. Eleven (3%) samples harboured L. monocytogenes. Although, a high incidence was observed in raw milk, floors from raw milk reception areas and untreated water, no positive sample was found among finished products such as cheese and butter (Franco-Abuín et al., 1996).

A retrospective study detected a listeriosis outbreak, involving 30 individuals, in Portugal between 2009 and 2012 and due to the consumption of cheese (queijo fresco) from cow and goat pasteurised milk. The L. monocytogenes isolates recovered from cheeses and the environment of a Portuguese manufacturer shared the same PFGE type serogroup IVb as the human isolates (Magalhães et al., 2015).

In Denmark between 1988 and 1992, 26 L. monocytogenes isolates were collected from cheeses (soft, feta, hard, white moulded and blue-veined), raw milk (2 isolates) and the environment (9 isolates) from four Danish dairies. MEE identified one predominant type among these isolates: ET 4 serogroup 1, followed by ET 2 serogroup 4 and ET 4 serogroup 4, which was the second most frequent among human isolates. ET 1 serogroup 4 was commonly identified in human isolates but was only found in one cheese isolate. In 1989–1990, ET 1 and ET 4 serogroup 4 caused most human listeriosis cases in Denmark (73%) and were involved in two outbreaks in Switzerland and Massachusetts, USA, in the 1980s (Nørrung & Skovgaard, 1993).

In a Swedish study, 333 cheese samples from twelve countries were collected between 1989 and 1993. Only cheeses from three countries harboured L. monocytogenes; 18 (10.3%) samples of French cheeses were contaminated, with sixteen samples sharing serogroup 1 and two serogroup 4, and one sample each from German and Italian cheeses shared serogroup 1. Of 30 French soft cheeses made of raw milk, 13 (43.3%) tested positive for L. monocytogenes. The majority of the positive French cheeses harboured < 100 cfu L. monocytogenes serogroup 1/2 per g; however, the serovar was 4b in two cheeses with the largest number (2300 and 100,000 cfu/g). The French cheeses positive for L. monocytogenes, Brie de Meaux, Pont l’Évêque, Livarot and Epoisses, were available on the Swedish market between 1989 and 1993 (Loncarevic et al., 1995; Personal communication: Loncarevic, March 2017). During this time, Brie de Meaux was one of the most popular soft raw milk cheeses sold in Sweden (Personal communication: Tham, 2017).

During 1980s and 1990s, large outbreaks and sporadic cases of food-borne listeriosis due to closely related strains belonging to serovar 4b were reported (Kathariou, 2002). Between 1983 and 1987, a large human listeriosis outbreak involving 122 individuals occurred in Switzerland due to contaminated soft cheeses (Bille & Glauser 1988). In 1985, a large outbreak involving 142 individuals was reported in California, USA, and was due to soft cheese (Linnan et al., 1988). A Danish outbreak involved 26 individuals and occurred during 1989–1990: the vehicle

45 of transmission was probably blue mould cheese (Jensen et al. 1994). In the French 1992 outbreak, the epidemic strain serovar 4b (pulsovar 2/1/3), which infected 247 individuals, was isolated from several RTE products, i.a. 12 cheeses. It also caused disease in previous years with 6 to 27 cases annually in France (Jacquet et al., 1995). The epidemic cheese-borne strains from Switzerland, California, Denmark and France belong to the ‘‘long Swiss epidemic phagetype’’ (known from the Swiss soft cheese outbreak) and were closely related regarding serotyping, ribotyping, phagetyping, MLE and PFGE (Buchrieser et al., 1993; Rocourt et al., 1993; Tham, 1993; Jacquet et al., 1995; Salvat et al., 1995; Kathariou, 2002). This phagetype was common among the Swedish 4b human isolates (63.5%) during the period 1976–1985 (Ericsson et al., 1996).

In Philadelphia, USA, a listerial outbreak in 1986–1987 was investigated: 36 individuals (32 non-pregnant adults and 4 pregnant cases) were involved and there were 16 fatalities (44%). L. monocytogenes was isolated from Brie cheese eaten by one patient that matched with patient’s isolate (Schwartz et al., 1989). Between 1986 and 1988, a surveillance study in the USA investigated the prevalence of Listeria species in dairy environments and products. Of 1370 plants inspected, 37 (2.7%) had dairy products contaminated with Listeria and 29 plants were contaminated with L. monocytogenes (Kozak et al., 1996).

Between 2001 and 2003 in New York State, USA, 495 L. monocytogenes isolates originating from human cases (120), food products (74), animal farms (221) and urban/natural environments (80) were collected. PFGE with AscI yielded 244 types and PFGE with ApaI yielded 266 types: a combination with two enzymes yielded 310 PFGE types. Of those, a majority (n=235, 47.5%) types were each represented by one isolate only. Two PFGE types serovar 4b were predominant, one of which was predominant among humans (16 isolates, 13.3%); however, this was only isolated from two different foods: 2 turkey breast samples and 2 white cheese samples. This type was responsible for a multi-state outbreak in the northeastern USA in 2002, where 46 individuals were affected due to consumption of sliced turkey meat. The second most common PFGE type was identical to the “long Swiss epidemic phagetype” and accounted for 15 isolates identified from all sources and during the entire study period. This strain caused multinational listeriosis cheese-borne outbreaks, e.g. in California (1985) and Switzerland (1983–1987) (Fugett et al., 2007).

2.9.3 Contamination in fish

One of the most frequently L. monocytogenes-contaminated RTE food is vacuum-packed gravad and smoked fish (EFSA 2011; Kramarenko et al., 2013, 2016). L. monocytogenes has been isolated from gravad, hot and cold-smoked rainbow trout (Oncorhynchus mykiss) and salmon (Salmo salar) around the world. Løvdal (2015) summarises results from scientific publications from 2000 and highlights a prevalence of L. monocytogenes in the retail sector for cold-smoked salmon of 0–61%, averaging 9.8%. The serovar most often encountered in these products is serovar 1/2a (Johansson et al., 1999; Rørvik, 2000; Vitas et al., 2004; Meloni et al., 2009; Kramarenko et al., 2013; Gyurova et al., 2015; Kramarenko et al., 2016).

During the 1990s, outbreaks of listeriosis occurred in Sweden (Ericsson et al., 1997) and in

Finland (Miettinen et al., 1999b) due to rainbow trout. This generated an increased global interest to determine the prevalence of L. monocytogenes in RTE fish products, processing plants and environments. For example in Belgium (Van Coillie et al., 2004; Uyttendaele et al., 2009), Brazil (Cruz et al., 2008), Canada (Kovačević et al., 2012), Estonia (Kramarenko et al., 2013, 2016), Finland (Lyhs et al., 1998; Johansson et al., 1999), France (Besse et al,. 2008; Beaufort et al., 2007), Greece (Soultos et al., 2014), Iran (Fallah et al., 2013), Ireland (Corcoran et al., 2006; Dass et al., 2011), Island (Gudmundsdottir et al., 2005), Italy (Latorre et al., 2007; Di Pinto et al., 2010), Japan (Inoue, et al., 2000; Nakamura et al., 2004), Nigeria (Salihu et al., 2008; Meloni et al., 2009), Poland (Medrala et al., 2003; Kwiatek, 2004), Spain (Dominguez et al., 2001; Vitas et al., 2004; Cabedo et al., 2008; Garrido et al., 2009; Gonzalez et al., 2013), Norway (Rørvik et al., 2000), Sweden (Peiris et al., 2009; Thisted Lambertz et al., 2012).

Gravad and cold-smoked salmons are vacuum-packed RTE products with a generous best before date of normally 4 to 5 weeks and constitute an optimal environment for the facultative anaerobic, psychrotrophic organism Listeria. The temperature of the cold-smoke, salt content and refrigeration storage will not prevent the growth of L. monocytogenes (Ben Embarek, 1994; Rørvik, 2000; Vaz-Velho et al., 2001). The near neutral pH values (6.0–6.5) and the high aw values (0.96–0.98) in smoked salmon create an environment suitable for L. monocytogenes (Rocourt et al., 2000; Uyttendaele et al., 2009). However, smoke, salt, and vacuum packaging extends shelf life and renders the food products attractive to consumers. Nevertheless, the occurrence of L. monocytogenes in vacuum-packed smoked fish products constitutes a serious food safety risk (Uyttendaele et al., 2009; Tocmo et al., 2014; Løvdal et al., 2015).

The prevalence of L. monocytogenes in raw fish is generally low (Autio et al., 1999; Johansson et al., 1999; Fonnesbech Vogel et al., 2001; Markkula et al., 2005). However, the possibility that raw fish is a source of contamination in the processing equipment and environment cannot be excluded (Rørvik, 2000; Gudmundsdottir et al., 2005). L. monocytogenes probably arrives at the plant via raw material and establishes itself in the processing equipment and processing environment (Huss et al., 2000; Fonnesbech Vogel et al., 2001; Tompkin 2002; Medrala et al., 2003; Cruz et al., 2008). Thus, L. monocytogenes will remain and contaminate the products in the final stages of fish processing during brining, slicing and packaging (Autio et al., 1999; Fonnesbech Vogel et al., 2001). Some strains of L. monocytogenes may be specially adapted to the processing plant environment, and are difficult to sanitise through standard hygiene procedures (Fonnesbech Vogel et al., 2001). Especially salting and slicing machines are reported as difficult to clean and keep Listeria-free. Cold-salted and smoked products are usually sliced, causing contamination of the slicing machine (Johansson et al., 1999). However, there are no differences in the adherence of persistent and non-persistent strains over extended cultivation times (Lundén et al., 2003).

As RTE fish products are often contaminated by L. monocytogenes, outbreaks would be expected to be more frequent. One reason for the absence of fish-borne outbreaks could be that counts of L. monocytogenes frequently follow the food safety criterion of 100 cfu/g during the product’s self-life. In addition, the long incubation period for listeriosis renders it difficult to determine the food source (Tompkin, 2002). Another theory presented by Rocourt et al. (2000) is that outbreaks due to fish consumption are rare because factories producing fish products are often smaller than factories processing milk and meat products.

Vacuum-packed gravad and cold-smoked salmon should be considered possible sources of listeriosis in Sweden. Since the mid 1990s, cold-smoked and gravad salmons have been more frequently contaminated with L. monocytogenes than cheeses and their consumption has increased in Sweden, and in several other countries (Loncarevic et al., 1998a; Tompkin et al., 2002; Knapp et al., 2007; Thisted Lambertz et al., 2012, 2013).

In 1980, an outbreak of listeriosis in New Zealand involved 19 perinatal cases and 3 adults. The L. monocytogenes isolates belonged to serovar 1/2a. Shellfish and raw fish were suspected as the sources of infection, as mothers frequently consumed these products, although no foods were analysed (Lennon et al., 1984; Sutherland et al., 2003). Another small outbreak in New Zealand, due to the consumption of smoked mussels produced in a local manufacturer, occurred in 1991– 1992. The L. monocytogenes isolates from four human cases and mussels shared indistinguishable ApaI and SmaI PFGE type serovar 1/2a. Analysis with MvLST indicated this type shared ST01-N, which is frequently identified in seafood, seafood-processing plants, and human cases in New Zealand (Brett et al., 1998; Cruz et al., 2014).

In Switzerland during 1988–1990, 990 imported fish products were collected and L. monocytogenes was isolated with high prevalence in gravad salmon (26.9%), cold-smoked fish (13.6%), and hot-smoked fish (8.9%). Serovar 1/2b was predominant (58.6%) in these products, 1/2a was 19.8% and 4b was 14.4% (Jemmi, 1990). In another study, 58 (8.4%) of 691 hot- smoked fish and 49 (11.3%) of 434 cold-smoked fish harboured L. monocytogenes. In hot- smoked fish samples, 50% of isolates shared serovar 1/2b, 39.6% shared serovar 1/2a and 1.7% shared serovar 4b. In cold-smoked fish samples, serovar 1/2b constituted 38.8% of isolates, 22.4% was serovar 1/2a and 26.5% was serovar 4b (Jemmi et al., 1992).

In 1993–1995 in Switzerland, 72 L. monocytogenes isolates were collected from diverse fish products from six countries and 12 producers: Norway (1), Denmark (2), New Zealand (1), Great Britain (4), France (3) and Germany (1). Only 23 fish isolates were compared with 47 human isolates collected in 1993–1994. Serovar 1/2a was found in 47 (65%) fish isolates, and in 17 (36%) human isolates. In contrast, L. monocytogenes isolates serovar 4b accounted for 21 (45%) isolates in humans and one (1.4%) in fish: even serovar 1/2b was prevalent in humans (8 isolates) and in fish (2 isolates). Serovar 1/2c (4 isolates) and 3a (2 isolates) were only found among fish isolates. Serovar 7 was found in 12 fish isolates. Human and fish isolates shared three electrophoretic types (ETs) that were common among human isolates. One of these ETs is frequently associated with meat and meat products in Switzerland. Among fish isolates, PFGE identified 13 ApaI types with only one type found in both fish and human isolates (Boerlin et al., 1997). During 1992–2000 in Switzerland, imported and exported meat and fish products of various kinds and from different production plants were analysed for L. monocytogenes. The products contaminated with L. monocytogenes were marinated fish (38%), fermented sausages (15%), and cold-smoked fish (14%). The level of L. monocytogenes was < 100 cfu/g with one exception; one smoked salmon sample harboured 1100 cfu/g. The dominant serovar in meat and fish samples was 1/2a (57%): serovar 4b was present in 10% of samples (Jemmi et al., 2002).

In a French study, 1010 samples of vacuum-packed sliced cold-smoked salmon, produced by nine French plants during 2001 to 2004 were collected: 104 (10.3%) samples harboured L.

48 monocytogenes. Altogether, 693 isolates were characterised and 72.4% shared serovar 1/2a, 16.7% shared serovar 1/2b, and 10.9% shared serovar 1/2c: no serovar 4b isolate was found (Beaufort et al., 2007). The number of invasive listeriosis cases due to cold-smoked salmon consumption in France is predicted to be 307 per year. Even if this calculation is uncertain, it is based on scientific observations and can be used to manage the risk linked to the consumption of contaminated cold-smoked salmon (Pouillot et al., 2009).

A national case-control study of sporadic listeriosis cases in England and Wales during 1990– 1992 revealed listeriosis was associated with high-risk foods including shellfish and cooked chicken (McLauchlin, 1996b). Furthermore, the United Kingdom requested a national sampling survey for analysing the presence of L. monocytogenes and among 1344 cold-smoked fish, 236 (17.4%) products harboured L. monocytogenes, all samples had a level of < 100 cfu/g (ACMSF, 2009). In Ireland, 26 L. monocytogenes isolates recovered from vacuum-packed smoked salmon products were characterised in 2001. PFGE identified six ApaI types and a single PFGE type serovar 1/2a accounted for 17 (65%) isolates. Only two isolates shared serovar 4b with identical PFGE type but different MLVA profiles (Corcoran et al., 2006; Murphy et al., 2007).

In Belgium, L. monocytogenes was detected in 27.8% (25/90) smoked fish samples (Uyttendaele et al., 2009). In Austria, among 41 human listeriosis cases occurring in 1997–2000, 31% had consumed cold-smoked salmon, raw milk products and soft cheeses. Among 41 L. monocytogenes human isolates, 37 pulsotypes were identified. The human isolates and 83 food isolates collected from 1986 onwards were compared. One pulsotype serovar 1/2a from salmon was identical to one human isolate. A second pulsotype serovar 1/2a from salmon was identical to two human isolates and a third pulsotype serovar 4b from raw salmon was identical to one human isolate (Wagner & Allerberger, 2003).

In a Spanish study, 30 L. monocytogenes isolates were collected from smoked salmon products between 1995 and 2005. In total, 17 (56.7%) isolates belonged to serovar 4b and 13 (43.3%) to serovar 1/2a. From 33 Spanish clinical isolates, 10 isolates sharing pulsotype 1 serovar 4b were identical to seven salmon isolates, and one human isolate belonging to pulsotype 16 serovar 1/2a was identical to five salmon isolates (Garrido et al., 2008).

Between 2002 and 2005, 72 L. monocytogenes fish isolates were analysed in Italy. From 17 raw fish isolates, 11 (65%) shared serovar 1/2a, 3 (18%) shared serovar 1/2b and 1 (6%) shared 4b. Among 55 RTE fish isolates, with 80% from smoked salmon, 34 (62%) shared serovar 1/2a, 6 (11%) serovar 1/2b and 10 (18%) 4b. Two AscI pulsotypes were predominant in RTE fish isolates and one was identical to a human isolate (Gianfranceschi et al., 2009).

In Japan, 95 RTE seafood products were examined for L. monocytogenes between 1999 and 2000. L. monocytogenes was isolated from 12 (13%) of cold-smoked salmon or trout products. The amount of L. monocytogenes in all products was < 100 cfu/g. The predominant serovar was 1/2a (Nakamura et al., 2004). In 2004–2008, 14 (12%) of 116 minced tuna samples, 15 (9%) of 164 cod roe samples, 7 (5.7%) of 123 salmon roe samples and 1 (3%) of 33 smoked salmon harboured the pathogen. Serovar 1/2a was predominant, followed by serovars 1/2b and 4b (Miya et al., 2010).

In Brazil, a gravad salmon processing plant that used raw fish from Chilean ocean farm-raised salmon was studied during 1999–2000. Of 415 samples, 48% of raw salmon samples and 36% of vacuum-packed samples were contaminated: 179 L. monocytogenes isolates were characterised. Most isolates shared serogroup 1 (73%) and serogroup 4 was mainly found in the final product. Restriction enzyme AscI generated 28 PFGE types, ApaI generated 30 PFGE types, and a combination of both enzymes yielded 61 types (Cruz et al., 2008).

In the Scandinavian countries, RTE vacuum-packed fish products are popular foods (Johansson et al., 1999; Noriega Orozco, 2000; Lukinmaa et al., 2003; Kvistholm Jensen et al., 2016). Between 1996 and 1998 in Finland, 110 samples of diverse vacuum-packed fish products from 12 producers were collected. Twenty-two samples from six producers harboured L. monocytogenes, either serovar 1/2a (19 isolates) or 4b (3 isolates). Five of 30 cold-smoked samples (17%), 16 of 32 cold-salted rainbow trout (50%), and one from 48 hot-smoked fish samples (2%) were positive for L. monocytogenes. As most samples from one producer contained > 100 cfu/g of L. monocytogenes, a further 200 samples were collected from the plant and products. Simultaneously, 55 samples of raw fish were collected from six farms supplying fish: no L. monocytogenes was isolated from raw fish samples. One dominant PFGE type was isolated from samples of four producers, and this type has been identified among human isolates in Finland (Johansson et al., 1999). In 1998, a febrile gastroenteritis outbreak (5 cases) occurred in Finland due to contaminated vacuum-packed cold-smoked rainbow trout containing 1.9 x 105 cfu/g of L. monocytogenes. Isolates from the implicated food and stool swabs belonged to serovar 1/2a and shared identical AscI and SmaI PFGE types (Miettinen et al., 1999b).

In 2001, the presence of L. monocytogenes in two freshwater fish farms in Finland was investigated and several samples were collected from earth canal, earth ponds, a concrete-plastic tank, slaughterhouses, and slaughtered fish. Serovar 1/2a was shared by all L. monocytogenes isolates from both farms. Five AscI pulsotypes were identified, four were isolated from farm A and the fifth from farm B. Similar pulsotypes were found in both primary food production facilities and in final fish products. Three pulsotypes from farm A had been previously isolated from fish products (Katzav et al., 2006).

During an outbreak of listeriosis in 2010 in Finland, 240 food samples, including 186 (78%) fish samples, were analysed and from 323 L. monocytogenes isolates, 162 (50%) were isolated from two fishery processing plants. The same year, an official survey collected 257 samples of various foodstuffs (i.e. gravad and cold-smoked fish samples) but only fish samples were positive for the presence of L. monocytogenes, 20 (16%) of 126 fish samples. Between these two studies, 307 L. monocytogenes isolates from 204 food samples collected in both studies were characterised, of which 162 (79%) were fish samples. The serovar 1/2a PFGE type (Lm96) that caused the outbreak was identified in 76 (37%) of the food samples and isolated from three fish processing plants, one of which was located in Finland (Nakari et al., 2014).

In Denmark, cold-smoked fishery products are considered an important source of listeriosis in humans (Kvistholm Jensen et al., 2016). During 2013–2015, two outbreaks involving 20 cases, including eight fatalities (40%), occurred in Denmark and were associated with smoked fish consumption. All cases belonged to known risk groups. The two epidemic strains involved were serotype IIa, ST391 and serotype IVb, ST6 (Gillesberg Lassen, 2016).

In Norway, in 1991–1992, the frequency of L. monocytogenes in vacuum-packed smoked salmon was 11%, and in raw fish was 17% at a salmon processing plant, slaughterhouse and smokehouse. MEE identified 11 ETs and one was predominant (ET-6). This ET was the most common type in Norway and caused a small outbreak due to consumption of vacuum-packed meat sausage in 1992 (Kolstad et al., 1992; Rørvik et al., 1995). Between 1992 and 1997, 261 isolates were collected from seafoods, seafood-processing plants and seawater (from 55 different producers) and compared with 44 human isolates collected in 1992–1996. Nine ET were detected in all sources and four ET belonging to serogroups 4 and 1 (2 ET each) were most commonly represented (Rörvik et al., 2000). In another Norwegian study, 65 L. monocytogenes isolates recovered from three fish-processing plants were characterised with MLVA. Of 15 MLVA profiles identified, nine, including profile 07-07-09-10-06, corresponded to MLVA profiles from Norwegian human isolates. MLVA profile 07-07-09-10-06 is regularly found in Norwegian (30%) and Swedish (4.3%) patients (Lindstedt et al., 2008).

In Iceland, 38 samples of vacuum-packed gravad salmon, cold-smoked salmon and trout from 10 different local companies were analysed. Five (13%) of all samples and four (28.6%) of 14 gravad salmon samples harboured L. monocytogenes (Noriega Orozco, 2000).

A case of gastrointestinal listeriosis occurred in 1993 in Sweden due to the consumption of gravad salmon. The PFGE type, with ApaI and SmaI enzymes, of L. monocytogenes faecal isolates were identical to the PFGE type of the L. monocytogenes isolates identified in salmon kept in the patient’s refrigerator. This PFGE type serovar 4b was previously identified in a Swedish salmon plant and was identical to the French clone that caused an outbreak due to consumption of pig tongue in aspic in 1992–1993 (unpublish data). In 1993–1994, the National Food Administration of Sweden analysed the occurrence of L. monocytogenes in RTE fishery vacuum-packed products. The products were from 15 manufacturers and purchased in Stockholm. Four (4%) samples out of 100 harboured L. monocytogenes: one gravad rainbow trout and three hot-smoked mackerel. Furthermore, 50 samples of raw fish from three countries were analysed: Sweden (31), Norway (17) and Denmark (2). One fish sample from Sweden and one from Norway were positive and both shared L. monocytogenes serogroup 4 (Westöö, 1994). During August and December 1994, 64 vacuum-packed fish samples from 15 different brands were collected in southern Sweden. Fifteen (23%) samples harboured L. monocytogenes (gravad, hot/ cold-smoked rainbow trout and hot-smoked mackerel): one hot-smoked mackerel sample contained 11,100 cfu/g (Detmer & Blomgren, 1995).

During 1993 and 1994, 150 samples of vacuum-packed fish purchased in Sweden were collected and 190 L. monocytogenes isolates from 16 positive samples were isolated. Five samples harboured isolates belonging to serogroup 4, nine samples were serogroup 1/2, and two samples serogroup 3, but one also harboured serogroup 1/2. Twelve (20.7%) of 58 gravad, 3 (11.5%) of 26 cold-smoked and one of 66 hot-smoked samples harboured L. monocytogenes (Loncarevic et al., 1996a). The first fish borne outbreak of listeriosis in the world that could be linked to vacuum-packed rainbow trout occurred in 1994–1995 in Sweden. Six non-pregnant cases were immunosupressed and three were pregnant. All rainbow trout fish isolates belonged to serovar 4b but different PFGE types (Ericsson et al., 1997).

In 2000 in Sweden, 14 vacuum-packed gravad and cold-smoked salmon/rainbow trout samples were collected from 9 different brands. Four samples (29%) from three processing plants harboured L. monocytogenes and one sample contained 1600 cfu/g. In total, 60 fish isolates (four isolates of positive samples and isolates submitted by national laboratories from five different plants) were characterised with PFGE. Most of the fish isolates shared serogroup 1/2 and PFGE identified 17 AscI types. The most contaminated sample with 1600 cfu/g harboured at least three PFGE types, and encountered in two plants. One PFGE type was found in three plants. A comparison of the fish PFGE types with human L. monocytogenes isolates collected during the same year found the three PFGE types in the most heavily contaminated fish sample were common among human isolates (Netterby, 2000). In autumn 2002, the prevalence of L. monocytogenes in vacuum-packed gravad, cold-smoked salmon was analysed; 36 fish products were purchased in Malmö and Lund, two in Stockholm and two in Uppsala. Ten of the samples harboured the pathogen and all isolates shared serovar 1/2a and belonged to eight different AscI PFGE types that were all identified among human isolates. In six salmon samples from the same manufacturer, a common human PFGE type, with 2000–4000 cfu/g, was identified. One gravad salmon sample harboured three PFGE types and one cold-smoked salmon two PFGE types (Mandorf, 2003).

In 2001 and 2010, the National Food Administration of Sweden requested two large national samplings of food products for analysis of L. monocytogenes in RTE foods (Rosengren, 2001; Rosengren & Lindblad, 2003; Thisted Lambertz et al., 2012). In the first national study, including 3439 samples, the presence of L. monocytogenes was higher in cold-smoked and gravad fish than in other product groups: gravad fish products (13.8%: 17/123), cold-smoked (7.2%: 6/82) and hot smoked fish (5.5%: 4/73) (Rosengren & Lindblad, 2003). In the second national study, which was part of an EU-wide baseline survey, 1590 RTE foods were sampled throughout the country (packaged gravad fish, hot-smoked fish, cold-smoked fish, soft cheeses, semisoft cheeses, and heat-treated meat products). Of 525 cheese samples, 2 (0.4%) tested positive for the presence of L. monocytogenes and among 507 heat-treated meat-products, 5 (1%) was positive. Of 558 fish samples, 66 (12%) tested positive and 14% of both gravad and cold- smoked fish samples was positive. The National Food Administration of Sweden concludes these products constitute the main problem of human listeriosis in Sweden (Thisted Lambertz et al., 2012).

In 2013, the National Food Administration of Sweden, presented results from a large study where isolates of L. monocytogenes obtained from human cases during 2005–2010 (183 isolates) were compared with isolates recovered in 2010 from RTE foods (73 isolates) and from 22 meat processing plants (26 isolates), three fish processing plants (5 isolates). However, the pathogen could not be isolated from six cheese-processing plants and one of the fish plants was not located in Sweden. Serovar 1/2a was dominant in all sources, i.e. 134 isolates (73%) in human, 70 isolates (96%) in food, and 27 isolates (87%) in the environment; whereas, serovar 4b was less common, 36 (20%) among human isolates, and only one food isolate, isolated from soft cheese. PFGE with AscI and ApaI enzymes identified 94 pulsotypes among 287 isolates. Six unique pulsotypes were identified among human isolates (42%) and fish isolates (59%). The most common human pulsotype is also the most common among fish isolates, and is found only in fish samples from non-Swedish plants (Thisted Lambertz et al., 2013). Serovar 1/2a is the serovar regularly found in cold-smoked and gravad salmon bought in Sweden (Thisted Lambertz

52 et al., 2013). PFGE types found in human cases of invasive listeriosis in Sweden are frequently encountered in vacuum-packed cold-smoked and gravad salmon/rainbow trout (Netterby, 2000; Mandorf, 2003; Kannius & Karlsson, 2010; Thisted Lambertz et al., 2013).

Listeriosis is still a topical foodborne disease. The world’s population is ageing and has a greater chance of developing debilitating chronic conditions. The demographic shift and the widespread use of immunosuppressive medications have increased the immunocompromised population that are susceptible to an increased risk for listeriosis (Allerberger & Wagner, 2010; Luber et al., 2011; Todd & Notermans, 2011). Consumer lifestyle has changed and less time is available for food preparation. Modern lifestyle has markedly changed eating habits worldwide, with a consequent increased demand for RTE foods (de Oliveira et al., 2010); therefore, more RTE and take away foods are consumed. Within the food industry, extended shelf life RTE foods and new RTE food types are an area of expansion, and as these foods pose the most concern for listeriosis, this area deserves more attention (Allerberger & Wagner, 2010). To be able to control L. monocytogenes in RTE products at risk, these products should be properly labeled with regard to time and temperature of storage, and consumers should be educated about food storage practices. In addition, the shelf life for some products with a known risk of L. monocytogenes contamination and growth (e.g. vacuum-packed gravad and cold-smoked salmon) should be restricted by authority regulations.

There is a concern that many Listeria outbreaks are reported from hospitals. Even low levels of L. monocytogenes pose a risk to immunocompromised people and RTE food is often served in hospitals. In the United Kingdom, it is recommended that food served to hospital patients is free from L. monocytogenes (Fretz et al. 2010; Coetzee et al. 2011; Yde et al., 2012). Large-scale delivery of precooked meals to a vulnerable population represents a threat if proper measures against listeriosis are not taken (Smith et al., 2011). It is vitally important that foods (especially cooked and chilled) delivered to hospitals, residential homes for senior citizens and elderly people are reheated to at least 72°C and that cold food, such as turkey deli meat and cold-smoked and gravad salmon should be free from L. monocytogenes.

Characterisation of L. monocytogenes isolates from human patients and foods with different methods has contributed to the identification of human listeriosis globally. Recently, whole genome sequencing (WGS) became a new molecular method for characterisation of clinical L. monocytogenes isolates in Sweden (SVA, 2014 and 2015).

3 AIMS OF THE THESIS

The main objective of the present work was to improve knowledge of the spread and prevalence of L. monocytogenes in humans and foods worldwide, and to elucidate the genetic diversity and relatedness of L. monocytogenes isolates in an extensive investigation to provide avenues for future research in this area. To achieve this aim, pulsed-field gel electrophoresis (PFGE) was used as a molecular tool for examining infectious routes and infectious sources of L. monocytogenes and for investigating any structure in the occurrence of PFGE types.

The specific aims of the thesis were:

1. to study infectious routes and infectious sources of L. monocytogenes through comparison of isolates from human cases and foods (Papers I and IV).

2. to explore the serovar diversity of L. monocytogenes isolates from human clinical cases over half a century (Paper I).

3. to investigate, through PFGE with AscI restriction enzyme, the genetic diversity of L. monocytogenes isolates from human clinical cases of invasive listeriosis in Sweden (Papers I and V).

4. to detect possible clustering of L. monocytogenes PFGE types in time or location during half a century in Sweden (Paper I).

5. to analyse the configuration of small bands with sizes < 145.5 kb (PFGE group) of L. monocytogenes AscI PFGE profiles, in order to establish the genetic relatedness of L. monocytogenes strains isolated from human cases (Papers II and III).

6. to determine whether there is any association between serovars, PFGE types and PFGE groups (Papers I, II and III).

7. to develop a new procedure for identifying L. monocytogenes profiles based on the configuration of detectable small bands of sizes < 145.5 kb, and in a later step, the larger band > 145.5 kb, in order to simplify and accelerate the analysis and interpretation of bacterial genomes and identify the source of infection faster without delay (Papers II and III).

4 MATERIAL AND METHODS

4.1 Bacterial isolates

The clinical and food isolates of L. monocytogenes used in the study (collected between 1958 and 2010) are described below.

4.1.1 Human isolates

L. monocytogenes isolates from 932 cases (mother-infant is represented by one isolate only) of invasive listeriosis in Sweden were collected between1958 and 2010. The isolates were stored in brain-heart infusion broth (BHI broth, Merck, Darmstadt, Germany) with 20% glycerol at -73ºC. The isolates were obtained from the Swedish Institute for Infectious Disease Control and from different county hospitals around Sweden. Most isolates were isolated from blood and cerebrospinal fluid, with a few from other sterile sites (Papers I, II, III and V).

4.1.2 Food isolates

Fifty-three samples of ready-to-eat, vacuum-packed salmon products were collected from retail stores in Sweden and a further three samples purchased in Germany during summer 2005. The samples obtained in Sweden were from 15 manufacturers and those bought in Germany from one manufacturer. The samples were analysed the day before the best-before date. Altogether, 56 L. monocytogenes isolates were isolated from 11 (three manufacturers) of 56 samples (Paper IV).

4.2 Isolation and detection methods for L. monocytogenes

The isolation and enrichment methods of the Nordic Committee on Food Analysis (NMKL, 2004) were used with some modifications (Paper IV).

Enrichment and culture From each food sample, 25g was mashed in a stomacher bag for a few minutes together with 225 ml enrichment broth I. The homogenate was incubated at 30ºC for 24 hours. Then, 1 ml of the enrichment culture was added to 10 ml enrichment broth II, with a higher concentration of antibiotics, and incubated for a further 24 hours at 37ºC. After incubation, 0.1 ml of enrichment broth II was streaked onto both PALCAM and Oxford agar media. Both plates were incubated at 37ºC for 24–48 hours, after which typical Listeria colonies were selected for further examination.

Quantification All samples were stored at -20ºC awaiting the enrichment procedure. From each L. monocytogenes positive sample, 10g was mashed in a stomacher bag together with 90 ml of peptone water. Tenfold serial dilutions of 1 ml were made from the suspension and then 0.1 ml was streaked onto blood, PALCAM and Oxford agar media. All plates were incubated for 48

55 hours at 37ºC, after which all typical L. monocytogenes colonies were counted. Five colonies taken from both the enrichment and the quantification plates were streaked onto blood agar for purity.

Confirmation Five typical colonies chosen from each plate were re-streaked onto blood agar and tested for Gram reaction, rhamnose and xylose fermentation. Motility test was in BHI broth incubated for 10 hours at 20ºC.

4.3 Conventional serotyping of L. monocytogenes

Serotyping was with commercial O-antigen Listeria antisera types I/II, I, IV, V/VI, VI, VII, VIII, IX and H-antigen Listeria antisera types A, AB, C, D (Mast Diagnostics, Mast house, Berby Road, Bootle, Liverpool, UK 201EA), according to the manufacturer’s instructions, with some modifications (Papers I and IV). Isolates from each of the freeze-stored bacterial cultures were streaked onto horse blood agar plate with a sterile plastic loop and incubated at 37ºC for 24 h. Then, 3 ml of 0.2% sodium chloride was added to the bacterial growth and mixed with a glass spatula. The suspension was transferred to a tube and boiled in a water bath at 100ºC for 1h, left at room temperature for 30 min before being centrifuged at 3700 rpm for 20 min. After resuspension in 1 ml of 0.2% sodium chloride, the pellet was tested for agglutination with the O antiserum, according to manufacturer’s instructions. To determine the H antigens, the isolates of each freeze-stored bacterial culture were streaked onto horse blood agar plate with a sterile plastic loop, and then incubated at 37ºC for 24 h. The cultures were passed three times through semi-solid nutrient agar plates at 30ºC for 24 h. Colonies removed from the plate were inoculated into 2ml of BHI broth with a sterile plastic loop and incubated at 30ºC for 24 h. Then, 2 ml of 0.9% sodium chloride was added and the suspension kept at 30ºC overnight. The tube agglutination test was with H antiserum and the prepared cell suspension, according to the manufacturer’s instructions.

4.4 Molecular typing by pulsed-field gel electrophoresis (PFGE) of L. monocytogenes

Each isolate was characterised by PFGE and restricted with AscI enzyme according to the Pulse Net standardised protocol described by Graves and Swaminathan (2001). Only 11 of 56 food isolates were analysed with ApaI restriction enzyme (Paper IV). Through serovars and AscI profiles, different PFGE types of L. monocytogenes were obtained (Paper I and IV). For each type, a reference strain was selected (Paper I). The protocol (Graves & Swaminathan, 2001) used was modified as follows.

4.4.1 Preparation of gel plugs

The isolates of each freeze-stored bacterial culture were streaked onto horse blood agar plates with a sterile plastic loop and incubated at 37ºC for 24 h. One well isolated colony was removed

56 from the plate and inoculated into 5 ml sterile BHI broth (Oxoid CM 225) with a sterile plastic loop and then incubated in a water bath at 37ºC for 24 h. The tube was vortexed for 3 seconds and kept in a water bath at 4–8ºC for 5–10 min. After centrifugation at 5700 rpm for 5 min, 4.5 ml of the supernatant was discarded and the pellet was vortexed to a homogeneous suspension. Thereafter, 5 ml of cold TN buffer (10 ml 1 M TrisHCl, pH 8; 20 ml 5 M NaCl and 970 ml sterile water) was added and the sample vortexed for 5 seconds. The suspension was cooled and kept in a water bath at 4–8ºC for 5–10 min and centrifuged at 5700 rpm for 5 min. Then 4.5 ml of the supernatant was discarded and the pellet was vortexed to a homogeneous suspension. The bacterial suspension was treated with 0.9 ml of lysozyme, Roche (1mg lysozyme/ 1ml TN buffer), vortexed 3 seconds and incubated in a water bath at 37ºC for 30 min. Fifteen ml of 1.2% agarose solution (180 g SeaKem Gold agarose, Cambrex, and 15 ml sterile water) was prepared and kept in a water bath at 55ºC for 10 min. Then 1.67 ml of ESP buffer (1g N-lauroylsarcosin, Merck; 100 ml 0.5 M EDTA, pH 8 and 200 mg pronase, Roche) was added to the agarose solution and vortexed for a few seconds. The bacterial suspension with lysozyme was transferred to the water bath at 55ºC, then mixed with 1.2 ml of agarose solution, vortexed for 3 seconds and incubated in a water bath at 55ºC for 30 min. The mixture was poured into three wells of a plug mould (Gene Navigator Pharmacia, Biotech, Uppsala, Sweden) with a sterile Pauster pipette and placed in the refrigerator for 10 min. The gel plugs were transferred to a sterile microcentrifuge tube containing 1 ml of ESP buffer and kept in a water bath at 55ºC for 1 h, after which, the ESP buffer was renewed and the test tube was incubated in a water bath at 55ºC for further 24 h. The ESP buffer was renewed twice during two days and the gel plugs were then stored in the refrigerator until ready for restriction.

4.4.2 Restriction digestion of DNA

All isolates were restricted with AscI restriction enzyme (New England Biolabs, Inc.). A plug slice, approximately 1.5 mm was placed into a micro centrifuge tube containing 0.5 ml of PEFA solution (3.5 mg Pefablock, Roche, and 10 ml TE buffer [10 ml 1 M Tris HCl, pH 8; 2 ml 0.5 M EDTA, pH 8 and 1000 ml sterile water]) and incubated in a water bath at 37ºC for 40 min. After which, the PEFA solution was renewed and the micro centrifuge tube was incubated in a water bath at 37ºC for a further 40 min. The solution was aspirated and replaced by 1 ml TE buffer and incubated twice during 40 min in a water bath at 55ºC. Then TE buffer was aspirated and replaced by 150 µl restriction solution (870 µl sterile water; 108 µl 10x NE4 buffer, New England Biolabs, Inc; 10 µl 10 mg/ml BSA, Promega, and 12 µl 10 U/µl AscI) and incubated at 37ºC for 15–18 h. The isolates restricted with ApaI restriction solution (870 µl sterile water; 98 µl 10x buffer A, Boeringer Mannheim; 10 µl 1 mg/ml BSA, Promega, and 22 µl 10 U/µl ApaI, Boeringer Mannheim) and incubated at 30ºC for 15 h.

4.4.3 Electrophoresis

The restriction solution was aspirated and replaced by 200 µl of 0.5 x TBE [9 ml sterile water and 1 ml 5 x TBE buffer (54 g 0.45M Trisbas, Amersham Biosciences; 27.5 g 0.45 M Boric acid, Merck; 20 ml 0.5 M EDTA, pH 8 and sterile water up to1000 ml)] and incubated at 37ºC for 30– 60 min. Then, 1.2% agarose solution was prepared (1.3 g SeaKem Gold agarose, 11 ml 5 x TBE

57 buffer and 99 ml sterile water) and kept in a water bath at 55ºC. The agarose solution for plugs restricted with ApaI was 1% (1.4 g SeaKem Gold agarose, 14 ml 5 x TBE buffer and 126 ml sterile water). The plug slice was removed carefully from the microcentrifuge tube and loaded onto comb teeth, and then the comb was transferred to a gel form. The plug was pushed gently to the bottom and covered with a dash of agarose. The remaining 1.2% agarose solution was poured into the gel form and left at room temperature for 15 min. The comb was removed from the gel and the wells were filled with agarose. A Lambda ladder PFGE Marker NO340S (New England Biolabs, Inc.) was used as molecular weight standard (50 to 1,000 kb). A Pharmacia Gene Navigator (Pharmacia LKB Biotecnology AB, Uppsala, Sweden) was used to separate DNA fragments in plugs restricted with AscI under the appropriate electrophoretic parameters: migration period was 24 h at 200 V: initial switch time 4.0 sec, final switch time 40.0 sec, and running buffer 0.5 x TBE at 8ºC. The plugs restricted with ApaI were separated by electrophoresis in 0.5 x TBE at 14ºC in CHEF MAPPER XA (BIO-RAD), run time being 20 h with initial switch time of 1.0 sec and final switch time of 15.0 sec. After electrophoresis, the gel was stained for 25 min in 1 L 0.5 x TBE buffer containing 100 µl ethidium bromide, Molbase (1 mg/ml) and then destained for 1 h in 1 L 0.5 x TBE buffer. Then, the banding patterns of the gels were visualised with short-wave ultra violet (312 nm) light and photographed with a Polaroid camera.

4.4.4 Analysis of PFGE profiles

The DNA profiles were analysed visually and the position of each restriction fragments of each PFGE profile was sized with both Lambda ladder and the universal marker Salmonella Braenderup H9812 (digested with XbaI, Roche) to establish the reference position. PFGE types were established based on the number and distribution of all detectable fragments in a DNA restriction profile (Paper I and IV), whereas, PFGE groups were established solely on the number and distribution of all detectable small fragments below 145.5 kb (Papers II and III). PFGE profiles were considered distinguishable if the banding patterns were different. PFGE profiles with identical banding patterns below 145.5 kb were considered as sharing the same PFGE group. The reference collection was created by selecting a reference profile from each PFGE type. The new reference standard profile was designated by a numerical code, for example the designation A:1/2a:4A indicated the strain belonged to PFGE group A, serovar 1/2a and represented the 4th AscI profile among the serogroup 1/2 isolates of the collection. The last letter indicates the AscI profile no 4 has various closely-related variants, in this case the strain is variant A (Tenover et al., 1995).

4.5 In silico analyses

Thirteen completely sequenced L. monocytogenes genomes belonging to lineage I (serovar 1/2b, 3b, 4b and 4e), and lineage II (serovar 1/2a, 1/2c, 3a and 3c) were obtained from either website insilico.ehu.es, or EMBL-EBL. Each sequenced genome was analysed in silico with PFGE and restriction enzyme AscI. The in silico program calculated the theoretical number of restriction fragments in the DNA (Papers II and III).

5 RESULTS AND DISCUSSION

5.1 Serovars of L. monocytogenes

The study period in Sweden, 1958–2010, could be divided into three periods: 1958–1971, where three serovars 1/2a, 1/2b and 4b were equally common; 1972–1995, where serovar 4b was prevalent; and, 1996–2010 where serovar 1/2a was the major L. monocytogenes serovar in human listeriosis in Sweden (Paper I: Table 2). The three periods are discussed below.

5.1.1 HumanL. monocytogenes isolates from 1958–1971 in Sweden

Since Linell presented the first case of human listeriosis in Sweden in May 1958 (Linell et al., 1959), a limited number of cases were reported in Sweden, as it was difficult to diagnose the disease (Meyer, 1961; Sepp & Roy, 1963; Seeliger & Finger, 1983). In the UK, McLauchlin (1990b) observed a similar pattern during 1967–1985 and considered improvement in isolation and identification of the pathogen influenced the reporting of the cases. The majority of listeriosis cases reported in the 1950s and 1960s in Sweden were associated with pregnancy; a trend also seen in other countries (Ekelund et al., 1962; Sepp & Roy, 1963; Seeliger et al., 1969; Becroft, et al., 1971; Guevara et al., 1979; Campbell, 1990; McLauchlin, 1990b). During the 1970s, between 10 and 15 human listeriosis cases were reported annually in Sweden; however, there is a lack of food isolates of L. monocytogenes available from this time. From 1958 to 1971, the three L. monocytogenes serovars 1/2a, 1/2b and 4b were equally common among human isolates, although based on a small number of available isolates (Paper I). Serovar 1/2a predominated in West Germany until 1959, whereas, from 1960 most human strains were serovar 4b (Seeliger et al., 1969).

5.1.2Human L. monocytogenes isolates from 1972–1995 in Sweden

In the period 1972–1995, serovar 4b was the most common serovar found in human cases of listeriosis in Sweden (58.1% of available isolates), and 32.5% of isolates shared serovar 1/2a. During the1970s, 1980s, and 1990s, the majority of human cases of listeriosis worldwide, were linked to lineage I serotype 4b L. monocytogenes isolates (Guevara et al., 1979; Campbell, 1990; Kathariou, 2002; Paper I). At the same time, L. monocytogenes serovar 4b was a common finding in soft and semi-soft cheeses in Europe (Ericsson et al., 1996). French cheeses purchased in Swedish retail outlets during 1987 and 1993 harboured L. monocytogenes serovar 4b, and two smear cheeses contained 2300 and 100,000 cfu per g (Danielsson-Tham, 1993; Loncarevic et al., 1995).

5.1.3Human L. monocytogenes isolates from 1996–2010 in Sweden

Among the available human L. monocytogenes isolates during this period, serovar 1/2a was predominant (68.7%) (Paper I), probably due to increased consumption of gravad and cold-

59 smoked fish as L. monocytogenes serovar 1/2a is common in these products (Paper IV). This observation is in agreement with other studies from Sweden (Loncarevic et al., 1996b; Rosengren, 2001; Rosengren &Lindblad, 2003; Thisted Lambertz et al., 2012; Thisted Lambertz et al., 2013). The decrease in cases of listeriosis caused by serovar 4b strains since 1996 in Sweden may be due to the measures taken at the RTE food level in France and other European countries. During a study from May-December 1999, Rudolf and Scherer (2001) report an improvement in processing hygiene, as only 4 (4.4%) out of 91 French soft raw milk cheeses (red smear) were positive for L. monocytogenes. However, the overall results from analyses of 329 European soft, semi-soft and hard red smear cheeses made from raw or pasteurised milk were not encouraging; 6.4% were positive for L. monocytogenes (Rudolf & Scherer, 2001). Thus, red smear cheeses should still be regarded as a considerable public health risk. In 2005, 12.9% of RTE vacuum-packed gravad salmon and 28% of cold-smoked salmons harboured L. monocytogenes: the highest level of L. monocytogenes was 1500 cfu/g from a cold-smoked salmon product (Paper IV). The most common PFGE types found in human cases of listeriosis in Sweden are also the types frequently encountered in vacuum-packed cold-smoked and gravad salmon/rainbow trout (Netterby, 2000; Mandorf et al., 2003). Since the early 2000s, the incidence of listeriosis has increased in Europe (SVA, 2007; Little et al., 2012).

5.1.4 The shift from serovar 4b to serovar 1/2a in human listeriosis

A shift from L. monocytogenes serovar 4b to serovar 1/2a occurred in the middle of the 1990s and the early 2000s in both Sweden and Europe. In 1987, 59% of 722 cases of listeriosis in Britain were reported as due to L. monocytogenes serovar 4b (McLauchlin, 1987), and in 1991, serovar 4b prevailed in human listeriosis in most of Europe, with serovar 4b accounting for 63.9% in France and 64% in United Kingdom (Farber & Peterkin, 1991). Generally, most foodborne listeriosis outbreaks appear to have been caused by serovar 4b (Buncic et al., 2001; Orsi et al., 2011; Cartwright et al., 2013). However, large outbreaks of listeriosis predominantly caused by serogroup 4b strains have become less frequent (Swaminathan & Gerener-Smidt, 2007). During an international Listeria conference (ISOPOL) in Australia, Gerner-Smidt et al. (1995) reported that until 1992 in Denmark, two-thirds of human L. monocytogenes isolates belonged to serogroup 4: the following year, 1993, serogroup 1 became predominant in Denmark. McLauchlin and Newton (1995) reported similar results from United Kingdom at the same conference and conclude that “The trends towards an increase in listeriosis amongst the immunocompromised, together with the increase in cases due to serogroup 1/2 mean that future resources will be directed at improving our ability to subtype within serogroup 1/2” (McLauchlin & Newton, 1995). The Danish and the British conference abstracts from 1995 were among the first reports of a shift from L. monocytogenes serogroup 4 to serogroup 1 in human listeriosis. In Sweden, serogroups 4 and 1/2 were equally common during the decade 1986 to 1996 (Loncarevic et al., 1998b); however, “the changes in serogroup distribution among human cases in Sweden … might be explained by changes in eating habits” (Loncarevic et al., 1998b).

Orsi et al. (2011) stress lineage II serotype 1/2a strains appear more common among human listeriosis cases in Northern Europe; which is confirmed by Rosef et al. (2012) from Norway, where 55.6% of human clinical isolates collected between 1992 and 2005 were lineage II, and 41.6% were lineage I with ribotyping. The change in the serotypes of human L. monocytogenes

60 isolates has also been observed in Finland in L. monocytogenes isolates from invasive infections during an 11-year period, 1990–2001. Since 1990, the number of cases caused by serotype 4b has remained constant, with some exceptions. However, the number of listeriosis cases caused by serovar 1/2a increased during 1990–2001 and exceeded the number of cases caused by serovar 4b since 1991 (Lukinmaa et al., 2003). Even during 2002–2004, serovar 1/2a was predominant in Finland (Lyytikäinen et al., 2006). In Denmark, during 2002–2012, 42% of human isolates belonged to lineage I and 58% to lineage II: the lineage II isolates belonged to serotype 1, predominantly serovar 1/2a, as determined by PCR (Kvistholm Jensen et al., 2016). Serovar 1/2a has been the dominant L. monocytogenes serovar in human listeriosis in Sweden since 1996 (Paper I).

The shift from serovar 4b to 1/2a is also seen in Switzerland, serogroup 1/2 became predominant among human strains after 1994 (Pak et al., 2002), and during 2011–2013, the number of human isolates of serovar 1/2a was more than twice the number of 4b isolates (Althaus et al., 2014). During 1990–1999, serovar 4b dominated (52%) among human strains in different regions in Italy, whereas, serovar 1/2a constituted only 27.2%. However, during 2002–2005, serovar 1/2a constituted 50.9% and serovar 4b 24.6% of human strains (Gianfranceschi et al., 2003, 2009). Serovar 1/2a was also the predominant serovar in the Lombardy region, Italy, between 2006 and 2010 (Mammina et al., 2013). Mammina et al. (2013) suggest serotype 1/2a has replaced serotype 4b worldwide as the leading serotype causing human listeriosis. In Canada, between 1995 and 2004, serotype 1/2a was the predominant serotype among human cases, with serotype 4b being predominant in cases associated with pregnancy and miscarriage. Among 722 isolates, serovar 1/2a (47.5%) and serovar 4b (30%) were identified (Clark et al., 2010); however, there is no evidence that lineage II strains are common in human listeriosis outbreaks (den Bakker et al., 2010).

Several countries have experienced a shift from L. monocytogenes serovar 4b to 1/2a in causing human infections. The increase in the incidence of human listeriosis among patients > 60 years old in England and Wales between 2001 and 2007 appears to be associated with cancer or other conditions requiring treatment to reduce stomach acid secretion (Gillespie et al., 2009). Swaminathan & Gerner-Smidt (2007) claim blood stream infection (bacteraemia) is now a more common clinical presentation in listeriosis than meningoencephalitis and serovars 1/2a and 1/2b are more common in blood stream infections than in meningoencephalitis. Thus, more blood stream infection will lead to more serovar 1/2a and 1/2b in listeriosis. Swaminathan & Gerner- Smidt (2007) suggest “the chance a blood stream infection will be detected has increased because the blood culturing systems have become more sensitive and the indications for drawing a blood culture have become broader the past 20 years.”

A simple reason for more 1/2a cases of listeriosis may be due to the frequent occurrence of this serovar in the increasingly popular ready-to-eat foods (Buncic et al., 2001; Gilbreth et al., 2005, Paper I). A link between isolates obtained from patients and isolates obtained from smoked fish is reported in Scandinavian countries (Sweden, Norway, and Finland) and in eastern Spain (Luber et al., 2011; Ariza-Miguel et al., 2015). In the 2000s, several outbreaks where serovar 1/2a was involved are reported: fresh cheese (Danielsson-Tham, 2004), sliced processed delicatessen turkey meat (Olsen et al., 2005), flat whipping cream (Pagotto et al., 2006), tomme cheese (Bille et al., 2006), Quargel-acid curd cheese (Fretz et al.,2010), delicatessen meats

(Garrido et al., 2010; Farber et al., 2011), and soft washed-rind cheese (Gaulin et al., 2012). Furthermore, later outbreaks of listeriosis are linked to L. monocytogenes serotype 1/2a (Tham and Danielsson-Tham, 2014; Ariza-Miguel et al., 2015; Public Health Agency of Sweden, 2017).

Although serovar 4b has previously been predominant, since the middle of the 1990s, there has been a shift in human listeriosis in Sweden and currently L. monocytogenes serovar 1/2a isolates are more frequently isolated than serovar 4b (SVA, 2011–2015; Personal communication: Hedemström, 2017)

5.2 Seasonal variation

From 932 human L. monocytogens isolates from 1958–2010, the majority of serovar 1/2a isolates (29.5%) were received during October to January, whereas, the majority of serovar 4b and 1/2b isolates (32.6%) were received during June to October (Paper I). This observation was in agreement with a study in the USA (Orsi et al. (2011), where more human serovar 1/2a isolates were found in water during autumn and winter, whereas, human serovar 4b was more often isolated during summer. In contrast, an Iranian study reported that among seafood products and environmental isolates (Fallah et al., 2013), serovar 1/2a isolates are dominant in warm periods and serovar 4b during cold seasons. Between 1967 and 1985, the seasonal peaks of human listeriosis in the UK and Scotland were in the late summer and autumn (Campbell, 1990; McLauchlin 1990a; McLauchlin 1990b) and Listeria bacteria were frequently isolated in faeces and soils during July to September (MacGowan et al., 1994). No significant seasonal variations were observed in Latvia during 1998–2007, except for a peak of listeriosis during September– December, 2000 (Bērzinš et al., 2009).

In Sweden, most pregnancy-associated isolates (n=27, 27.6%) arrived at the laboratory during summer (July–Sept) and the least (n=16, 16.3%) during autumn (Oct–Dec: Table 4). In the north of Sweden (Jämtland, Västerbotten and Norrbotten), cases of listeriosis are registered almost entirely during January–March, whereas, in the south of Sweden (Skåne), the majority of cases are registered during summer and autumn. According to Danish researchers, the prevalence of L. monocytogenes is higher during the summer due to the increase in production that hinders control measures in the meat processing plants. A similar trend has been observed in fish processing plants, although during November and December (Tompkin et al., 1992).

Table 4. Serovars and seasonal distribution of Listeria monocytogenes isolates in pregnancy- associated cases in Sweden, per decade, between 1958 and 2010. $0<49/ =96,>0= $<028,8> .346/ &0<9@,< &0,=98

8 8 , - - *48>0< &:<482 &?770< ?>?78 89/,>, G G G G G '9>,6 *Non-pregnant and pregnancy-associated isolates. **Percentage of pregnancy-associated cases

5.3 Geographical variation

All L. monocytogenes isolates of human cases come from the three regions of Sweden: Götaland (Southern: n=526, 56%), Svealand (Central: n=294, 32%)) and Norrland (Northern: n=108, 12%). The geographical distribution indicated the majority of cases of listeriosis occurred in Southern of Sweden, where most of the cases were from Skåne (n=225, 24%), Västra Götaland, in the southwest (n=173, 19%) and Stockholm in the central region (n=141, 15%). In Västernorrland county there were less cases of listeriosis (n=56, 6%) (Table 5). Most cases occurred in the cities of Stockholm (15%), Göteborg (13%), Malmö (11%) and Lund (8%): the differences in percentage appear related to population density of the counties.

isolates in Sweden, per county, during during county, in Sweden, isolates per Listeria monocytogenes Listeria . Numbers and serovars of human of and serovars . Numbers Table 5 1958–2010. 9?8>40=&5D80 $9:?6,>498 =96,>0= 8 , - &0<9@,<= . - - 6054820,66,8/)C=><,E>,6,8/9>6,8/ <989-0<2 ,67,< E85E:482B=>0<2E>6,8/ B<0-<9&E/0<7,86,8/(::=,6, &>9.53967 )C<76,8/,6,<8, C@60-9<2 )C=>0<89<<6,8/ )C=>7,86,8/ C7>6,8/)C=>0<-9>>08 "9<<-9>>0889/,>, '9>,6

64 5.4 Age and gender

In the early 1960s, Seeliger (1961) reports no particular gender association, except susceptibility to listeriosis during pregnancy, or age correlation. However, females aged 20–49 years were more associated with listeriosis than males in the same age group (Table 5 in Paper I). Although this is a normal pattern due to female association with pregnancy, the incidence of listeriosis in males aged 50–89 years was high (59.4%) and generally listeriosis is more frequent in males in Sweden. A predominance of listeriosis in males is reported from England (56%), Switzerland (58%), France (62%), Israel (64%) and Portugal (62%) (Jones et al., 1994; Büla, 1995; Goulet & Marchetti, 1996; Siegman-Igra et al., 2002; Almeida et al., 2006). The number of cases associated with pregnancy has decreased over time in Sweden (Table 4), as the increased health focus on pregnant women may have reduced exposure to the Listeria pathogen. A similar trend is observed in other countries (McLauchlin, 1996b; Antal et al., 2007). Conversely, non-pregnant cases have increased globally. Bertrand et al. (2016) observe an increase among non-pregnant cases in Belgium and suggest “probably due to the rise of highly susceptible patients in an aging population...” and “...can be attributed to significant increase in incidence of isolates belonging to serovar 1/2a”. The National Food Administration in Sweden has provided advice on food to pregnant women since the early 1990s through the website and brochures in maternity health care centres. Despite dietary recommendations being translated into other languages, 5 of 7 pregnant cases in2010 were women with foreign backgrounds (Ivarsson, 2010), and food-safety advice concerning risk food for listeriosis may not reach pregnant women belonging to ethnic minorities (Mook et al., 2010). Although the incidence of listeriosis cases associated with pregnancy has remained stable in Norway and other European countries, there is a clear preponderance of serovar 4b within pregnancy-associated isolates. Among 102 isolates from the period studied, 70 (68.6%) shared 4b, 24 (23.5%) isolates belonged to serovar 1/2a, and 7 (6.9%) to serovar 1/2b: these results presented similar patterns to pregnancy-associated cases reported in other studies (McLauchlin, 1990a; Doorduyn et al., 2006; Goulet et al., 2008; Clark et al., 2010).

Between 2000 and 2010, 75% of the listeriosis cases in Sweden were aged >60 years (Paper I: Tables 5 and 6). In England and Wales between 2001 and 2007, the number of cases in those ≥ 60 years with bacteraemia and without involvement of central nervous system has increased. The increase relates especially to digestive-organ malignancies (cancer) or other underlaying conditions and in whom treatment included acid-suppressing medication such as proton pump inhibitors. Patients with CNS infections have fewer underlying conditions than those with bacteraemia (Gillespie et al., 2009; Allerberger & Wagner, 2010). In Denmark, Kvistholm Jensen et al. (2016) also report an increase in blood stream infections among patients > 60 years old and during 2005–2009, lineage II isolates were the main cause of the increase. The increased number of listeriosis cases in France 2006–2007, was mainly due to a rise in cases in people > 60 years old, regardless of any underlying condition (Goulet et al., 2008). Serovar 1/2c was prevalent (66.7%) among male isolates, and the association of males with the less-common serovars 1/2c is noted in other studies (Andersson, 1980; Lukinmaa et al., 2003). Andersson (1980) concludes this trend “may be explained by consumption of Steak tartar or other raw meat especially appreciated by males”. Among 133 L. monocytogenes isolates from meat products collected in France, serovar 1/2c accounted for 37.6%, whereas, serovar 1/2a was found in 25.6%, serovar 1/2b in 15% and serovar 4b accounted for 12.8% of isolates (Rocourt et al., 1992). In Finland, 5% of 180 male isolates shared one PFGE type belonging to serovar 1/2c

(Lukinmaa et al., 2003). In the catering industry, comprehension about hygiene (kitchen, equipment and personal) is lower among male employees than among female employees (Çakıroğlu & Uçar, 2008). Al-Shabib et al., (2017) observed males have riskier behaviour regarding cleaning kitchens than females; therefore, differences regarding gender and listeriosis can be partly to do with food safety awareness, which is stronger among females (Jevšnik et al., 2008).

5.5 Pulsed-field gel electrophoresis (PFGE) types and PFGE groups

A major goal for public health scientists during investigations of food-borne cases and outbreaks is the analysis and interpretation of bacterial genomes to assist in identification of sources and origin of contamination in order to control and presentation measures to be implemented. Therefore, initial screening and comparison of genomes requires simplification. PFGE with restriction enzyme AscI is efficient in distinguishing L. monocytogenes PFGE groups (based on the number and distribution of all detectable small fragments below 145.5 kb), and PFGE types (based on the number and distribution of all detectable fragments above 145.5 kb). Although L. monocytogenes isolates are from different sources, times and places, their small fragments are genetically more conserved than the major fragments. The small fragment configuration reveals which PFGE group, lineage and serovar the isolate belongs to (Papers II and III). The AscI PFGE group identifies relatedness of two different PFGE types that belong to a same PFGE group, even if profiles differ more than three bands above 145.5 kb: this is supported by figures (DNA profiles) published by Burall et al. (2016), Chen et al. (2016b), Chen et al. (2017b) and Li et al. (2017)

The 63 PFGE types of L. monocytogenes identified among 427 clinical isolates belonging to lineage I could be further divided into 17 PFGE groups, based on the position of distinctly detectable bands below 145.5 kb. Among 334 isolates of serovar 4b, 30 PFGE types were identified, which were further divided into 8 PFGE groups (I, J, Q, R, X, Z, Ö-4 and Ö-5). Furthermore, the 90 serovar 1/2b isolates were distributed into 32 PFGE types and nine PFGE groups (D, G, O, P, T, U, Ö-1, Ö-2 and Ö-3). PFGE group G also included a PFGE type belonging to serovar 3b (3 isolates). Five of the 17 PFGE groups were each represented by only one isolate in each group (Ö-1 to Ö-5). Small fragments below 33.3 kb were non-detectable in L. monocytogenes lineage I isolates (Paper II): the absence of small fragments is reported in other studies (Brosch et al., 1994; Johansson et al., 1999; Miettinen et al., 1999a).

Altogether 504 clinical isolates belonged to lineage II including 483 serovar 1/2a isolates distributed into 114 PFGE types and 21 serovar 1/2c isolates distributed into 9 PFGE types were further divided into 21 PFGE groups. L. monocytogenes isolates belonging to PFGE groups A, B, C, E, F, H, K, L, M, S, V, W, Y, Ö-6 to Ö-12 shared serovar 1/2a, with one exception: PFGE group E also included two PFGE types sharing serovar 1/2c and four PFGE types belonging to either serovar 1/2a or 1/2c. Isolates belonging to PFGE group N shared only serovar 1/2c. Generally, all L. monocytogenes isolates displaying one PFGE type belonged to the same serovar. No serovar 1/2a isolates shared restriction profiles with serovar 4b isolates: these observations are corroborated by others (Johansson et al., 1999; Autio et al., 2002;

Gianfranceschi et al., 2009). In contrast to lineage I isolates, small fragments < 33.3 kb were visible in all L. monocytogenes isolates belonging to lineage II (Paper III).

The average number of human isolates was 5.1 per PFGE type, i.e. 932 human L. monocytogenes isolates were distributed into 183 PFGE types. The highest average belonged to serovar 4b (10.8 isolates), whereas, the average serovar 1/2a isolates was 4.2 isolate per PFGE type (Paper I). Among 559 human L. monocytogenes isolates collected during 2002–2012 in Denmark, 122 AscI profiles were identified, i.e. an average of 4.6 isolates per pulsotype (Kvistholm Jensen et al., 2016). In The Netherlands, 134 L. monocytogenes isolates were characterised from human cases between 1999 and 2003 and PFGE identified 58 genotypes, an average of 2.3 isolates per genotype (Doorduyn et al., 2006). In Finland, 81 AscI PFGE were identified from 314 human L. monocytogenes isolates, an average of 3.9 isolates per PFGE type (Lukinmaa et al., 2003). In Switzerland, 93 L. monocytogenes isolates were analysed from human cases collected between 2011 and 2013 (Althaus et al., 2014): PFGE yielded 70 different AscI types, an average of 1.3 isolates per PFGE type. The authors conclude “the population of Swiss L. monocytogenes strains is representative of the known diversity of this species and demonstrates the presence of all major L. monocytogenes clones in Switzerland” (Althaus et al., 2014). The average of isolates per PFGE type among 932 human L. monocytogenes isolates (Paper I) indicated this species is genetically heterogeneous, which was in concordance with other studies (Cantinelli et al., 2013; Haase et al., 2014).

The highest diversity of PFGE types was identified in four counties: Skåne 79 of 183 types (43%), Västra Götaland (72 types (39%), Stockholm (64 types (35%) and Västernorrland (29 types (16%) types. One of the most common PFGE types in Sweden is A:1/2a:4A (58 cases), was first identified in 1982, and was still isolated at the end of the study period in 2010, with 11 human cases. This type was detected in 17 counties, but mainly in Skåne (9 cases), Västra Götaland (11 cases), Stockholm (9 cases) and Västernorrland (8 cases). In contrast, the closely related type A:1/2a:4B (40 cases) was identified later in 1988 and was isolated from six human cases at the end of study period. This type was detected in 14 counties, mostly in Skåne (15 cases: Table 6). Both types, A:1/2a:4A and A:1/2a:4B, were not present at the beginning of the study period, but appeared in the early 1980s. Similarly, in Canada, human isolates sharing subtype LMACI.0009:LMAAI.0234 serovar 1/2a were not identified before 2002, but have been frequently isolated since 2002 (Clark et al., 2010). The two closely related PFGE types I:4b:3 (first isolated in 1976) and I:4b:6 (first isolated in 1971) were the most common types in the study period with 67 cases each. Both PFGE types were isolated from 1 (I:4b:3) and 5 (I:4b:6) human cases in 2010 (Papers I and II) and were detected in 13 (I:4b:3) and 11 (I:4b:6) counties, mostly in Skåne and Västra Götaland (Table 6). PFGE type J:4b:5 (64 cases) was present during a long period (1965–22008) and was found in 16 counties, mostly in Skåne (14 cases) and Västra Götaland (16 cases: Table 6). PFGE type E:1/2a:9B (36 cases) was first identified in 1993 (1 case) and in 2010 (8 cases) and was detected in 12 counties, mainly in Skåne (8 cases) and Västernorrland (7 cases). The population structure of L. monocytogenes was mainly clonal and stable over time and space. Furthermore, the results suggest some L. monocytogenes PFGE types may be adapted to specific niches that facilitate their diffusion with high prevalence in Sweden.

Generally, L. monocytogenes isolates collected during 1958–2010 clustered into two lineages, I (serovar 1/2b, 3b, 4b) and II (serovars 1/2a, 1/2c). In lineage I, two groups were prevalent. PFGE group I (193 isolates, serovar 4b) was the largest PFGE group among human isolates that included L. monocytogenes isolates identified during the period 1965–2010, and was distributed across all counties. PFGE group J (95 isolates, serovar 4b) was isolated from 1958 to 2008 and identified in 17 counties, mostly in Skåne (25 cases) and Västra Götaland (20 cases). PFGE group D (41 isolates, serovar 1/2b) was present during the entire study period (1958–2010) and was predominant in Skåne (18 cases), but identified in 10 counties. In lineage II, two groups were present during the entire study period: PFGE group E (159 isolates, all serovar 1/2a, except 17 1/2c isolates) was distributed in 19 counties and was the most frequent lineage II PFGE group, followed by PFGE group A (135 isolates, serovar 1/2a) distributed in 20 counties. PFGE group A was observed every year from 1987 and at high frequency (1–20 cases per year). PFGE group B (57 isolates, serovar 1/2a) was first identified in 1963 and remained present to the end of the study period, and was detected in 14 counties but rarely in Norrland region (Table 7; Tables 3 in Papers II and III). These observations suggested few PFGE groups were frequently associated with human listeriosis in Sweden, because of their high prevalence in sources or specific for a single source.

Lukinmaa et al. (2003) compared 314 human isolates collected during 11 years and found several closely related PFGE types, which are further grouped into major clusters. The PFGE types within a cluster differ in three or fewer fragments and these clusters of closely related PFGE types have been present every year since the 1990s. According to the dendrogram (Lukinmaa et al., 2003), the PFGE types are similar in the region of the small fragments within each cluster. All these clusters have been identified among PFGE groups in Swedish human strains (Papers II and III). A surveillance study of human listeriosis between 1985 and 2014 in Belgium reports that among L. monocytogenes serovar 1/2a isolates, the most frequent human pulsovar belong to ST8 and are the only type isolated every year: this type is related to CC8 strains that caused outbreaks in Switzerland, Denmark, Germany and Canada (Bertrand et al., 2016).

In another study (Michelon et al., 2015), three different PFGE standard operating procedures (SOP) were compared and all PFGE profiles gained were similar in quality and indistinguishable except one profile. Although PFGE profiles “... are both similar and comparable...” (Michelon et al., 2015), the surveillance system needs to be improved to be able to compare PFGE profiles from both human and food isolates (Michelon et al., 2015). However, the European Union Reference Laboratory for L. monocytogenes (EURL for Lm) that uses PFGE profile interpretation, according to SOP, does not recommend the analysis of profiles below the 33 kb limit (Felix et al., 2013). This omits an important part of the bacterial genome, and although small fragments below 33.3 kb were non-detectable in L. monocytogenes lineage I isolates, those small fragments were present in all lineage II isolates (Papers II and III). Thus, small bands can be used to differentiate between lineage I and lineage II strains. Several sequenced L. monocytogenes strains belonging to both lineages analysed in silico with PFGE and AscI corroborated this observation (Papers II and III). With in silico analysis, it is possible to detect the presence of a small fragment (1454 bp) in all L. monocytogenes lineage I strains. Although whole genome sequencing is an increasingly used subtyping method, it is not accessible in all microbiological laboratories (Camargo et al., 2016; Kramarenko et al., 2016).

PFGE types found in Swedish studies A retrospective characterisation of L. monocytogenes strains isolated in published and unpublished Swedish studies from outbreaks and some sporadic cases are described below, along with L. monocytogenes PFGE types from foodstuffs analysed in previously published and unpublished Swedish studies. This is done in order to put PFGE types, found in Sweden during the 53 year period, into a broader context. The PFGE type nomenclature is according to the type designation formulated in Papers II, III and section 4.4.4 in this summary (Analysis of PFGE profiles).

Data from the retrospective studies (Papers I, II and III) indicated five L. monocytogenes isolates of patients in the suspected outbreak in 1981 (reported by Bernander in 1983 and described in Section 2.8.2) belonged to different lineages, PFGE groups, and PFGE types. These were lineage I PFGE types I:4b:3 (an adult and an infant case), I:4b:1, J:4b:5 and lineage II PFGE type K:1/2a:134. At least three cases shared the same PFGE group (I), and the PFGE types (I:4b:1 and I:4b:3) were closely related and probably part of a local outbreak. These two types were isolated

71 from 23 and 67 human cases in the study period. PFGE type J:4b:5 belonged to PFGE group J, the fourth largest PFGE group with 95 isolates/cases (Paper II), where the first case was identified in 1958, and included 9 PFGE types. Bernander (1983) found all clinical isolates in the suspected outbreak belong to 4b, however, one of the human isolates was identified as serovar 1/2a (PFGE type K:1/2a:134), thus, the sample may have contained two different strains (Tham et al., 2002; Paper III). PFGE type J:4b:5 was isolated from two fish products in 2002 and from a milk product in 1991. PFGE group K is the seventh most common PFGE group in the period studied, with 34 human cases, and included 10 PFGE types. The PFGE group K was identified in the early 1980s (Paper III).

The first documented food-borne case of listeriosis in Sweden occurred in 1993 and was due to medwurst (Loncarevic et al., 1997). The L. monocytogenes strain was identified as PFGE type E:1/2a:31, and this type was involved in 17 cases during the study period (1958–2010): 8 cases from 1984 to 1992 and 9 cases between 1993 and 2010. This PFGE type belonged to lineage II, PFGE group E, and was the second largest PFGE group among human isolates (159 cases), with a distribution into 28 PFGE types (Paper III).

The rainbow trout outbreak occurring in Värmland in 1994–1995 (Ericsson et al., 1997) involved PFGE types Q:4b:10 (six cases), I:4b:3 (one case) and I:4b:16 (one case). The ninth human isolate shared PFGE type I:4b:6 and was not considered part of the outbreak (Papers I and II). The four AscI PFGE types belonged to lineage I, and serovar 4b and three PFGE types belonged to PFGE group I and one to PFGE group Q. PFGE type Q: 4b:10 was isolated from 12 human cases between 1974 and 1995, including eight human cases during the rainbow trout outbreak (1994–1995). This type was the most common within PFGE group Q and included six PFGE types (Paper II). The PFGE type I:4b:6 was isolated from three fish products (two products in 1993 and one product in 1998).

A sporadic case of gastroenteritis occurred in 1993 was due to consumption of gravad salmon from brand X. The L. monocytogenes isolates serovar 4b from human patient faeces sample and a salmon sample shared identical PFGE patterns with ApaI and SmaI enzymes. This PFGE type was found previously in a Swedish salmon plant (brand X) and was identical to the French epidemic type causing an outbreak due to consumption of pig tongue in aspic in 1992–1993 (Personal communication: Jacquet, 1994). The PFGE type found in both the pig tongue in aspic and in salmon samples was I:4b:6. However, this long Swiss epidemic phagetype is the epidemic cheese-borne strain from Swiss, Californian, Danish and French outbreaks during the 1980s and 1990s (Tham et al., 2000; Tompkin et al., 2002). In Japan in 1991, serovar 4b was predominant (60%) among human L. monocytogenes isolates, whereas, serovar 1/2b only accounted for 30%. The long Swiss epidemic phagetype was identified among 102 Japanese L. monocytogenes isolates (82 human isolates from 1978–1993 and 20 isolates from food sold in Japan 1989– 1993). With PFGE and three restriction enzymes, one isolate from raw sausage and 17 of 82 human Japanese L. monocytogenes isolates were found to be indistinguishable from the long Swiss epidemic phagetype (Nakama et al., 1998). Of 107 L. monocytogenes human isolates collected in Sweden during 1976–1985, 47 shared the long Swiss epidemic phagetype. Of those, 19 isolates showed the same characteristics (restriction with ApaI, SmaI and XhoI) as the epidemic strains from Switzerland (1983–1987) and Denmark (1985–1987 & 1989–1990; Ericsson et al., 1996). Although, this indistinguishable PFGE type (I:4b:6) was identified in

72 different sources globally, it appears cheeses are the main source as large cheese outbreaks due to this epidemic type were reported from several countries during the 1980s and 1990s.

In the two sporadic cases associated with cheese and Chinese cabbage in 1998 in Västernorrland (Tham et al., 1998, Section 2.8.2), the AscI PFGE type A:1/2a:4A was found in all samples from patients and food. This type corresponds to the AscI PFGE type 0038 (EFSA nomenclature) and ST7. Type A:1/2a:4A was the most common lineage II PFGE type in the study period. The PFGE group A was the third most common and included 17 PFGE types (Papers I and III). In Finland, PFGE “type 1” closely related to the Swedish type A:1/2a:4A (unpublished data) and has been common in sporadic cases from 1994 onwards (Lukinmaa et al., 2003). PFGE type A:1/2a:4A has been isolated from RTE fish products between 2000 and 2010 (Netterby, 2000).

The AscI PFGE type encountered in all samples from the non-invasive outbreak in Hälsingland (2001) was A:1/2a:4B (Carrique-Mas et al., 2003; Danielsson -Tham et al., 2004). Moreover, the epidemic strain from the meat-borne outbreak in 2013–2014 in Sweden, involving 49 human cases, belonged to the AscI PFGE type 0039 and ST7 (Public Health Agency of Sweden, 2015 and 2016; Dahl et al., 2017) and was identical to PFGE type A:1/2a:4B. This PFGE type, with MLVA profile 07-07-09-10-06, is also regularly isolated from clinical cases in Norway (30%) and has been involved in two listeriosis outbreaks in Norway: from a meat slicer at Ålesunds hospital in 2005 and from ecological camembert cheese at Rikshospitalet in 2007 (Lindstedt et al., 2008; Johnsen et al., 2010: Lunestad et al., 2013; Personal communication: Lin Thorstensen Brandal, 2017; Paper I). The PFGE type A:1/2a:4B was isolated from cheese in 2001–2002 and from RTE fish products in 2000–2002 (Mandorf, 2003). The two closely related PFGE types (A:1/2a:4A and A:1/2a:4B) belong to PFGE group A and ST7. The PFGE type A:1/2a:6 belongs to PFGE group A, and to ST19, has been implicated in the Swedish fish-borne outbreak between 2013 and 2015 (Public Health Agency of Sweden, 2015 and 2016). This PFGE type (A:1/2a:6) was isolated from five human cases in the study period (1958–2010), the first case in 1995 and then four cases during 2000–2010 (Paper III). According to MLST analysis, among these five L. monocytogenes isolates, four belong to ST19 and one to ST7 (Personal communication: Hedenström, 2017).

In 2000–2002, two research projects analysed the prevalence of L. monocytogenes in vacuum- packed gravad and cold-smoked salmon/rainbow trout (Section 2.9.3). In the first study (Netterby, 2000), the sample with the largest cfu/g harboured three PFGE types (A:1/2a:4A, B:1/2a:3A and G:1/2b:2A) and these types were found in two different plants and were common among human isolates in the same year. An additional PFGE type (K:1/2A:25) was found in one fish processing plant (Netterby, 2000). In the second study, Mandorf (2003) analysed 40 fish products from nine fish plants: four fish plants were contaminated with the pathogen. All isolates belonged to serovar 1/2a distributed into eight different AscI PFGE types (all identified among human clinical isolates). In six fish samples from plant A, the AscI PFGE type E:1/2a:9A was identified (see below). One gravad salmon sample from plant E harboured three PFGE types belonging to two different PFGE groups (B:1/2a:114, B:1/2a:125 and F:1/2a:13) and one cold- smoked salmon sample from plant A harboured two PFGE types (E:1/2a:9A and E:1/2a:12A). The PFGE type G:1/2b:2A was involved in 19 human cases during the study period. This PFGE type belonged to lineage I, PFGE group G, and was the tenth most common PFGE group among human isolates (27 cases) and was distributed into 5 PFGE types (Paper II).

The PFGE type B:1/2a:3A was isolated during 1996–2003 in six human cases and the closely related PFGE type B:1/2a:3B was identified in 1981, and until 2007, among seven cases. In 2000, both types were involved in three (B:1/2a:3A) and four human cases (B:1/2a:3B: Paper III). The PFGE type B:1/2a:3A was isolated from RTE fish products in 2000–2001 and the B:1/2a:3B type was isolated from RTE fish products in 2001 and from meat products (hot dogs) in 2011 (Netterby, 2000). PFGE type B:1/2a:22 was isolated from humans only once in 2001 (Paper III), although this type was isolated from RTE fish products between 1989–2002 and from soft cheeses in 1988 and 1993 (Loncarevic et al., 1998a; Netterby, 2000; Mandorf, 2003). The PFGE type B:1/2a:114 was isolated from only 4 human cases in the study period, even though it was first identified in 1972 and reappear in 2006–2007 with three isolates/cases (Paper III), but was isolated from fish products between 2000–2010 and Gorgonzola cheese in 2001 (Netterby, 2000; Mandorf, 2003). The PFGE type B:1/2a:125, belonging to the same PFGE group (B), was identified in one human case in 1963 (Paper III) and in RTE fish products during 2000–2002 (Mandorf, 2003). All these PFGE types belonged to PFGE group B, that is the fifth most common PFGE group, with 57 cases and included the largest number of PFGE types (26) (Paper III).

The PFGE type F:1/2a:13 was involved in 6 cases between 1988 and 2006 and belonged to lineage II, PFGE group F, and was the eleventh most common PFGE group with 26 cases and was distributed into 8 PFGE types (Paper III). This type was isolated from RTE fish products during 1988–2002 (Loncarevic et al., 1998a; Netterby, 2000). The PFGE type E:1/2:12A included two serovars (1/2a and 1/2c) was first isolated in 1962, and was isolated from 15 human cases up to 2006. PFGE type E:1/2:12A was closely related to PFGE type E:1/2:12B which also shared two serovars but was found in only six human cases between 1989 and 2005 (Paper III). PFGE type E:1/2:12A was identified in RTE fish products in 2001 and in 2011 and in meat products in 1993–1994 (Netterby, 2000). The PFGE type K:1/2a:17 was first identified in two human cases in 1981 and was involved in 11 human cases during the study period, with five cases in 1988–1989 and three cases in 2000–2001 (Paper III). This PFGE type was identified in fish products between 1988 and 2002 (Loncarevic et al., 1998a ; Netterby, 2000; Mandorf, 2003). PFGE type K:1/2A:25 shares the same PFGE group as K:1/2A:17 and has been only seen in three human cases: it was isolated from RTE fish products in 2000–2001 (Netterby, 2000). These two PFGE types belonged to lineage II and PFGE group K was the seventh most common PFGE group among human isolates (34 cases) and was distributed into 10 PFGE types (Paper III).

The PFGE type E:1/2a:9A was seen in 20 human cases (1994–2010), and was the seventh most common type, with 14 cases in 2002–2003. The closely related PFGE type E:1/2a:9B was isolated in 36 human cases (1993–2010) and involved in 30 human cases between 2003 and 2010 (Papers I and III). Both PFGE types were isolated from RTE fish products, the PFGE type E:1/2a:9A, in 2002, and the PFGE type E:1/2a:9B, in 2001, 2002 and 2010 (Mandorf, 2003). These two PFGE types corresponded to the most common human pulsotypes in Denmark, AscI 40-ApaI 42, PFGE type E:1/2a:9A (82 isolates) and AscI 38-ApaI 48, PFGE type E:1/2a:9B (40 isolates), and were isolated from human cases in Denmark between 2002 and 2012. These closely related pulsotypes differ by only two bands in the AscI profiles and one band in the ApaI pattern (Kvistholm Jensen, 2016). In Finland, an increase in listeriosis in 2010 was partly caused

74 by a specific AscI type, Lm96, which caused 19% of human cases during 2010 and was the most prevalent type found in food isolates in Finland in 2010. The type Lm96 was identical to the Danish AscI profile 38, the second most common AscI profile belonging to CC8 in Denmark, and was found in a specific fishery production plant in Finland and in cold-smoked rainbow trout products from the plant (Nakari et al., 2014, Kvistholm Jensen, 2016). Consequently, the Swedish AscI PFGE type E:1/2a:9B, the Danish 38-48, and the Finnish Lm96 have identical AscI profiles.

Among L. monocytogenes isolates found in cold-smoked salmon and gravad salmon packed under modified or vacuum atmosphere, five AscI PFGE types were identified (Paper IV). Three PFGE types were isolated from manufacturer A (B:1/2a:114, E:1/2a:49 and Q:4b:2), whereas, PFGE types E:1/2a:93 and F:1/2a:48 were from different manufacturers. The PFGE type E:1/2a:49 was first identified in a human patient in 1985 and was implicated in 18 human cases during the study period (1958–2010), with 4 cases in 2007 and 12 cases during 2009–2010 (Paper V). This PFGE type was isolated from RTE fish products in 2005 and 2010 (Paper IV). The PFGE type E:1/2a:93 was only seen in four human cases during 1985–2009 (Paper III) and was isolated from fish products in 2001 and 2005 (Paper IV). The PFGE type F:1/2a:48 was implicated in eight human cases during 2003–2010 (Paper III).

5.6 Relationship between PFGE group, PFGE type and serovar

All L. monocytogenes isolates within a given PFGE group in both lineages I and II shared the same serovar i.e. PFGE groups were homogeneous with respect to serovar, except lineage I PFGE group G and lineage II PFGE group E, which both included two serovars. In lineage I, five PFGE types belonged to PFGE group G sharing serovar 1/2b and only one PFGE type sharing serovar 3b, G:3b:2B (Paper II). Revazishvili et al. (2004) also identified isolates with the same PFGE type that sometimes belonged to different serovars (3b and 1/2b). However, in lineage II, the majority of isolates belonging to PFGE group E were serovar 1/2a. Four PFGE types were distributed on either serovar 1/2a or 1/2c. These were PFGE types E:1/2a:12A (6 isolates) and E:1/2c:12A (9 isolates); E:1/2a:12B (4 isolates) and E:1/2c:12B (2 isolates); E:1/2a:93(2 isolates) and E:1/2c: 93 (2 isolates); and, E:1/2a:105 (1 isolate) and E:1/2c:105 (1 isolate). Furthermore, PFGE group E also included two PFGE types sharing only serovar 1/2c, E:1/2c:36 (2 isolates) and E:1/2c:95 (1 isolate). However, PFGE group N (lineage II) included all three PFGE types of serovar 1/2c, indicating this group was more genetically homogeneous (Paper III). Gianfranceschi et al. (2009) report nearly complete correlation between pulsotypes and serotypes: only 5% of all isolates with the same pulsotype belonged to different serotypes. Lukinmaa et al. (2003) report a few L. monocytogenes isolates with indistinguishable PFGE profiles displaying different serotypes (1/2a and 3a). These data suggest PFGE groups provide adequate discrimination between serotypes.

Although the L. monocytogenes isolates were from different sources, time and location, the diversity of PFGE types within PFGE groups and homogeneity of PFGE groups among PFGE types were observed during fifty-three years (1958–2010) in Sweden. Seventeen (44.7%) of 38 PFGE groups were each represented by only one PFGE type, six groups by two types and two groups by three types. Three L. monocytogenes PFGE groups contained higher numbers of PFGE

75 types among human isolates than other PFGE groups during the study period: PFGE group B, serovar 1/2a (26 PFGE types), PFGE group A, serovar 1/2a (17 PFGE types) and PFGE group D, serovar 1/2b (15 PFGE types) (Papers II and III), suggesting these small bands have high conservation within PFGE groups. Similarly, Cantinelli et al. (2013) observe the homogeneity of MvLST gene sequences of L. monocytogenes within clonal complexes (CCs) among isolates collected from 13 countries in Europe, America and Asia between 1963 and 2000. Numerous PFGE types have been involved in large national and multinational outbreaks and these types, called epidemic clones (EC), have been defined based on MEE, ribotyping and PFGE, and later by MvLST. Some representative epidemic clones such as ECI and ECII, both belonging to serovar 4b, and ECIII, belonging to serovar 1/2a, have been included in major groups through MLST and are called Clonal Complex (CC), e.g., ECI was included in CC1, ECII in CC6, and ECIII in CC11 (Cantinelli et al., 2013). Most CC of L. monocytogenes have persisted over decades and only a small number of new CC have been identified in both lineages I and II (Haase et al., 2014). Similarly, few new PFGE groups have appeared among human isolates in Sweden during the period 1958 to 2010. A limited number of PFGE groups have increased in frequency over decades and have been responsible for several cases of human listeriosis in Sweden, possibly due to some L. monocytogenes strains persisting during long periods in food processing plants and continuously contaminating RTE foods.

The recombination rate of L. monocytogenes is one of the lowest in bacterial species (Ragon et al., 2008). Among MLST genes, recombination has only been identified at gene dapE in lineages I and II. In MvLST genes, recombination in lineage II has been identified at genes actA, intA and dal, whereas, in lineage I, recombination has only been distinguished at inlB locus. Moreover, the effect of recombination is higher in lineage II than in lineage I (den Bakker et al., 2008; Cantinelli et al., 2013). At the same time, lineage I is more affected by positive selection, which occurs among MvLST genes actA and inlC and MLST loci dapE and ldh (Cantinelli et al., 2013). These observations confirmed housekeeping genes are more conservative than virulence genes (Nightingale et al., 2005; den Bakker et al., 2010; Nightingale, 2010). However, there was more of a difference in the average number of isolates between lineages I and II based on PFGE types (Papers II and III). In lineage I, the average number of isolates was 6.8 isolates per PFGE type, and in lineage II, the average number of isolates was 4.2. In contrast, the average difference was low, based on the PFGE group. In lineage I, the average number of isolates was 25 and in lineage II, the average number of isolates per PFGE group was 24 (Papers II and III). The L. monocytogenes genome is highly conserved (Kuenne et al., 2013); although, within L. monocytogenes clonal groups, there is some degree of diversity at whole genome level (Chen et al., 2016a).

5.7 PFGE and L. monocytogenes genetic variants

Burn (1935) described the occurrence of three L. monocytogenes variants in cultures from animal organs and cultures that had been transferred several times in immune serum (Burn, 1935). One of the strains lost the property of haemolysing blood in agar and broth media, but the other biochemical, morphological and serological characteristics remained unchanged. The second variant lost its morphological characteristics in broth medium, but not the property of haemolysing blood. The third variant appeared in meat infusion broth after six to eight weeks

76 and although the biochemical characteristics remained unchanged, the morphological characteristics differed from the original strain (Burn, 1935).

Several studies cover cases and outbreaks associated with two or more closely related L. monocytogenes genetic variants, where the AscI PFGE profiles usually differ by only two bands above 250 kb and are similar in the region below 145.5 kb (Kathariou et al., 2006; Gilmour et al., 2010; Lomonaco, et al., 2013; Tham et al., 2013; Kvistholm Jensen, et al., 2016). In the early 1990s, Austrian and French researchers considered “such differences in profiles might be due to recent mutations during the outbreak, during isolation procedures or during storage of the cultures, or might in contrast be due to sporadic cases superimposed on the epidemic” (Buchrieser et al., 1993). Two closely related L. monocytogenes strains were isolated from blood samples from two human cases (Paper V). In the first case, both strains belonged to PFGE group E but shared different PFGE types. Similarly, in the second case, the two strains belonged to the same PFGE group F, but shared different PFGE types, i.e. DNA pattern above 145.5 kb.

In the USA, two outbreaks of listeriosis were associated with RTE meat products with hot dogs (1998–1999) and turkey deli meats (2002) being implicated. The AscI PFGE profiles from the two outbreaks were closely related and belonged to serovar 4b, ECII, MvLST 19, ST6 and CC6 (Kathariou et al., 2006; Chen et al., 2007; Chen et al., 2016a ). The PFGE profiles differed in the largest bands, but not in the small bands below 145.5 (Kathariou et al., 2006). The differences were detected in prophage and internalin-like gene sequences produced by a single point mutation. In a French study (Charlier et al., 2012), three 4b L. monocytogenes strains were identified in the same synovial fluid from a joint sample. Analysis with PFGE and AscI restriction enzyme identified three PFGE types, whereas, MLST analysis identified two sequence types (ST): two strains belonged to ST160 and the third to ST161. The difference in ST was related to a single mutation in the amplified ldh gene fragment. In the two strains belonging to the same ST160, the strains were identical in the AscI PFGE profiles below 145.5 kb, but differed by three bands above 145.5 kb (Charlier et al. (2012) assume “these three isolates derive from a unique clone but have likely evolved within the patient’s infected joint”. The two closely related strains in the studies by Kathariou et al. (2006) and Charlier et al., (2012) appear identical in the AscI restricted fragments < 145.5 kb i.e., the isolates belonged to the same PFGE group.

In Finland, during six years, 37 L. monocytogenes isolates were collected from an ice cream processing environment and equipment and 11 serovar 1/2b PFGE types were identified with three different restriction enzymes: PFGE type II was predominant (26 isolates) and was even found in the ice cream. PFGE type II persisted during six years, whereas, the other 10 types “...were single findings or appeared twice during the same year...” and appear to be a “...mutant of the PFGE type II, since most of them differed only by one or two bands...” (Miettinen et al., 1999a). Similarly, in the USA, several L. monocytogenes strains were detected with closely related AscI profiles belonging to serovar 1/2b or 3b, CC5 and ST5 in contaminated ice cream facilities, which caused an outbreak in 2010–2015. SNP-based subtyping identified two clusters and each cluster had two clades. The two clusters identified were consistent with AscI PFGE groups, but each PFGE group contained three AscI PFGE types. In clades Ia and IIa, AscI PFGE also identified one AscI PFGE profile in each clade, but in clades Ib and IIb, AscI PFGE identified two AscI PFGE profiles in each clade. Isolates of serovar 1/2b cluster I differed in the

77 presence or absence of three intact prophages, which were visible among AscI PFGE profiles (Chen et al., 2017b). In another Finnish study, the occurrence of L. monocytogenes in RTE fish products was studied by Johansson et al. (1999) during 14 months. With the use of two restriction enzymes, nine PFGE types serovar 1/2a were identified in a fish processing plant, however, only four types of serovar 1/2a were identified in the fish products from retail markets and two were not found in the plant. PFGE type A was dominant among the isolates and was persistent, whereas, the other types were found sporadically. These types “...differed by only 1–2 bands indicating a close relationship of the strains” (Johansson et al., 1999). Although various PFGE types were detected in an outbreak involving a manufacturer, both AscI PFGE and WGS were consistent to cluster related clonal groups.

Two closely related AscI PFGE types (38 and 40), were identified among 122 human clinical cases collected between 2002 and 2012 in Denmark. Both PFGE types belonged to serovar 1/2a, CC8 and ST8. (Kvistholm Jensen et al., 2016). These two PFGE types corresponded to the Swedish PFGE types E:1/2a:9A and E:1/2a:9B, which were isolated in 56 human cases between 1993 and 2010 (Paper I and III). The two AscI profiles differed by only two bands above 400 kb (Fig. 1). In Canada in 2008, two closely related AscI PFGE patterns (08-5578 and 08-5923), serovar 1/2a, caused a large outbreak due to L. monocytogenes contaminated RTE meat products. These two similar but distinct PFGE types were collinear, harboured a 50 kbp putative mobile genomic island encoding translocation, resistance and regulatory determinants, but differed by 28 SNPs and three insertion/delitions. The differences in AscI restriction patterns were two bands above 400 kb that were due to the presence of a 33 kb prophage in profile 08-5578 (Gilmour et al., 2010). The two closely related strains in both the Danish and Canadian studies belonged to serovar 1/2a and CC8 (Gilmour et al., 2010; Kvistholm Jensen et al., 2016), and appear identical in the AscI restricted fragments <145.5 kb i.e., the isolates are considered to belong to the same PFGE group. Multiple closely related PFGE types among L. monocytogenes isolates sharing similar PFGE groups have been observed, where WGS confirmed they evolved from a common ancestor albeit prophage diversification in the genome was identified (Chen et al., 2017b).

Generally, throughout Sweden, only one isolate of L. monocytogenes from patients with invasive listeriosis is submitted by clinical laboratories to the Public Health Agency. As more than one serovar or PFGE type can be isolated from a single sample (i.e. clinical, animal and food), this complicates the epidemiological investigation of the sources and routes of Listeria infection (Loncarevic et al., 1996a; Tham et al., 1999; Paper V). Three gravad salmon samples purchased in Sweden, harboured two PFGE types each from different lineages (E:1/2a:49 and Q:4b:2: Paper IV). In a mother-baby pair, Seeliger (1962) identified L. monocytogenes serovar 4b in the baby and L. monocytogenes serogroup 3 in the mother. Furthermore, in one chinchilla both L. monocytogenes serovar 4b and L. monocytogenes serogroup 1 were isolated (Seeliger, 1962). Therefore, for investigating sources and pathways of L. monocytogenes infection, several isolates from each sample need to be characterised and isolates from humans and food need to be compared in order to minimise misleading and erroneous results. Simultaneously, the discriminatory power of a subtyping method should be carefully evaluated as excessive discrimination can lead to illogical relations between isolates and a genetic relationship cannot be determined (Chen et al., 2017b).

Acquisition or loss of bacteriophages in bacterial genomes changes all genomes, and generates genetic variants. Thus, random genetic events, including point mutations, insertions and deletions of bacterial DNA can alter the PFGE patterns. PFGE analysis with AscI is suitable for identifying gain or loss of prophages in L. monocytogenes genomes usually located at the larger restriction fragments. Even with differences in SNP results, the same conclusions were reached with PFGE, as strains were related. PFGE with restriction enzyme AscI appeared appropriate for grouping PFGE types into PFGE groups and enabled the definition of 932 human L. monocytogenes isolates into at least 38 PFGE groups, based on the small bands below145.5 kb, with 17 groups belonging to lineage I and 21 groups to lineage II.

Fig.1. Closely related PFGE types. AscI profiles of L. monocytogenes lineage II, Clonal Complex 8 and ST8. Lane 1 (PFGE type E:1/2a:9b); Lane 2 (PFGE type E:1/2a:9a); Lane 3 (Salmonella Braenderup H9812). (Kvistholm Jensen et al., 2016). Published with permission by Kvistholm Jensen 2019.

79 6 CONCLUSIONS

1. Generally, in Sweden, the incidence of listeriosis is higher in males and in an aging population (> 60 years), as has been observed in other countries. Pregnancy-associated cases have decreased over time. The primary route of human listeriosis is ingestion of contaminated RTE foods. In several studies around the world, L. monocytogenes has been isolated in high frequencies from soft cheeses, hot- or cold-smoked or gravad fish and deli-meat products, and this trend is similar in Sweden. Food contamination occurs through the processing environment and from the raw material to the final product. L. monocytogenes can persist over decades in processing plants.

2. The conventional serotyping by agglutination identifies five main L. monocytogenes serovars belonging to lineage I (4b, 1/2b and 3b), and lineage II (1/2a and 1/2c). Although, there is no association between serovars, gender and age, the prevalence of serovar 1/2c among males, and serovar 4b in pregnancy cases is reported globally, including Sweden. A shift from L. monocytogenes serovar 4b to serovar 1/2a among clinical human L. monocytogenes isolates occurred in the mid 1990s and early 2000s in Europe, including Sweden.

3. Pulsed field gel electrophoresis (PFGE) with restriction enzyme AscI is efficient in distinguishing L. monocytogenes subtypes and variants (based on the number and distribution of all detectable fragments in a DNA restriction profile). Among 932 human isolates, 183 PFGE types were identified, indicating relatively low genetic diversity.

4. Some human L. monocytogenes PFGE-types were present over extended periods during the study period (1958–2010). Others were isolated only during limited periods. Six PFGE types belonging to serovar 1/2a and 4b have persisted at least between 17 to 47 years and are frequently associated with human listeriosis in Sweden. The two most common PFGE types are distributed across 16 to 17 counties. Some PFGE types have been identified globally, indicating certain L. monocytogenes PFGE types may be adapted to specific niches and are thus widely distributed with a high prevalence.

5. Based on the number and distribution of all detectable small fragments below 145.5 kb, L. monocytogenes PFGE types could be further divided into PFGE groups. Although the L. monocytogenes isolates are from different sources, time and location, the small fragments below 145.5 kb are genetically more conserved than the larger fragments, supporting the utility of the genomic region of small fragments in grouping L. monocytogenes. The distribution of these small fragments establishes the relatedness of strains and defines genetic markers for both lineages I and II. The three most common PFGE groups are distributed across 19, 20 and 21 counties, suggesting specific PFGE groups may be frequently associated with human listeriosis in Sweden. As a new PFGE group rarely appears, it is reasonable to assume the most important PFGE groups of L. monocytogenes have been identified in Sweden.

6. L. monocytogenes PFGE types are homogeneous with respect to serovar. The genomic region of small fragments below 145.5 kb establishes an association between PFGE

group and serovar. The number of PFGE types is considerably higher in lineage II than in lineage I, whereas, the numbers of PFGE groups in both lineages are equivalent. In contrast to lineage II, L. monocytogenes isolates, small DNA fragments below 33.3 kb are nonvisible in all isolates belonging to lineage I. Thus, lineage I isolates and lineage II isolates do not share any PFGE group. In consequence, the small fragments are able to differentiate the lineages and confirm the relatedness of strains.

7. A new procedure for improving the identification of a L. monocytogenes isolate is proposed. First, the smallest DNA fragments below 145.5 kb are analysed to identify the AscI PFGE group, and then the large fragments above 145.5 kb are used to determine the PFGE type. As the small DNA fragments in the AscI PFGE profiles are relatively conservative, they can be used for an initial rough division of profiles. PFGE with AscI restriction enzyme is still a valuable method for genomic analysis and comparison for identifying genomic relatedness among L. monocytogenes isolates. Consequently, the application of PFGE in silico subtyping enhances the WGS analysis through first identifying the PFGE group and then the PFGE type, which allows rapid screening of genomes. However, the genetic structures of the small restriction fragments in AscI PFGE groups of L. monocytogenes genomes remain to be determined.

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Human isolates of Listeria monocytogenes in Sweden during half a century (1958–2010)

G. LOPEZ-VALLADARES1,W.THAM1*, V. SINGH PARIHAR1, S. HELMERSSON2,B.ANDERSSON3, S. IVARSSON4, C. JOHANSSON4, H. RINGBERG5,I.TJERNBERG3,B.HENRIQUES-NORMARK4 1 AND M.-L. DANIELSSON-THAM 1 Department of Restaurant and Culinary Arts, Örebro University, Grythyttan, Sweden 2 Department of Environmental Assessment, SLU, Uppsala, Sweden 3 Department of Bacteriology, Malmö University Hospital, Malmö, Sweden 4 The Swedish Institute for Infectious Disease Control and MTC, Karolinska Institutet, Solna, Sweden 5 Regional Centre for Communicable Disease Control and Prevention, Malmö, Sweden

Received 22 April 2013; Final revision 19 November 2013; Accepted 11 December 2013; ﬁrst published online 20 January 2014

SUMMARY Isolates of Listeria monocytogenes (n=932) isolated in Sweden during 1958–2010 from human patients with invasive listeriosis were characterized by serotyping and pulsed-ﬁeld gel electrophoresis (PFGE) (AscI). Of the 932 isolates, 183 different PFGE types were identiﬁed, of which 83 were each represented by only one isolate. In all, 483 serovar 1/2a isolates were distributed over 114 PFGE types; 90 serovar 1/2b isolates gave 32 PFGE types; 21 serovar 1/2c isolates gave nine PFGE types; three serovar 3b isolates gave one PFGE type; and, 335 serovar 4b isolates gave 31 PFGE types. During the 1980s in Sweden, several serovar 4b cases were associated with the consumption of European raw soft cheese. However, as cheese-production hygiene has improved, the number of 4b cases has decreased. Since 1996, serovar 1/2a has been the dominant L. monocytogenes serovar in human listeriosis in Sweden. Therefore, based on current serovars and PFGE types, an association between human cases of listeriosis and the consumption of vacuum-packed gravad and cold-smoked salmon is suggested.

Key words: Cheese, Listeria monocytogenes, PFGE, serotyping, salmon.

INTRODUCTION and/or meningitis, and abortion in pregnant women. Listeria monocytogenes is a pathogen associated with Listeriosis primarily affects pregnant women, new- food and is mainly transmitted through ready-to-eat borns, and individuals with weakened immune systems. The overall case-fatality rate from invasive food such as soft cheese, vacuum-packed gravad and cold-smoked salmon, salads, raw milk, and uncooked listeriosis is from 20% to >50% [1]. ﬁ L. monocytogenes meat. The clinical picture consists of septicaemia In Sweden, the rst isolation of was from rabbits in 1910 [2], and in 1960, human listeriosis became a notiﬁable disease [3]. Sweden is divided into 21 counties and surveillance is based on * Author for correspondence: Dr W. Tham, Department of these administrative regional units (Fig. 1). During Restaurant and Culinary Arts, Örebro University, Grythyttan, Sweden. 2009, Sweden had the second highest number of cases (Email: [email protected]) reported (n=73), and had the second highest number 2252 G. Lopez-Valladares and others

Isolates the incidence rate is around 0·3/100000 population [5]. Scandinavian countries have consistently higher rates 4–8 than other countries, which is possibly due to the con- 9–19 sumption of smoked ﬁsh [5]. 20–36 L. monocytogenes isolates from human patients 37–56 in Sweden suffering from invasive listeriosis during 57–225 BD 1986–2007 have been characterized through serotyping and pulsed-ﬁeld gel electrophoresis (PFGE) [6]. The aim of the present study was to continue the characterization of human L. monocytogenes isolates for the periods before (1958–1985) and after (2008– AC 2010). A second aim was to relate genotypic results to epidemiological information, and to identify possible clustering of L. monocytogenes genotypes in Z time, season, location, age, or gender. In the present – Y paper, results from the entire 53-year period (1958 2010) are presented and discussed.

MATERIALS AND METHODS X L. monocytogenes isolates Isolates of L. monocytogenes (n=932) from patients W with invasive listeriosis were received from clinical C laboratories throughout Sweden and represented S U 64% of the 1460 human listeriosis cases reported AB T during the entire 53-year period: a mother–child pair D was considered as one case. The overall case-fatality rate was estimated as 30%. Isolates were from blood E (n=531), cerebrospinal ﬂuid (n=137) or other sources O (n=68) of sporadic cases, and from one known out- F I break in 1994/1995 involving eight patients [7]. Data H regarding source was lacking for 196 isolates and N G duplicate isolates from patients were disregarded. − K The isolates were frozen at 75 °C in 80% brain M heart infusion (Merck, Germany) and 20% v/v glycerol until analysis by serotyping and PFGE.

Fig. 1 [colour online]. Number of isolates collected from the different counties in Sweden. Götaland region: Skåne Serotyping (M); Blekinge (K); Halland (N); Västra Götaland (O); All L. monocytogenes isolates were serotyped with Gotland (I); Kronoberg (G); Kalmar (H); Jönköping (F); Listeria O antiserum types I/II, I, IV, V/VI, VI, VII, Östergötland (E). Svealand region: Örebro (T); Söder- manland (D); Västmanland (U); Uppsala (C); Stock- VIII, IX and H antiserum A, AB, C, D (Mast holm (AB); Värmland (S); Dalarna (W). Norrland region: Diagnostics, UK) according to the manufacturer’s Gävleborg (X); Västernorrland (Y); Jämtland (Z); Väster- instructions, with some modifications [6]. botten (AC); Norrbotten (BD). For four isolates there was no information. PFGE of reported cases within the EU (0·78 cases/100000). Each isolate was restricted with the AscI enzyme and During 2013, Sweden had the highest number of cases subsequently subjected to PFGE, according to Graves (n=93) since 1960, i.e. 0·97 cases/100 000 people [4]. & Swaminathan [8] with some modifications [6]. Gels In developed countries where listeriosis is documented, with profiles were visualized by short-wave ultraviolet Human Listeria isolates in Sweden, 1958–2010 2253

(312 nm) light and photographed with a Polaroid camera. The pictures were analysed visually and the DNA restricted fragments were sized against lambda ladder PFG marker no. 340 S (New England Bio- Labs Inc., USA). Image analysis software was not used for comparison. PFGE types were established based on the number and distribution of bands in each profile. The profiles were considered distinguishable if there was a difference of one band or more. Each unique PFGE strain was designated in a specific way. For example, serovar, AscI profile, and variant; thus, the designation 1/2a:4A indicated the strain belonged to serovar 1/2a, AscI profile 4, and variant A. 2010 – RESULTS AND DISCUSSION Results from a previous characterization of human 58 40 36 67 67 64 Most common PFGE type Swedish L. monocytogenes isolates from 1986 to 2007 by Parihar et al.[6], are included in the present

) study. Thus, the present paper covers the characteriza- n

† tion of L. monocytogenes isolates from 932 human

PFGE types ( patients with invasive listeriosis in Sweden during the period 1958–2010.

Characterization Of the 932 human L. monocytogenes isolates, 183 different PFGE types were identiﬁed, of which 83 PFGE types were each represented by only one isolate. The number of isolates, serovars, number of PFGE types, and the six most common PFGE types of L. monocytogenes isolates per region during 1958– 2010 are presented in Table 1. The distribution of L. monocytogenes isolates, serovars, and the six most common PFGE types per year (1958–2010) are pre- Serovars 1/2a 1/2b 1/2c 3b 4b 1/2a:4A 1/2a:4B 1/2a:9B 4b:3 4b:6 4b:5 isolates in Sweden by serovar, PFGE type and region during 1958 sented in Table 2. Thus, 483 serovar 1/2a isolates were distributed in 114 PFGE types (an average of 4·24 isolates per PFGE type); 90 serovar 1/2b isolates ed. ﬁ ) gave 32 PFGE types (2·81 isolates per PFGE type); n Isolates ( 21 serovar 1/2c isolates gave nine PFGE types (2·33 isolates per PFGE type); three serovar 3b iso-

L. monocytogenes lates gave one PFGE type; and 335 serovar 4b isolates gave 31 PFGE types (10·80 isolates per PFGE type) (%) ]. n

54 (Table 3). –

eld gel electrophoresis. The period studied (1958 2010) was divided into ﬁ Population 2011* three periods: 1958–1971, when three serovars 1/2a,

Distribution of 1/2b, and 4b were equally common; 1972–1995, when serovar 4b was the main L. monocytogenes serovar in human listeriosis; and 1996–2010, when Total number of PFGE types identi No dataTotal 9482855(100·0) 932 483 4 90 1 21 0 3 335 0 183 0 3 4 0 0 0 0 0 1 Table 1. Region PFGE, Pulsed- * Statistics Sweden [ GötalandSvealandNorrland 4542263(47·9) 3787796(39·9) 1152796(12·2) 526 294 108 255 154 58 73 26 13 6 6 3 2 0 197 0 108 141 27 99 30 47 14 27 14 10 17 3 11 44 8 18 46 20 5 39 18 1 6 † serovar 1/2a was the main L. monocytogenes serovar 2254 G. Lopez-Valladares and others

Table 2. Serovars, PFGE types and the six most common PFGE types of L. monocytogenes isolates in Sweden, per year, during 1958–2010

Serovars Most common PFGE types Isolates PFGE Year (n) 1/2a 1/2b 1/2c 4b 3btypes (n) 1/2a:4A 1/2a:4B 1/2a:9B 4b:3 4b:6 4b:5

1958 5 2 1 1 1 4 1959 1 1 1 1960 0 1961 0 1962 3 2 1 3 1963 5 3 2 4 1964 0 1965 3 1 2 3 1 1966 2 1 1 2 1 1967 3 1 1 1 3 1968 1 1 1 1969 4 2 2 4 1 1970 3 2 1 3 1971 4 2 2 3 2 1972 9 3 6 4 1 5 1973 6 1 1 4 4 2 1974 15 1 14 9 7 1 1975 15 15 4 6 7 1976 13 13 4 3 8 1 1977 16 1 1 14 8 5 4 2 1978 9 3 1 5 7 1 1979 11 11 5 5 3 1 1980 11 1 5 1 4 9 1 1 1981 13 5 8 9 2 3 1982 12 3 1 8 7 1 6 1 1983 11 1 1 9 5 3 3 3 1984 7 3 0 4 2 4 1985 16 6 3 2 5 14 1 3 1986 2 1 1 2 1 1987 33 9 1 23 11 3 9 7 1988 41 24 3 1 13 22 8 1 3 1 4 1989 23 10 1 1 11 14 1 3 6 1990 27 11 1 1 14 10 6 3 6 1991 17 5 1 1 10 14 1 1 1 1992 23 9 2 12 16 1 4 2 1993 24 17 1 6 15 1 2 1 3 3 1994 28 11 4 1 12 17 2 2 1 1995 21 9 12 13 1 1 4 4 1996 20 14 6 13 1 2 2 1997 16 8 2 6 13 1 1 1 1998 21 12 4 5 16 3 1 1 1 1999 22 11 4 1 6 15 2 1 1 2000 42 27 8 1 6 24 5 1 2001 46 27 7 1 10 1 25 2 7 3 2 4 2002 38 26 9 1 2 26 2 2 1 2003 48 40 4 4 27 1 3 2 1 1 2004 28 17 1 2 8 21 1 2 2 1 1 2005 17 10 5 2 15 1 1 2006 24 16 1 2 5 17 3 3 1 2007 29 25 0 0 4 21 1 1 3 1 2008 32 20 2 0 10 23 2 6 1 2 1 2009 49 40 2 0 7 27 2 7 5 2 2010 63 47 4 0 10 2 28 11 6 8 1 5 Total 932 483 90 21 335 3 58 40 36 67 67 64

PFGE, Pulsed-ﬁeld gel electrophoresis. Human Listeria isolates in Sweden, 1958–2010 2255

Table 3. Serovar and PFGE type distribution of Sweden, shared the same PFGE type (4b:6) as the L. monocytogenes isolates in Sweden during 1958–2010 epidemic cheese-borne strains from Switzerland, Denmark, and USA [15, 20, 21]. Serovar Isolates n (%) PFGE types Average*

1/2a 483 (51·8) 114 4·24 1/2b 90 (9·7) 32 2·81 Raw milk soft cheeses and L. monocytogenes 1/2c 21 (2·3) 9 2·33 serovars 3b 3 (0·3) 1 3·00 One hypothesis was a majority of human L. mono- 4b 335 (35·9) 31 10·80 Total 932 (100·0) 187† cytogenes serovar 4b isolates in Sweden, during the period 1972–1995, came from soft raw milk cheeses PFGE, Pulsed-field gel electrophoresis. imported from European countries. In 1987, Beckers * Average number of isolates per PFGE type. et al. [22] reported nine (65%) of 14 French soft raw † 183 PFGE types were identified. Four PFGE types are milk cheeses purchased in The Netherlands were posi- shared by serovars 1/2a and 1/2c. tive for L. monocytogenes. Eppert et al.[23] found 15 (46·9%) of 32 French soft raw milk cheeses sampled (Table 4). As the present study was retrospective, the on the German market between 1992 and 1994 were data about the sources were not supported by positive for L. monocytogenes. Of 30 French soft Swedish epidemiological investigations of outbreaks raw milk cheeses purchased during 1989–1993 in or case-control studies of sporadic cases. However, Sweden, 13 (43·3%) were positive for L. monocyto- food sampling has been conducted several times in genes [9]. Sweden during different periods [9–13]. In 1995, the first documented outbreak of listeriosis in France, which involved 36 patients, was linked to soft raw milk cheese, Brie de Meaux: the epidemic L. monocytogenes isolates from 1958 to 1971 strain belonged to serovar 4b [24–26]. In 1997, From 1958 to 1971, serovars 1/2a, 1/2b, and 4b were another 14 people became ill after consuming the ’ equally common, although based on a small number soft raw milk cheese Livarot, Pont l Évêque (serovar of available human isolates (n=34). During a possible 4b) [25, 27] and in 1999, three more cases were due outbreak of listeriosis in Uppsala at the beginning to the soft raw milk cheese Epoisses (serovar 4b) of the 1960s, bacteriological investigations on eggs [26]. These cheese brands were available on the – and poultry failed to identify Listeria bacteria [14]; Swedish market during 1989 1993 [9]. In Belgium in however, this investigation was conducted two dec- 1997, a case of listeriosis in a 73-year-old immuno- ades before the foodborne route for L. monocytogenes compromised man was associated with the consump- was established. tion of a French Camembert cheese bought a few days before he became ill: the isolates from both patient and cheese shared serovar 1/2a [28]. In 1998, ‘ L. monocytogenes isolates from 1972 to 1995 de Valk et al.[29] stated soft cheese may account for a substantial proportion of sporadic listeriosis’, Epidemic cheese-borne strains and in a case-control study in metropolitan France Of the available human isolates (n=403) from 1972 to during 1997, 49% of the sporadic cases of listeriosis 1995, serovar 4b was shared by 234 (58·1%) and sero- studied could be attributed to eating soft cheese [29]. var 1/2a was only shared by 131 isolates (32·5%) A majority of the decrease in L. monocytogenes in (Table 4). During the 1980s, large outbreaks and spor- ready-to-eat foods, including cheese, in France during adic cases of foodborne listeriosis due to closely re- the 1990s is probably related to stricter control mea- lated strains sharing serovar 4b were reported from sures implemented during food production [30]. In both the USA and Europe [15]. An indistinguishable cheese production, milking hygiene has improved PFGE type was identified in a Swiss soft cheese out- and cows with L. monocytogenes mastitis are not break between 1983 and 1987 [16], a Danish outbreak used for milk production: several further precaution- in 1985–1987 [17] and again in 1989–1990 [18], the ary measures have been implemented in raw milk latter probably due to blue mould cheese and, in cheese preparation [25, 26, 31]. The number of samples California in 1985 due to fresh cheese [19]. One of from dairy products contaminated at 5100 c.f.u./g the most common PFGE types during 1972–1995 in decreased by 41% between 1993/1994 and 1995/1996 2256 G. Lopez-Valladares and others

Table 4. Serovar and PFGE type distribution of L. monocytogenes isolates in Sweden, by period, during 1958–2010

1958–1971 1972–1995 1996–2010

Isolates PFGE Isolates PFGE Isolates PFGE Serovar n (%) types (n) Average* n (%) types (n) Average* n (%) types (n) Average*

1/2a 12 (35·30) 11 1·1 131 (32·5) 51 2·6 340 (68·7) 87 3·1 1/2b 10 (29·4) 7 1·4 27 (6·7) 16 1·7 53 (10·7) 21 2·5 1/2c 2 (5·9) 2 1·0 11 (2·7) 7 1·6 8 (1·6) 4 2·0 3b 0 (0) 0 0 0 (0) 0 0 3 (0·6) 1 3·0 4b 10 (29·4) 6 1·6 234 (58·1) 21 11·1 91 (18·4) 25 3·6 Total 34 (100·0) 403 (100·0) 495 (100·0)

PFGE, Pulsed-ﬁeld gel electrophoresis. * Average number of isolates per PFGE type.

[27]. As ready-to-eat products became less contami- the laboratory and at the end of their shelf-life. nated, the number of listeriosis cases in France Across the entire EU, the prevalence in fish samples decreased by 68% between 1987 and 1997 [27]. In was 10·4% at the time of sampling and 10·3% at the France, the annual number of listeriosis cases per mil- end of shelf-life. The proportion of samples with a lion population, fell from 5·2 in 1995 to 3·8 in 1996 L. monocytogenes count exceeding 100 c.f.u./g was [27]. Since 1996, serovar 4b has been reduced to the 1% at the time of sampling and 1·7% at the end of second or third most common serovar in human clin- shelf-life [35]. ical cases in Sweden. Gravad and cold-smoked salmons are vacuum- packed ready-to-eat products with a lengthy best- before date of 4–5 weeks: therefore, they constitute L. monocytogenes isolates from 1996 to 2010 an optimal environment for the facultative anaerobic, Cold-smoked and gravad fish and L. monocytogenes psychrotrophic organism Listeria. The consumption serovar 1/2a of gravad and cold-smoked salmons increased mark- There has been a shift in human listeriosis in Sweden edly in Sweden during the 1990s [36]. In 1996 in and L. monocytogenes serovar 1/2a isolates are now Sweden, 20·7% of gravad and 11·5% of cold-smoked fi more frequent than serovar 4b. In Finland, serovar vacuum-packed sh purchased, especially salmon, 1/2a isolates became dominant in 1991 [32], but 1/2a harboured L. monocytogenes [10]. In the UK, a survey fi was also the major serovar in Sweden in 1988 and of retail cold-smoked sh found 236/1344 (17·4%) pro- 1993. In Canada, a predominant L. monocytogenes ducts were positive for L. monocytogenes, all samples serovar 1/2a clone caused human listeriosis cases and had a level of <100 c.f.u./g [37]. The current PFGE outbreaks during 1988–2010 [33]. types found in human cases of invasive listeriosis in Serovar 1/2a is the serovar regularly found in Sweden are frequently encountered in vacuum-packed cold-smoked and gravad salmon purchased in cold-smoked and gravad salmon/rainbow trout – Sweden and, L. monocytogenes has been found in gra- [11 13]. In Spain, a cluster of L. monocytogenes isolates vad and cold-smoked fish samples. In a recent study in smoked salmon share pulsotype 1, a type recently iso- 14% of each group were positive for the presence lated from clinical cases in Spain [38]. In Brazil, 41% of of L. monocytogenes, and the National Food salmon samples in a gravlax salmon processing line Administration of Sweden concluded these products were positive for L. monocytogenes [39]. ‘constitute the main problem’ [34]. A European A decrease or stabilization in the number of human Union (EU)-wide survey on L. monocytogenes during isolates sharing serovar 4b and an increasing number fi 2010 and 2011 aimed to estimate the prevalence of of serovar 1/2a isolates have also been identi ed in L. monocytogenes in hot- or cold-smoked or gravad other countries, e.g. Italy [40] and Switzerland [41]. fish within the EU. Packaged (not frozen) hot- or L. monocytogenes serovar 4b cold-smoked or gravad fish (3053 batches) from 3632 retail outlets in 26 EU member states and one In Sweden, the decrease in the number of listeriosis non-EU country were sampled, both on arrival at cases caused by serovar 4b strains since 1996 might Human Listeria isolates in Sweden, 1958–2010 2257

Table 5. Distribution of L. monocytogenes isolates in Sweden by gender and age during 1958–2010

Age (years)

Gender n* (%) 1–910–19 20–29 30–39 40–49 50–59 60–69 70–79 80–89 90–99 No data

Female 354 (42·6) 4 6 14 23 30 40 73 87 65 10 1 Male 449 (54·1) 6 5 4 11 18 58 120 115 98 12 2 No data 27 (3·3) 0 0 0001 1 00025 Total 830 (100·0) 10 11 18 34 48 99 194 202 163 22 28

* Pregnant and newborns are excluded.

Table 6. Serovar distribution of L. monocytogenes isolates in pregnant-associated cases and the elderly in Sweden, by decade, during 1958–2010

Serovars Serovars

Period Isolates n (%) Pregnant/child (n) 1/2a 1/2b 4bElderly 560 years (n) 1/2a 1/2b 4b

1958–1969 27 (3·0) 7 0 2 4 3 1 2 0 1970–1979 101 (10·8) 33 2 1 30 34 3 4 26 1980–1989 169 (18·1) 22 5 1 16 85 33 8 38 1990–1999 219 (23·5) 22 6 1 15 145 80 8 52 2000–2010 416 (44·6) 18 11 2 5 314 233 33 43 Total 932 (100·0) 102 24 7 70 581 350 55 159 be due to precautionary measures taken during cheese L. monocytogenes; the first in 2001 [45, 46] and the production in France and other European countries. second in 2010 [34]. Products such as pig tongue in In a study from May to December 1999, only four aspic and rillettes were not tested by the National (4·4%) of 91 French soft raw milk cheeses (red Food Administration nor sampled by the different smear) were positive for L. monocytogenes [42]. Health Boards in these studies, as they are not popular Although this represented an improvement, the over- foods and not generally available from retail outlets. all results from analyses of 329 European soft, semi- soft, and hard red-smear cheeses made of raw or pasteurized milk were not encouraging, as 6·4% were Historical, seasonal, and geographical variation, – positive for L. monocytogenes. Thus, the authors con- 1958 2010 cluded that red-smear cheeses should still be regarded In the collection of 932 human L. monocytogenes as a considerable public health risk. A EU-wide sur- isolates from 1958 to 2010, the majority (29·5%) of vey in 2010 and 2011 sampled 3452 cheeses at the serovar 1/2a isolates were isolated during October– end of their shelf-life from retail outlets in 26 EU mem- January, with a peak in October, whereas, most ber states and one non-EU country. The prevalence (32·6%) of the serovar 4b and 1/2b isolates were across the entire EU was only 0·47%. The EU-level pro- isolated during June–October, with a peak in August. portion of cheese samples with a L. monocytogenes In the USA, more serovar 1/2a isolates are found in count exceeding 100 c.f.u./g was 0·06% at the end of water during autumn and winter, whereas, serovar 4b shelf-life [35]. During the 1990s, other food products are more often isolated during summer [47]. were carriers of L. monocytogenes serovar 4b and In the north of Sweden (Fig. 1), i.e. Jämtland (Z), caused outbreaks in France, such as pork tongue Västerbotten (AC) and Norrbotten (BD), cases of lis- in jelly in 1992 [43] and rillettes in 1993 [44]. teriosis are registered almost entirely during January– However, to our knowledge, those products were not March, and in the south of Sweden, i.e. Skåne (M), generally imported into Sweden. The National Food the majority of cases are registered during summer Administration of Sweden requested two large and autumn (Fig. 1). Of the most common PFGE national samplings of food products for analysis of types, 1/2a:4A was distributed across 17 counties 2258 G. Lopez-Valladares and others and 4b:5 was distributed across 16 counties. recommendations from the National Food Admin- Conversely, PFGE types 4b:6, indistinguishable from istration which has provided advice on food to preg- the epidemic cheese-borne strains in Switzerland, nant women since the early 1990s. According to the Denmark, and USA (1983–1990), and PFGE types dietary advice, pregnant women should avoid gravad 4b:3 and 1/2a:4b were concentrated in Götaland and smoked fish at the end of its shelf-life, cheeses (south region of Sweden), possibly due to its proximity made from unpasteurized milk, and even some soft to the European continent. In Norrland (north region cheeses made from pasteurized milk, and sliced cold of Sweden), PFGE type 4b:6 was reported in only cuts and cold foods at the end of their shelf-life. The one county, type 4b:3 was reported in three counties preponderance of serovar 4b in pregnancy-associated and 1/2a:4B in two counties (Table 1). isolates is in agreement with other studies [51–53]. Some PFGE types are present over long periods, However, there was an increase in listeriosis in Sweden such as 1/2b:75A (1970–2010) and 4b:5 (1965–2008), in the older age group (560 years) which constituted whereas, others appear during limited periods, such 11·1% of all cases reported during 1958–1969; 33·7% as 1/2a:53 (1985–1995) and 1/2a:11B (1990–1994). in 1970–1979; 50·3% in 1980–1989; 66·2% in 1990– The two currently dominant PFGE types in Sweden, 1999; and 75·5% in 2000–2010 (Table 6). Even in 1/2a:4A and the closely related 1/2a:4B, were almost other European countries there is an increased inci- non-existent during the 1960s and 1970s (Table 2). dence of listeriosis in the elderly population aged Lukinmaa et al. [32] suggest a PFGE closely related >60 years [51]. to the Swedish type 1/2a:4A was common in sporadic cases in Finland from 1994 onwards. Similarly, Clark et al.[48] state isolates of one unique type were not CONCLUSION seen before 2002 in Canada, but have been detected Between 1972 and 1995, human listeriosis in Sweden in each subsequent year. Emerging PFGE types was mainly caused by serovar 4b, possibly due to fl could re ect a change in dietary habits [32]. the consumption of raw soft cheese imported from European countries. The number of cases caused by serovar 4b has decreased due to improved cheese- Relationship between PFGE type and serovar production hygiene, but since 1996, serovar 1/2a has Generally, all L. monocytogenes isolates belonging been the dominant serovar. Based on the serovars to one PFGE type share the same serovar. However, and the PFGE types identified, an association between isolates within four PFGE types were distributed in human cases of listeriosis and the consumption of either serovar 1/2a or 1/2c, i.e. 1/2:12A (six 1/2a, vacuum-packed gravad and cold-smoked salmon is nine 1/2c isolates), 1/2:12B (four 1/2a, two 1/2c), suggested. 1/2:93 (two 1/2a, two 1/2c), and PFGE type 1/2:105 (one 1/2a, one 1/2c). Although correlation between ‘pulsotypes and serotypes’ is reported [40], few ACKNOWLEDGEMENTS L. monocytogenes isolates with indistinguishable This study was financially supported by the Research PFGE profiles displaying different serovars (1/2a Foundation of Ivar and Elsa Sandberg, to whom we and 3a [32], 3b and 1/2b [49]) have been identified. express our gratitude.

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FOODBORNE PATHOGENS AND DISEASE Volume 12, Number 5, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2014.1880

Division of Human Listeria monocytogenes Pulsed-Field Gel Electrophoresis (PFGE) Types Belonging to Lineage I (Serovar 4b, 1/2b, and 3b) into PFGE Groups

Gloria Lopez-Valladares,1 Marie-Louise Danielsson-Tham,1 Richard V. Goering,2 and Wilhelm Tham1

Abstract The 63 pulsed-field gel electrophoresis (PFGE) types identified among 427 clinical isolates of Listeria monocytogenes that were characterized in a previous study by serotyping and PFGE (AscI) could be further divided into 17 PFGE groups. While the 63 PFGE types, all part of lineage I, were established based on the number and distribution of all bands in each DNA profile, the 17 PFGE groups were based on the configuration of small bands with sizes < 145.5 kb. The 30 PFGE types of L. monocytogenes serovar 4b isolates (n = 334) were divided into 8 PFGE groups; the 32 PFGE types of serovar 1/2b isolates (n = 90) and the serovar 3b isolates (n = 3, 1 PFGE type) were divided into 9 PFGE groups. An association was observed between PFGE groups and serovars. L. monocytogenes isolates belonging to PFGE groups I, J, Q, R, X, Z, O¨ -4, and O¨ -5 all shared serovar 4b, whereas isolates from PFGE groups D, G, O, P, T, U, O¨ -1, O¨ -2, and O¨ -3 shared serovar 1/2b. Small fragments < 33.3 kb were nonvisible in all L. monocytogenes isolates. From the results of the present study, a procedure for accelerating the identification of PFGE types when analyzing new PFGE profiles can be suggested. Therefore, we propose a stepwise procedure to PFGE profiling by first identifying the PFGE group using the smaller band patterns < 145.5 kb, and then determining PFGE types based on the band patterns > 145.5 kb.

Introduction been reported since 2000 (Tham and Danielsson-Tham, 2014). However, serotyping is a poorly discriminating he infectious disease listeriosis is primarily caused method for the epidemiological characterization of bacterial Tby the bacterial species Listeria monocytogenes, which isolates associated with foodborne outbreaks. is a food- and feedborne pathogen causing infection in both Pulsed-field gel electrophoresis (PFGE) using restriction humans and animals (Peiris et al., 2009; Lambertz et al., enzyme AscI is well established as an effective molecular 2013; Lopez-Valladares et al., 2013). The main manifesta- subtyping method for characterizing L. monocytogenes tions of human listeriosis are meningoencephalitis, septice- (Brosch et al., 1996; Kerouanton et al., 1998; Lopez- mia, and abortion. L. monocytogenes is a Gram-positive rod Valladares et al., 2014) with utility in the identification of that grows and multiplies intracellularly, and the bacteria are human listeriosis outbreaks (Buchrieser et al., 1993; Ericsson ubiquitous with the ability to grow at low temperatures and et al., 1997; Goulet et al., 1998; Danielsson-Tham et al., low pH values (Seeliger and Jones, 1986). The incidence of 2004; Knabel et al., 2012). L. monocytogenes isolates from listeriosis is lower than with other bacterial diseases; how- human patients suffering from invasive listeriosis in Sweden ever, the case-fatality rate is high, between 20% and 40% in between 1958 and 2010 have been effectively characterized humans (McLauchlin, 1996). The high case-fatality rate is by serotyping and PFGE (Lopez-Valladares et al., 2014). associated with individuals who have an underlying im- However, examination and assignment of PFGE types to munosuppressed condition (McLauchlin et al., 1991). specific profiles is a delicate and tedious process that would During the 1970s, 1980s, and 1990s, the global incidence benefit from simplification. of human listeriosis was mainly caused by L. monocytogenes Therefore, the main aim of this study was to accelerate the serovar 4b (Guevara et al., 1979; Kathariou, 2002; Lopez- identification of PFGE profiles first by grouping the band Valladares et al., 2014), although several 1/2a outbreaks have patterns < 145.5 kb and then by subtyping those > 145.5 kb

1School of Hospitality, Culinary Arts and Meal Science, O¨ rebro University, Grythyttan, Sweden. 2Department of Medical Microbiology and Immunology, Creighton University Medical Center, School of Medicine, Omaha, California.

447 448 LOPEZ-VALLADARES ET AL.

Table 1. Distribution of Listeria monocytogenes were considered distinguishable if there was a difference of Lineage I Isolates in Sweden by Serovar, one band or more. Serovar 4b isolates (n = 334) were dis- Pulsed-Field Gel Electrophoresis (PFGE) tributed over 30 PFGE types; 90 serovar 1/2b isolates gave 32 Types, and Groups from to PFGE 1958 2010 PFGE types and 3 serovar 3b isolates gave 1 PFGE type PFGE PFGE (Table 1). For storage, isolates were maintained in brain- Isolates type group heart infusion broth (Merck, Darmstadt, Germany) with 20% Serovar (n) (%) (n) Average (n) Average glycerol at - 70C.

1/2b 90 21.1 32 2.81 9 10.0 Grouping of L. monocytogenes PFGE types 3b 3 0.7 1 3.0 1a 3.0 4b 334 78.2 30 11.13 8 41.75 In a previous study, the 63 PFGE types of L. mono- Total 427 100.0 63 17 cytogenes were established based on the number and distribution of all fragments in a DNA restriction profile aShared the same PFGE group (G) as some serovar 1/2b isolates. (Lopez-Valladares et al., 2014). The AscI restriction fragments of each PFGE type were analyzed visually and sized against a Lambda ladder PFGE marker and the universal using PFGE band patterns of 427 L. monocytogenes isolates marker Salmonella Braenderup H9812. In the present study, belonging to lineage I (serovars 4b, 1/2b, and 3b) as the data PFGE groups were established solely on the number and source. Possible association between PFGE groups and ser- distribution of small bands with sizes < 145.5 kb. Thus, each ovars was also attempted. PFGE group included several different PFGE types with identical configuration of bands with sizes < 145.5 kb. The Materials and Methods PFGE groups were designated by capital letters. Each isolate of L. monocytogenes was then assigned a multipart L. monocytogenes isolates code. For example, the designation D:1/2b:75A indicates Lineage I L. monocytogenes (n = 427) isolated during that the isolate belonged to PFGE group D, serovar 1/2b, and 1958–2010 from human invasive listeriosis in Sweden was represented the 75th AscI profile within the serogroup 1/2 selected for the present study. All of the isolates were pre- portion of the collection, respectively. The last character viously characterized by serotyping and PFGE with restric- (i.e., ‘‘A’’) refers to variation among the AscI profiles. AscI- tion enzyme AscI according to Graves and Swaminathan profile no. 75 comprises closely related variants (A, B, C) (2001) with some modifications (Lopez-Valladares et al., (Tenover et al., 1995). The AscI profiles of L. mono- 2014). PFGE types were established based on the number and cytogenes isolates representing the different PFGE groups distribution of all bands in each DNA profile. The profiles are presented in Figure 1.

FIG. 1. AscI proﬁles of Listeria monocytogenes lineage I, human strains. Electrophoretic conditions: 21 h, pulse times 4– 40 s, 120 angle, and 14C. Lane 1 (Lambda ladder PFGE marker No. 340 S); Lane 2 (Salmonella Braenderup H9812); Lane 3 (group G); Lane 4 (group D); Lane 5 (group O); Lane 6 (group P); Lane 7 (group T); Lane 8 (group U); Lane 9 (group O¨ - 1); Lane 10 (group O¨ -2); Lane 11 (group O¨ -3); Lane 12 (group I); Lane 13 (group J); Lane 14 (group Q); Lane 15 (group R); Lane 16 (group X); Lane 17 (group Z); Lane 18 (group O¨ -4); Lane 19 (group O¨ -5); Lane 20 (Salmonella Braenderup H9812); and Lane 21 (Lambda ladder PFGE Marker No. 340 S). DIVISION OF L. MONOCYTOGENES PFGE TYPES INTO PFGE GROUPS 449

Table 2. Serovar, Pulsed-Field Gel Electrophoresis Table 2. (Continued) (PFGE) Group, and PFGE Type Distribution of Human Listeria monocytogenes Lineage I Isolates (n) Serovar PFGE group PFGE type Isolates in Sweden from 1958 to 2010 6 4b X 4b:4 Isolates (n) Serovar PFGE group PFGE type 2 4b Z 4b:23 14bO¨ -4 4b:13 7 1/2b D 1/2b:5 14bO¨ -5 4b:14 3 1/2b D 1/2b:29 2 1/2b D 1/2b:32A 2 1/2b D 1/2b:32B 2 1/2b D 1/2b:37 Results and Discussion 3 1/2b D 1/2b:55 The 63 PFGE types previously identified in 427 clinical 9 1/2b D 1/2b:75A L. monocytogenes isolates could be further divided into 2 1/2b D 1/2b:75B 17 PFGE groups based on the position of distinctly detectable 1 1/2b D 1/2b:77 < < 1 1/2b D 1/2b:104 bands 145.5 kb. Small fragments 33.3 kb were nonvisible 4 1/2b D 1/2b:119 in all L. monocytogenes isolates (Fig. 1). Five of the 17 PFGE 1 1/2b D 1/2b:121 groups were each represented by only 1 isolate, and were 1 1/2b D 1/2b:124 designated as capital letter O¨ followed by a number (e.g., O¨ - 1 1/2b D 1/2b:127 1). The 30 PFGE types of L. monocytogenes serovar 4b iso- 2 1/2b D 1/2b:129 lates (n = 334) were divided into 8 PFGE groups (an average 19 1/2b G 1/2b:2A of 41.75 isolates per PFGE group); the 32 PFGE types of 2 1/2b G 1/2b:23 serovar 1/2b isolates (n = 90) were divided into 9 PFGE 1 1/2b G 1/2b:71 groups (10 isolates per PFGE group). One of the nine serovar 1 1/2b G 1/2b:89 1 1/2b G 1/2b:110 1/2b PFGE groups also included the three serovar 3b isolates 10 1/2b O 1/2b:15 sharing one PFGE type (Table 1). The number of L. mono- 1 1/2b O 1/2b:59 cytogenes isolates, serovars, PFGE types, and PFGE groups 1 1/2b O 1/2b:122 are presented in Table 2. Numbers and PFGE groups of L. 1 1/2b P 1/2b:21 monocytogenes isolates, per year, from 1958 to 2010, are 1 1/2b P 1/2b:62 shown in Table 3. In Sweden, L. monocytogenes isolates 2 1/2b T 1/2b:84 belonging to the two most widespread PFGE groups over 2 1/2b T 1/2b:130 time (i.e., D and J) have been isolated from human cases over 2 1/2b U 1/2b:78 six decades. However, L. monocytogenes PFGE group I is 2 1/2b U 1/2b:90 currently the dominant PFGE group within lineage I in 1 1/2b O¨ -1 1/2b:106 1 1/2b O¨ -2 1/2b:108 Sweden and has been responsible for 193 cases of listeriosis 1 1/2b O¨ :3 1/2b:102 during 1958–2010. One of the most common PFGE types 3 3b G 3b:2B (4b:6) belonging to PFGE group I was identical to the epi- 23 4b I 4b:1 demic cheeseborne L. monocytogenes strain in Switzerland, 67 4b I 4b:3 Denmark, and the United States during 1983–1990 (Lopez- 67 4b I 4b:6 Valladares et al., 2014) (Table 3). 6 4b I 4:7 An association was observed between PFGE groups and 1 4b I 4b:12 serovars. L. monocytogenes isolates belonging to PFGE 3 4b I 4b:16 groups I, J, Q, R, X, Z, O¨ -4, and O¨ -5 all shared serovar 4b. 18 4b I 4b:18 Isolates from PFGE groups D, G, O, P, T, U, O¨ -1, O¨ -2, and O¨ - 6 4b I 4b:25 1 4b I 4b:26 3 shared serovar 1/2b. With one exception, PFGE group G 1 4b I 4b:27 (which included PFGE type G:3b:2B) was associated with 64 4b J 4b:5 serovar 3b (Table 2). The occurrence of atypical combina- 3 4b J 4b:9 tions of serotypes and grouping based on different molecular 1 4b J 4b:11 typing methods has also been observed in other studies 3 4b J 4b:15A (Buchrieser et al., 1991; Nadon et al., 2001; Revazishvili 7 4b J 4b:15B et al., 2004). A portion of our L. monocytogenes collection 11 4b J 4b:20 has been characterized by multilocus variable-number tan- 3 4b J 4b:24 dem-repeat analysis with the resulting clusters generally 1 4b J 4b:28 agreeing with both the data generated by PFGE as proposed 2 4b J 4b:31 9 4b Q 4b:2 here and serotyping data (Lindstedt et al., 2008). A study of 3 4b Q 4b:8 Staphylococcus aureus has shown that the configuration of 12 4b Q 4b:10 small PFGE bands (10–85 kb) correlates better with the 5 4b Q 4b:21 phylogenetic typing method multiprimer random amplified 1 4b Q 4b:22 polymorphic DNA analyses than larger PFGE bands ( > 85– 1 4b Q 4b:32 700 kb). The authors propose that this is due to a greater 5 4b R 4b:19 propensity for size instability in larger fragments since the frequency of random genetic events increases with the size of (continued) the DNA fragment (Blanc et al., 2002). In another 450 LOPEZ-VALLADARES ET AL.

Table 3. Numbers and Pulsed-Field Gel Electrophoresis (PFGE) Groups of Listeria monocytogenes Lineage I Isolates in Sweden, per Year, from 1958 to 2010 PFGE groups Year D G O P T U O¨ -1 O¨ -2 O¨ -3I J Q RXZO¨ -4 O¨ -5 1958 1 1 1959 1960 1961 1962 2 1963 2 1964 1965 1 1 1 1966 1 1 1967 1 1 1968 1969 2 1970 2 1971 2 1972 1 5 1973 1 4 1974 9 3 1 1 1975 6 9 1976 11 2 1977 1 10 3 1 1978 3 1 4 1979 8 3 1980 3 2 2 2 1981 4 4 1982 1 7 1 1983 1 6 3 1984 4 1985 3 1 4 1986 1 1987 14 7 1 1 1988 2 1 6 5 2 1989 1 4 6 1 1990 1 12 2 1991 1 5 2 1 2 1992 1 1 9 2 1 1993 3 3 1994 4 4 2 6 1995 9 1 2 1996 6 1997 1 1 5 1 1998 1 3 4 1 1999 4 4 1 1 2000 1 5 1 1 4 1 1 2001 1 5 2 3 4 3 2002 4 2 3 2 2003 1 3 3 1 2004 1 2 4 2 2005 1 1 1 1 1 1 1 2006 1 3 1 1 2007 2 1 1 2008 1 1 4 3 2 1 2009 1 1 2 2 3 2010 1 2 1 1 1 8 2 Total 41 27 12 2 4 4 1 1 1 193 95 31 5 6 2 1 1 Table 4. In Silico Asc I Pulsed-Field Gel Electrophoresis (PFGE) Proﬁles of Sequenced Listeria monocytogenes Strainsa Year of isolation 1985–1987 1983–1987 1995 1999 2002 2002 1956 1967 1994 Country of isolation USA Switzerland USA USA France USA USA USA USA USA Source Cheese Cheese Poultry Animal Cheese Human Food Human Environment Chinchilla Chocolate milk Serovar 4b 4b 4e 4b 4b 4b 4b 4b 4b 3b 1/2b 1/2b 1/2b Strain F2365 LL195 SLCC2378 J2-1091 WSLC1042 Clip80459 L312 J1776 J1926 SLCC2540 N1-011A SLCC2755 R2-502 729598b 729233 730060 730493 730497 848765 849032 844505 827326 906997 947668 898966 944845 461301 461288 461287 590088 461278 600876 600450 785237 802410 389826 400219 400167 399973 354158 354145 389912 370814 389916 463947 463788 325311 325311 370462 366587 354598 394624 330803 330805 331348 261113 331185 322146 322165 314811 314810 323212 354880 325120 275313 236971 236979 237000 237022 237036 240103 240059 246828 246826 234918 236097 238249 267657 235616 235468 235014 235616 235522 232447 236320 238229 168120 120878 120880 120878 119797 119798 119798 119798 119796 116627 116631 116625 116626 116626

451 115860 115859 115859 115859 115857 113887 113888 112074 112074 111772 111772 109739 109224 109224 109224 107384 78053 69921 69921 69921 69921 69921 69935 69934 69919 69919 69956 69966 69226 69906 62644 62643 64405 63684 63683 63685 53299 53299 53584 53584 53584 53585 53586 53582 48739 48739 48740 48740 47230 47230 47229 47079 47079 47081 47080 47079 47079 47079 47079 47078

46877 46877 46877 46877 46876 43345 43345 39125 39125 39124 39125 39125 39125 39124 37104 37024 37100 37100 8833 8833 8833 8833 1454 1454 1454 1454 1454 1454 1454 1454 1454 1454 1454 1454 1454 aAscI restriction fragment sizes were calculated from L. monocytogenes whole genome sequences obtained from either GenBank or EMBL-EBI. bNumber of base pairs in restriction fragment. 452 LOPEZ-VALLADARES ET AL. staphylococci study, PFGE showed good concordance with monocytogenes and their correlation with serovar. Appl En- multilocus sequence typing, with authors suggesting that viron Microbiol 1994;60:2584–2592. PFGE is a reliable method for long-term, nationwide epide- Brosch R, Brett M, Catimel B, Luchansky JB, Ojeniyl B, Ro- miological surveillance studies (Hallin et al., 2007). court J. Genomic fingerprinting of 80 strains from the W. H. In a small study, Brosch et al. (1991) digested L. mono- O. multicenter international typing study of Listeria monocytogenes, serovar 4b isolates (n = 42), with ApaI producing cytogenes via pulsed-field gel electrophoresis (PFGE). Int J 16 PFGE types that could be further divided into 4 PFGE Food Microbiol 1996;32:343–355. groups as defined by the close relatedness of 9–12 lower- Buchrieser C, Brosch R, Rocourt J. Use of pulsed-field gel molecular-weight fragments. Later, Brosch et al. (1994) electrophoresis to compare large DNA-restriction fragments compared low-molecular-weight AscI restriction fragments of Listeria monocytogenes strains belonging to serogroups 1/ (23–60 kb) of L. monocytogenes isolates (n = 176) and iden- 2 and 3. Int J Food Microbiol 1991;14:297–304. Buchrieser C, Brosch R, Catimel B, Rocourt J. Pulsed-field gel tified 2 genomic divisions that correlated with the flagellar electrophoresis applied for comparing Listeria mono- (H) antigen type: division I (1/2a, 1/2c, 3a, 3c) and division II cytogenes strains involved in outbreaks. Can J Microbiol (1/2b, 3b, 4b, 4d, 4e). Their results, however, cannot be di- 1993;390:395–401. rectly compared with the results from the present study, as Danielsson-Tham M-L, Eriksson E, Helmersson S, Leffler M, different analytical approaches were used. In silico analyses Lu¨dtke L, Steen M, Sørgjerd S, Tham W. Causes behind a (PFGE with restriction enzyme AscI) of different sequenced human cheese-borne outbreak of gastrointestinal listeriosis. L. monocytogenes strains belonging to lineage I are presented Foodborne Pathog Dis 2004;1:153–159. in Table 4 (GenBank or EMBL-EBI). Although the strains Ericsson H, Eklo¨w A, Danielsson-Tham M-L, Loncarevic S, are from different sources, time, and location, their AscI re- Mentzing LO, Persson I, Unnerstad H, Tham W. An outbreak striction fragments below the size of 145.5 kb are more of listeriosis suspected to have been caused by rainbow trout. conserved than larger fragments, supporting the utility of J Clin Microbiol 1997;35:2904–2907. smaller fragments in grouping L. monocytogenes. Interest- Goulet V, Rocourt J, Rebiere I, Jacquet C, Moyse C, Dehau- ingly, a small fragment (1454 bp) was present in all L. mont, P, Salvat G, Veit P. Listeriosis outbreak associated monocytogenes strains. with the consumption of rillettes in France in 1993. J Infect From the results of the present study, a procedure for im- Dis 1998;177:155–160. proving the identification of PFGE types when analyzing new Graves LM, Swaminathan B. PulseNet standardized protocol PFGE profiles is proposed. First the smallest banding patterns for subtyping Listeria monocytogenes by macrorestriction below 145.5 kb are analyzed to determinate the PFGE group. and pulsed-field gel electrophoresis. Int J Food Microbiol Then, within the identified PFGE group, the banding patterns 2001;65:55–62. above 145.5 kb are analyzed to determine the PFGE type. Guevara JM, Pereda J, Roel S. Human listeriosis in Peru. Tropenmed Parasit 1979;30:59–61. Hallin M, Deplano A, Denis O, De Mendonca R, De Ryck R, Conclusions Struelens MJ. Validation of pulsed-field gel electrophoresis L. monocytogenes PFGE groups were established for each and spa typing for long-term, nationwide epidemiological isolate based on the configuration of small AscI restriction surveillance studies of Staphylococcus aureus infections. J fragments below the size of 145.5 kb. Within the identified Clin Microbiol 2007;45:127–133. PFGE group, the banding patterns above 145.5 kb were uti- Kathariou S. Listeria monocytogenes virulence and pathoge- lized to determine the PFGE type. This process simplifies the nicity, a food safety perspective. J Food Prot 2002;65:1811– identification of PFGE types. Moreover, it is possible to es- 1829. tablish an association between PFGE groups and serovar 4b, Kerouanton NA, Brisabois A, Denoyer E, Grour J, Salvat G, 1/2b, and 3b (i.e., lineage I). Picard B. Comparison of five typing methods for the epidemiological study of Listeria monocytogenes. Int J Food Mi- crobiol 1998;43:61–71. Acknowledgment Knabel SJ, Reimer A, Verghese B, Lok M, Ziegler J, Farber J, This study was supported by the Stiftelsen Grythytte Pagotto F, Graham M, Nadon C, Gilmour M. Sequence typ- Akademi Stipendiefond, to whom we express gratitude. ing confirms that a predominant Listeria monocytogenes clone caused human listeriosis cases and outbreaks in Canada Disclosure Statement from 1988 to 2010. J Clin Microbiol 2012;50:1748–1751. Lambertz ST, Ivarsson S, Lopez-Valladares G, Sidstedt M, No competing financial interests exist. Lindqvist R. Subtyping of Listeria monocytogenes isolates recovered from retail ready-to-eat foods, processing plants References and listeriosis patients in Sweden 2010. Int J Food Microbiol Blanc DS, Francioli P, Hauser PM. Poor value of pulsed-field 2013;166:186–192. gel electrophoresis to investigate long-term scale epidemi- Lindstedt BA, Tham W, Danielsson-Tham M-L, Vardund T, ology of methicillin-resistant Staphylococcus aureus. Infect Helmersson S, Kapperud G. Multiple-locus variable-number Genet Evol 2002;2:145–148. tandem-repeats analysis of Listeria monocytogenes using Brosch R, Buchrieser C, Rocuort J. Subtyping of Listeria multicolour capillary electrophoresis and comparison with monocytogenes serovar 4b by use of low-frequency-cleavage pulsed-field gel electrophoresis typing. J Microbiol Methods restriction endonucleases and pulsed-field gel electrophoresis. 2008;72:41–48. Res Microbiol 1991;142:667–675. Lopez-Valladares G, Danielsson-Tham M-L, Tham W. The Brosch R, Chen J, Luchansky J. Pulsed-field fingerprinting of prevalence of listeriosis in South American camelids. J Camel Listeriae: Identification of genomic divisions for Listeria Pract Res 2013;20:129–132. DIVISION OF L. MONOCYTOGENES PFGE TYPES INTO PFGE GROUPS 453

Lopez-Valladares G, Tham W, Parihar VS, Helmersson S, An- Seeliger HPR, Jones D. Genus Listeria. In: Bergey’s Manual of dersson B, Ivarsson S, Johansson C, Ringberg H, Tjernberg I, Systematic Bacteriology, Volume 2. Sneath PHA, Mair NS, Henriques-Normark B, Danielsson-Tham M-L. Human iso- Sharpe ME, Holt JG (eds.). Baltimore: Williams & Wilkins, lates of Listeria monocytogenes in Sweden during half a cen- 1986, pp. 1235–1245. tury (1958–2010). Epidemiol Infect 2014;142:2251–2260. Tenover F, Arbeit R, Goering R, Mickelsen P, Murray B, Per- McLauchlin J. The relationship between Listeria and listeriosis. sing D, Swaminathan B. Interpreting chromosomal DNA Food Contr 1996;7:187–193. restriction patterns produced by pulsed-ﬁeld gel electropho- McLauchlin J, Hall SM, Velani SK, Gilbert RG. Human listeriosis resis: Criteria for bacterial strain typing. J Clin Microbiol and paˆte´: A possible association. Br Med J 1991;303:773–775. 1995;33:2233–2239. Nadon CA, Woodward DL, Young C, Rodgers FG, Wiedmann Tham W, Danielsson-Tham M-L. Listeria monocytogenes— M. Correlations between molecular subtyping and serotyping Very foodborne bacteria. In: Food Associated Pathogens. of Listeria monocytogenes. J Clin Microbiol 2001;39:2704– Tham W, Danielsson-Tham M-L (eds.). Boca Raton, FL: 2707. CRC Press/Taylor & Francis Group, 2014, pp. 124–140. Peiris IP, Lopez-Valladares G, Parihar VS, Helmersson S, Barbuddhe SB, Tham W, Danielsson-Tham M-L. Gravad and cold-smoked salmon, still a potential source of listeriosis. J Address correspondence to: Foodservice 2009;20:15–20. Wilhelm Tham, VMD Revazishvili T, Kotetishvili M, Stine CO, Kreger AS, Morris School of Hospitality JG, Sulakvelidze A. Comparative analysis of multilocus se- Culinary Arts and Meal Science quence typing and pulsed-ﬁeld gel electrophoresis for char- O¨ rebro University acterizing Listeria monocytogenes strains isolated from SE-712 60 Grythyttan, Sweden environmental and clinical sources. J Clin Microbiol 2004; 42:276–285. E-mail: [email protected]

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FOODBORNE PATHOGENS AND DISEASE Volume 14, Number 1, 2017 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2016.2173

Lineage II (Serovar 1/2a and 1/2c) Human Listeria monocytogenes Pulsed-Field Gel Electrophoresis Types Divided into PFGE Groups Using the Band Patterns Below 145.5 kb

Gloria Lopez-Valladares,1 Marie-Louise Danielsson-Tham,1 Richard V. Goering,2 and Wilhelm Tham1

Abstract Among 504 clinical lineage II isolates of Listeria monocytogenes isolated during 1958–2010 in Sweden, 119 pulsed-field gel electrophoresis (PFGE) types (AscI) have been identified based on the number and distribution of all banding patterns in each DNA profile. In this study, these types were further divided into PFGE groups based on the configuration of small bands with sizes <145.5 kb. The 504 isolates included 483 serovar 1/2a isolates distributed into 114 PFGE types and 21 serovar 1/2c isolates distributed into 9 PFGE types; these were further divided into 21 PFGE groups. PFGE group, that is, configuration of small bands below 145.5 kb, and serovars were correlated. L. monocytogenes isolates belonging to PFGE groups A, B, C, E, F, H, K, L, M, S, V, W, Y, and O¨ -6 to O¨ -12 shared serovar 1/2a, with one exception. PFGE group E also included two PFGE types sharing serovar 1/2c and four PFGE types belonging to either serovar 1/2a or 1/2c. Isolates belonging to PFGE group N shared serovar 1/2c. In contrast to lineage I isolates, small fragments <33.3 kb were visible in all L. monocytogenes isolates belonging to lineage II. In the results from both the present and previous studies, the genomic region of small bands was genetically more conservative than in large bands. The distribution of these small bands established the relatedness of strains and defined a genetic marker for both lineages I and II, while also establishing their serogroup. The division of L. monocytogenes PFGE types into PFGE groups is advan- tageous as the profile of every new isolate can be identified easily and quickly through first studying the PFGE group affiliation of the isolate based on the smaller band patterns <145.5 kb, and then identifying the PFGE type based on the band patterns >145.5 kb.

Keywords: PFGE type, PFGE group, Listeria monocytogenes, serovar, lineage II

Introduction Little et al., 2009; Knabel et al., 2012; Mammina et al., 2013; Lopez-Valladares et al., 2014; Kvistholm Jensen et al., isteria monocytogenes is a bacterium widely distrib- 2016). Since 2000, several 1/2a outbreaks have been reported Luted in the environment and has been isolated from (Tham and Danielsson-Tham, 2014). floors, walls, and equipment surfaces in food processing Pulsed-field gel electrophoresis (PFGE) has been used plants (Unnerstad et al., 1996; Carpentier and Cerf, 2011; since the 1990s as an effective tool for identifying subgroups Nakari et al., 2014). The main route for Listeria infection is of L. monocytogenes (Brosch et al., 1996; Gianfranceschi the consumption of contaminated food and since the 1980s, a et al., 2009; Knabel et al., 2012; Lopez-Valladares et al., large number of sporadic cases and outbreaks of listeriosis 2014; Kvistholm Jensen et al., 2016). Outbreaks involving have been reported globally. During the 1990s, human lis- epidemic strains, with identical PFGE profiles, have been teriosis underwent a shift and now, L. monocytogenes serovar identified, for example, cheese-borne outbreaks in Switzer- 1/2a (lineage II) has become more common in several land and California (Buchrieser et al., 1993), fish-borne countries than lineage I, serovar 4b (Garrido et al., 2008; in Sweden (Ericsson et al., 1997), hot dogs and turkey

8 LINEAGE II LISTERIA MONOCYTOGENES STRAINS 9 delicatessen meats in United States (Kathariou et al., 2006), with both a concatenated Lambda ladder and Salmonella soft washed-rind cheese in Canada (Gaulin et al., 2012) and Braenderup H9812 (digested with XbaI) as reference stan- ready-to-eat salad in Switzerland (Stephan et al., 2015). In dards. The PFGE groups were established based on the Sweden, L. monocytogenes lineage II isolates have been number and distribution of small bands below 145.5 kb. characterized by serotyping and PFGE with restriction en- PFGE profiles with identical banding patterns below zyme AscI from human patients suffering from invasive lis- 145.5 kb were considered the same PFGE group. For each teriosis between 1958 and 2010 (Lopez-Valladares et al., PFGE group, a reference profile was selected for creating a 2014). From 504 L. monocytogenes isolates, 483 shared reference collection with groups being denoted by capital serovar 1/2a, these were partitioned into 114 PFGE types, and letters. Each L. monocytogenes isolate was then assigned a 21 serovar 1/2c isolates yielded 9 PFGE types (Table 1). The numerical code. For example, the designation A:1/2a:4A PFGE types were established from the number and distribu- denoted the isolate that belonged to PFGE group A. The tion of all fragments in each DNA profile. The profiles were second figure indicated that the isolate belonged to serovar considered distinguishable if there was a difference of one 1/2a, the third figure represented the consecutive number of band or more. However, as examination of DNA profiles PFGE type for serogroup 1/2 (i.e., 4th AscI profile), and the obtained by PFGE and identification of PFGE types is delicate fourth figure denoted the isolate belonged to variant A (i.e., and monotonous work, the initial screening and comparison closely related variants were termed A, B, C, etc.: Tenover of DNA profiles require simplification (Lopez-Valladares et al., 1995). The AscI profiles of L. monocytogenes isolates et al., 2015). representing the different PFGE groups are presented in The first aim of this study was to gather 119 L. mono- Figure 1. cytogenes PFGE types belonging to lineage II, namely serovars 1/2a and 1/2c, into PFGE groups based on the number In silico analyses and distribution of small bands below 145.5 kb. This would facilitate and improve the interpretation and analysis of PFGE In silico means ‘‘research is conducted by computer simu- profiles during investigations of food-borne cases and out- lations with models closely reflecting the real world’’ (Apache breaks. A second aim was to investigate the genetic related- Software Foundation, 2016). Some sequenced L. mono- ness within PFGE types, and the third aim was to identify cytogenes strains belonging to lineage II were analyzed in si- possible association between PFGE groups and serovars. lico with PFGE and restriction enzyme AscI(Bikandiet al., 2004). By selecting a specific strain of L. monocytogenes with known sequence (http://insilico.ehu.es), it is possible to make a Materials and Methods theoretical cleavage (in silico) by selecting a desired restriction L. monocytogenes isolates enzyme, for example, AscI, on the same site. Five hundred four isolates of L. monocytogenes lineage II from cases of human invasive listeriosis in Sweden during Results and Discussion 1958 to 2010 were selected for the study. All isolates had L. monocytogenes PFGE groups been previously characterized by serotyping and PFGE with restriction enzyme AscI according to Graves and Swami- In a previous study (Lopez-Valladares et al., 2014), 483 nathan (2001) with some modifications (Lopez-Valladares human L. monocytogenes serovar 1/2a isolates were distrib- et al., 2014). Among these 504 L. monocytogenes isolates, uted into 114 PFGE types and the 21 serovar 1/2c isolates 483 serovar 1/2a isolates were identified and based on the were distributed into 9 PFGE types. In this study, all the number and distribution of all fragments in each DNA profile, PFGE types were further divided into 21 PFGE groups based partitioned into 114 PFGE types. A further 21 serovar 1/2c on the number and distribution of small bands below isolates yielded 9 PFGE types (Table 1). The isolates were 145.5 kb. The 114 PFGE types of L. monocytogenes serovar kept at -70C in brain–heart infusion broth (Merck, Darm- 1/2a could be divided into 20 PFGE groups and the 9 PFGE stadt, Germany) with 20% glycerol. types of serovar 1/2c into two PFGE groups (Table 1). PFGE group E harbored both 1/2a and 1/2c isolates (Table 2). Small restriction fragments with sizes <33.3 kb were visible in all Grouping of L. monocytogenes isolates L. monocytogenes isolates (Fig. 1). Seven PFGE types re- The DNA profiles were analyzed visually and the position presented by only one isolate were identified from among the of each restriction fragment of each PFGE type was sized 114 different PFGE types belonging to serovar 1/2a (Table 2).

Table 1. Distribution of Listeria monocytogenes Lineage II Isolates in Sweden by Serovar, PFGE Types, and PFGE Groups from 1958 to 2010 Serovar Isolates (n) % PFGE type (n) Average PFGE group (n) Average 1/2a 483 95.8 114 4.2 20 24.2 1/2c 21 4.2 4a + 5 2.3 1a + 1 10.5 Total 504 100.0 119 21

The smallest banding patterns below 145.5 are analyzed to determine the PFGE group. The banding patterns above 145.5 kb are analyzed to determine the PFGE type. aShared the same PFGE types and PFGE group as some serovar 1/2a isolates. PFGE, pulsed-ﬁeld gel electrophoresis. 10 LOPEZ-VALLADARES ET AL.

FIG. 1. AscI proﬁles of Listeria monocytogenes lineage II, human strains. Electrophoretic conditions: 21 h, pulse times 4– 40 s, 120 angle, and 14C. Lane 1 (Lambda ladder PFG Marker No. 340 S); lane 2 (Salmonella Braenderup H9812); lane 3 (group A); lane 4 (group B); lane 5 (group C); lane 6 (group E); lane 7 (group F); lane 8 (group H); lane 9 (group K); lane 10 (group L); lane 11 (group M); lane 12 (group N); lane 13 (group S); lane 14 (group V); lane 15 (group W); lane 16 (group Y); lane 17 (group O¨ -10); lane 18 (group O¨ -12); lane 19 (group O¨ -11); lane 20 (group O¨ -6); lane 21 (group O¨ -8); lane 22 (group O¨ -7); lane 23 (group O¨ -9); lane 24 (Salmonella Braenderup H9812); and lane 25 (Lambda ladder PFG Marker No. 340 S).

These seven PFGE types all had different configuration, even Association between PFGE groups and serovars of small bands below 145.5 kb; thus, forming seven PFGE There was an association between PFGE groups and ser- ¨ ¨ L. monocytogenes groups (O-6 to O-12, Table 2). Although ovars. L. monocytogenes isolates belonging to PFGE groups sharing PFGE group O¨ -8 was isolated in 1987, this group had ¨ ¨ ¨ ¨ ¨ A, B, C, E, F, H, K, L, M, S, V, W, Y, O-6, O-7, O-8, O-9, not been subsequently identified: the other O groups have been O¨ -10, O¨ -11, and O¨ -12 shared serovar 1/2a, with one excep- isolated in recent years (Table 3). tion. PFGE group E also included two PFGE types sharing L. monocytogenes isolates representing PFGE groups A, serovar 1/2c (1/2c:36 and 1/2c:95) and four PFGE types be- C, and E have been common among human cases over six longing to either serovar 1/2a or 1/2c (1/2:12A, 1/2:12B, decades in Sweden, that is, the groups remain. PFGE 1/2:93, and 1/2:105, Table 2). All isolates from PFGE group groups A and E are still the dominant PFGE groups within N shared serovar 1/2c (Table 2). However, a few L. mono- lineage II and isolates from these groups have been re- cytogenes isolates with indistinguishable PFGE profiles dis- sponsible for 294 cases (out of 932) of invasive listeriosis playing different serovars are reported (Nadon et al., 2001; in Sweden during 1958–2010 (Table 3). In addition, two of Lukinmaa et al., 2003; Revazishvili et al., 2004; Gian- the most common PFGE types (1/2a:4A and 1/2a:4B) be- franceschi et al., 2009; Lopez-Valladares et al., 2015). longing to PFGE group A have been identified in various countries within Europe (unpublished data). PFGE groups L. monocytogenes isolates belonging to lineages A, C, and E have persisted over a few decades and may I and II versus PFGE groups signify that there is a common marker among the strains within each of the PFGE groups in the small fragments and PFGE band patterns (AscI) <145.5 kb have been used to the strains have a common ancestor. Isolates representing identify groups within L. monocytogenes lineage I (serovars sixdifferentPFGEgroups(M,N,S,V,W,andY)were 1/2b and 4b), with 334 serovar 4b isolates clustered into 30 observed sporadically throughout the period studied. Iso- PFGE types and 8 PFGE groups (Lopez-Valladares et al., lates representing PFGE group H, first identified in 1988, have 2015). In this study, 483 serovar 1/2a isolates clustered into appeared with an increasing trend (Table 3) and since 2000, only 114 PFGE types and 20 PFGE groups. Division of serovar 4b three new PFGE groups (M, V, and Y) have been identified, (lineage I) isolates in a smaller number of clusters, unlike each represented by two isolates per group. Therefore, it is serovar 1/2a (lineage II) isolates, has also been observed reasonable to assume that the most important PFGE groups of L. (Bibb et al., 1989; Aarts et al., 1999). In a comparison of the monocytogenes in food-borne listeriosis have been identified in grouping of lineage I isolates and the grouping of lineage II Sweden as new PFGE groups rarely appear (Table 3). Most isolates, there was a difference between the two lineages clonal complexes (CC) of L. monocytogenes have persisted over based on the small bands below 145.5 kb. In contrast to the decades and only a small number of new CC have been identified PFGE banding pattern for lineage I isolates, small fragments within lineages I and II (Haase et al., 2014). A 1/2a/CC8 strain <33.3 kb were visible in all L. monocytogenes isolates be- was causing listeriosis throughout Canada for at least two de- longing to lineage II. Thus, lineage I isolates and lineage II cades (Knabel et al., 2012). isolates did not share any PFGE group. Hence, the small Table 2. Serovar, PFGE Group, and PFGE Type Table 2. (Continued) Distribution of Human Listeria monocytogenes Lineage II Isolates in Sweden from 1958 to 2010 PFGE Isolates (n) Serovar group PFGE typea PFGE Isolates (n) Serovar group PFGE typea 3 1/2a E 1/2a:51 4 1/2a E 1/2a:56 58 1/2a A 1/2a:4A 5 1/2a E 1/2a:57A 40 1/2a A 1/2a:4B 1 1/2a E 1/2a:57B 1 1/2a A 1/2a:4C 3 1/2a E 1/2a:91 4 1/2a A 1/2a:4E 2 1/2a E 1/2a:93 5 1/2a A 1/2a:6 1 1/2a E 1/2a:98 2 1/2a A 1/2a:14 1 1/2a E 1/2a:105 4 1/2a A 1/2a:41 1 1/2a E 1/2a:111 1 1/2a A 1/2a:46 1 1/2a E 1/2a:132 4 1/2a A 1/2a:58 1 1/2a E 1/2a:135 6 1/2a A 1/2a:64 9 1/2c E 1/2c:12A 1 1/2a A 1/2a:70 2 1/2c E 1/2c:12B 1 1/2a A 1/2a:80 2 1/2c E 1/2c:36 1 1/2a A 1/2a:88 2 1/2c E 1/2c:93 4 1/2a A 1/2a:100 1 1/2c E 1/2c:95 1 1/2a A 1/2a:112 1 1/2c E 1/2c:105 1 1/2a A 1/2a:131 6 1/2a F 1/2a:13 1 1/2a A 1/2a:137 5 1/2a F 1/2a:16 6 1/2a B 1/2a:3A 1 1/2a F 1/2a:24 7 1/2a B 1/2a:3B 8 1/2a F 1/2a:48 1 1/2a B 1/2a:3C 3 1/2a F 1/2a:79 1 1/2a B 1/2a:10 1 1/2a F 1/2a:82 1 1/2a B 1/2a:22 1 1/2a F 1/2a:85 5 1/2a B 1/2a:26 1 1/2a F 1/2a:118 1 1/2a B 1/2a:27 12 1/2a H 1/2a:1 3 1/2a B 1/2a:30 2 1/2a H 1/2a:8 3 1/2a B 1/2a:38A 2 1/2a H 1/2a:72 1 1/2a B 1/2a:38B 1 1/2a H 1/2a:139 1 1/2a B 1/2a:39A 5 1/2a K 1/2a:11A 1 1/2a B 1/2a:39B 9 1/2a K 1/2a:11B 2 1/2a B 1/2a:52 1 1/2a K 1/2a:11C 2 1/2a B 1/2a:54 11 1/2a K 1/2a:17 3 1/2a B 1/2a:63 3 1/2a K 1/2a:25 2 1/2a B 1/2a:69 1 1/2a K 1/2a:97 2 1/2a B 1/2a:109A 1 1/2a K 1/2a:99 2 1/2a B 1/2a:109B 1 1/2a K 1/2a:134 1 1/2a B 1/2a:109C 1 1/2a K 1/2a:136 4 1/2a B 1/2a:114 1 1/2a K 1/2a:146 1 1/2a B 1/2a:116 11 1/2a L 1/2a:19 3 1/2a B 1/2a:117 1 1/2a L 1/2a:74 1 1/2a B 1/2a:123 1 1/2a L 1/2a:76 1 1/2a B 1/2a:125 1 1/2a L 1/2a:128 1 1/2a B 1/2a:126 2 1/2a M 1/2a:34 1 1/2a B 1/2a:143 1 1/2c N 1/2c:73 3 1/2a C 1/2a:28 2 1/2c N 1/2c:83 7 1/2a C 1/2a:53 1 1/2c N 1/2c:92 5 1/2a C 1/2a:60 10 1/2a S 1/2a:7 9 1/2a C 1/2a:67 1 1/2a S 1/2a:140 2 1/2a C 1/2a:94 1 1/2a V 1/2a:43 1 1/2a C 1/2a:96 1 1/2a V 1/2a:81 1 1/2a C 1/2a:142 3 1/2a W 1/2a:20A 1 1/2a C 1/2a:148 2 1/2a W 1/2a:20B 20 1/2a E 1/2a:9A 2 1/2a Y 1/2a:115 36 1/2a E 1/2a:9B 1 1/2a O¨ -6 1/2a:18 6 1/2a E 1/2a:12A 1 1/2a O¨ -7 1/2a:33 4 1/2a E 1/2a:12B 1 1/2a O¨ -8 1/2a:68 17 1/2a E 1/2a:31 1 1/2a O¨ -9 1/2a:138 8 1/2a E 1/2a:35 1 1/2a O¨ -10 1/2a:141 1 1/2a E 1/2a:40 1 1/2a O¨ -11 1/2a:145 4 1/2a E 1/2a:44 1 1/2a O¨ -12 1/2a:147 1 1/2a E 1/2a:45 18 1/2a E 1/2a:49 The smallest banding patterns below 145.5 are analyzed to determine 4 1/2a E 1/2a:50 the PFGE group. The banding patterns above 145.5 kb are analyzed to determine the PFGE type. aLopez-Valladares et al. (2014). (continued) PFGE, pulsed-field gel electrophoresis. 11 Table 3. Numbers and PFGE Groups of Human Listeria monocytogenes Lineage II Isolates in Sweden, Per Year, from 1958 To 2010 PFGE groups Year A B C E F H K L M N S V W Y O¨ -6 O¨ -7 O¨ -8 O¨ -9 O¨ -10 O¨ -11 O¨ -12 1958 2 1 1959 1 1960 1961 1962 1 1963 1 1 1 1964 1965 1966 1967 1 1968 1 1969 1 1 1970 1971 1 1 1972 1 2 1973 1 1974 1 1975 1976 1977 1 1978 1 1979 1980 1 1 1981 1 3 1 1982 1 1 1 1983 1 1984 3 1985 1 5 2 1986 1 1987 3 5 1 1 1988 10 1 5 2 1 3 3 1989 3 1 1 1 2 2 1 1990 6 3 1 2 1991 1 1 3 1 1992 2 2 1 2 1 1 1993 5 1 9 1 1 1 1994 3 2 1 3 3 1995 3 1 2 1 1 1 1996 5 1 7 1 1997 1 2 1 2 1 1 1998 4 1 5 1 1 1999 4 2 1 1 1 2 1 2000 8 8 2 2 2 4 2 2001 9 2 5 1 3 3 2 1 1 1 2002 6 7 2 8 1 1 1 1 2003 6 3 25 2 2 1 1 2004 5 2 1 6 2 1 2 2005 2 1 5 2 2006 5 1 9 2 1 2007 6 5 10 3 1 2008 2211012 1 1 2009 11 4 2 15 2 1 2 1 1 1 2010 20 3 16 3 2 1 1 1 Total 135 57 29 159 26 17 34 14 2 4 11 2 5 2 1 1 1 1 1 1 1 The smallest banding patterns below 145.5 are analyzed to determine the PFGE group. The banding patterns above 145.5 kb are analyzed to determine the PFGE type. PFGE, pulsed-field gel electrophoresis.

12 Table 4. In Silico AscI PFGE Profiles of Sequenced Listeria monocytogenes Strains Year of isolation 2008 2008 2000 1968 1960 1996 Country of Canada Canada USA Finland USA USA USA England isolation Source Human Human Turkey Food Human Human Animal Human Serovar 1/2a 1/2a 1/2a 3a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2c 1/2c 3c Strain 08-5578 08–5923 J0161 SLCC7179 Finland 199 C1-387 10403S SLCC5850 EGD J2-031 EGD-e FSL R2-561 SLCC2372 SLCC2479 NC NC NC NC NC NC NC NC NC NC NC NC NC NC 018589 013766 013768 017545 018593 017547 021823 017544 018592 022568 021837 003210 017546 018588 872,390a 872,417 841,707 821,409 818,911 818,421 819,505 821,121 821,080 820,870 776,542 774,781 779,223 775,780 778,227 562,980 562,698 471,719 448,035 480,997 450,592 450,591 450,589 460,164 458,928 458,935 458,828 438,402 429,316 432,560 432,556 402,387 402,704 405,980 404,145 403,233 403,232 390,623 392,329 391,848 392,337 383,089 380,046 381,188 383,218 374,849 356,240 366,961 362,888 362,870 362,841 362,854 341,155 340,836 342,924 343,577 335,660 337,664 337,672 337,423 326,282 326,287 326,918 252,758 264,818 246,423 246,423 236,462 242,878 243,443 243,560 242,748 242,738 242,747 243,524 243,499 243,416 13 224,642 216,143 216,209 215,812 167,664 167,685 118,692 118,691 118,690 112,438 113,720 113,716 108,271 106,103 104,517 104,517 104,515 104,515 104,512 104,557 104,516 104,515 104,514 104,516 104,516 104,508 104,517 104,515 92,698 71,306 71,306 70,009 70,008 70,009 71,306 71,331 71,322 71,323 62,437 62,436 64,148 56,818 45,089 45,089 45,087 45,060 45,106 45,113 45,106 45,086 45,081 45,081 45,081 45,081 40,342 41,361 41,361 41,361 41,361 36,643 36,643 38,190 38,222 38,152 38,151 38,152 38,219 38,195 38,195 38,195 38,195 27,101 27,101 27,100 27,101 27,101 27,101 27,099 27,101 27,100 27,101 27,101 23,862 22,975 22,975 22,771 22,771 22,771 23,418 17,361 17,361 17,364 17,365 17,367 17,367 17,367 17,368 17,361 17,361 17,361 17,361 6708 6708 6708 6708 408 612 612 612 612 204 204 204 204 204 204 Total length of 3,032,288 2,999,054 3,000,464 2,882,234 2,874,431 2,988,947 2,903,106 2,907,142 2,907,193 2,958,908 2,944,528 2,973,801 2,972,810 2,972,172 genome, bp AscI restriction fragment sizes were calculated from L. monocytogenes whole genome sequences obtained from either www.insilico.ehu.es (GenBank) or EMBL-EBI. aNumber of base pairs in restriction fragment. Length of sequence (sorted). PFGE, pulsed-field gel electrophoresis. 14 LOPEZ-VALLADARES ET AL. bands were able to confirm the relatedness of strains and sequence typing (MvLST). PFGE has the advantage that it differentiate lineages I and II. Some isolates from the can be performed in the absence of detailed information L. monocytogenes collection were analyzed by multiple- about the genome (Cooper and Feil, 2004). Although, PFGE locus variable-number tandem-repeat analysis (MLVA) and typing is labor intensive, it probes the entire genome, the clusters were generally in good agreement with data whereas, for example, MLST only analyzes nucleotides generated by both PFGE groups and serotypes (Lindstedt within specific genes (Revazishvilli et al., 2004). In a com- et al., 2008). parison of results from PFGE, MLST, and MvLST among L. In study on Staphylococcus aureus (Blanc et al., 2002), monocytogenes clonal groups, Cantinelli et al. (2013), ob- the configuration of small PFGE bands (10–85 kb) corre- served that MvLST did not provide more discrimination than lated better with the phylogenetic typing method RAPD MLST, and PFGE has a higher discriminatory power than (multiprimer random amplified polymorphic DNA) than MLST. Besides WGS, all available molecular approaches to with large PFGE bands (>85–700 kb). The authors (Blanc genomic comparison represent incomplete information et al., 2002) conclude that larger bands, because of their (Goering, 2010). Three different PFGE protocols have been size, should have a greater propensity to change at a faster compared by Michelon et al. (2015) with all PFGE profiles rate than smaller bands, and proposed this is due to a greater being similar in quality and indistinguishable except one propensity for size instability in larger fragments, as the profile: the PFGE profiles ‘‘are both similar and comparable’’ frequency of random genetic events increases with the size (Michelon et al., 2015). Even though WGS is an appropriate of the DNA fragment. In another staphylococci study subtyping tool for replacing all other subtyping methods, it is (Hallin et al., 2007), PFGE had good concordance with not accessible in all reference laboratories. Therefore, PFGE multilocus sequence typing (MLST), suggesting that PFGE is still the gold standard method. is a reliable method for long-term, nationwide epidemiological surveillance studies. Conclusions Some sequenced L. monocytogenes strains belonging to lineage II analyzed in silico with PFGE and restriction en- The results from this study for L. monocytogenes lineage II zyme AscI (Bikandi et al., 2004) are presented in Table 4. The strains and the results previously obtained for lineage I strains in silico analyses indicated that the AscI restriction fragments (Lopez-Valladares et al., 2015) reveal that the genomic re- below the size of 145.5 kb were more preserved than the gion of small bands is genetically more homogeneous than in larger fragments. This observation supported the approach of large bands. The distribution of these small bands establishes using small fragments for grouping PFGE types into PFGE the relatedness of strains and defines genetic marker for groups based on the number and distribution of small bands both lineages I and II, while determining their serogroup. In below 145.5 kb. addition, the results confirm the advantage of dividing Several studies covering cases and outbreaks have ob- L. monocytogenes PFGE profiles, first into PFGE groups and served closely related PFGE types (with AscI) that are then into PFGE types. However, further analyses on the se- identical in the region below 145.5 kb. Tham et al. (2007) quence of small bands are required to determine in which report two L. monocytogenes strains isolated from a blood genes the small bands are located and clarify the relatedness sample of one human case of invasive listeriosis; the strains of strains. belonged to different PFGE types, but shared the same PFGE group E. In a second case of listeriosis, another two strains Acknowledgment were isolated from the bloods simple; similarly, the two strains belonged to different PFGE type, but shared the same This study was supported by the Stiftelsen Grythytte PFGE group F. In the United States, two outbreaks of liste- Akademi Stipendiefond, to whom we express gratitude. riosis are associated with ready-to-eat meat products contaminated with L. monocytogenes serovar 4b occurring in Disclosure Statement 1998–99 (hot dogs) and 2002 (turkey deli meats: Kathariou et al., 2006). The PFGE profiles (AscI) of the isolates from the No competing financial interests exist. 1998–1999 and 2002 outbreaks were closely related (Ka- thariou et al., 2006). The closely related isolates appeared References identical in the AscI-restricted fragments <145.5 kb, that is, the isolates belonged to the same PFGE group. In Denmark, Aarts HJ, Hakemulder LE, Van Hoef AM. Genomic typing between 2002 and 2012, two closely related PFGE types of Listeria monocytogenes strains by automated laser fluo- rescence analysis of amplified fragment length polymor- among 122 L. monocytogenes clinical isolates cases represent phism fingerprint patterns. Int J Food Microbiol 1999;49: a possible outbreak (Kvistholm Jensen et al., 2016). The two 95–102. PFGE types both belong to clonal complex 8 and sequence Apache Software Foundation. What Is ‘In Silico’ Experi- type 8 and these closely related isolates are identical in the mentation? Available at https://taverna.incubator.apache.org/ < AscI-restricted fragments 145.5 kb, that is, the isolates be- introduction/what-is-in-silico-experimentation, accessed July long to the same PFGE group. Although these data suggest 3, 2016. a common ancestor among strains sharing a PFGE group, Bibb WF, Schwartz B, Gellin BG, Plikaytis BD, Weaver RE. ultimately this must be confirmed through methods such as Analysis of Listeria monocytogenes by multilocus enzyme whole genome sequencing (WGS). electrophoresis and application of the method to epidemi- Several typing methods have been compared with ologic investigations. Int J Food Microbiol 1989;8:233– PFGE including, MLVA, MLST, and multivirulence-locus 239. LINEAGE II LISTERIA MONOCYTOGENES STRAINS 15

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Research Article

20th Anniversary Volume Gravad (Gravlax) and cold-smoked salmon, still a potential source of listeriosis

Inoka Priyadarshani Peiris,* Gloria Lopez-Valladares,* Vishal Singh Parihar,*† Seved Helmersson,‡ Sukdevo Barbuddhe,§ Wilhelm Tham,* and Marie-Louise Danielsson-Tham* *Department of Restaurant and Culinary Arts, Örebro University, Sörälgsvägen 2, S-712 60 Grythyttan, Sweden; †Department of Clinical Medicine, Örebro University, S-701 82 Örebro, Sweden; ‡Department of Environmental Assessment, Box 7050, SLU, S-750 07 Uppsala, Sweden; §ICAR Research Complex for Goa, Old Goa, India

Abstract

Correspondence: Gravad (Gravlax) and cold-smoked salmon are associated with human list- Wilhelm Tham, eriosis in Sweden. The present investigation of Listeria monocytogenes in Department of salmon was a follow-up of a similar study in the middle of the 1990s. Since Restaurant and Culinary our first study, there has been an increasing focus on food hygiene in general Arts, Örebro University, Sörälgsvägen 2, S-712 60 and specially on self-inspection and Hazard Analysis and Critical Control Grythyttan, Sweden. Points from the authorities and food producers. L. monocytogenes was Tel: +46 (0)19 303000; isolated from 11 (three manufacturers) of the 56 products analyzed. The Fax: +46 (0)591 14443; highest level of L. monocytogenes was 1500 cfu/g from a cold-smoked E-mail: [email protected] salmon product; however, the level was low (<100 cfu/g) in most products. Keywords: Serovar 1/2a was predominant, followed by 4b. restriction enzyme analysis/ gravlax, gravad, Listeria, pulsed-field gel electrophoresis typing of the 56 salmon isolates identified five self-inspection, types of L. monocytogenes. One type was identical to a human type, whereas HACCP two other were closely related. These findings suggest that gravad and cold- smoked salmon are still possible sources of listeriosis in Sweden.

Food-borne outbreaks of listeriosis in humans Introduction are associated with a wide variety of food prod- Since the 1980s, Listeria monocytogenes has been ucts, mainly cold, ready-to-eat foods (Farber & recognized as a food-borne pathogen that causes Peterkin 1991). Certain pre-packed, ready-to-eat listeriosis in human beings. Human listeriosis is foods with a long shelf life and stored at refrig- relatively rare, with an incidence ranging from eration temperature are foods at high risk, as they 0 to 7.5 cases per million of the population in promote the growth of L. monocytogenes (Guyer Europe, but the fatality rate of listeriosis can be as & Jemmi 1991; Loncarevic et al. 1996). The high as 20–30% (de Valk et al. 2005). L. mono- hazard with these products is that they are con- cytogenes is of major concern for the foodservice sumed without prior heat treatment. Examples of industry because of its ability to adapt to and such food items are gravad and cold-smoked tolerate a wide range of temperatures and pH salmon/rainbow trout, which are popular ready- values, and to form biofilms (Jeong & Frank to-eat dishes in Sweden. Gravad fish is prepared 1994; Bansal 1996; Lundén & Korkeala 2004). from raw salmon/rainbow trout; fillets are rubbed

© 2008, The Authors 15 Journal compilation © 2009, Wiley Periodicals, Inc. Journal of Foodservice, 20, pp. 15–20 DOI: 10.1111/j.1748-0159.2008.00118.x 16 Gravad (Gravlax) and cold-smoked I. P. Peiris et al.

with a mixture of sugar, salt and pepper and products purchased in Sweden were kept under covered with dill and stored in a refrigerator for 2 refrigeration (4°C) and analyzed on the recom- days in a plastic bag. They are then packaged, mended best-before day. The products bought in sliced or whole, under vacuum or modified atmo- Germany were frozen for 16 h and kept at room sphere. In the preparation of cold-smoked fish, temperature for 10 h during transportation to fillets are rubbed with salt or the cure is injected Sweden, and refrigerated until analysis. with multiple needles into the fillet, which is then smoked at 25–30°C for 2–3 h, and packed sliced Enrichment and cultural procedures or whole under vacuum or modified atmosphere. The recommended storage for these products is Enrichment and cultural procedures for isolation 3 weeks at 4°C. However, sometimes both the and detection of L. monocytogenes were accord- storage time and temperature will be longer/ ing to NMKL (2004), with some modifications. higher. Such products have also been associated From each product, 25 g was transferred to a with an outbreak of listeriosis in Sweden and sterile stomacher bag and macerated with the help Finland (Ericsson et al. 1997; Loncarevic et al. of a rolling pin. First, an enrichment broth (Listeria 1998; Miettinen et al. 1999). enrichment broth I [LBI] with nalidixic acid and The present investigation is a follow-up of a acriflavine) was added, and the suspension was similar study conducted in the middle of the incubated at 30°C. After 24 h, a second enrich- 1990s, where we found that 12 of 58 (20.7%) ment broth (Listeria enrichment broth II [LBII]; gravad and 3 of 26 (11.5%) cold-smoked fish same composition as LBI but higher acriflavine harboured L. monocytogenes (Loncarevic et al. concentration) was performed and incubated at 1996). Since our first study, there has been an 30°C for 24 h. From the second enrichment, increasing focus on food hygiene in general and 0.1 mL was inoculated onto the surface of Palcam specially on the self-inspection and Hazard and Oxford agar. Plates were incubated at 37°C Analysis and Critical Control Points from the and observed for growth after 24–48 h. authorities and food producers. Therefore, we were interested to investigate if the occurrence Quantification and level of L. monocytogenes in gravad and cold-smoked salmon (Salmo salar) products Products determined as L. monocytogenes- packed under vacuum or modified atmosphere positive by the enrichment procedure were stored have been reduced. In addition, the isolated in a freezer (-20°C) to await the quantification L. monocytogenes strains were characterized by procedure. From each product, a 10-g fish sample serotyping and restriction enzyme analysis (REA) was macerated in a stomacher bag with the help with pulsed-field gel electrophoresis (PFGE). of a rolling pin, and 90 mL of peptone water was Finally, the genotypes of the fish isolates were added. Ten-fold serial dilutions of 1-mL portions compared with the genotypes for human strains of the suspension were prepared in peptone water. isolated in Sweden (Parihar et al. 2008). The original suspension and each dilution were surface-plated onto Oxford, Palcam and horse- blood agar plates. All plates were incubated at Materials and methods 37°C for 48 h and presumptive L. monocytogenes colonies were counted. Sample collection Fifty-six salmon (Salmo salar) products were Confirmation purchased from retail stores between June and September 2005. Fifty-three products were bought From each sample enrichment, five presumptive in Sweden and three in Germany. The purchases L. monocytogenes colonies, or all when fewer were from 16 manufacturers and included 30 were present, were picked from the platings. In vacuum-packed gravad, one modified-atmosphere addition, one to seven presumptive L. monocyto- gravad, 19 vacuum-packed cold-smoked and six genes colonies were picked from the platings modified-atmosphere cold-smoked products. All of each quantification procedure. Colonies were

© 2008, The Authors Journal compilation © 2009, Wiley Periodicals, Inc. Journal of Foodservice, 20, pp. 15–20 Gravad (Gravlax) and cold-smoked I. P. Peiris et al. 17 streaked onto blood agar for purity and presence and profiles of salmon and human isolates were of haemolytic activity. Isolates were tested for indistinguishable with both enzymes, they were Gram reaction and motility (20°C for 10 h in considered as belonging to the same L. monocy- brain–heart infusion). L. monocytogenes were togenes type, whereas a two to three fragment identified with ramnose and xylose fermentation difference with one or both enzymes was consid- tests. ered closely related, a four to six fragment difference was possibly related, and seven or more fragment differences were unrelated (Tenover Serotyping et al. 1995). All L. monocytogenes isolates were serotyped with Listeria O Antiserum types I/II, I, IV, V/VI, Results VI, VII, VIII, IX and H Antiserum A, AB, C, D (Mast Diagnostics, Liverpool, UK) according to Occurrence and level manufacturer’s instructions, with some modifications (Parihar et al. 2008). L. monocytogenes was isolated from 11 (19.6%) of 56 products analyzed. The occurrence of L. monocytogenes was 12.9% in gravad (three PFGE manufacturers, A, B and C) and 28.0% in cold- All 56 salmon isolates of L. monocytogenes were smoked salmons (one manufacturer, A). The level characterized by REA with PFGE with Asc I. In of L. monocytogenes contamination found were addition, 11 of these isolates (one representative between <100 and 1500 cfu/g. strain from each of the five Asc I types, plus three more from strains with Asc I type 1/2 : 48 and Character of L. monocytogenes three more from strains with 4 : 2) were characterized with Apa I. The procedure used is based Of 56 isolates analyzed, 43 (76.8%) belonged on the PulseNet standardized protocol (Graves & to serovar 1/2a and 13 (23.2%) to serovar 4b. Swaminathan 2001) with modifications. Pharma- Isolates obtained by enrichment from product cia Gene Navigator (GE Healthcare, Uppsala, number 28 yielded two serovars: 1/2a and 4b. Sweden) was used for plugs cleaved with Asc I, Isolates obtained by enrichment from product and CHEF MAPPER XA (Bio-Rad Laboratories, number 3 yielded serovar 4b, and the isolates Hercules, CA) was used for plugs cleaved with from quantification yielded serovar 1/2a. Seven Apa I. The migration period for Apa I was 20 h isolates obtained from the quantification of with an initial and final pulse time of 1.0 and 5.0 s product 47 yielded five isolates belonging to respectively. For Asc I, the migration period was serovar 1/2a and two isolates belonging to 24 h, and initial and final pulse times were 4.0 serovar 4b: each serovar with a unique PFGE and 40 s respectively. The photographs were ana- profile. lyzed visually. REA/PFGE typing of the 56 salmon isolates Through serogroups and Asc I profiles, differ- identified five types. L. monocytogenes type ent types of L. monocytogenes were obtained. For 1/2 : 48 was identical to human type 1/2 : 48, each type, a reference strain was selected, e.g. the whereas salmon L. monocytogenes types reference strain type 1/2 : 48 belonged to sero- 1/2 : 114 and 4 : 2 were closely related to group 1/2 and to Asc I profile number 48. The human types. The remaining salmon types reference strain was also restricted with Apa I, (1/2 : 49 and 1/2 : 93) were unrelated to human although the Apa I profiles were not designated. types. Thus, all isolates sharing serogroup 1/2 and Asc I profile number 48, as well as having the same Apa Discussion I profile, were regarded as identical with the reference strain. Reference strain was compared Salmons were bought in retail stores and, to our with previously characterized human strains iso- knowledge, none of these outlets has been lated from listeriosis cases in Sweden. If serovar involved in a case or outbreak of human listerio-

sis. L. monocytogenes type 1/2 : 48 found in The highest level of L. monocytogenes con- salmon has been identified in five human patients, tamination found in the present study was type 4 : 2 in three and type 1/2 : 114 in two since 1500 cfu/g in cold-smoked salmon (number 47), the year 2000 (Parihar et al. 2008). Therefore, it but the level was low (<100 cfu/g) in most prod- could not be excluded that some of the salmon ucts. Loncarevic et al. (1996) reported <100 cfu/g may have caused human listeriosis. One must to 25 400 cfu/g contamination level in cold- remember that the source is actually found in a smoked salmon and a range from <100 cfu/g to few outbreaks. The majority of human cases of 3400 in gravad. listeriosis are sporadic, with unknown sources of Treatment of raw salmon/rainbow trout with the route of infection. The incubation period for salt, sugar, pepper, dill (gravad fish) or with the disease has been reported to vary from 20 h to 2.5–3.5% NaCl and cold-smoked at 25–30°C 90 days (Linnan et al. 1988; Dalton et al. 1997). (cold-smoked fish) is not sufficient to kill L. mono- Therefore, in many cases, the interviews with cytogenes. Hence, if the fish are contaminated patients are made several months after eating con- from the beginning or get contaminated during the taminated food and the patients have forgotten production, the L. monocytogenes will remain. what they have consumed. In the present study, The bacterium can grow both aerobically and L. monocytogenes was isolated from 12.9% of anaerobically at temperatures as low as -0.4°C gravad salmon (three manufacturers, A, B and C) (ICMSF 1996). It has no specific nutrient require- and from 28.0% of cold-smoked salmon (one ments. Fish products have high water activity, e.g.

manufacturer, A). In the study of Loncarevic et al. smoked salmon has an aw value of about 0.97 (1996) 20.7% of the samples of the gravad and (Dillon et al. 1992), and thus the condition for 11.5% of the cold-smoked salmon/rainbow trout multiplication of L. monocytogenes is good. L. were positive for L. monocytogenes. Considering monocytogenes occurs in a faecally contaminated the rather high percentage for cold-smoked environment and may enter the food-processing salmon in the present investigation, it must be plant via utensils, persons, birds, naturally con- kept in mind that all positive salmons were pro- taminated raw products, etc. Re-circulating brine duced by the same manufacturer (producer A). is another source for contamination of the prod- They were processed in a European country ucts as L. monocytogenes could grow in NaCl and distributed to other European countries, e.g. solutions up to 10%. As L. monocytogenes has the Sweden and Germany, which shows that one pro- ability to attach to surfaces and produce biofilms cessing plant can distribute and introduce patho- (Bansal 1996; Blackman & Frank 1996), it can genic L. monocytogenes strains into several colonize, multiply and persist in the food- countries. Because of the findings in the present processing environment (Boerlin et al. 1997; Vogel study, producer A was released from their pro- et al. 2001; Medrala et al. 2003). For example, duction commissions to a European chain of Unnerstad et al. (1996) reported the survival of the food stores. same type of L. monocytogenes in a dairy for at L. monocytogenes isolated from cold-smoked least 7 years. fish of manufacturer A harboured two serovars Producers, retailers and foodservice operators (1/2a and 4b), each with unique Asc I profiles, endeavour to achieve a longer shelf life for those implying that serotyping and genotyping of foods by packaging them in vacuum. This means several isolates from each food sample is impor- long storage at refrigerator temperature before tant (Loncarevic et al. 1996; Ericsson et al. 1997; consumption. Vacuum-packed gravad or cold- Autio et al. 1999; Dauphin et al. 2001). Further- smoked fish that are initially contaminated with a more, products from the same manufacturer (A) small number of L. monocytogenes might there- purchased in both Germany and Sweden con- fore harbour large numbers of bacteria at the tained L. monocytogenes strains with the same day of consumption. And, ready-to-eat foods Asc I and Apa I profiles, indicating a common are sometimes kept in consumers’ refrigerators source of contamination, which could be the raw beyond the recommended best-before day that fish, employees or the environment in the process- will give L. monocytogenes opportunity to mul- ing plant. tiply. The amount of bacteria needed to cause

© 2008, The Authors Journal compilation © 2009, Wiley Periodicals, Inc. Journal of Foodservice, 20, pp. 15–20 Gravad (Gravlax) and cold-smoked I. P. Peiris et al. 19 infection in humans has not been established. It References seems to vary considerably, depending on the virulence of the strain and on the patient’s Autio T et al. (1999). Sources of Listeria monocytoge- immune status (Farber & Peterkin 1991). Patients nes contamination in a cold-smoked rainbow trout have been ill after ingestion of foods harbour- processing plant detected by pulsed-field gel electrophoresis typing. Applied And Environmental Micro- ing levels of L. monocytogenes ranging from biology 65:150–5. < 9 0.3 cfu/g to 10 cfu/g (Swaminathan et al. Bansal NS (1996). Development of a polymerase chain 1995). Even though the contamination levels are reaction assay for the detection of Listeria monocy- usually high in food that are responsible for epi- togenes in foods. Letters of Applied Microbiology demics and sporadic food-borne cases, low con- 22:353–6. tamination levels (<100 cfu/g) cannot be excluded Blackman IC, Frank J (1996). Growth of Listeria monocytogenes as a biofilm on various food- as non-infective (Rocourt et al. 2000). In Sweden, processing surfaces. Journal of Food Protection during the period 1986–1999, 50.2% of L. 59:827–31. monocytogenes isolates belonged to serovar 1/2a Boerlin P, Boerlin-Petzold F, Bannerman E, Bille J, and 43.9% belonged to serovar 4b. However, Jemmi T (1997). Typing Listeria monocytogenes iso- during the period 2000–2007, 71% of isolates lates from fish products and human listeriosis cases. Applied and Environmental Microbiology 63:1338– belonged to serovar 1/2a and only 12.8% to 43. serovar 4b (Parihar et al. 2008). Dalton CB et al. (1997). An outbreak of gastroenteritis As seafood is popular and associated with and fever due to Listeria monocytogenes in milk. human listeriosis (Ericsson et al. 1997; Miettinen New England Journal of Medicine 336:100–5. et al. 1999; Farber et al. 2000; Rørvik et al. Dauphin G, Ragimbeau C, Malle P (2001). Use of 2000), prevalence studies are important in the PFGE typing for tracing contamination with Listeria monocytogenes in three cold-smoked salmon pro- identification of health hazards and to prevent cessing plants. International Journal of Food human disease (Nakamura et al. 2004; Vitas & Microbiology 64:51–61. Garcia-Jalon 2004; Gudmundsdottir et al. 2005). de Valk H et al. (2005). Surveillance of Listeria infections in Europe. Eurosurveillance 10:251–5. Dillon R, Patel T, Ratnam S (1992). Prevalence of Conclusion Listeria in smoked fish. Journal of Food Protection 55:866–70. Isolation of identical or closely related L. mono- Ericsson H et al. (1997). An outbreak of listeriosis sus- cytogenes strains from human clinical cases of pected to have been caused by rainbow trout. Journal listeriosis and gravad and cold-smoked salmon of Clinical Microbiology 35:2904–7. Farber JM, Daley EM, Mackie MT, Limerick B (2000). in retail stores suggest that these types of A small outbreak of listeriosis potentially linked to product are still possible sources of listeriosis in the consumption of imitation crab meat. Letters in Sweden. Even though the number of L. mono- Applied Microbiology 31:100–4. cytogenes containing salmons in the retail store Farber JM, Peterkin PI (1991). Listeria monocytogenes, seems to have decreased, these products should a food-borne pathogen. Microbiology and Molecular Biology Reviews 55:476–511. be considered risk products for human listerio- Graves LM, Swaminathan B (2001). PulseNet stan- sis. Prevalence studies are important and allow dardized protocol for subtyping Listeria monocyto- products contaminated with L. monocytogenes genes by macrorestriction and pulsed-field gel to be identified and recalled, thus preventing electrophoresis. International Journal of Food human infections. Also, education in food Microbiology 65:55–62. hygiene is necessary for producers and food- Gudmundsdottir S, Gudbjornsdottir B, Lauzon HL (2005). Tracing Listeria monocytogenes isolates service operators. from cold-smoked salmon and its processing environment in Iceland using pulsed-field gel electrophoresis. International Journal of Food Acknowledgements Microbiology 101:41–51. Guyer S, Jemmi T (1991). Behavior of Listeria mono- This study was financially supported by the Foun- cytogenes during fabrication and storage of experi- dation of Stadsveterinär Billström, to which we mentally contaminated smoked salmon. Applied and express our gratitude. Environmental Microbiology 57:1523–7.

ICMSF (1996). Microorganisms in Foods 5: Character- NMKL(Nordic Committee on Food Analysis) (2004). istics of microbial pathogens. Blackie Academic & Listeria monocytogenes. Detection in foods. No 136, Professional: London. 3rd. ed. Danish Institute for Food and Veterinary Jeong DK, Frank JF (1994). Growth of Listeria mono- Research: Søborg, Denmark. cytogenes at 10°C in biofilms with microorganisms Parihar VS et al. (2008). Characterization of human isolated from meat and dairy processing environ- invasive isolates of Listeria monocytogenes in ments. Journal of Food Protection 57:576–86. Sweden 1986–2007. Foodborne Pathogens and Linnan MJ et al. (1988). Epidemic listeriosis associated Disease 5. with Mexican-style cheese. New England Journal of Rocourt J, Jacquet Ch, Reilly A (2000). Epidemiology Medicine 319:823–8. of human listeriosis and seafoods. International Loncarevic S, Danielsson-Tham M-L, Gerner-Smidt P, Journal of Food Microbiology 62:197–209. Sahlström L, Tham W (1998). Potential sources of Rørvik LM, Aase B, Alvestad T, Caugnat DA (2000). human listeriosis in Sweden. Food Microbiology Molecular epidemiological survey of Listeria mono- 15:65–9. cytogenes in seafoods and seafood-processing plants. Loncarevic S, Tham W, Danielsson-Tham M-L (1996). Applied and Environmental Microbiology 66:4779– Prevalence of L. monocytogenes and Listeria spp. in 84. smoked and ‘gravad’ fish. Acta Veterinaria Scandi- Swaminathan B et al. (1995). Update on listeriosis in navica 37:13–8. the United States: shifting paradigms. XII Interna- Lundén J, Korkeala H (2004). Adherence of Listeria tional Symposium on Problems of Listeriosis, Perth, monocytogenes strains to stainless steel surface after Australia, Promaco Conventions Pty Ltd, p. 489. short and long contact times. In: Proceedings of XV Tenover FC et al. (1995). Interpreting chromosomal International Symposium on Problems of Listeriosis: DNA restriction patterns produced by pulsed-field 5–12 September 2004; Uppsala (ed. W Tham), pp. y gel electrophoresis: criteria for bacterial strain abstract no. 142. typing. Journal of Clinical Microbiology 33:2233–9. Medrala D et al. (2003). Persistence of Listeria mono- Unnerstad H et al. (1996). Prolonged contamination of cytogenes strains isolated from products in a Polish a dairy with Listeria monocytogenes. Netherland fish-processing plant of a 1-year period. Food Micro- Milk and Dairy Journal 50:493–9. biology 20:715–24. Vitas AI, Garcia-Jalon VAI (2004). Occurrence of List- Miettinen MK et al. (1999). Molecular epidemiology eria monocytogenes in fresh and processed foods of an outbreak of febrile gastroenteritis caused by in Navarra (Spain). International Journal Food Listeria monocytogenes in cold-smoked rainbow Microbiology 90:349–56. trout. Journal of Clinical Microbiology 37:2358– Vogel BF, Huss HH, Ojeniyi B, Ahrens P, Gram L 60. (2001). Elucidation of Listeria monocytogenes con- Nakamura H, Hatanaka M, Ochi K (2004). Listeria tamination routes in cold-smoked salmon processing monocytogenes isolated from cold-smoked fish prod- plants detected by DNA-based typing methods. ucts in Osaka City. Japan. International Journal of Applied and Environmental Microbiology 67: Food Microbiology 94:323–8. 2586–95.

Epidemiol. Infect. (2007), 135, 854–856. f 2006 Cambridge University Press doi:10.1017/S0950268806007503 Printed in the United Kingdom

SHORT REPORT More than one variant of Listeria monocytogenes isolated from each of two human cases of invasive listeriosis

W. THAM 1*, G. LOPEZ VALLADARES1, S. HELMERSSON2,A.O¨STERLUND3 1 AND M.-L. DANIELSSON-THAM

1 Department of Restaurant and Culinary Arts, O¨rebro University, Sweden 2 Department of Environmental Assessment, SLU, Uppsala, Sweden 3 Department of Communicable Disease Prevention and Control, Lulea˚, Sweden

(Accepted 5 October 2006; ﬁrst published online 17 November 2006)

SUMMARY Two variants of Listeria monocytogenes were isolated from blood cultures from each of two patients with listeriosis. Each variant displayed a two-band difference in DNA profile from the other by pulsed-field gel electrophoresis. Although this difference in profile is insufficient to distinguish clearly between the variants, the possibility of co-infection with different strains of L. monocytogenes needs to be considered. We suggest that more than one colony should be selected for molecular typing to aid interpretation during investigation of the sources and routes of Listeria infection.

Listeria monocytogenes is a foodborne Gram-positive the evolution of these variants was the consequence bacterium that can cause severe sepsis particularly of a single gene insertion event in the genome of the meningitis, encephalitis and spontaneous abortions. index straintr over time. The first report of a foodborne outbreak of listeriosis We describe here the recovery of PFGE variants [1] led to a number of epidemiological investigations within the same blood samples from each of two into the reservoirs and transmission of L. mono- patients with listeriosis. Blood samples (10 ml) were cytogenes and the description of a variety of molecu- taken from the two patients (A and B) and inoculated lar methods for the typing of strains [2]. Pulsed-field into BacT/ALERT FA bottles (Organon Teknika gel electrophoresis (PFGE) of DNA digests generated Corp., Durham, NC, USA). The bottles were sent by with rare-cutting restriction endonucleases has be- regular post without cooling and arrived the next day come the ‘gold standard’ for the typing of L. mono- at our laboratory, where they were kept refrigerated cytogenes, and many other bacterial species owing until analysis within 1 week; 0.1 ml from each sample to its reproducibility and discriminatory power [2]. was spread on 5% bovine blood agar plates and There are a number of reports on the diversity of incubated at 37 xC for 24 h. Forty-six colonies of L. monocytogenes strains isolated from humans, food L. monocytogenes were isolated from patient A and 50 and the food environment. Boerlin et al. [3] found colonies from patient B. All isolates were frozen that a limited number of clonally similar and poss- in brain heart infusion broth (Merck, Darmstadt, ibly related PFGE subtypes emerged slowly over a Germany) containing 20% glycerol, at x70 xC, for 5-year outbreak in Switzerland and concluded that later characterization. Each isolate was characterized by restriction en- * Author for correspondence: Dr W. Tham, Department of zyme analysis with AscI, followed by PFGE accord- Restaurant and Culinary Arts at O¨rebro University, So¨ra¨lgsva¨gen 2, S-712 60 Grythyttan, Sweden. ing to the PulseNet standardized protocol [4] with (Email: [email protected]) some modifications. Pharmacia Gene Navigator PFGE variants of L. monocytogenes 855

1 2 3 4 5 6 7 kbp Patient B also exhibited two variants that differed in two bands (37 isolates of variant 1 and 13 of variant 2) (Fig.). All isolates were confirmed as L. monocytogenes serovar 1/2a. The two-band difference in 582·0 both pairs of variants was also found with ApaI re- 485·0 striction. However, all isolates from patient A were identical in SmaI digests but this enzyme confirmed 388·0 the presence of two variants in patient B. Of the 16 291·0 isolates recovered after 6 years’ refrigeration, 14 were identified as variant 1 and the remainder, variant 2. 194·0 Boerlin et al. [3] analysed five isolates of L. monocytogenes from different body sites of a single patient,

97·0 sampled the same day, and all gave the same PFGE ApaIandSmaI patterns thus confirming the validity of macrorestriction analysis for short-term epidemiological tracing of this species. In the long term Swiss epidemic outbreak PFGE profiles were found to vary Fig. Pulsed-field gel electrophoresis patterns from AscI over time which was probably to be expected par- digests of DNA of Listeria monocytogenes isolates. Lanes 1, ticularly as a result of rearrangements involving in- 4, 7, lambda ladder; lane 2, SLU2916 (patient A, variant 1); lane 3, SLU2918 (patient A, variant 2); lane 5, SLU3267 sertion, deletion, or transposition of genes [3]. The (patient B, variant 1); lane 6, SLU3268 (patient B, present study, however, shows that more than one variant 2). Note: The thick band slightly above the 388 kbp variant of L. monocytogenes can be recovered from a marker band of isolate SLU3267 (lane 5) was separated into single blood sample. This finding may be interpreted two bands when the gel was run for another 24 h. as either genetic instability of a single strain population or infection with mixed populations of the organism; the latter scenario is feasible if they were (Pharmacia, Uppsala, Sweden) was used with a run acquired through ingestion of food contaminated time of 24 h, an initial pulse time of 4 s and final pulse with multiple strains. time of 40 s; a lambda ladder PFG marker no. 340 S Baxter et al. [5] found that multiple isolates of (New England Bio-Labs Inc., Beverly, MA, USA) L. monocytogenes from a single site of a sheep had was used as molecular size markers. Isolates showing identical enzyme electrophoretic types as did isolates variation in profile were examined further by restric- from different tissues within an individual sheep. tion with ApaI and SmaI and with different migration Wiedmann et al. [6], however, found that RAPD protocols. DNA was prepared three times from patterns for brain isolates from a goat were different isolates representing each variant and analysed by (the degree of difference is unknown) from the pattern PFGE to ensure repeatability. In addition, the BacT/ for the cerebrospinal fluid isolates from the same ALERT FA bottle from patient A was kept for 6 animal. In contradiction of their own results, they years in the refrigerator (+6to+8 xC) and a further discounted co-infection with two different L. mono- 16 colonies were isolated after this time and charac- cytogenes strains as being unlikely as in such a case terized by restriction with AscI. One representative both strains would be expected to be present in the isolate of each of the PFGE variants was confirmed as brain. However, two different strains of L. mono- L. monocytogenes by their catalase and haemolysin cytogenes in a human [7] and an animal case of lis- production, Gram reaction and fermentation of teriosis [8] have been described. In each of those cases, rhamnose and xylose. The isolates were serotyped the strains were clearly unrelated by PFGE and with Listeria O and H antisera (Mast Diagnostics, thought to have been ingested through food and feed Bootle, UK), according to the manufacturer’s in- respectively. structions. Although a considerable degree of strain diversity L. monocytogenes was recovered in pure culture in PFGE profiles exists in L. moncytogenes popu- from each sample. Two PFGE variants, differing by lations [2] the number of band differences required to two bands in AscI digests, were isolated (43 isolates discriminate between different strains is contentious. of variant 1 and three of variant 2) from patient A. Strains differing by several bands are clearly distinct 856 W. Tham and others from each other but some strains from temporally and 2. Brosch R, et al. Genomic fingerprinting of 80 strains geographically distinct sources may exhibit few dif- from the WHO multicentre international typing study ferences in DNA profile and by the Tenover criteria of Listeria monocytogenes via pulsed-field electrophoresis (PFGE). International Journal of Food Micro- could be considered epidemiologically related [9]. biology 1996; 32: 343–355. However, the latter criteria have not been validated 3. Boerlin P, et al. Subtyping Listeria monocytogenes for typing L. monocytogenes and studies addressing isolates genetically related to the Swiss epidemic clone. the variation of populations in single specimens Journal of Clinical Microbiology 1996; 34: 2148–2153. are thus necessary. The evidence for dual infection 4. Graves LM, Swaminathan B. PulseNet standardized protocol for subtyping Listeria monocytogenes by in the two patients presented here is admittedly cir- macrorestriction and pulsed-field gel electrophoresis. cumstantial but the study does highlight the fact International Journal of Food Microbiology 2001; 65: that an approach of typing a single isolate from a 55–62. clinical site of patient may pose problems for in- 5. Baxter F, et al. Characterization by multilocus enzyme terpretation of DNA profiles when investigating electrophoresis of Listeria monocytogenes isolates in- sources and routes of Listeria infection. The examin- volved in ovine listeriosis outbreaks in Scotland from 1989 to 1991. Applied and Environmental Microbiology ation of multiple isolates from blood cultures and 1993; 59: 3126–3129. other patient sites and from mother–baby pairs is 6. Wiedmann M, et al. 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