9/18/2016 MPN Index Enrichment Techniques 100ml Lower Upper Zero tolerance or 0 cells per 25 grams of sample is 225734 517 required for food safety against Salmonella, Listeria,and Escherichia coli0157:H7 Must be able to find one or more cells among millons of other bacteria 350050504 91001000 10 How? -Pre-enrichment using non-selective media Use selective media to isolate the pathogen 060000 Confirm identity via biochemical tests and/or genetic authentification Preenrichment allows the bacteria to repair, recover and regain resistance to selective agents. Physiological States of Bacteria Traditional flowchart for isolation of Salmonella Outside of the lab,bacterial communities are species from foods highly dynamic Each community may involve the following physiological states /↓ 1.Injured and unable to grow on selective W书限K media 2.Viable but non-culturable 3.Biofilm-structured communities 4.Coordination of activities as a population via cell-cell communication
9/18/2016 6 21 Enrichment Techniques • Zero tolerance or 0 cells per 25 grams of sample is required for food safety against Salmonella, Listeria, and Escherichia coli O157:H7 • Must be able to find one or more cells among millons of other bacteria • How? - Pre-enrichment using non-selective media - Use selective media to isolate the pathogen - Confirm identity via biochemical tests and/or genetic authentification • Preenrichment allows the bacteria to repair, recover and regain resistance to selective agents. 22 Traditional flowchart for isolation of Salmonella species from foods 23 Physiological States of Bacteria • Outside of the lab, bacterial communities are highly dynamic • Each community may involve the following physiological states 1. Injured and unable to grow on selective media 2. Viable but non-culturable 3. Biofilm – structured communities 4. Coordination of activities as a population via cell-cell communication 24
9/18/2016 Injury Bacterial Injury following Mild Stress Non-selective medium Defined-inability of cells exposed to sublethal stress to grow on selective media,while lethality retaining culturability on non-selective media Selective 4 n山y medium Sub-lethal levels of heat,radiation,acid,or sanitizers may injure rather than kill cells Injured cells are less resistant to selective agents or have increased nutritional requirements (minutes) Recovery of injured cells may be needed before growth on selective media Injured bacteria are not detected on the selective media Cell Injury is a Threat to Food Safety How are Bacteria Injured? 1.If injured cells are classified as dead Heating,freezing,and detergents during heat resistance determination, Injury:damage membranes and leak then the heat process will be ineffective cytoplamic components 2.Injured cells that escape detection may Repair:membrane integrity is repair before food is eaten and cause reestablished illness ·Osmoprotectants 3.Selective agents may be common food ingredients like salt or organic acid that prevent injury prevent repair and lead to underestimation of the microbial levels
9/18/2016 7 Injury • Defined – inability of cells exposed to sublethal stress to g , row on selective media, while retaining culturability on non-selective media • Sub-lethal levels of heat, radiation, acid, or sanitizers may injure rather than kill cells • Injured cells are less resistant to selective agents h d l or have increased nutritional requirements • Recovery of injured cells may be needed before growth on selective media 25 Bacterial Injury following Mild Stress Non-selective medium lethality Selective injury medium Injured bacteria are not detected on the selective media 26 Cell Injury is a Threat to Food Safety 1. If injured cells are classified as dead during heat resistance determination during heat resistance determination, then the heat process will be ineffective 2. Injured cells that escape detection may repair before food is eaten and cause illness 3. Selective agents may be common food ingredients like salt or organic acid that prevent repair and lead to underestimation of the microbial levels 27 How are Bacteria Injured? • Heating, freezing, and detergents - Injury: damage membranes and leak cytoplamic components - Repair: membrane integrity is reestablished • Osmoprotectants prevent injury 28
9/18/2016 Oxygen toxicity damages nucleic acid Repair of Injured Bacterial Cells Recovery: -add detoxifying agents such as catalase to Requires RNA and protein synthesis the medium Lag phase is prolonged -Grow anaerobically Environmental factors influence the extent and rate of repair ·Example -Listeria monocytogenes injured at 55C,20 mins Starts repair immediately at 370C and is complete in 9h 冒 园 At 40C,repair is delayed for 8-10 days and is complete in 16-19 days Viable but Non-Culturable (VBNC) Viable but Non-Culturable (VBNC) Defined-VBNC bacteria Most often induced by nutrient cannot be cultured on any limitation medium but still cause Also induced by salt concentration disease exposure to hypochlorite,and shifts in Vegetative cells differentiate temperature into a dormant cell as a How the VBNC state occurs is mostly survival strategy for non- unknown sporulating bacteria Estimated that 99%of the bacteria in VBNC cells are found in the biosphere may be VBNC marine,soil,and gastrointestinal environments
9/18/2016 8 • Oxygen toxicity – damages nucleic acid • Recovery: - add detoxifying agents such as catalase to the medium - Grow anaerobically 29 Repair of Injured Bacterial Cells • Requires RNA and protein synthesis • Lag phase is prolonged • Environmental factors influence the extent and rate of repair • Example - Listeria monocytogenes injured at 550C, 20 mins - Starts repair immediately at 370C and is complete in 9h - At 40C, repair is delayed for 8-10 days and is complete in 16-19 days 30 Viable but Non-Culturable (VBNC) • Defined - VBNC bacteria cannot be cultured on an y medium but still cause disease • Vegetative cells differentiate into a dormant cell as a survival strategy for nonsporulating bacteria • VBNC cells are found in marine, soil, and gastrointestinal environments 31 Viable but Non-Culturable (VBNC) • Most often induced by nutrient limitation • Also induced by salt concentration, exposure to hypochlorite, and shifts in temperature • How the VBNC state occurs is mostly unknown • Estimated that 99% of the bacteria in the biosphere may be VBNC 32
9/18/2016 Bacterial Cellular Communication Quorum Sensing Communication via small chemical molecules to coordinate Two different gene regulation activities as a population instead of as single cells mechanisms 1.Quorum sensing -population size Low Cell Dersity High Cell Density 2.Signal transduction-environment 飞 Tw-Cop Bacterial Quorum Sensing ⊙a LNI CDABE6 autoirduction Two-Component Signal Transduction response to environmental stress/stimuli Role of Quorum sensing in Food Microbiology Speculated that QS has a role in food spoilage Not much data to support this Signal molecules are found in food products such as milk,chicken soup, bean sprouts Components of food may mimic or alter Phasphorylated response QS signaling systems Modulates gene Probiotics may inhibit QS signaling 9
9/18/2016 9 Bacterial Cellular Communication • Two different gene regulation mechanisms 1. Quorum sensing – population size 2. Signal transduction - environment 33 Quorum Sensing Communication via small chemical molecules to coordinate activities as a population instead of as single cells Low Cell Density Hi h C ll D nsit High Cell Density Light luxR luxI C D A B E G LuxR LuxR LuxI Acyl-ACP SAM luxR luxI C D A B E G LuxI LuxR unstable inactive Luciferase autoinduction 34 Two-Component Signal Transduction response to environmental stress / stimuli Signal does not diffuse across the membrane Kinase transmits signal across the membrane via a conformational change Modulates gene expression in respone to the environment Phosphorylated response regulator activates gene expression 35 Role of Quorum sensing in Food Microbiology • Speculated that QS has a role in food spoilage • Not much data to support this • Signal molecules are found in food products such as milk, chicken soup, bean sprouts • Components of food may mimic or alter QS signaling systems • Probiotics may inhibit QS signaling 36