9/18/2016 Food Learning Objectives 1.Distinguish among different methods of culturing foodborne microbes Chapter 2 AN INTRODUCTION 2.Recognize intrinsic and extrinsic factors used to control microbial growth 3.Water activity concept Microbial Growth, 4.Affect of water activity on microbial growth Survival,and Death and ecology in Foods 5.Qualitatively predict the size of microbial populations and their rate of growth 6.Relate biochemistry to energy production and metabolic products of foodbo ne bacteria Food Microbiology Agencies Involved in Food Safety USDA-US Department of Agriculture Regulated by numerous government dplir sofety rd agencies -pressures food processors to initiate "voluntary"recall Requires understanding of microbes as FDA-Food and Drug Administration well as complex food systems .regulates all foods except meat,poultry and alcohol issues mandatory recalls and quorantines foods - regulates drugs and medical devices CDC-Center for Disease Control and Prevention for obreoks of fodbarre =@ oversees PulseNet,a DNA fingerprinting clearinghouse that links geographically dispersed cases to a common outbreak EPA-Envirormental Protection Agency .insecticides,fungicides,herbicides
9/18/2016 1 Chapter 2 Microbial Growth, Surv val, and Death ival, and Death in Foods 1 Learning Objectives 1. Distinguish among different methods of culturing foodborne microbes 2. Recognize intrinsic and extrinsic factors used to control microbial growth 3. Water activity concept 4. Affect of water activity on microbial growth and ecology 5. Qualitatively predict the size of microbial populations and their rate of growth 6. Relate biochemistry to energy production and metabolic products of foodborne bacteria 2 Food Microbiology • Regulated by numerous government agencies • Requires understanding of microbes as well as complex food systems Campylobacter is the second-most reported cause of food-borne illness. 3 Agencies Involved in Food Safety USDA – US Department of Agriculture • regulates meat and poultry via Food Safety and Inspection Service (FSIS) • pressures food processors to initiate pressures food processors to initiate voluntary recall ”voluntary” recall FDA – Food and Drug Administration • regulates all foods except meat, poultry and alcohol • issues mandatory recalls and quarantines foods • regulates drugs and medical devices CDC – Center for Disease Control and Prevention • provides epidemiological expertise for outbreaks of foodborne illnesses • oversees PulseNet, a DNA fingerprinting clearinghouse that links geographically dispersed cases to a common outbreak EPA – Environmental Protection Agency • insecticides, fungicides, herbicides 4
9/18/2016 Hurdle Technology Food as Ecosystems Interaction of intrinsic and The food environment consists of: extrinsic factors determines which intrinsic factors-inherent to the food microbes grow in an ·pH environment ·water activity Hurdle technology ·nutrients uses multiple extrinsic factors-external to the food environmental factors ·temperature to inhibit microbial ·gaseous environment growth ·other bacteria important preservation Both can be manipulated to preserve food strategy Microbial Ecology Heterogeneity of Food the study of the interactions between Heterogeneous on a scale of the chemical,physical,and structural micrometers aspects of a niche and the composition Gradients of pH,oxygen,and nutrients of its specific microbial population Example-food poisoning outbreak "interactions"emphasize the dynamic associated with aerobic foods caused by complexity of food ecosytems "strict anaerobe"Clostridium botulinum Oxygen is driven out by cooking and diffuses in slowly so that most of the product remains anaerobic
9/18/2016 2 Hurdle Technology Interaction of intrinsic and extrinsic factors d i hi h determines which microbes grow in an environment Hurdle technology - uses multiple environmental factors to inhibit microbial growth - important preservation strategy 5 Food as Ecosystems The food environment consists of: intrinsic factors - inherent to the food • pH • water activity • nutrients extrinsic factors - external to the food • temperature • gaseous environment • other bacteria Both can be manipulated to preserve food 6 Microbial Ecology • the study of the interactions between th h i l h i l d t t l th e c hemical, p hysical, and s truc tural aspects of a niche and the composition of its specific microbial population • ”interactions” emphasize the dynamic complex complex ty of food ecosytems ity of food ecosytems 7 Heterogeneity of Food • Heterogeneous on a scale of micrometers • Gradients of pH, oxygen, and nutrients • Example - food poisoning outbreak associated with aerobic foods caused by ”strict anaerobe” Clostridium botulinum • Oxygen is driven out by cooking and diffuses in slowly so that most of the product remains anaerobic 8
9/18/2016 Plate Count Methods for Enumeration of Bacteria Plate Counts Classical estimation method for counting bacterial Used to quantify bacterial populations cell numbers ->250 CFU ml for liquids Assumes every cell forms one colony and every colony originates from one cell ->2,500 CFU ml for solids colony forming unit-CFU Food sample is homogenized 1:10 in buffer followed by serial dilutions Limitations: not all bacteria grow under the same conditions not all bacteria grow as single cells samples require dilution or enrichment Serial Dilution Spread Plate vs Pour Plate 只兴兴兴夏 9 mL water 9 mL water mL water 3
9/18/2016 3 Plate Count Methods for Enumeration of Bacteria • Classical estimation method for counting bacterial cell numbers • Assumes every cell forms one colony and every colony originates from one cell colony forming unit - CFU • Limitations: not all bacteria grow under the same conditions not all bacteria grow as single cells samples require dilution or enrichment 9 Plate Counts • Used to quantify bacterial populations - >250 CFU / ml for liquids - >2,500 CFU / ml for solids • Food sample is homogenized 1:10 in buffer followed by serial dilutions 10 Serial Dilution 11 Spread Plate vs Pour Plate 12
9/18/2016 Plate Count Simple Right? Used to be referred to as the total plate count This is not accurate,Why? 35 degrees C and aerobic growth were standard but do not detect anaerobes or bacteria growing at 15 degrees C -important pathogens may go undetected Variations of the standard aerobic plate count would increase specificity for certain types of bacteria 6.5×105 Countable plate 25-250 colonies Selective and Differential Media Mannitol Salt Agar Staphylococci Selective media Selective agent-7.5%salt inhibits growth of most includes a component that favors the microbes growth of the desired microbe Mannitol fermentation -may contain an ingredient that inhibits differentiates between pathogenic and non- the growth of competing microbes pathogenic staphylococci ·Differential media 5.aureus ferments mannitol turning phenol -a component is added to the media that red to yellow enables colonies of the target microbe 5.epidermidis cannot to look different to other colonies ferment mannitol and appears red
9/18/2016 4 Plate Count 6.5 x 105 Countable plate 25–250 colonies13 Simple Right? • Used to be referred to as the total plate count • This is not accurate Why? This is not accurate, Why? - 35 degrees C and aerobic growth were standard but do not detect anaerobes or bacteria growing at 15 degrees C - important pathogens may go undetected • Variations of the standard aerobic plate count would increase specificity for certain types of bacteria 14 Selective and Differential Media • Selective media - includes a component that favors the growth of the desired microbe - may contain an ingredient that inhibits the growth of competing microbes • Differential media - a component is added to the media that enables colonies of the target microbe to look different to other colonies 15 Mannitol Salt Agar • Staphylococci • Selective agent - 7.5% salt inhibits growth of most microbes • Mannitol fermentation differentiates between pathogenic and nonpathogenic staphylococci - S aureus S. aureus ferments mannitol turning phenol red to yellow - S. epidermidis cannot ferment mannitol and appears red 16
9/18/2016 Thiosulfate-Citrate-Bile salts-Sucrose Xylose Desoxycholate(XLD)Agar ·Vibrio ·Enteric species like Selective agents-sodium Salmonella and Shigella citrate,sodium thiosulfate, Selective agents-sodium oxgall wh desoxycholate inhibits gram- alka positive bacteria ￥ee nella from other enteric bacteria vibrios Xylose fermentation V.cholerae ferments sucrose turning pH stimulates Salmonella-black centers icator brom thymol blue Lysine decar ces alkaine ylation are due to hydrogen yellow pro sulfide production Other enteric bacterio Hydrogen sulfide and appears blue-green production ow or red 1.0ml 1.0ml Most Probable Number Methods rcem Selective agents are added to a broth medium ” mite of smple is rnferred to the 签P11111 1111计iiii训 tctate2re1o-fodseraldltiorsare 、 Turbid growth is recorded MPN is derived from a statistical chart 血i诚诚 After incub MPN/100 ml of water on a statistical table
9/18/2016 5 Thiosulfate-Citrate-Bile salts-Sucrose • Vibrio • Selective agents – sodium citrate, sodium thiosulfate, oxg p all which provide an alkaline pH of 8.6 and slows growth of other microbes • Sucrose fermentation differentiates between vibrios - V. cholerae ferments sucrose turning pH indicator brom thymol blue yellow - V. parahaemolyticus cannot ferment sucrose and appears blue-green 17 Xylose Desoxycholate (XLD) Agar • Enteric species like Salmonella and Shigella • Selective agents – sodium desoxy g cholate inhibits grampositive bacteria • Three reactions differentiate Salmonella from other enteric bacteria - Xylose fermentation stimulates Salmonella black centers - Lysine decarboxylation produces alkaline conditions which stimulate - Hydrogen sulfide production Salmonella - black centers are due to hydrogen sulfide production Other enteric bacteria remain acid yellow or red without a black center 18 Most Probable Number Methods • Used to estimate MPN of organisms in a sample when low numbers of bacteria are exp( ) ected (<30 CFU / ml) - Selective agents are added to a broth medium - One milliliter of sample is transferred to the broth - At least three 10-fold serial dilutions are inoculated - Turbid growth is recorded - MPN is derived from a statistical chart 19 After incubation, a 3-digit number is produced based on the number of positive tubes per set. The number corresponds to a MPN/100 ml of water on a statistical table. 20
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
9/18/2016 Biofilms Defined-a community of microbes associated with a surface and typically encased in an extracellular polymeric substance matrix or EPS d Biofilm:a Multi-Step Process Cells in a biofilm are more resistant to heat,chemicals,and sanitizers than are planktonic cells First recognized in 1684 by Antonie van Leeuwenhoek 1-Planktoric bocteria 2-Surfoce attachment 3-Microcolony Dental caries as the biofilm Vinegar as the antimicrobial + Planktonic cells showed no motility -After vinegar wash,plaque bacteria still motile 4-Macrecolory 5-Detachment vater filled voids heterogeneity.stobilized coordinates activities Dental plaque 10
9/18/2016 10 Biofilms Defined - a community of microbes associated with a surface and typically encased in an extracellular polymeric substance matrix or EPS biofilm on a piece of lettuce 37 Campylobacter cells attached to chicken juice 38 Biofilm: a Multi-Step Process - motility - random contacts - appendages - EPS matrix - nutrient dependent growth - cell-cell binding interactions 1-Planktonic bacteria 2-Surface attachment 3-Microcolony water filled voids, heterogeneity, stabilized by the EPS matrix, quorum sensing coordinates activities 4-Macrocolony 5-Detachment 39 Cells in a biofilm are more resistant to heat, chemicals, and sanitizers than are planktonic cells First recognized in 1684 by Antonie van Leeuwenhoek - Dental caries as the biofilm - Vinegar as the antimicrobial - Planktonic cells showed no motility - After vinegar wash, plaque bacteria still motile Dental plaque40