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

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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 1 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 2 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 3 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). 4 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; 5 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,
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