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Cheuzeville Lisa.Pdf (4.845Mb) Faculty of Science and Technology MASTER’S THESIS Study program/ Specialization: Spring semester, 2015 Biological Chemistry Open /Restricted access Writer: CHEUZEVILLE Lisa ………………………………………… (Writer’s signature) Faculty supervisor: LILLO Cathrin Thesis title: Rapid detection of microorganisms in the dairy value chain by MALDI-TOF MS Credits (ECTS): 60 Key words: MALDI-TOF MS Pages: 80 Identification + enclosure: 77 Species Yeasts Bacteria Stavanger, June, 12th 2015 Milk Cheese Mastitis Resistance Β-lactamase Front page for master thesis Faculty of Science and Technology Decision made by the Dean October 30th 2009 Master thesis in Biological Chemistry Rapid detection of microorganisms in the dairy value chain by MALDI-TOF MS CHEUZEVILLE Lisa 2014-2015 Supervisors: FLIEGEL Daniel, PhD Tine FoU, Fagleder hurtiganalyser Tine R&D, Division leader Analytical Instrumentation LILLO Cathrin University of Stavanger, TN-IMN Centre for Organelle Research List of contents List of contents ................................................................................................................................ List of Figures.................................................................................................................................. List of Tables ................................................................................................................................... Acknowledgements ....................................................................................................................... 1 Abstract.......................................................................................................................................... 2 Abbreviations ................................................................................................................................ 3 Introduction ................................................................................................................................... 5 1. Scope of the study .................................................................................................................. 5 2. Theoretical background .......................................................................................................... 7 2.1. History of the identification of microorganisms: from GRAM staining to MALDI-TOF Mass Spectrometry ............................................................................................................................ 7 2.2. Milk ............................................................................................................................... 8 2.3. Definitions ..................................................................................................................... 9 2.4. Mastitis ........................................................................................................................ 12 2.5. Cheeses: hard white type ............................................................................................ 13 2.6. HACCP (Hazard Analysis Critical Control Point) ......................................................... 15 Materials and Methods ................................................................................................................ 16 1. Sampling and cryoconservation ............................................................................................ 16 1.1. Thermoduric bacteria and spore-formers bacteria from the raw milk ........................... 16 1.2. Psychrotrophic bacteria in consumer’s milk (end of the shelf-life) ................................ 19 1.3. Yeasts on the cheese surfaces ................................................................................... 20 1.4. Purification and collection ............................................................................................ 21 2. Mastitis samples ................................................................................................................... 24 2.1. Sampling and registration of the raw milk from the cow ............................................... 24 2.2. Microbial testing and collection .................................................................................... 24 3. Matrix Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry (MALDI-TOF MS) identification......................................................................................................................... 25 3.1. Principle of the MALDI-TOF MS .................................................................................. 25 3.1.1. Identification of microorganisms ............................................................................ 27 3.1.2. Detection of resistance mechanisms: B-lactamase ............................................... 28 3.2. Methods for microorganisms identification by MALDI-TOF MS .................................... 31 3.2.1. Bacterial and yeasts isolates from the freezer or fridge ......................................... 31 3.2.2. Identification of microorganisms ............................................................................ 31 3.2.3. Detection of the resistance mechanisms by MALDI-TOF MS ................................ 35 Results ......................................................................................................................................... 37 1. Identification of thermoduric bacteria and spore-from raw milk by MALDI-TOF MS .............. 37 1.1. Identification of the spore-formers bacteria: target Bacillus cereus group .................... 37 1.2. Milk analysis (deposit of milk on the target), target: Bacillus cereus ............................ 40 1.3. Identification of spore-formers bacteria: target Clostridium species ............................. 41 1.4. Identification of mesophiles and thermophiles bacteria: target Lactobacillus species .. 45 1.5. Target: Escherichia coli ............................................................................................... 47 1.6. Identification of total viable bacteria on mPCA............................................................. 47 2. Psychrotrophic bacteria in the commercialized milk at the end of the shelf-life ..................... 50 2.1. Identification of spore-formers bacteria, target: Bacillus cereus ................................... 50 2.2. Target: Enterobacteriaceae ......................................................................................... 52 2.3. Target: Pseudomonas ................................................................................................. 53 2.4. Identification of total viable bacteria ............................................................................. 54 3. Identification of yeasts on the cheese surfaces by MALDI-TOF MS ..................................... 57 4. Identification of mastitis samples and detection of the β-lactamase resistance ..................... 59 4.1. Identification of bacteria collected from the mastitis laboratory in Molde ...................... 59 4.2. Determination of B-lactamase resistance .................................................................... 63 5. Bacterial stains comparison between the different milks products ........................................ 67 Discussion ................................................................................................................................... 70 Conclusion ................................................................................................................................... 74 Perspectives ................................................................................................................................ 74 References ................................................................................................................................... 75 Appendices ...................................................................................................................................... List of Figures Figure 1: Cycle of production of spores by spore-forming bacteria as Bacillus and Clostridium ...... 10 Figure 2: The stages of cheese production. .................................................................................... 13 Figure 3: Preparation of serial dilutions by using of Dilucups shook on a Dilushaker. ..................... 19 Figure 4: Principle of the MALDI-TOF MS. ..................................................................................... 26 Figure 5: Comparison at strains level of spectra of 4 different microorganisms............................... 27 Figure 6: Comparison of strains of Bacillus cereus, using MALDI Biotyper V3.0 software. ............. 28 Figure 7: β-lactam core................................................................................................................... 29 Figure 8: β-lactam antibiotic hydrolyzed by β-lactamase................................................................. 30 Figure 9: Creation of own MSP. ...................................................................................................... 34 Figure 10: Comparison of MALDI-TOF MS spectra at strain level of 2 strains ................................ 39 Figure 11: Comparison at species level of Bacillus cereus of 3 strains. .......................................... 40 Figure 12: Comparison at a strain level of Clostridium tyrobutyricum ATCC 25755 strains. ............ 42 Figure 13: Comparison of Clostridium strain against a strains from own MSP. ..............................
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