MOLECULAR MONITORING OF MEAT SPOILING PSEUDOMONAS SPECIES AND ANALYSIS OF STAPHYLOCOCCAL ENTEROTOXIN EXPRESSION AND FORMATION Dóra Márta Doctoral Thesis Corvinus University of Budapest Faculty of Food Science Department of Microbiology and Biotechnology Budapest, 2011 ii According to the Doctoral Council of Life Sciences of Corvinus University of Budapest on 29 th November 2011, the following committee was designated for defence. Committee: Chair: Tibor Deák, D.Sc. Members: Péter Biacs, D.Sc. László Abrankó, Ph.D. Judit Tornai-Lehoczki, Ph.D. Judit Beczner, C.Sc. Opponents: Adrienn Micsinai, Ph.D. László Varga, Ph.D. Secretary: László Abrankó, Ph.D. iii “I am among those who think that science has great beauty. A scientist in his laboratory is not only a technichian: he is also a child placed before natural phenomena which impress him like a fairy tale.” Marie Curie (1867-1934) iv CONTENTS LIST OF ABBREVIATION............................................................................................................vii 1. INTRODUCTION..........................................................................................................................1 2. LITERATURE REVIEW..............................................................................................................3 2.1. Characterization of red meat.....................................................................................................3 2.1.1. Microbiological aspects of food spoilage especially on pork............................................4 2.2. Food spoilage-causing Pseudomonas species and their role on aerobically stored meat .........7 2.2.1. The genus Pseudomonas ....................................................................................................8 2.2.1.2. Impact of proteolytic and lipolytic activities of Pseudomonas species during spoilage ....................................................................................................................................9 2.2.2. Detection of Pseudomonas species..................................................................................11 2.2.2.1. Traditional culture-based methods............................................................................11 2.2.2.2. Molecular methods for identification and typing of food-borne bacteria.................14 2.3. Food-borne pathogens on meat which connected to food-borne diseases..............................19 2.3.1. The genus Staphylococcus ...............................................................................................20 2.3.1.1. Staphylococcal enterotoxins and their relevance in SFP ..........................................22 2.3.1.2. Characterization of prophage-encoded sea ...............................................................24 2.3.1.3. Regulatory system of sed ..........................................................................................27 2.3.2. Molecular methods used for studying gene expression ...................................................29 2.3.2.1. Real-time PCR and qRT-PCR...................................................................................29 2.3.2.2. Immunological methods for investigation of staphylococcal enterotoxin production ................................................................................................................................................34 3.OBJECTIVES ...............................................................................................................................37 4. MATERIALS AND METHODS ................................................................................................38 4.1. Bacterial strains.......................................................................................................................38 4.2. Media and broths.....................................................................................................................40 4.3. Solutions..................................................................................................................................42 4.3.1. Solutions for DNA extraction ..........................................................................................42 4.3.2. Solutions for gel electrophoresis......................................................................................42 4.3.3. Solutions for RNA extraction ..........................................................................................43 4.3.4. Solutions for ELISA.........................................................................................................44 4.3.5. Reagents for ELISA.........................................................................................................45 4.3.6. Processed pork products for Staphylococcus aureus experiments...................................45 4.4. Methods...................................................................................................................................46 4.4.1. Methods for the studies of Pseudomonas and Chryseobacterium species.......................46 4.4.1.1. Isolation and characterization of bacterial strains from pork chops .........................46 4.4.1.2. Detection of proteolytic and lipolytic activities........................................................46 4.4.1.3. Genomic DNA isolation............................................................................................47 4.4.1.4. Pseudomonas genus-specific PCR assay..................................................................47 4.4.1.5. Typing of bacterial isolates by RAPD-PCR .............................................................48 4.4.1.6. 16S rDNA-RFLP analysis of the Pseudomonas isolates ..........................................49 4.4.1.7. rpoB -RFLP analysis of the Pseudomonas isolates ...................................................49 4.4.1.8. Sequencing of 16S rDNA and rpoB amplicons and constructing phylogenetic trees ................................................................................................................................................49 v 4.4.1.9. Identification of P. lundensis , P. fragi isolates by species-specific PCR .................50 4.4.1.10. Cultivation and characterization of growth of Chryseobacterium antarcticum at different temperatures ............................................................................................................50 4.4.1.11. Competition analysis between C. antarcticum and P. fragi strains........................51 4.4.2. Methods for the studies with Staphylococcus aureus ......................................................51 4.4.2.1. Cultivation of Staphylococcus aureus SA45 ............................................................51 4.4.2.2. Cultivation of S. aureus SA45 in situ on meat products...........................................52 4.4.2.3. RNA extraction and reverse transcription.................................................................52 4.4.2.4. Primer and probe design ...........................................................................................53 4.4.2.5. The real-time PCR assay...........................................................................................54 4.4.2.6. Relative quantification ..............................................................................................54 4.4.2.7. ELISA .......................................................................................................................54 5. RESULTS AND DISCUSSION ..................................................................................................56 5.1. Characterization of Pseudomonas isolates..............................................................................56 5.2. Molecular characterization of Pseudomonas isolates .............................................................61 5.2.1. Testing the applicability of a Pseudomonas genus-specific primer pair .........................61 5.2.2. Molecular typing of Pseudomonas isolates by RAPD-PCR analysis..............................63 5.2.3. Results of 16S rDNA-RFLP analysis of Pseudomonas isolates......................................65 5.2.4. Identification of Pseudomonas species using carA specific primers...............................68 5.2.5. Results of sequencing the 16S rDNA and rpoB genes.....................................................69 5.2.6. Results of rpoB -RFLP......................................................................................................73 5.3. Evaluation of lipolytic and proteolytic activities of the Pseudomonas isolates at different temperatures...................................................................................................................................75 5.4. Chryseobacterium antarcticum and investigation its spoiling potential.................................80 5.4.1. Microscopic and colony morphology of Chryseobacterium antarcticum F1445/3.........81 5.4.2. Characterization of growth of C. antarcticum F1445/3 at different temperatures..........84 5.4.3. Characterization of the lipolytic and proteolytic activities of C. antarcticum strains .....86 5.4.4. Detection of competition between C. antarcticum and P. fragi strains...........................88 5.5. Expression and production of enterotoxin A and D of Staphylococcus aureus SA45 ...........91 5.5.1. Growth and enerotoxin A and D expression in meat products ........................................91 5.5.1.1. Boiled ham ................................................................................................................93