Midterm I Flashcards
What is an ecosystem?
Series of interactions between the environment & organisms that inhabit it
Impacted by generation & utilization of energy
Food ecosystems affect host ecosystems
What are the two types of food ecosystems compositions?
Intrinsic: factors inherent to the food - pH - Water activity - Nutrients Extrinsic: factors external to the food - Temperature - Gaseous atmosphere Can change over time as bacteria grow & produce bi-products Can be heterogenous on a micrometer scale
What are the 4 stages of bacterial growth & division?
- Lag phase
- log/exponential growth phase
- Stationary phase
- Death/ log decline phase
What is needed for bacterial growth & division?
Bacteria must have carbon (Anabolism)
- To build the array of chemical substances they are composed of
Bacteria must have a source of energy (catabolism)
Bacteria must have a source of electrons
- To use in energy conservation & biosynthetic reactions
Bacteria must have an electron acceptor
What are the ways bacteria gain carbon?
Autotrophic: carbon from Co2 through carbon fixation
Heterotrophic: carbon from organic compounds
Mixotrophic: autotrophic + heterotrophic
What are the sources of energy for bacteria?
Chemotrophic: energy is obtained from external chemical compounds
Phototrophic: energy is obtained from light
What are the sources of electrons?
LIthotrophic: reducing equivalents come from inorganic compounds
Organotrophic: reducing equivalents come from organic compounds
What are the different electron acceptors?
Aerobic organisms: use oxygen
Fermentation: use organic carbon (anaerobes)
- Other forms of fermentation = succinate fermentation or oxalate fermentation
- The other forms have very low energy yield
- Other anaerobic TEAs not common in food
Facultative anaerobes: can use oxygen or not
What are the non-taxonomic groupings of bacteria?
Lactic acid bacteria Proteolytic bacteria Lipolytic bacteria Thermophilic bacteria Thermoduric bacteria Psychrotrophic bacteria Cryophilic bacteria Halophilic bacteria Aciduring bacteria Osmophilic bacteria Spore formers Coliforms Fecal coliforms Enteric pathogens
If bacterial growth can occur, what will be the highest number of microbes?
Those that can grow optimally in the conditions provided (intrinsic & extrinsic)
Raw meat:
Carcasses after slaughter have 10^1-3cells/square inch (not many)
Meat is stored chilled
- Psychotropic organisms are main issue (Listeria)
Ground meat:
Can have 10^4-5cells/g
Lunch meat is heat processed & contains several chemicals to reduce bacteria & increase shelf life (50+ days)
- Heat processing can reduce microbes to 10^1-2cells/g
Stored anaerobically
- Psychotropic facultative anaerobes are main issue
Raw milk:
Contains 10^3cells/mL
- Higher in cows with mastitis
High in carbohydrates (lactose) & protein
- Great sources of carbon & energy for microbial growth
Refrigerated to preserve
- Psychotrophs still grow
Pasteurized milk:
Pasteurization kills all pathogens & most microbes, but milk quickly becomes contaminated from equipment, handling, & air
- Contains 10^4cells/mL
- Thermodurics survive pasteurization
Psychotrophs cause spoilage if milk is properly stored
Fish & shellfish:
Rich in protein & low in fat & carbs Microbes come from harvest environment - Based on water pollution & temperature - Can grow rapidly from Aw, pH, C & N availability Marine environment (high salt): - Halophiles are common Human waste polluted waters: - Several varied pathogens (including Vibrio cholerae)
Vegetables, fruit, & nuts:
High in carbs pH - Vegetables = 5.0-7.0 - Fruits = 4.5- Stored at room temp Consumed after minimal processing This all equals good growth for bacteria, yeasts, & mold Sources of contamination - Soil, water, air, animals, insects, birds, equipment, harvesters... Cell count: - Vegetables = 10^4-7cell/g - Fruits = 10^3-4cells/g
Canned food:
Hermetically sealed containers given heat treatment
pH:
- If below 4.6, mesophiles can germinate (produce toxins such as Clostridium)
- If above 4.6, mesophiles can’t grow but if cans are temperature abused, thermophilic spores grow & spoil food
Staphylococcus: can’t grow, but toxins can survive canning process
- If S. grows before canning, toxins survive & cause poisoning from consumption
What is the difference between coliforms and fecal coliforms?
Both: used as an index for sanitation
Fecal coliforms: also indicates presence of fecal contamination
What is the cell membrane responsible for?
Solute transport
Electron transport
Establishment of electrochemical gradients
ATP synthesis
Synthesis of lipids
Secretion of proteins
Secretion & uptake of intercellular signals
What is the cell wall (peptidoglycan) responsible for?
Turgor pressure (protects for cell bursting)
- Bacteria live in environments more dilute than the cytoplasm (causes net influx of water)
Defining feature of gram + vs -
What does the cell wall of gram + bacteria contain?
Peptidoglycan cell wall
- With teichoic acids that comprises 30-60% of dry cell weight (several functions)
- With fimbrae & pili found on some bacteria
Plasma membrane
What does the gram - cell wall of bacteria contain?
Outer lipid membrane (lipopolysaccharide)
- Consists of lipid A, core, & repeating oligosaccharide (O-antigen)
With fimbrae & pili found on most bacteria
Peptidoglycan cell wall
Plasma membrane
How does the gram stain work?
Apply crystal violet (purple dye) Apply iodine (mordant) (traps crystal violet in peptidoglycan) Alcohol wash (decolorization of gram neg) Apply safranin (counterstain) (for gram neg) *Differentiates cell wall by physical properties (thickness/amt of peptidoglycan)
What is a bacterial capsule made of?
Polysaccharide
Multiple roles
What are fimbrae & pili?
Protein fibrils extending from the cell surface
Found in most gram -
Found in some gram +
Function not always known
- Mediate attachment to other cells or surfaces
- Adhesive pili have adhesins (proteins)
- Some mediate genetic exchange
- Sex pili used for bacteria to attach to each other & transmit DNA
Where are sex pili found?
Different from other pili
Found in enteric bacteria
Ex. E. coli: coded by a conjugative transmissible plasmid (F plasmid)
What are bacterial flagella?
Not the same as eukaryotic flagellum in composition, structure, & mechanisms of action
Stiff, helical filament
Rotates like a propeller
What are serotypes?
Groups within a single species of microorganisms that share distinctive surface structures
What are the different physiological & physical states of food bacteria?
Vegetative Injured Viable but not culturable Communicating in structured communities Sporulated
What are injured bacterial cells?
Caused by sublethal stressors
Unable to grow on selective media
- Can be more sensitive to selective agents
- Can have increased nutritional requirements
Repair: process by which cells recover from injury
Why are injured bacteria important?
If injured cells appear dead, the thermal resistance will be underestimated & the true “kill” values will be low
Injured cells escape routine detection post-processing which leads to faster spoilage or a safety problem
The “selective agent” may be common food ingredient
How do bacterial cells repair from injury?
Requires de novo RNA & protein synthesis May seem an extended lag phase Membrane integrity is reestablished Adding a peroxide detox agent can repair damage from oxygen toxicity Temperature is critical
What are viable but not culturable (VBNC) bacteria?
A state in which cells can’t be cultured even if viable
Survival strategy for non-sporulating species
Morphologically different from a normal vegetative cell
- Rod-shaped cells shrink & become spherical
Most often induced by nutrient limitation
- Also by salt conc., exposure to chemicals, temp shifts
Can be repaired by temp shifts & gradual return of nutrients
Up to 99% of bacteria may be unculturable
How can viability of VBNC bacteria be tested?
Cytological methods
Substrate responsive metabolism
What are the cytological methods of VBNC testing?
Structural integrity of cytoplasmic membrane indicates life
- Fluorescent NA stains (red) are incubated cells
- Counterstain (green) stains cell wall
- If bacteria stains green, cell wall is intact; if bacteria stains red, the membrane is damaged
What are the substrate responsive metabolism methods of VBNC testing?
Cells can be incubated in a nutrient & an antibiotic that inhibits cell division
- LIve cells will elongate, but no divide
Specific method for Lm
What is Quorum Sensing?
Regualtes genes that would be useless to isolated cells but useful to large populations
Low pop = extracellular conc. of signal molecule is low & signal molecule diffuses away from cell
High pop = Extracellular conc increases & all signal molecules diffuse back into cells causing:
- phenotypic response
In gram neg bacteria: N-acyl homoserine lactones (AHLs) act as the signaling molecule
Waht are the two cellular communication types?
Signal transduction: two component regulatory systems where one protein spans the membrane, picks up a signal, & reacts based on a response regulator protein
Quorum sensing: systems built on autoinducers which diffuse through the cellular membrane to initiate a specific response when a certain pop density has been reached
Difference: quorum sensing trigger molecule diffuses into the cell but not in signal transduction
What is signal transduction?
A way to communicate between bacteria
Consist of a histidine kinase receptor & a response regulator
Signal molecule binds to N-terminus on outside of membrane spanning kinase
- Protein kinase transduces signal across membrane using conformation change that increases kinetic activity on the inside
- Phosphorylates a response regulator protein that changes:
- gene expression
- enzymatic activity
- flagellar rotation
- another phenotype
What does Pleiotrophic mean?
Occurs when one gene influences two or more seemingly unrelated phenotypic traits
What is a biofilm?
An aggregation of cells (can be multiple species) into a heterogeneous complex structure that attaches to a solid surface
Composed by microcolonies hta have defined boundaries allowing fluid channels to run through the biomatrix
uses cell-to-cell communication (most likely Quorum sensing) so undifferentiated cells don’t fill the channels (don’t clog the pores)
- Channels needed to bring in nutrients & carry out waste
More resistant to heat, chemicals, sanitizers
Waht are endospores?
Differentiated cells that are very resistant to heat & can’t be easily destroyed (even by chemicals)
Found in most soil samples
What are the genetics of bacteria?
Most ahve one chromosome (main genomic element) that contain most of the genes
Usually circular chromosomes
Plastic (combinations of genes present different in each individual)
What are the different types of genomes?
Core genome: genes shared by all members of a certain group (species)
Pangenome: genes present in any member of a certain group
Accessory genome: genes present in a subset of members of a certain group
What are the mobile genetic elements of bacteria?
Bacteriophage
Plasmids
Pathogenicity Islands
Transposons
What is lysogenic conversion?
A prophage changing a non-pathogenic bacteria to a pathogen
What are defective prophages?
Bacteriophage that can’t form infective phage due to loss of essential genes
DNA elements of bacteria strains are suspected to be defective prophages
What is a plasmid?
Circular molecules of dsDNA which are self replicating
- Carry genes which encode for products that can benefit the bacteria under certain circumstances (but aren’t essential)
Can be few thousand to few hundred-thousands bp
Each cell can have multiple plasmids
Passed horizontally & vertically
What are pathogenicity islands?
Large DNA elements integrated into bacterial chromosomes containing genes for special properties on the bacteria that carry them
- Bacteria carrying them can occupy special ecological niches
Not carried by all strains & not integrated plasmids or prophages
Can move between bacteria
Have an integrase gene allowing them to integrate into a region of the host DNA
Have a repeated sequence at the ends of their sequences, involved in integration
What are transposons?
DNA elements able to hop between places in chromosomal DNA
Encodes its own transposaes, so it carries the ability to hop
Types:
- Insertion Sequence (IS) elements: single transposon
- Composite transposons: multiple acting as vehicle
What are insertion sequence (IS) elements?
Smallest transposons, only encoding the transposase
Can interrupt gene function by inserting in middle of gene
Usually have inverted repeat at either end
What are composite transposons?
When two IS elements bracket other genes & carry those genes with them when they move
What are the categories of foodborne diseases?
Intoxication
Infection
Toxicoinfection
What is intoxication?
Occurs after ingestion of a bacterial or mould toxin
Toxin must be present in active form
Once toxin is producing viability, microbe isn’t needed
Toxins are better able to withstand processing better than the organism
What is infection?
Caused by viable bacterial cells or viruses
Viable cells are able to multiply in the digestive tract
What are toxicoinfections?
Caused by ingestion of large numbe rof viable cells of some pathogenic bacteria
Cells either sporulate, colonize, or die, releasing the toxins
What are the human factors for illness?
Infants, old, sick, pregnant, immunocompromised more susceptible
Due to how much eaten, & how many organisms in that serving
What are the basic sources of pathogen contamination?
Processed food can gain microbes from processing environment while nonprocessed, healthy, fresh has sterile internal tissues
Natural:
- Surface of cut fruit, damaged tissues, skin, feathers, GI tract
External
- Air, soil, sewage, water, feed, human, ingredients, equipment, packages, insects
Sources of pathogen contamination in plants:
microbes on surface
present from environment (ex. raw sewage in soil)
Reduce bacterial load via:
- Treated sewage
- Washing with clean water
- Storage at low temp (if viruses there before frozen, can survive)
- Workers practice good hygiene
Sources of pathogen contamination in animals:
Carry microbes in their body
Food producing animals can carry human pathogens without showing symptoms
Meat can be contaminated from GI tract during slaughter
Dairy contamination due to fecal material on udder surafces, eggs
Reduce bacterial load via:
- Effective husbandry
- Avoid overcrowding
- Use clean food/water
- Test flocks for presence of pathogens before slaughter
- Careful removal of GI tract
- Harvest fish from clean water
What are sources of pathogen contamination from the air?
Microbes in dust & moisture droplets Mostly bacterial or mold spores, yeast, some gram + bacteria Air in highly contaminated areas can contain pathogens Reduce by: - Removing potential sources - Controlling dust particles - Using positive air pressure - Reducing humidity - Installing UV lights
What are sources of pathogen contamination in sewage?
Used as fertilizer
Particularly enteropathogenic bacteria
What are human sources of pathogenic contamination?
Carrying environmental contamination into processing plants
Sick workers
What are sources of pathogenic contamination from water?
Contaminated water
Seafood washed or maintained in contaminated seawater
use presh potable water
What are sources of pathogenic contamination from equipment?
Contaminated by any source
It then spreads the contamination to future batches
What are the factors that influence microbial growth?
Temperature
Redox potential
pH
Water activity
How does temperature influence microbial growth?
most improtant environmental factor
For every 10C rise in temperature, catalytic rate of an enzyme doubles (vice versa)
- Microbial growth is accomplished by enzymatic rxns
There is a maximal possible rate for enzymatic rxns (optimal temp)
- If it goes too high, protein denaturation & lysis occurs
- If it goes too low, membrane gelling occurs & transport processes are too slow for growth
Psychotrophs: can grow at refrigerated temps, but grow faster at room temp
How does the redox potential effect microbial growth?
Redox potential is the measurement of the potential energy difference in a system generated by a coupled rxn in which one substance is oxidized & a second is reduced (primary energy generator) Reducing agent is electron donor Oxygen in best electron acceptor Influenced by: - Chemical comp - Processing treatment - Storage conditions Fresh food spoils when cellular respiration stops & O diffuses in
What are the different types of redox potentials
Aerobes: need free O2
Anaerobes: only transfer electrons via fermentation (die in O2)
- Lack superoxide dismutase needed for O2)
Facultative anaerobes: can use free O2 or bound O2
Microaerophiles: grow better with low O2
Aerotolerant anaerobe: tolerate O2, but don’t grow in it
What are the types of atlering atmosphere packaging to preserve food?
Controlled atmosphere packaging (CAP)
Modified atmosphere packaging (MAP)
Vacuum packaging (VP)
What is controlled atmosphere packaging?
Atmosphere in storage facility is altered & gases are controlled
Expensive
Used for long-term storage for fruits & vegetables
What is modified atmosphere packaging (MAP)?
Food is enclosed in a high gas barrier packaging material
Air is removed, then gases are added & package is hermetically sealed
Stops enzymatic & respiratory activities of fresh foods
- Anaerobes can grow
Extend life of products (non-meat)
What is vacuum packaging (VP)?
Air is removed from package & package is hermetically sealed
Extend shelf life of meat products
- No gases added so anaerobes can’t grow
Why is pH important for microbial growth?
Bacteria = pH 2-3 - Gram + grow at lower pH Acidophiles = 1-4.5 Neutrophiles = 5.5-8.5 Akaliphiles = 7.5-11.5 (rare) Weak acids (have lower dissociation rate) are more problematic for bacteria
Why are weak acids bad for bacteria?
Undissociated molecules rae lipophilic (generate H+ in cytoplasm)
Causes inc in cellualr pH, destroying the proton gradient, cell can’t generate energy
What is acetic acid?
Vinegar, pickling
Effective against bacteria (not yeast0
Denatures proteins, neutralized electrochemical gradient of PM
Used as a carcass wash
What is propionic acid?
Used as salts in bread, cheeses, jams
Effective agains molds & bacteria
Acidifies cytoplasm, destabilize PM proton gradients
What is lactic acid?
Used as acid or sodium Found in carbonated drinks, salad dressing, pickling Effective against bacteria Neutralizes proton gradient Low effectiveness
What is citric acid?
Acid in drinks, jams, paking products, sauces…
Antibacterial activity is unknown
What is sorbic acid?
used as acid, salts, or calcium in drinks, fruits, veggies, ice cream, mayo…
Effective against models & yeasts
Inhibits function of enzymes, synthesise of cell wall, protein, RNA, DNA
What is benzoic acid?
Used as acid or salt in drink, fruits/veggies, ice cream, mayo…
Inhibits action of enzymes for oxidative phosphorylation
Why is water activity important for microbial growth?
0 = dry; 1 = pure water
Water usually moves into cells
Most bacteria can’t live in low aw
- Most food processing technologies use high solute conc (low aw)
What are the ways of removing water from food?
Dehydration
Crystallization
Adding solutes
What is the hurdle concept?
Applying multiple methods of controlling microbial growth at lower levels
- Reduces growth, prevents growth, or kills microorganisms
Most effective when combining two stressors that act by different mechanisms
What are additive hurdle effects?
Need an equal amount of both (fall on the line)
What are synergistic hurdle effects?
need a lower number of both (fall below the line)
What are antagonistic hurdle effects?
Need more of both (fall above the line)
What are the general characteristics of foodborne intoxication?
Toxin is produced by a pathogen while growing in the food
Toxin can be heat liable or stable
Ingestion of a food containing active toxin, not cells, is necessary for poisoning
Symptoms occur quickly (as early as 30 mins)
Febrile symptoms are not present (fever)
Staphylococcus aureus general
Before 1980s: common source of outbreaks
- Declined due to better refrigeration, improved sanitation
Gram positive
Cocci
Single cell or grape like clusters
Nonmotile, noncapsular
Non-spore forming, sensitive to heat treatment
Poor competitors
Common infections, but foodborne cause is usually intoxication
- Food treatment usually removes bacteria, but not toxin
Same distribution between mammals, reptiles, & avians
All S.a enterotoxins are also exotoxins
Infectious dose = 10^5cells/g; 1ng/g of toxin
S. aureus grows best at:
Facultative anaerobes ( but grow rapidly with O2)
Mesophiles (can grow 7-48C, good at 20-37C, best at 37-40C)
- Only takes 3 hrs for sufficient toxin production
- Toxin is heat stable, can’t be destroyed by boiling or canning
Low Aw (.86), low pH (4.8)
High salt & sugar conditions
What is Multilocus sequencing typing (MLST)?
Common to define the sequence type or allelic profile based on the 7 housekeeping genes found in all strains of S. aureus
- Extract the sequence of the 7 genes from the whole genome sequence
If at least 5/7 alleles are the same, the group can be called a clonal complex (CC)
- Gene transfer between clonal complexes is not possible due to an advanced restriction modification system
What clonal complexes are common in human colonization?
CC1 CC5 CC8 CC30 CC45
What types of genes are found in S. aureus?
Accessory genome: made of multiple mobile genetic elements (transferred horizontally) - Consist of 20% of the S.a genome Prophage genomes: 1-4 found in Sa genome - Have enterotoxins Enterotoxins are needed for intoxication
What are enterotoxins?
A toxin that acts on the GI tract
Found on mobile genetic elements
Expressed by:
- made throughout most of the exponential growth phase
- Can be inhibited by presence of glucose
- May be involved in quorum-sensing in Sa
What are endotoxins?
A toxin taht is structurally associated with the bacterial cell (LPS)
Only on gram - bacteria
What are exotoxins?
A toxin that is usually secreted into the extracellular fluid but can be intracellular or injected directly into eukaryotic cells
What are exfoliative toxins?
Dermolytic exotoxins that cause impetigo or scalded skin syndrome
Produced exclusively by S.a
What is Methicillin-Resistant Sa (MRSA)?
Carry mecA gene
Causative agent of soft tissue & skin infections
Found in .4-12% of retail meat
- Carry the enterotoxin gene
Could be source of extraintestinal infection
What are the environmental sources of Sa?
People
- Found on skin, inside of nose (main site)
- Spread by direct contact skin fragments or respiratory droplets
- Most contamination of food is traced to humans during food preparation
Animals
- Causes bovine mastitis + others
- Toxin can survive pasteurization
Lives intracellularly
Can survive extreme environments
Can acquire resistance to heavy metals & antibiotics
What are contributing factors to Sa food contamination
Inadequate refrigeration Preparing food too soon Poor personal hygiene Inadequate cooking Prolonged use of warming plates
What are the characterstics of Sa disease?
Average incubation is 4.4 hrs (between 30 mins-10hrs)
Self-limiting
Recover in 24-48hrs
Outbreaks are seasonal
No fever, death is rare
Vomiting, nausea, diarrhea, abdominal pain
- Stimulates vagus nerve in stomach
How can Sa be prevented?
Hand washing, glove wearing, no active skin infections on food prep workers
Adequate refrigeration or continued heating of prepared food
- reheating doesn’t help
Preservatives can stop growth of organism
How many healthy individuals have enterotoxin containing Sa in their gut?
50%
What are the four illness causes of Clostridium botulinum?
Intoxication: ingestion (or injection) of toxin
Infant botulism: ingestion & interstinal proliferation of organism, internal production of the toxin (an infection)
Wound botulism: deep wound results in anaerobic environment
Bioterrorism: deliberate delivery of toxin to large pop.
Why are all strains of Cb toxic?
Non-toxic variant strains belong to different species
What is the toxin of Cb?
Botulinum neurotoxin (BoNT)
An A-B toxin
Inactive after production, activating once in the stomach
Absorbed by the gut and spread by blood to peripheral nerves
- Acts as protease, cleaves SNARE proteins, causing flaccid paralysis
- Did they evolve from proteases?
What is the difference between proteolytic Cb & nonproteolytic Cb?
Nonproteolytic: host’s stomach acid cleave the toxin, activating it
Proteolytic: bacteria produces a protease that activates the neurotoxin
What are SNARE proteins?
Connect axon terminal of the neuron with muscle tissue, causing contraction
What are the different types of BoNT?
A-G
Four groups based on physiologicla differences (don’t correlate to the type of toxin)
Group 1: Cb A & proteolytic strains of B & F (in humans)
Group 2: Cb E & nonproteolytic strains of B & F (in humans)
Group 3: Cb C & D (in animals)
Group 4: Cb G
How is Cb toxin tested?
- Prepare food, feces, serum, or gastric contents
- Dispense filtrate into 3 tubes
- No treatment tube
- Tube boiled or 10 mins
- Trypsin added to tube (trypsin is an enzymes that aids in digestion) - Inject 2 mice with each tube (6 total)
- Test positive if 2/2 mice have pinched waist, labored breathing, or die
- Test for which toxin with antiserums from the no treatment tube mice
The Cb genome:
1 circular chromosome, between 3.6-4.1 Mb
- G+C content of 27-28%
Contain plasmids that vary in size
Toxin can be carried on chromosome or plasmid
- Matters bc if on plasmid, can move between bacteria (can potentially be taken in by C. difficile)
Why is it thought that all bont genes came from the same ancestor?
Similarity of the different BoNTs
Ability of different species to produce BoNT
Some strains contain combinations of bont genes
* This also shows that bont gene moves around (on plasmid)
What are the environmental factors of Cb?
Found in soil
Secrete hydrolytic enzymes then transport the degraded products into the cell to survive
- Over 300 transport genes
Lack genes needed for amino acid synthesis
Outcompeted by other organisms in nature
- Grow in canned goods
General characteristics of Cb:
Strict anaerobe
Has fermentative metabolism
- prefer proteinous compounds for fermentation
Use amino acids as sole source of C & N
Absorb nutrients by secreting hydrolytic enzymes
Have spores
Best conditions for Cb:
Sensitive to low pH (can’t germinate below 4.6)
Sensitive to high salt (can’t germinate over 5.5%)
Spores resistant to heat (above 115C)
- But toxin isn’t
Best growth in canned goods
- No competition
How to remove Cb toxins from canned goods:
Heat liable: boil for 10 mins
Cans given low pH or high salt content
What are the best foods for Cb?
Low acid fruits & vegetables (Type A & B) Fermented fish (Type E)
Why do waterfowl Cb outbreaks occur?
Ingest toxins, not cells Due - Human sewage in water - Inc environmental temp - Inc number of rotting aquatic animal carcasses
Infant botulism:
90% of cases in infants below 6mth - No gI flora Symptoms: - Not eating - Loss of head control - Visual problems - Dry mouth - Generalized weakness - Constipation - Paralysis - Respiratory failure No fever Recover Honey is an important reservoir
Inadvertant/wound botulism:
Depends on concentration fo toxin
Diluted toxin is used as a therapeutic cure to chronic muscle tension disorder, or cosmetics (wrinkle removal)
Anaerobic growth can occur in deep wounds
General characteristics of Salmonella enterica:
Gram neg Non-sporulating Facultative anaerobe Motile Rods
What are the optimum conditions for growth of S. enterica?
Mesophilic (grow between 5-54C, optimum between 35-37C)
Sensitive to low pH (grow between 4.5-9.5)
Doesn’t multiply below Aw .94
Heat liable (easily killed by pasteurization)
- Lower Aw = higher heat resistance
- Solutes influence heat resistance
Can survive in frozen or dried states
What is the only species/subspecies of Salmonella to cause illness in humans?
Salmonella enterica enterica
Has over 2,000 serovars based on somatic, flagellar, & capsular antigens
- Serovars Typhimurium & Enteritidis are most commonly associated with foodborne illness
How does S. enterica serotyping occur?
By reactions with antiserums Based on whole genome sequence - More common Serovars named after geographic origin Serovars are clonal
Serotype Thyphimurium:
Serotype of S. enterica enterica
Found in many food animals and pets
Serotype Enteritidis:
Serotype of S. enterica enterica
Infects ovaries of poultry
- Transmitted through eggs & undercooked poultry
Salmonella enterica vehicles:
Food animals & pets (Typhimurium) Poultry & eggs (Enteritidis) - Can only stop by highly cooking eggs (gross) Fish & shellfish - From polluted aquaculture areas Fruits & vegetables
S. enterica symptoms:
Begin after 8-72 hrs Self-limiting Non-bloody diarrhea Abdominal pain Resolve in 5 days - Only need supportive therapy
What happens to immunocompromised people that get S. enterica?
non-typhoidal salmonella infection can manifest as an invasive disease
Characterised by:
- High bacteremia
- High mortality rate
How does S. enterica infect?
Exploits phagocytic intestinal cells
- M cells
- Dendritic cells
Forces uptake into non-phagocytic epithelial cells
- Intestinal cells (attach using fimbriae)
Invade other cells from basolateral side
- Stays localized normally (remain in lumen)
Host’s inflammatory response causes gastroenteritis
In lumen, gain selective advantage over resident microbiota due to host’s inflammatory response
Invading supopulation is eventually cleared by host neutrophils
What are Salmonella Pathogenicity Islands (SPIs)?
Contain virulence factors that are required for infection
Not all strains have same SPIs
What happens during the ingestion & adhesion of S. enterica?
After ingestion, S. activates acid tolerance response (ATR)
- To maintain intracellular pH in acidic stomach
Strains express functional flagella
- Inc. chance of encountering the epithelium
Adheres to intestinal cells using SPI-3 & 4 adhesins
- SPI-3 encodes MisL, binding to fibronectin on epithelial cells
- SPI-3 encodes MgtCB, involved in intramacrophage survival
- SPI-4 contain 6 ORFs, encoding SiiE, which mediates attachment
What happens during the invasion of S. enterica?
SPI-1 encodes a T3SS, delivering multiple virulence effector proteins into host cell
- Effectors force uptake of pathogen & change host cell signaling pathways (specifically inflammatory response pathways)
- Induce rearrangement of host cell actin cytoskeleton, causing membrane ruffles
- Ruffles engulf the pathogen into Salmonella-containing vacuoles (SCVs)
- SCVs go near Golgi apparatus to intercept vesicles & get nutrients
- *In immunocompromised individuals, SCV can migrate to basolateral side & move into the blood
SPI-2 used for intracellular replication in epithelials & macrophages while inside the SCVs
Host response & inflammation due to invasion cause diarrhea
What is a type 3 secretion system?
Protein appendage found in gram - bacteria
Used as a sensory probe to detect presence of eukaryotic cells
Can inject effector proteins in host cells
- Effectors exert efforts to help pathogen survive & escape immune response
What is pyroptosis?
A highly inflammatory form of programmed cell death
Occurs mostly from infected intracellular pathogens
Form of antimicrobial response
AvrA gene stops this in S. enterica
What genes does SPI-1 use?
AvrA: inhibit apoptosis, inhibit proptosis
SipA: stablize & localize actin filaments during invasion
SipB: Adhesion to epithelial cells
SipC: Adhesion to epithelial cells
SptP: disruption of actin cytoskeleton
What are the genes of SPI-2?
SsaB: Disrupt Golgi apparatus & lysosomes, inhibit SCV-lysosome fusion
SsaE, F, & G and SscA & B: chaperone
SseF: SCV perinuclear migration
SseG: SCV perinuclear migration
How does inflammation occur in S. enterica?
SPI-5 encodes SopE, SopE2, & SigD
- Leads to cytoskeletal remodeling & induction of proinflammatory response
Results in GI dysbiosis
Inflammation favors Salmonella growth since respiratory electron acceptors are produced
Neutrophils form new electron acceptor: tetrathionate
- SPI-2 encodes ttr operon, allowing Salmonella to use tetrathionate as TEA
How does diarrhea occur in S. enterica?
Water is blocked from entering epithelial layer or transported from epithelium to lumen
- Salmonella can modify tight junctions
- Salmonella regulate actin filament dynamics to induce membrane ruffling
Uses SipA, SopB, SopE, & SopE2 to disrupt tight junctions
Why are salmonella strains multidrug resistant?
Antibiotics are used in feed for animals & birds
- Up to 8-9 antibiotic resistance
What is the difference between typhoid & non-typhoid S. enterica?
Typhoid:
- serovars typhi, paratyphi
- found in humans only
- systemic (uses immune cells to get to lymphatic system, where they hide)
- causes enteric fever (fever, abdominal pain, transient, diarrhea, rash) after 7-28 days
- More serious disease; need antibiotics (but many are resistant)
Non-typhoid:
- serovars typhimuirum, enteriditis
- broad-range of hosts
- causes gastoenteritis (abdominal pain, vomiting, inflammatory diarrhea)
S. enterica has what antigens?
O antigen
H antigen
Listeria monocytogenes general:
Listeria genus only has 2/17 pathogenic species - L. monocytogenes - L. ivanovii (cows only) Gram positive Psychotrophic Facultative anaerobic Nonsporulating Motile Small rod Flagella Recent pathogen (determined from serotype lineages)
Serotypes of Lm:
There are 13 - 4 lineages - 63 CCs - Lineage 1Lm is most virulent (tropism for NS & placenta) 12 CCs account for 80%b of infections 95% of outbreaks are caused by: - 1/2a - 1/2b - 4b
Optimal growth of Lm:
Motile only at 20-30C Can grow from 4-45C - Outcompetes bacteria in fridge pH 4.4 or higher Salt content up to 10% - Heat resistance increases as salt content decreases Sensitive to pasteurization - Can survive if in white blood cells Fridge slows growth, doesn't stop it Found in moist environments
How do Listeria survive in processing plants?
Last for years once established due to:
- Biofilms
- Found in moist areas
What are the sources of Lm?
Raw milk (pasteurization reduces Lm cells)
- Survives during raw cheese manufacturing from low temp & high salt
- Second highest risk
Cold meats (ready-to-eat meats)
- Highest risk
Infection of Lm:
Uncommon
Mortality is high: 20-30%
Pathway:
- Intestine -> lymph node -> bloostream -> liver & spleen -> brain & placenta
- LIver Kupffer cells phagocytose Lm cells
- Can cross the placental barrier & blood brain barrier
Lm is an intracellular pathogen
- Taken by non-phagocytic or phagocytic cells
What are the symptoms of Lm?
Gastroenteritis mostly At risk people: - Sepsis - Meningitis - Fetus infection
Epithelial cell infeciton of Lm:
Enters through receptor-mediated endocytosis
- Requires Internalin A (lnlA) & Internalin B (lnlB) to bind to cell receptors E-cadherin & Met, respectively
Inside, Lm uses listeriolysin O (LLO) & phospholipases A & B (PlcA & PlcB) for vacuole rupture
- Survives & divides in cytosol
Induces changes
- LLO changes mitochondrial morphology & function, increases ER stress, causes permeabilization of cell
Polymerizes actin to move between cells
Goblet cells infected by Lm:
Goblet cells form goblet cell associated antigen passages to deliver luminal substances to underlying lamina propria (LP) antigen-presenting cells (induces adaptive immune response)
lnlA binds to E-cadherin, leading to receptor clustering
- Phosphorylation & ubiquitylation of E-cadherin causes uptake of the bacteria
Lm remains in vacuole & rapidly moves through cell
Macrophages infected by Lm:
Phagocytize Lm cells
Inside, Lm can replicate in spacious Listeria containing phagosoems (SLAPs)
- Lm expresses LLO, interfering with pH gradient required for acidification of phagosome
Trophoblast infection by Lm:
Trophoblasts are the cells forming the placenta
lnlB binds Met & induces phosphorylation & ubiquitylation and receptor-mediated endocytosis
- Binding lnlB to complement component (C1QBP) causes receptor clustering & speeds uptake
Over 200 unique virulence factors upregulated for this
What does listeriolysin S (LLS) do?
Restricts growth of common commensals in teh intestine to promote Lm growth
How does Lm interact with the immune system?
Peyer’s patches are small masses of lymphatic tissue responsible for immune surveillance in small intestine
- They are follicles that contain B cells surrounded by T cells (may also contain M cells)
IgA is also secreted, interrupting the ability of Lm to bind to cells
How are virulence genes of Lm regulated?
PrfA promoter
- Regulates genes located on Listeria PIs and three other regulated loci
- Regulates Lm virulon (main virulence gene)
- Includes genes necessary for invasion & vacuole escape
PrfA is under a positive feed-back loop to regulate expression under infection conditions
Low temperature causes weak transcription of PrfA
- Not translated
In host, temp increases, leading to rapid translation
Campylobacter general:
Gram negative
Curved s-shaped rod
Mitle with single polar flagellum at one or both ends
Microaerophilic (survives in reduced O2 environments)
Peak in the summer
How is Campylobacter transmitted to humans?
Undercooked or contaminated food
Contact with animals
Tap, bore, & pond water
Person-to-person (fecal-oral or fomite transmission)
What is the most commone Campylobacter species?
C. jejuni
Reservoir: poultry, pigs, bulls, dogs, cats, birds, minks, rabbits, insects
Diseases: gastroenteritis, speticemia, meningitis, abortion, Guillain Barre Syndrome
What are the clinical manifestations of Campylobacter?
Gastroenteritis (most common) Brain Abscesses (rare) Periodontiis (rare) Guillain-Barre syndrome (GBS) & Miller Fisher Syndrome Inflammatory Bowel Diseases (IBD) Esophageal Diseases Bacteremia/Septicemia
What is gastroenteritis in Campylobacter?
Most common 90% of cases cause by C. jejuni (10% caused by C. coli) - Clinically indistinguishable Occurs 24-72 hrs after infection Symptoms: - Diarrhea (loose or bloody) - Fever - Chillds - Headache - Abdominal chills
What are brain abscesses in Campylobacter?
Rare Causes nonspecific febrile illness - Fever - Confusion - Unsteadiness - Painful legs - Diarrhea - Fecal incontinence Patient develops bilateral lower-leg erythema & lower-back pain Death
What is periodontitis in Campylobacter?
Rare
Found in C. rectus, C. curvus, C. showae
Reach bottom of periodontal pockets with flagellum
- Major pathogenic factor
What is Guillain-Barre syndrome (GBS) & Miller Fisher Syndrome in Campylobacter?
Miller Fisher Syndrome is a variant of GBS
Extraintestinal infection
Symptoms:
- Motor & sensory deficits in lower extremities (spread)
- First nerve groups affected by paralysis are in head
- Other forms of paralysis begin in the legs
From C. jejuni
- Develop antibodies against certain antigens in LPS
What are inflammatory bowel diseases in Campylobacter?
Chronic inflammatory conditions of GI tract
- Includes Crohn’s disease (CD) & ulcerative colitis
Have lesions that occur at different sites along GI track
Dysregualted immune response leads to chronic inflammation
Factors:
- GI dysbiosis
- Host genetic factors
- Disruption of GI epithelium
What are esophageal diseases in Campylobacter?
Includes: - Gastroesophageal reflux disease (GERD) - Barrett's esophagus (BE) - Esophageal adenocarcinoma Caused by C. concisus - Due to strong immune response in esophagus
What is bacteremia/speticemia in Campylobacter?
Bacteremia: presence of viable bacterial cells in bloodstream
Septicemia: blood poisoning by bacteria
Caused by C. jejuni, C. coli, & C. fetus mostly
Most cases occur in immunocompromised
10-15% die within 30 days
Epidemiology of Campylobacter:
Thought that exposure early in life leads to life long protection
- Although adults can have asymptomatic Campylobacter
In developed countries, most common from:
- Poultry
- Water
In developing countries:
- Endemic
Pathogenesis of Campylobacter jejuni:
Uses flagella to swim towards mucosa
Adheres to host cell with many known & unknown factors
- CadF: fibronectin binding protein
- PEB1: periplasmic binding protein
- J1pA: surface exposed lipoprotein
* Correlation between how it adheres & how virulent it is
Export of bacteria caused by main virulence factor:
- CiaB
- Strains with low CiaB are less invasive
T4SS: found on plasmid pVir; causes bloody diarrhea
What are the toxins of Campylobacter?
Cytoleathal distending toxin (CDT)
- A tripartite complex toxin
- Coded for by cdtA, cdtB, & cdtC
- cdtA & C bind to cell membrane & facilitate entry of cdtB
- cdtB is transported by the Golgi to the ER to the nucleus where is cause DNA damage & stops mitosis
Causes fragmentation of the nucleus, cellular distension, & cell death
- Increases permeability of epithelium (results in diarrhea)
E. coli general:
Gram neg Motile Nonsporulating Rod-shaped Facultative anaerobe
Nonpathogenic vs pathogenic E. coli:
E. coli is part of normal microbial population in intestinal track
Most strain are non-pathogenic
Found in large intestine
- Used as an indicator organism for fecal contamination
What are the pathotypes of E. coli?
Based on ability to produce toxins, to adhere to epithelial cells, to invade epithelial cells 8 pathotypes - Adherent Invasive E. coli (AIEC) - Enterotoxigenic E. coli (ETEC) - Enteroaggregative E. coli (EAEC) - Shiga toxin producing E. coli (STEC) - Enteroinvasive E. coli (EIEC/shigella) - Diffusely Adherent E. coli (DAEC) - Enteropathogenic E. coli (EPEC) - Uropathogenic E. coli (UPEC)
What are the serotypes of E. coli?
O antigen (defines serogroup) - Can fall into one or multiple pathotypes H antigen (defines serotype) K antigen
What are the E. coli strain groups?
Based on phylogeny: A B1 B2 D E * EIEC forms a different one
What are basics to the E. coli virulence factors?
Very plastic genome
- Can bary by a million bp (unique)
- 1,700 genes in core genome, 16,400 genes in pangenome
- Prophages, PIs, & plasmids regularly move in & out of genome
Most virulence factors are from mobile genetic elements
Phenotype & disease caused is determine dby which virulence factors have been picked up
* Important virulence factors in humans can also be important for environmental survival
Enteropathogenic E. coli (EPEC)
Can form lesions on intestianl epithelial cells
Carries locus of enterocyte effacement (LEE) genes)
- Each of the four lineages of EPEc have acquired LEE differently
Mainly affects children in developing countries
- Serious risk of death
Transmitted by fecal-oral route
Humans are the only carrier
Prevention: clean fresh water
No enterotoxins generally
Causes diarrhea (how is unknown)
- Efface microvilli (can increase water in small intestine)
- Tight junctions may be disrupted
Shiga toxin producing E. coli (STEC)
Presence of Shiga toxin 1 or 2 (stx1/2) - Acquired from bacteriophages - Carried on lysogenic phages which can become lytic from stress (antibiotics not recommended) Causes: - Mild to blood diarrhea - Haemolytic uremic syndrome (destruction of RBCs, cloggin the kindeys) Has LEE genes & forms lesions on microvilli - STEC without LEE is EHEC O157:H7 is most common serotype Transmits through fecal oral route - Infectious dose is low - Contaminate food & water Cattle are main reservoir Cause large outbreaks
Enteroinvasive E. coli (EIEC)/shigella
Facultative intracellular pathogens Shigella is historically a genus Transmit via fecal oral route - From contaminated water or food Invasive & divide in inside intestinal cells Leads to tissue lesions Shigella has increased virulence compared to EIEC - Also has Stx toxins Symptoms: - Mild diarrhea (with blood & mucus later) - Fatigue - Malaise - Fever - Anorexia - Abdominal cramps - HUS (in this case, antibiotics work) Self limiting (more serious in developing world)
Cell invasion of EIEC/Shigella:
- Cells penetrate epithelial barrier
- Induce macrophage cell death
- Invade intestinal epithelial cells
- Engage in intra & intercellular movement
- Degrade epithelial integrity (destabilize tight junctions)
- Cause cell death
Enteroaggregative E. coli (EAEC)
Worldwide, most common diarrhea cause (even in NA)
Endemic in many places
- Causes persistent diarrhea in children/immunocompromised here
Transmit by fecal oral route
- Through food & water (salads, desserts, salsa, livestock)
Infect by:
- Cells adhere to intestinal mucosa
- Cells produce enterotoxins & cytotoxins
- Mucosa becomes inflamed due to toxins & immune system
* virulence factors needed for this found on pAA plasmid
Strains cause variability in virulence factors & toxins
Enterotoxigenic E. coli (ETEC)
Major cause of traveler’s diarrhea (30% in some continents)
Endemic in underdeveloped countries
- Has symptomatic & asymptomatic carriers
- High mortality in children (325,000/year)
Can produce heat-liable or heat-stable enterotoxin
- Induce chloride & fluid secretion into the lumen (cause diarrhea)
* Heat-stable toxin is defining characteristic, only E. coli with it
No virulence factors in core genome
Acquire plasmid-borne toxins & virulence factors (driving force behind pathogenesis)
- Different ones in different geographic regions
Infects pigs
Transmit by fecal oral route
- Contaminated food & water
Symptoms of ETEC:
Mild to severe diarrhea (non-bloody) Dehydration Headaches Fever Abdominal cramping Nausea Vomiting Rapid onset (after 5 hrs to 1-2days) Lasts 3-5days
Diffusely Adherent E. coli (DAEC)
Adheres over entire surface of epithelial cell
- Induces actin rearrangement & destroyed microvilli
- TIght-junctions become leaky
Unknown how transmitted, reservoir, or prevalence
Virulence factor:
- Afa/Dr family of adhesin (allow diffuse binding)
Most strains are antibiotic resistant
- Treatment is rehydration
Adherent Invasive E. coli (AIEC)
Causative agent of Crohn’s disease (causes IBD)
- 30% of patients with Crohn’s have AIEC
No common virulence factors
Unknown proinflammatory & invasive phenotype
Most related to UPEC
- UPEC doesn’t have same adhesion, invasion, & intracellular replication traits
Uropathogenic E. coli (UPEC)
Most related to AIEC
- Different adhesion, invasion, & intracellular replication traits
