Riber, Leise

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Downloaded from orbit.dtu.dk on: Dec 19, 2017Salmonella genes involved in attachment on carcass surface and pork meatcontaminationRiber, LeisePublication date:2009Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Riber, L. (2009). Salmonella genes involved in attachment on carcass surface and pork meat contamination.Poster session presented at BIOTRACER Dissemination Conference and Third General Meeting, Berlin,Germany.Salmonella genes involved in attachment on carcass surface and pork meat contamination  Leise Riber & Jeffrey Hoorfar National Food Institute, DTU, Copenhagen, Denmark E-mail: leiri@food.dtu.dk Collaborators: KU-LIFE, DK & IFR, UK          Human infections caused by Salmonella from contaminated food is a common problem. Consequently, there is a need for optimizing food production environment in order to reduce any threat to human health caused by the food chain.   METHODS   REFERENCES Brocklehurst TF, et al. (1995).  JFM 27:45-60.  Datsenko KA & Wanner BL (2000). PNAS 97:6640-6645.   - Identify genes involved in attachment to meat  - Study the influence of the “history” of Salmonella           on attachment potential  - Measure the expression of selected genes over time for     Salmonella on a meat surface    BACKGROUND 1. Identify genes involved in attachment to meat - Genes are selected from literature studies and expression data from   gel cassette system (KU-Life) - Selected genes: - flagellar genes (fli, flj, flh, flg, mot) - fimbriae and curli genes (agf, fim, pef, lpf, std) - genes related to extracellular matrix (prg, bap, mis, shd) - genes involved in cellulose biosynthesis (yhj, bcs) Datsenko & Wanner, 2000 - Knock-out mutants are constructed using   lambda red system (Datsenko &    Wanner, 2000) and P22 transduction - Strain: S. Typhimurium 4/74 - The ability to attach to meat for mutant   cells and wild type cells is compared 2. Attachment studies – “history of cells” - Influence of physiological state of     Salmonella on adhesion is studied:  - Liquid state – broth  - Immobilized state – gel cassette    Gel cassette ready for inoculation medium (IFR)  - Gel cassette system developed by IFR   (Brocklehurst et al., 1995) 4. Time-course of gene expression on meat - Isolate RNA from cells strongly attached to meat over time:  - Qiagen RNeasy mini kit using combined enzymatic lysis and   Proteinase K digestion - Quality check using Bioanalyzer - Conversion of total RNA into cDNA - Preparing for expression analysis either by whole genome   microarray technology or by quantitative RT-PCR - Genes of interest: attachment, virulence and lag-phase AIM  Establish a new approach to identify and use genes of Salmonella involved in the attachment on carcass surfaces and in pork chain contamination. With the design of a new meat surface model, we will analyze a number of meat and non-meat isolates for their ability to express the attachment genes. Subsequently, we will look at the  time-course of the expression profile of attachment, virulence and lag-phase genes to see if specific probes can be designed to be used for ”early-warning” assays. This may give us an idea of what kind of food products and pro- duction environments that facilitate expression of lag-phase genes. STRATEGY - Growth experiments in broth + gel:  - Temperature: 25 oC - Medium (gel): LB + 22% pluronic  - Medium (broth): LB   - Directly from broth and gel cassette cells are applied to a meat model   at the same inoculation level (~ 108 CFU/ml)  3. Attachment studies – pork meat surface model Growth curves for S. Typhimurium 4/74  Broth     Gel Salmonella cells from broth and gel are diluted ten times in sterilized maximum recovery diluent (MRD) and inoculated onto pieces (9.6 cm2) of pork fillets (meat model) Inoculation level (CFU/ml) Strain Adhesion time (min) Temperature Treatment Attachment potential ~ 107  500 μl spread on meat surface S. Typhimurium 4/74: wt vs. mutant gel vs. broth 0, 5, 10, 30, 60, 120 Room temp. Loosly attached: wash in 40 ml MRD for 1 min Strongly attached: stomach in 40 ml MRD  for 1 min Calculated as: Strongly Strongly+Loosly Initial test study of attachment potential of Salmonella to pork meat  Microarray lab at National Food Institute, DTU - Optimization needed: - Minimize background flora? - Increase amount of cells extracted   from the meat surface? Attachment curve for S.  typhimurium 4/740,30,350,40,450,50,550,60 20 40 60 80 100 120 140Time (min)Attachment potentialTime (hours) Log 10 CFU/ml Meat  model 

Salmonella genes involved in attachment on carcass surface and pork meat contamination

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Salmonella genes involved in attachment on carcass surface and pork meat contamination

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