Exam 1

The good, the bad, and the ugly

Good = ferment (yummy)
Bad = make you sick :(
Ugly = spoilage (yucky)

Pasteur is the father of microbio

Types of microbes

Microbes are too small to see as individuals by the unaided eye

Bacteria: prokaryotes, no membrane-bound organelles, single cell

Fungi: single, multi-cellular, or filamentous; eukaryotic → have membrane-bound nucleus and organelles

Viruses: infectious agent consisting of nucleic acids wrapped in a protein coat, not living

Parasites: organism that lives off of host organism to hosts expense

Parasite vs pathogen

Pathogen: cause disease in host, defined by outcome: causes harm to host (may kill them!), simple, often single host life cycle
Ex: viruses, bacteria, fungi, prions, some parasites

Parasite: live on or in host at host expense, eukaryotes (protozoa, helminths, ectoparasites), defined by relationship: dependence on host for survival → usually keeps hot alive to continue sapping resources
Complex life cycles, often multiple hosts, but not always

Naming

3 domains of life: bacteria, archaea, eukaryotes

We often focus on Family, Genus, Species

Rules:
Genus species or can be underlined instead
Abbreviated: G. species
subspecies is unitalicized and not capitalized

How are microorganisms classified

Morphology → how it looks
Biochemical characteristics → what it eats and synthesizes
Physiology → key attributes of the cell
Genetic relatedness → based on DNA

Morphology

Colony (form, elevation, margin) vs cell (shape + organization)
Ex: form = how it looks overall → circular, irregular, filamentous
Margin is like if you took a slice of it how does it look
Organization is how the cells are in relation to each other

Reproductive structures → mainly for fungi
Anamorph = asexual
Teleomorph = sexual
Holomorph = everything (freaky)

Biochemical characteristics

Ex: carbohydrate fermentation
Indicator products (catalase or oxidase)

Based on what substrates they use, enzymes they produce, and metabolites (products) they make

Physiology

Cell envelope → gram + or gram -
Anaerobic vs aerobic
Toxin production → not defined genetically, but has relevance for clinical
Pathotypes - have same pathology on given host (similar ways of causing disease)
Spore formation
Serovars or serotypes → defined based on cell surface antigens and the serological response they generate

DNA and genetic relatedness

Central dogma: DNA → RNA → Protein
DNA encodes info for proteins “gene expression”

G+C% same species have >90% similarity
DNA-DNA hybridization (seeing if their DNA will zipper together) (>70%)

Single locus sequence identity (>98% for 16S)
locus = region, not each region is a gene
16S used for bacteria because highly conserved rDNA sequences
rDNA = DNA encoding rRNA or spacers
ITS used for fungi
PCR → sequence → database → compare to ID

Whole genome sequencing
More complex guys will have more genes
ANI = average nucleotide identity → similarity index b/w given pair of genomes → they will compare orthologous genes

Determines taxonomic assignments

Quantifying microbial growth

Exponential growth is ideal
We use a log scale because it is easier to interpret results than a linear scale and you can see the relative or full differences in data points??

Fresh produce

Fruits and vegetables → leaves, stems, roots

Produce commodities are most often related to outbreaks

Risk factors:
Typically grown outside in field (huge contamination risk)
No “kill step” → only chemical sanitizers used
Sold as ready to eat consumer product

Gold axiom of food safety

1. dookie happens

2. dookie flows downstream

3. we all live downstream (ruh roh)

Since all fresh produce and foods can never be risk-free, we must mitigate the risk as much as possible

Food safety programs

Food safety modernization act (FSMA)
established produce safety rule (PSR) → science-based minimum standards for safe production and harvesting of fresh produce based on foundation of good agricultural practices → this is law

Good agricultural practices (GAPs) → collection of principles applied on-farm and post-harvest to promote food safety in fresh produce → these are voluntary guidelines

Contamination risks

included under the PSR
Biological soil amendments, worker health/hygiene/training, agricultural water (production and post-harvest), domesticated and wild animals, equipment/tools/buildings/sanitation

It can be harder to remove contaminants if they get into crevices, calyx, core, cuts, webbing → “internalization”

There are lots of different pathogens on lots of different produce because there are so many routes for contamination to occur

Soil amendment contamination

material added to soil to improve its quality → compaction, aeration, nutrients

A source of pathogens:
Highest risks → lowest risk
Raw animal manure → raw green (plant) manure → aged animal manure → compost teas → meals and vermicompost → composted animal manure → composted plant material

For raw manures we use national organic program standards:
If edible portion likely to contact → apply 120 days before harvest to allow pathogens to die off
If not likely → 90 days before harvest

Composting phases

Mesophilic: dominated by, you guessed it, mesophilic organisms! They are releasing a lot of CO2, H2O, heat → T rises

Thermophilic: when pathogens are killed

Maturation: nutrients for microorganisms are depleted and compost cools down → susceptible to contamination so we must store it away from production fields, packing houses, processing → store it down hill of production fields and away from water sources

Wildlife and domestic animals contamination

all manure carries pathogens:
Domestic: livestock, cats, dogs, rabbits, horses
Wildlife: like everything else and insects

We must keep the wildlife out with physical barriers, noise makers, decoys, netting, strings across open water sources, falcons, bait stations

And also identify when they do get in → look for signs of contamination (tracks, poop, damage), flag, and do not harvest contaminated produce within a 5ft radius

We also gotta keep the domestic guys out of growing rows at least 7 days before harvest and working animals out of everywhere the food goes

Personnel contamination

caused by workers directly or indirectly due to clothing, personal hygiene, etc.

basically use your common sense: handle raw manure after you touch the plants and do not wear clothing that you used to handle livestock

Farms must also provide the proper infrastructure to maintain worker hygiene
Ex: bathrooms away from production fields but close by (w/in 10 min walk) and enough for everyone (1 per 20 workers), handwashing stations

Equipment and utensil sanitation

Goal: prevent cross contamination from surfaces into produce

4 step process
pre rinse → clean/wash → rinse → sanitize (rinse*)

Clean/wash = removing foreign material from a surface through friction
Sanitation = applying a disinfecting chemical to clean surface to kill bacteria rather than physically removing it → you cannot sanitize a dirty surface

Tool design and harvest containers are also important in mitigating risk of cross - contamination

Agricultural water contamination

source and treatment pose different risk
Overhead more risky than drip irrigation because overhead will touch the edible portion (therefore must use potable H2O and apply in the morning to allow it to dry), while drip only reaches the root system

Highest → lowest risk
Surface water → recycled water → ground water → Municipal water

Flood water bad… exposed to many contaminant and large volumes increase risk
Based on FDCA, if it directly contacts produce (edible portion) we cannot use it. If it is indirect (splash), it requires a kill step

Produce labeling

by region, not by farm → the supply chain is complex and hard to trace back to farm

water quality assessment

Physical and chemical: T, pH, salinity, turbidity

Biological: indicator microorganisms used to evaluate presence of pathogens and monitor conditions
Coliforms are typically present on produce so they are not great indicators for produce??

Interpreting results:
Top is lab and client info, sample details, date
then physical and chemical parameters, then biological parameters
Results must provide an actual count (presence or absence not acceptable) and they use generic E. coli count

And the fda changed the psr to be a systems-based assessment??

Post harvest microbial water standards

Post harvest requires potable water used for washing produce, cleaning equipment and surfaces, and cooling

To prevent cross-contamination, sanitizers are used for wash water: Chlorine-based, Peroxyacetic acid/PAA is common

Only use EPA registered products at recommended rates and monitor sanitizer effectiveness via concentration, pH (for chlorine), T, turbidity, oxidation reduction potential (ORP)

Spoilage biota

vegetable soft rot caused by gram negative bacteria
Acidic fruit spoilage caused by LAB, yeast, and molds

Leads to lots of food waste
But remember, plant pathogens = food spoilage, but does not necessarily equal human pathogens → spoilage mainly decreases quality, not safety

Processing of fruits and vegetables

fresh-cut, drying, fermentation, thermal processing, freezing, HPP, UV

History of dairy microbiology

Raw milk is widely associated with disesases → tuberculosis, typhoid fever, diphtheria, scarlet ever, gastrointestinal distress

Milk pasteurization introduced in late 1800s

Initially controversial and slow to be adopted as commercial practice → was not more widespread until 1920s

Microbial growth in milk

Milk has lots of fat, protein, sugars → good for microbial growth
Also lots of vitamins and ash (minerals)

Notice how the poopy booty hole is next to the udders? yeah… contamination is highly likely
Also, the dairy supply chain is long:
Farm → milking parlor → transport and bulk storage → processing → stores
So, there are many places for things to go wrong

Dairy farm

conditions cow lives in → feed conditions, bedding
not the cleanest…

Bedding types: sawdust, rubber mattresses, water beds, sand, straw

Milk producing animals: dairy cows (diff from beef cows), dairy goats, camels. water buffalo
Dairy cow breeds vary in volume and composition (fat and protein content) of milk they produce

Cow life cycle

born → 15-18 months impregnated → birth → repeat ~5 times before milk production slows and they are slaughtered :(
Cows are milked everyday, multiple times a day ~6gal/day per cow
But they get to relax and eat for most of the day

Contributors to milk microbiology

Milk is virtually sterile when secreted into the alveoli, but beyond this stage, introduction of microbes can occur based on external factors:

• tails

• bedding

• udder sanitation

• sanitation of environment

• cow hygiene

• equipment cleanliess

Raw milk leaving the udder from healthy cows has relatively low bacterial counts

Dairy cow udder has 4 teats = quarters

Mastitis

inflamed udders → can increase microbial counts in milk

Cause by various bacteria
Ex: Staphylococcus aureus, Streptococcus agalactiae, Mycobacterium bovis, Klebsiella spp.,Brucella spp., E. coli

Antibiotics on dairy farms

• Dairy cattle receive antibiotics to treat illness

• Cattle on antibiotics are segregated from the heard so their milk is not collected

• A withdrawal period to flush antibiotic residues from the cow’s system is required before milking

• Antibiotic containing milk cannot enter the food supply

Milking parlors

several different styles
Cows will willingly go to get milked because they feel the pressure or something, so certain systems allow cows to go get milked when they feel ready → Ex: tandem, rotary are fully automated robotic system open 24/7

Automated milking with manual management 2x a day → parallel and Herringbone

Dairy rules and regulations

Pasteurized milk ordinance (PMO)
- Grade A PMO is a set of minimum standards established by FDA for production, processing, and packaging of Grade A milk
- updated every 2 years by vote across 50 states and determined by FDA → however it is not a federal requirement and is voluntarily adopted by states

Standards of identity
- Requirements related to nature, composition, and essential characteristics of a food that must be met in order to be marketed under a specific name

PMO microbial quality standards

pre-pasteurized standards for comingled milk are less strict than individual producer milk for total bacteria counts
Raw milk is the strictest on total bacteria counts
PMO is adopted by NYS

Microbial testing and indicators used in the dairy industry

Standard plate count (SPC)
Somatic cell count (SCC)
Coliforms
Enterobacteriaceae (EB)
Total yeast and mold counts (Y&M)
Methylene blue reduction test
Alkaline phosphatase test (ALP)

SPC in dairy

measure of total aerobic bacteria in milk
Use standard methods agar → incubate 32C 48 hours → count colonies
More stringent quality thresholds exist than the ones specified in PMO

SCC in dairy

= immune cells → increases due to poor cow health
Quantified via microscopy or flow cytometry

Coliform counts and enterobacteriaceae in dairy

Coliform: gram negatives, non-spore formers that can ferment lactose
Found in feces, vegetation, aquatic environments → indicators after pasteurization but not before since its typically from environment

EB: gram -, indicators of microbial hygiene in food processing, includes E. coli, Salmonella, Shigella

Total yeast and mold counts, Methylene blue reduction test, alkaline phosphatase test (ALP)

Total yeast and mold counts (Y&M) → spoilage biota for cultured dairy products

Methylene blue reduction test → blue dye disappears if lots of microbial metabolism

Alkaline phosphatase test (ALP) → phosphatase broken down by heat so yellow only disappears if it is still active - this test depends on the processing technique you’re using

Raw milk sales

Rules vary by state
NYS allows direct to consumer sales, with permit and mandatory animal testing

Herd share = people buy shares in a milking animal or herd, entitling them to milk from those animals

Raw milk and cheese account for almost all dairy foodborne illness (96%), despite being a minority of all dairy products sold

How do you know which microorganism to target with pasteurization conditions?

There are lots of little guys found in milk that cause disease.
Toxin producers - whether disease occurs depends on product handling

We target the most heat resistant guy: Coxiella burnetti that causes Q fever → this is what we want to see the 5 log reduction in

Types of pasteurization

118F is the baseline temperature when proteins denature

Low temp/Vat: 145F → more enzymes and proteins remain in tact below this T

High temp, short time (HTST) → kills enzymes, healthy microorganisms, denatures proteins. this is harder to digest

Ultra pasteurization → this combined with aseptic filling (involving direct heating with steam injection) is for shelf stable milk
This kills harmful bacteria, but also damages vitamins, minerals and other nutrients

Thermization → enough to kill less heat-resistant microbes before cheese is made… not pasteurization

Higher T = shorter time
May utilize plate heat exchangers

Fluid milk spoilage

Coming from Raw milk: spore formers, especially because they survive pasteurization → reduced through on-farm practices

Post-pasteurization re-contamination: gram -, which are introduced from the environment after pasteurization → reduced through food plant sanitation

Clean in place

method of automated cleaning for interior surfaces of pipes and equipment

Sanitation fluids are circulated through equipment

Compared to manual cleaning: more effective, less time, less water use

Dairy foods and pathogens + spoilage microbes of concern

Ice cream:
Pathogen: Listeria monocytogenes
Spoilage: Non-microbial quality defects (ice crystal formation)

Yogurt:
Pathogen: Listeria, low risk
Spoilage: Yeast and Mold → ex: mucor, CO2 production
Fermentative: LAB → Lactobacillus delbruekii sbusp. bulgaricus, Streptococcus thermophilus

Cheese:
Pathogen: Listeria
Spoilage: Yeast and mold
Fermentative: LAB (milk → curds), yeast and mold (aging rinds, color)
Coagulants: acid, rennin

Dairy powders:
Pathogen: salmonella, cronobacter sakazakii
Spoilage: non microbial quality defects (clumping, discoloration)

Dairy industry challenges

Declining consumption: much less dairy farms
Narrow profit margin
Supply chain resiliency
Spoilage and consumer food waste: just behind fruits and veg
Generation and management of waste streams: acid whey

History of meat microbiology

The Jungle by upton Sinclair was a story of Chicago’s stock yards and led to public outcry about the sanitary conditions of meat production → led to:
Pure food and drug act → with prohibited sale of misbranded or adulterated food and drugs and was the foundation for the FDA
Meat inspection act → prohibited sale of misbranded or adulterated livestock + meat packing reform, mandated by USDA

Other big event: Jack in the box E. coli outbreak caused by inadequate cooking of hamburgers → led to stronger emphasis on microbial controls via HACCP + finished product testing scheme

Overall: public uproar led to stronger regulation

USDA vs FDA

USDA regulates meat, poultry, liquid egg products

FDA regulates shell eggs, everything else
Seafood, except farmed catfish
Multicomponent products containing <3% raw meat or <2% cooked meat, like pizzas

Other regulations:
Federal meat inspection act (FMIA) requires all meat sold commercially be inspected and passed by USDA inspector to ensure it is safe and wholesome
Meat processors have HACCP plans

Meat supply chain

farm → slaughterhouse → processing → retail → consumer

Note: consumers can be in charge of the final kill step, so education is important!

meat vs poultry vs seafood

all are animal tissue, but their source differs

Meat: mainly muscle from mammals used as food

Poultry: from chickens and other domestic poultry

Seafood: from fish and other fishery products

Meat, poultry, seafood farms

Livestock feeding operations → animal feeding operations (AFOs)
→ final stage of livestock farming, nutrient dense rations
→ antemortem inspections to ensure sick or injured animals don’t enter food supply, required for all livestock presented for slaughter

Poultry farms: hatcheries produce chicks
Meat chickens (broilers) raised in houses, floor raised on litter
Laying hens are provided egg collection boxes or cages
Lots of controls around biosecurity → Chickens may have Salmonella and other diseases without symptoms → you can contract this with contact!!!

Seafood farm: use aquaculture, antibiotics are heavily managed
Wild-caught fish must consider chemical and biological hazards from the environment such as toxins (chemical, EX: ciguatera from algae) or waste pollution (biological, like human waste)

Abattoir/slaughterhouse process

• Stunning and bleeding

• Dehiding, scalding/plucking, or scaling

• Evisceration

• Postmortem inspection

• Rinsing, chilling

• Cutting, deboning

• Packaging

Stunning and bleeding

- Stunning: induce immediate insensibility for humane slaughter, animal stress ↓quality and is inhumane
Methods: mechanical (captive bolt gun), electrical, CO2

- Bleeding: sever carotid artery (livestock) or remove head (poultry) to kill the animal

Dehiding, scalding/plucking, or scaling

Hide removal (livestock): remove hide and head, hides contain pathogens like E. coli that can contaminate meat

Scalding and plucking for poultry to remove feathers
Plucking is mechanically done with rubber fingers

Evisceration and post mortem inspection

Evisceration: removal of internal organs, huge risk of contamination from GI tract

Post Mortem inspection: uses FS-2 food safety standard → zero tolerance for contamination (feces, digestive matter, milk) → how fast is too fast for line speed controversy
Inspections done by USDA inspector
Carcasses are stamped after passing inspection

Antimicrobial sprays and chilling

Antimicrobial sprays are used to reduce pathogen levels on surfaces
→ treat red meat carcasses after trimming
→ often uses organic acids or PAA
→ provides about 2 log reduction
→ improves food safety and shelf-life extension

Chilling:
Meat is chilled → hung in a cold room and aged (beef)
Rigor mortis → muscles stiffen and loose elasticity, muscle becomes meat
Poultry carcasses can be chilled in tanks of cold water → huge source of cross-contamination, so often includes antimicrobials like PAA (peroxyacetic acid)

Meat pathogens

Raw meat: E. coli, Salmonella, Campylobacter, parasites
Processed meat: Listeria monocytogenes, Clostridium perfringens
Ex: steak vs hamburger
Steak → pathogens mainly on surface
Hamburger → pathogens on surface may be ground and mixed internally → so must be fully cooked

Poultry: Salmonella, Campylobacter

Seafood: parasites, viruses (filter feeders), others

Food safety inspection service (FSIS)

agency of USDA responsible for meat, poultry, egg product safety

Establishes “performance standards” for pathogen testing in raw product
→ Rates of pathogen contamination

Standards indicate: type of product (carcass vs parts), pathogen, sampling scheme, passing criteria

Adulterants

Overall: anything harmful, unapproved, unsanitary, died other than by slaughter, exposed to radiation, misbranded

Pathogens may be considered adulterants depending on the product
Ex: Listeria is an adulterant in cooked or RTE meat
Big six E coli serotypes are adulterants in raw ground beef
Salmonella is adulterant in breaded and stuffed raw chicken → to reduce salmonellosis cases

Meat spoilage organisms

Depends on product and packaging type:
Unmodified atmosphere: gram negatives → these guys are aerobic and can cause odors, slimes, quality defects
Vacuum packed: LAB → anaerobic conditions

Spoilage: Psychrotrophs which grow <10C or 50F

Meat processing

Raw meat sold fresh or frozen

Processing can involve curing, smoking, drying, fermentation, thermal

Curing (add salt, sugar, nitrites for preservation), smoking (cooking method to add flavor), drying (lower a_w), and fermentation (lowers pH due to LAB growth all must control for:
Inactivation of pathogens on raw meat via temperature or pH
Inhibition of S. aureus toxin formation during processing
Decrease water activity if shelf-stability is desired

Thermal: make sure to check internal temps properly (in the center)
Alt to discontinuous checks: continuous or in line monitoring

Meat cooling

meat must be chilled rapidly or else pathogen growth can occur in temperature danger zone
For meat, C perfringens

Models like USDA’s ComBase can be used to estimate growth over time base on meat type, pH, and a_w

Egg processing

Eggs in the US are washed, sanitized, and dried

– Decreases microbial counts, but also removes the cuticle

• Makes them more porous, refrigeration is required

– Other countries do not wash their eggs, cuticle is intact

• Salmonella vaccines may be given to chickens to decrease salmonella cases

• Egg refrigeration not required

Salmonella transmission to eggs

Internal contamination (vertical transmission)

- Contamination in the reproductive tract

- Directly into egg contents

- Before expulsion of the egg from the oviduct

External contamination (horizontal transmission)

- Contamination from the environment (feces)

- After expulsion of the egg from the oviduct

- Onto the outer surface of the eggshell

- Internalization through the shell

Salmonella Enteritidis is the serovar most associated with eggs

– Known for contamination via vertical transmission

– Other serovars contaminate eggs through horizontal transmission

• More commonly found in the egg white

– Salmonella grows faster in the yolk than in the white

– The albumen (egg white) has antimicrobial properties

• Estimated 1 in 10,000-20,000 eggs are contaminated

with Salmonella