USDA Food Safety and Inspection Service 

= US Offi\RTf,ENHlf AGA!CU.TLRE 

Overview 

A review of basic microbiological concepts. 

Review relationship between bacterial adulterants and meat 

and poultry products during processing. 

General hurdle technologies used and some general good 

food/meat manufacturing safety practices. 

Basics of reporting and use of whole genome sequencing by FSIS applicable to role of the EIAO. 

What Are Bacteria? 

Single-celled or unicellular microorganisms. 

Extremely small - you need a microscope to see them. 

Living organisms - cany out metabolism through respiration 

or fermentation. 

Prokacyotic cells - do not have a nucleus. 

Live in evecy environment on the earth (e.g., volcanos, at the 

bottom of the ocean, and in extreme cold temperatures - - Arctic). 

Enclosed by a cell membrane. 

Some bacteria have a thick cell wall others have a thin cell wall 

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The Size of Bacteria Relative to Other Types of Cells 

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Basic Shapes of Bacteria 

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Pneumococd 

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Streptococci Pseudomona

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Staphylococci 

t Mycobocter

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tuberculosis 

~~>--- Flogello 

Salmonella typhi 

i if\ 1/ Clostridium tetanl 

Thick cell walls of Gram-positive 

bacteria retain stain 

and will not decolorize, resulting in a purple-blue 

bacteria seen under 

light microscope. 

Thin cell walls of Gram-negative 

bacteria allow stain 

to be decolorized and counterstained 

with safranin, 

resulting in a 

pinkish bacteria seen under li t 

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Fullerenes -

Molecules of

carbon atoms

spores

Color of Bacteria After Gram Staining Depends

on Thickness of the Bacterial CellWall

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GRAM•POSITIVE GRAM·NEGATIVE 

Examples of ~ Fixation 

~ Examples of Gram-Positive D Gram- Bacteria _, -> Negative 

Bacillus ., QystalVIOlet Bacteria 

CJostridium D Campylobacter 

Listeria 

-=- Iodine Treatment 

-=- E. coli / STEC 

Staphylococcus D Pseudomonas 

~ Salmonella 

-=- Oecolonsation 

D 

-=- CounterstaJnwlth 

Sal1anin ii" 

Based on staining characteristics of the bacterial cell wall, bacteria arc classified as Gram-positive or Gram-negative 

Ciram Positive Bacteria Ciram Negative Bacteria 

Process of Cellular Respiration in Bacteria - Needed to 

Breakdown Nutrients, to Gain Energy to Grow 

One of Life's "'°""' -- preferred basic nutrients 

::] Bacteria use the same 

respiratory mechanisms shown here in 

mitochondrion. 

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NADH NAO.+ 

ntallOf'l 

Respiration occurring 

aerobically uses oxygen. In 

contrast, during anerobic 

respiration oxygen is absent. 

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The Gram Staining Method Used to Divide Bacteria

Into Gram-Positive or Gram-Negative Bacteria

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Micrographs of Gram-Stained Bacteria

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Bacteria Use Four Major Mechanisms for Respiration 

1. Obligate aerobes - Requires oxygen to grow (e.g., Mycobacterium, fungi) 

2. Obligate anaerobes - Cannot grow in the presence of oxygen. Oxygen is toxic to these bacteria (e.g., Clostridium spp.) 

3. Facultative anaerobes - These bacteria can go under low and high oxygen conditions (e.g.,£. coli, STEC, Salmonella, other enterics within the Enterobacteriaceae, and some yeasts) 

4. Microaerophilic (2-6% oxygen) - Grows best in low oxygen situations (e.g., Campylobacter spp.) 

Bacteria Use Four Major Mechanisms for Respiration 

Aerobic -

bacterial growth 

with 02• 

Most of the 

growth is where 

most of the 0 2, 

is located. 

Microaerophile 

most of the 

bacterial growth 

is in areas of the broth with 

reduced 02 • 

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Temperatures at Which Specific Types of Bacteria Can Grow 

Growth temperature Growth Temp 

classification min / optimum range Examples of representative bacteria 

/max('F) 

Psychrophlles (cold-loving) 23 / 32-59 / 68 Achromobacter,Alcaligenes, 

Planococcus, Colwellia 

Psychrotrophs (tolerate cold} 32 / 68-77 / -100 Brochothrix, Listeria, Pseudomonas 

Mesophiles (love moderate Campylobacter, Clostridium, 

50 / 86-113 / 122 £nterobacter, Lactobacillus, Listeria, temp) Pseudomanas, Salmonella 

Deinococcus, 

Thermophiles (heat-loving) -110 / 113-122 / >125 Geobacillus stearothermaphilus, Thermus aquaticus 

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https://en.wikipedia.org/wiki/File:Anaerobic.png

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https://en.wikipedia.org/wiki/File:Anaerobic.png

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Note: Some species of bacteria can survive across a wide range of temperatures 

and fall within two categories, e.g. Listeria and Pseudomonas.

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Growth of Bacteria and Bacterial Growth Requirements 

A Typical Bacterial Growth Curve 

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Time to complete the 

bacterial growth cycle In a specific 

environment. 14 

Describing Food-related Factors Affecting Bacterial 

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Bacteria are alive and healthy, but

nutrients are limited so stopped

growing rapidly and are stationary.

Bacteria has adapted to the

environment and is growth Bacteria are dying no nutrients exponentially in Log phase. are available.

Bacteria getting

ready to grow,

adjusting to the

environment.

Intrinsic factors – related to the food itself; characteristics “inside” of the food

Extrinsic factors – related to the environment where the food is or what is added to it;

characteristics “outside” of the food

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Intrinsic Factors That Affect Bacterial Growth 

Intrinsic factors - inherent characteristics of food 

pH - >6.5-7.2; depends on amount of lactic acid produced in 

muscle after slaughter of animal. 

Water activity - raw meat ~0.98. 

Nutrient content - sugars, proteins, amino acids, organic acids. 

Physical structure - whole muscle, comminuted, chunked/formed. 

Naturally occurring antimicrobial components present in animal 

prior to slaughter. 

Oxidation-Reduction Potential (Redox potential) - measure of how 

easy it is for a molecule to accept or lose electrons; more oxygen = higher potential; inner side of meat anaerobic surface is aerobic; 

even after death respiration continues with formation of lactic acid. 

Extrinsic Factors That Affect Bacterial Growth 

Conditions for Bacterial Growth 

Food Acidic environment Temperature Time Oxygen Moisture (FATTOM) 

Food - meat and poultry are good sources of nutrients for bacteria 

Acidic environment - most all foods have a pH<? 

low acid foods have a pH>4.6 (aw>0.85) 

high acid foods have a pH s 4,6 

Temperature - 40 to 140°F = the Danger zone; allows bacterial growth 

Time - food in the Danger zone for more than .... 4-6 hours 

example - 1 bacteciai doubling every 20 min for 6 hours in the 

danger zone = 18 divisions = 5,4 log10 bacteria 

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Extrinsic factors – characteristics that modify the properties of a food 

• Storage temperature – frozen, refrigerated, room temperature.

• Relative humidity – affects speed of drying and if the meat dries properly.

• Presence of other bacteria – affects growth of pathogens; naturally occurring spoilage bacteria or 

the addition of lactic acid bacteria can prevent Staphylococcus growth.

• Gases in the environment – modified air packaging; meat that is vacuum-packed or flushed with 

other gases (carbon dioxide, carbon monoxide, nitrogen, etc.).

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Conditions for Bacterial Growth 

Food Acidic environment Temperature Time Oxygen Moisture (FATTOM) 

Oxygen 

Obligate aerobes, facultatively anaerobes, and microaerophilic organisms require 

oxygen to grow well. 

Obligate anaerobes will grow in the presence of oxygen. 

Moisture 

Moisture content is a measurement for food quality. 

Water activity (aw) is a measurement for food safetyj aw >0.85 

What is pH? 

pH is the measurement of the concentration of positively-charged hydrogen ions 

[H+]. 

Definition - the negative logarithm of the concentration of hydrogen ions [H+ ]. 

Logarithmic scale - each change in a whole number represents a tenfold change in 

[H +] concentration. 

A sample at a pH of 4.0 has 10x the concentration of (H+ 1 ions as a sample at pH 

of 5.01 and 1oox the concentration of [H+] than a sample with a pH of 6.o. 

Raw meat and poultry has an approximate pH between 5.4 - 6.8. 

lncreasinrJy 

AQdlc 

What is pH? 

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M.-BIIOS-...., & """-­

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"'"'" High ac,d foods 

pH sea l~ and common food types 

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M-\',pt;.obln. «:: 

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..... °"'"...,,.." 

Mu••Vwlln =-

•-"-' "" Low add foods 

tpHHlwdMllhtfl•Jgll-.dd ,,_ .. ~,_,,, 

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https:/lextension.okstate.edu/fact-sheets/the-lmportance-of-food - ph-ln-commerdal-cannlng-operatlons.html 

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pH 4.60 pH 4.61

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pH of Some Common FSIS-regulated Foods 

Food pH 

egg solids, whole 7.1- 7,9 

egg whites 7.0 - 9,0 

beef (unaged) 7-0 

egg yolk 6.4 

egg solids, whites 6.5 - 7.5 

chicken 6.5 - 6.7 

ham 5.9 - 6.1 

turkey (roasted) 5.7 - 6.8 

fish (most fresh) 6.6 - 6.8 

veal 6.o 

beef (aged) 5.8 

lamb 5.4-6.7 

beef (ground) 5.1 - 6.2 

sausage, dry 5-3 - 5-5 

sausage, semidry 5-3 - 5.0 

Definition of Water Activity (aw) 

aw is the ratio between the vapor pressure of the food and the vapor pressure of distilled water under identical conditions 

•MIOUllf.f:l ,-,,,11rwat• .-ld •"'"°"!ltd~-•••~ _,__tf!lltt'll~l&abl'I. 

dllleWlli!rlftl~(~J 

https:/fwww.freund-vector.rom/freund-group-lnttoduces-water-activlty-meter/ 

Water Activity (clw) 

Water that is strongly bound to ingredients cannot be used for bacterial growth. 

Ingredients used in food process that bind water: NaCl, phosphates, gums, 

humectants, surface effect of the substrate, sugar (requires much higher concentration than salt). 

Water and solutes (NaCl, acetic acid, phosphates) move along a gradient to 

reach an equilibrium. 

Low water activity outside a bacteria causes osmotic stress - cannot take up water and becomes dormant (not killed). 

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Adaptedfrom L.R.Beuchat,Cereal FoodsWorld,26:345(1981) 

How i1w Affects Biological Systems 

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J ii !/!fl! ii •1 J ff : •• , i I i ~ ,~,,;. 1/ , •• .: ,. 'ii ... -· I ,+ ,:/ t ! " ~ 

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.,,, .,,,. .,- o/ / ",._/ 0 l 

0.1 0.., 0.., 0.4 0.0 o.e .. , 0, .. WAT!R ACTIVITY 

http://www.fao.org/3/Y43S8E/y43S8eo6.htm 

How Bacterial Foodborne-Pathogen Cross- contamination 

of Food Products Occur 

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Pseudomonas, Escherichia, Proteus, Shigella, Klebsiella, Bacillus, some 0.98 – 0.95 raw meat, raw poultry, fish, milk, canned fruits, vegetables yeasts, C. perfringens

Some molds & yeasts, Salmonella, Pediococcus, Lactobacillus, V. 0.95 – 0.91 cured meat (ham, cold cuts), some firm cheeses, bread parahaemolyticus, C. botulinum

0.91 – 0.87 Many yeasts (Candida, Torulopsis, Hansenula), Micrococcus fermented sausage (salami), sponge cakes, dry cheeses, margarine

Most molds (mycotoxigenic penicillia), S. aureus, most Saccharomyces beef jerky, most fruit juice concentrates, syrups, jams/jellies, soft 0.87 – 0.80 (bailii), Debaryomyces pet food

0.80 – 0.75 Most halophilic bacteria, mycotoxigenic aspergilli beef jerky, marmalade, marzipan, glacé fruits

Xerophilic molds (Aspergillus chevalieri, A. candidus, Wallemia), molasses, raw cane sugar, some dried fruits, nuts, snack bars, 0.75 – 0.65 Saccharomyces bisporus snack cakes

dried fruits containing 15-20% moisture; some toffees and 0.65 – 0.60 Osmophilic yeasts and some molds caramels; honey, candies

dry pasta, spices, rice, confections, wheat

whole egg powder, chewing, gum, flour

0.60 – 0.50

No microbial proliferation cookies, crackers, breakfast cereals, dry pet food, peanut butter

0.30 – 0.20 whole milk powder

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Beef Processing Flow Diagram 

Cattle 

Receiving - I Stunning f--1 Sticking/Bleeding I- Hide and Holding Removal 

Carcass 

Rinse 

Organic Acid 

Application 

(CCP2) 

Trimming/ 

Final Rail (CCPt) 

Carcass 

splitting Evisceration 

Harvest Variety 

---------

1 

Chilling 1---- Meat (tongue, liver, kidney, etc.) i ~-~----,-..____ -~ 

Bacteria may be introduced: Sticking 

Hide removal Evisceration 

How Contamination of Meat Occurs During Processing 

Carcasses are contaminated during hide removal and evisceration. 

Bacterial adulterants and fecal contaminates such as coliforms, aerobic bacteria, and generic £. coli and enteric pathogens such as Salmonella and Shiga 

Toxin-Producing E.coli (STEC) can be deposited on carcass during 

evisceration. 

Bacteria are main cause for concern because they can multiply and cause illness if under-cooked products are consumed. 

Cross contamination can occur via: Knives and tools used during killing/processing of carcasses. Employees' smocks, hands. 

Food contact surfaces and the environment. 

Antibacterial Intervention Controls Commonly 

Used in the USA 

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• Hot water/steam

• Organic acids – lactic, lactic/citric mixtures, peroxyacetic, lactate/diacetate, nitrite

• Inorganic compounds - acidified sodium chlorite, trisodium phosphate, chlorine

• Microbiological – bacteriophage (viruses that infect bacteria) bacteriocins 

(proteins/peptides produced by bacteria to inhibit or kill bacteria, lactic acid 

bacteria, food spoilage bacteria

• Chilling, freezing, cooking

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Growth of Aerobic and Facultative Anaerobes & 

Their overlap 

Meat-associated Foodborne Bacterial Pathogens 

Two Major Methods Used by Foodborne Pathogens to 

Cause Illness 

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Aerobic plate count

or Total plate count

• Infection – ingestion of pathogen and growth of pathogen results in

physiological damage without enterotoxins. Bacteria grow within the

intestinal tract and cause pathological damage resulting in illness.

(e.g., Campylobacter, Listeria, Salmonella)

• Intoxication

o Ingestion of food containing a pre-formed toxin.

(e.g., Bacillus cereus, Clostridium botulinum, Staphylococcus aureus)

o Bacterial growth and toxin production in the intestinal tract. The toxin

made during infection is the major virulence factor responsible for the

symptoms and pathological effect of illness.

(e.g., Clostridium perfringens, STEC)

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Meat-associated Foodborne Pathogens - Bacillus cereus 

Widespread in nature - found in soil, hide/skin/feathers, cereals, 

herbs, spices, and unpasteurized milk. 

Food-associated sources - can be found on meat; also found on 

starchy foods such as cooked rice, potatoes, puddings. 

Characteristics: 

Gram-positive rods, spore forming facultative anaerobe 

Temperature growth range min/max - 41°F / 131°F 

Optimum temp range - 82 °F to 120°F 

lower pH limit - pH 4-9 

lower aw limit - 0.93 

Survival in salt - 7.5% 

Meat-associated Foodborne Pathogens - Bacillus cereus (continued) 

Foodborne illness - B. cereus food poisoning Infective dose 10s CFU/g 

Causative agents 

Intoxication; Diarrheal toxin (heat-labile) 

Diarrheal toxin - diarrhea and abdominal pain 

Emetic toxin (cereulide; heat-stable); both toxins mimic S. 

aureus enterotoxin 

Emetic toxin - nausea and vomiting 

Meat-associated Foodborne Pathogens - Campylobacter spp. ( C. jejuni, C. lari, C. coli) 

Widespread in nature - intestinal tract of animals, birds (e.g., cattle and poultry). 

Food sources - poultry and meat 

Characteristics: 

Gram-negative spiral-shaped and microaerophilic (5%) 

Temperature min/max - 86 °F / 122°F 

Optimum temp range - 98 °F to to8°F 

lower pH limit - pH 5.0 

lower aw limit - 0.98 

Sutvival in salt - No 

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Meat-associated Foodborne Pathogens -

Campylobacter spp. ( C. jejuni, C. lari, C. coli; continued) 

Food.borne illness - Campylobacteriosis 

Infective dose 500 CFU/g 

Illness Gastroenteritis - host cell invasion, toxin production, 

inflammation, and epithelial cell destruction. mimics S. 

aureus enterotoxin leading to nausea and vomiting. 

Sensitive to freezing, drying, acidic conditions (pH< 5.0), 

and salt. 

Meat-associated Foodborne Pathogens -

Clostridium botulinum 

Widespread in nature - soil, intestinal tract, and hides of animals. 

Food sources - cooked meat and poultry products (e.g., roast beef). 

Characteristics: 

Gram-positive rods obligate spore forming anaerobes 

Temperature min/max - 50 / 122°F 

Optimum temp range - 95 - 104 °F 

lower pH limit - pH 4. 7 

lower aw limit - 0.93 

Survival in salt - 10% 

Nitrite - 100 ppm inhibits growth and toxin productionj use with cure accelerator (sodium erythorbate or ascorbate) or a high salt concentration 

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Phosphate and lactate/ctiacetate - can have an inhibitory effect 38 

Meat-associated Foodborne Pathogens -

Clostridium botulinum (continued) 

Foodborne illness - botulism Infective dose ,o, CFU/g 

Illness Intoxication caused by a neurotoxi.n ingested with food. 

Controlling growth of C. perfringens will control C. botulinum and B. cereus 

FSIS indicates is there must be no multiplication of C. botulinum 

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Meat-associated Foodborne Pathogens - Clostridium perfringens 

Widespread in nature - soil, intestinal tract and hides of animals. 

Food sources - cooked meat and poultry meat from beef or pork 

and poultry cooked with sauce highest risk. (e.g., roast beef). 

Characteristics: 

Gram-positive rods spore-forming obligate anaerobe 

Temperature min/max - 43 / 126°F Optimum temp range - 109.4 - 117 °F 

lower pH limit - pH 5.0 

lower a,, limit - 0.93 

Survival in salt - 7% 

Nitrite - 100 ppm inhibits growthi use with cure accelerator (sodium erythorbate or ascorbate) or a high salt concentration 

Phosphate and lactate/diacetate - can have an inhibitory effect 

Meat-associated Foodborne Pathogens -

Clostridium perfringens (continued) 

Foodborne illness - gastroenteritis 

Infective dose 10s CFU/g 

Illness 

Intoxication -Toxin produced in the gut after ingestion of vegetative 

cells. 

Controlling C. perfringens growth will control C. botulinum and B. cereus due to the fast growth rate of C. perfringens, as fast as 15 minutes. 

No more than 1-log10 multiplication of C. perfringens to comply with 

9 CFR 318.17(a)(2). 

Meat-associated Foodborne Pathogens -

Listeria monocytogenes 

Widespread in nature - environment, intestinal tract and hides of animals, soil 

Food sources: 

undercooked or under-processed foods (fermented, dried, and ready-to-eat 

foods). 

Post-lethality contaminated meat such as cooked deli meats, hotdogs, fermented 

sausages/meats. 

Characteristics: 

Gram-positive rods facultative anaerobes 

Temperature min/max - 32 / 113°F 

Optimum temp range - 86 - 99 °F; grows at refrigerator temperatures 

lower pH limit - pH 4.4 (sutvives in low acid) 

lower aw limit - 0.92 

Survival in high salt - 10% 

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Meat-associated Foodborne Pathogens - Listeria monocytogenes (continued) 

Food.borne illness - listeriosis 

Infective dose -<100 CFU in a 25g sample 

Mortality rate as high as 30% in immunocompromised persons, neonates, pregnant women, elderly and other people with medical issues. 

Illness Invades and grows inside intestinal epithelial cells (enterocytes) and can move 

inside cells from cell-to-cell. 

Can cross the gut epithelial barrier into blood stream. Can lead to inflammation 

of the membranes and fluid surrounding the brain (meningitis). 

Can persist in establishments (harborage) due to formation of biofilm and spreads 

by cross contamination events by water (splashes/aerosols). 

Very difficult to eradicate after it establishes itself in a biofilm. 

Meat-associated Foodborne Pathogens - Salmonella 

Salmonella spp. - Gram-negativej rodi facultatively anaerobic; two species: 

Salmonella enterica - human pathogen; >2500 serovarsj six subspecies. 

Six subspecies (ssp.) of Salmonella enterica: enterica, indica, salamae, 

houtenae, diarizonae, and arizonae 

Proper nomenclature: 

Salmonella enterica subspecies enterica serovar Enteriditis 

Generally written as Salmonella Enteritidis 

Salmonella enterica subspecies enterica serovar Typhimurim 

Generally written as Salmonella Typhimurium 

Meat-associated Foodborne Pathogens - Salmonella (continued) 

Widespread in nature - environment, beef, pork, and poultry. 

Food sources 

Raw meat and poultry undercooked and under-processed RTE products (fermented or dried meat 

products) made with beef, pork, and poultry to achieve a .... 6-log10 

reduction. 

Characteristics: 

Gram-negative rod-shaped facultative anaerobes 

Temperature min/max - 36 F/129 F (can become heat resistant) 

Optimum temp range - 95 F - 99 F lower pH limit - pH 4.1 (can become acid resistant) 

lower aw limit - 0.93 

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Meat-associated Foodborne Pathogens - Salmonella (continued) 

Food.borne illness - salmonellosis 

Infective dose 1os-106 CFU/g 

lllness Gastroenteritis - diarrhea, fever, abdominal cramps, and vomiting. 

Secondary illness - reactive arthritis can last months or years and can 

lead to chronic arthritis. 

Can persist for several weeks on foods with low aw 

Gerky aw=0.80i egg powder aw=0.5) and can survive at aw<0.5. 

Meat-associated Foodborne Pathogens - Salmonella (continued) 

FSISpresentation "Salmonella:StateoflheScienceM{Sepu, 2020) 

Meat-associated Foodborne Pathogens - Shiga Toxin-Producing E.coli (STEC) 

Widespread in nature -

Food sources -

Characteristics: 

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Salmonella serovars that cause foodborne illness:

o Enteriditis

o Newport

o Typhimurium

o Javiana

o I:4, [5],12:i:-

• intestinal tract, fecal contamination of animal hides of bovine

• pork, leafy greens, vegetables, and acidic fruits (e.g., apples/apple cider).

•

• Characteristics:

o Gram-negative rods facultative anaerobes and psychroterant and grow at low temperatures

(1 to 45 °C = 33°F to 113°F)

o Temperature min/max - 40 °F / 113 °F

o Optimum temp range – 98.6 °F

o lower pH limit - pH 3.3

o lower aw limit - 0.90

o Survival in salt – 3.5%

o lactate/diacetate – can have an inhibitory effect

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Meat-associated Foodborne Pathogens - Shiga Toxin-Producing E.coli (STEC; continued) 

Food.borne illness - gastroenteritis Infective dose 1-100 CFU 

lllness Gastroenteritis, hemorrhagic colitis (bloody diarrhea), severe cramping 

(abdominal pain), vomiting, low-grade or no fever. 

Survives fermentation , drying, storage, and 69 ppm nitrite. 

Can become heat and acid resistant 

Designated by O antigen (somatic) and H antigen (flagella), 

(i.e., E.coli O157:H7). 

Seven serovars that FSIS consider adulterants: 

026, 045, 0103, Om, 0121, 0145, O157:H7 

Meat-associated Foodborne Pathogens -

Staphylococcus aureus 

Narrow source in nature - skin and nasal passages, not all strains are pathogenic. 

Food sources - improperly handled foods due to poor worker 

hygiene or lack of process controli meat carcasses. 

Characteristics: Gram-positive cocci facultative anaerobes 

Temperature min/max - 45 F / 118 F 

Optimum temp range - 95 F - 104 F 

pH limit - pH 4.2 

aw limit - 0.83 (tolerates low water activity) 

High salt tolerance (15%); nitrite has little inhibitory effect 

Does not grow well in the presence of competing bacteria 

Meat-associated Foodborne Pathogens -

Staphylococcus aureus (continued) 

Food.borne illness - gastroenteritis due to Staphylococcus Enterotoxins {SET) 

Infective dose 106 CFU/g 

Illness - nausea, vomiting, abdominal pain, diarrhea. 

Can survive improper fermentation (Summer sausage). 

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S. aureus - not affected by high salt concentration (-15%) or nitrite, can grow below aw 

0.91. Does not grow well when other bacteria are present. 

SET not affected by cooking or proteases. SEA 80%, SEB 10%, SED 37% (SEC and SEE also implicated). 

If S. aureus growth is suspected, you must look for SET (not for S. aureus) because S. 

aureus could be dead (or non-culturable) but the SET will be present (and make people 

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Comparison of Foodborne Pathogen Characteristics 

BadUusareus(saltM'll>;ITandTI) 

Compylobaaerspp. (5'11>0,; IN) 

Clostrldlum botullnum {salt 10'll>; NS; IT) 

Clostridlumpe,fr!ngms (salt ?"JI,; NS; Tl) 

ShlgaToxln-productng£.coli(salt}.5%;TI) 

Listeri<I mom,cytogenes (salt 0 -25%; IN) 

.......... , min/optimum range/ mu(~) 

41/81-104/01 

86/98-108 /Ul 

S0/95-104/lll 

43/109.4-117/126 

39/95-99/113 

32/86-99/113 

..... pH 11m1, 

, .. 

, .. 

, .. 

0.93 

.... 

0.93 

0.93 

lnfectlou.sdose 

!OI CFIJ/g 

SOOCFU/g 

toxln: 1ng/kg bodyweight 

106 CFU/g 

1- IOOCFU/g 

1000 cro 

Salmo11dlaspp.{IN) 36/95-99/129 0.93 10• - 106CFU/g 

Staphylococrusaureus(saltlS'll>;NlS;IT) 45/95- 104/118 

IN " Infection; IT = Intoxication preformed toxin; Tl = lntoJ:ication toxin made during infection in situ; NS" nitrite-sensitive; NlS " nitrite 

Insensitive 

General Measures to Prevent Bacterial Outgrowth 

Preventing food.borne illness 

Cooking or Lethality step - refer to Appendix A 

Ensure food is maintained either at a temperature above 

140°F or refrigerated below 40°F (outside the danger zone) 

Proper stabilization - refer to Appendix B; cool cooked foods 

that will not be immediately consumed to below 40°F. 

Hurdle Technology in Meat and Poultry Processing 

Hurdle technology involves several inhibitory factors used together to control or eliminate pathogen growth, which would 

be ineffective if used alone (Leistner, 19761 1992). 

Deliberately combines existing and new preservation techniques 

(salting, curing, drying, etc.) to establish a series of preservative 

factors that microorganisms are unable to overcome. 

Hurdle technology combines attempts to disrupt one or more 

homeostatic mechanism of bacteria to cause the microbes to 

become inactive or die. 

The best plans use multiple small hurdles to disturb several 

bacterial homeostatic mechanisms. 

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Hurdle Technology: Use of Multiple Food Safety 

Interventions 

}_ 

Graphic Depiction of Hurdle Technology 

I 

A single intervention may not be enough to kill or prevent bacteria from growing. 

Combining multiple interventions, where each alone may have with a 

minor contribution, but together have an additive or synergistic effect by disrupting one or more physiological pathways. 

Example: Food Code - Interaction of Hurdles: pH and i1w 

FDAFoodCode2017 

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Processesfor making foodssafe

Heating high temperature

Chilling refrigeration Freezing

Freeze-drying

Drying

Salting aw Sugar-addition*

Curing competitive flora

Fermentation pH preservatives Acidification

Smoking

Oxygen removal oxidation-reduction (redox) potential

*sugar is not as effective at controlling aw as salt

F = heating

t = refrigeration

aw = water activity

pH = acidification

Eh = Redox potential

pres. = preservatives

Leistner (1995)

Interaction of pH and aw for control of spores in FOOD

heat-treated to destroy vegetative cells and subsequently PACKAGED

Interaction of pH and aw for control of vegetative cells and spores in FOOD

not heat-treated or heat-treated but not packaged

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Cold Storage Deviation - An Example 

Heat Treated, Not Fully Cooked, Not Shelf Stable sausage (vacuum-packed) and Heat Treated, Fully Cooked, Not Shelf 

Stable (vacuum-packed) 

Bacon 

Head cheese Pork sausage, low sodium sausage, sausage with jalapeno and cheese, andouille, hot pork 

During an FSA, the cooler temperature at the coldest part was 

48 F. 

Internal product temperatures varied between 48 and 53 F. 

Previous month's temperature log showed temperatures 

fluctuated between 45.1 and 59.2 F. 

Cold Storage Deviation - An Example 

--.o ,~01• ----~ 

. 

I 

'-1- .__ ...... T_ 

- o.~--. ..,_ »»Gf4100toOII 

JUMMOOOlllltOO 

L ::'.:="-~ 

r! t· 

11 11(.., ... ..,,.. 

........ 

....... { II~ Ni 't, -- '~'\ 11~~' I l -·- ' 1 1 -- ""' 

. 

Cold Storage Deviation - An Example 

. 

. 

'~ ~~ 

Tw.Z-,. C-W..,_,.T_ 

.......,, 0.1'-. ...... 

....,.,000000 

~=-

~ WM~\vWtJi 

KeyVeNn 

....... 

-·-

-·-

---·-

""' 

~ • ~----y58 

,., 

. .. 

., 

'" 1) 

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Cold Storage Deviation - An Example 

Concerns with -

Heat-treated, Not Fully Cooked, Not Shelf Stable (vacuum-packed) 

Clostridium perfringens (C. botulinum) 

Listeria monocytogenes 

Salmonella 

Staphylococcus aureus enterotoxins 

Concerns with -

Heat-treated, Fully cooked, Not Shelf 

Stable (vacuum-packed) 

Clostridium perfringens (C. botulinum) 

Listeria monocytogenes 

Staphylococcus aureus enterotoxins 

Multiple discussions between OFO, OPPD and OPHS. 

Decided on multiple tests to be performed. 

Note: Every situation is different and must be handled 

individually. 

Whole Genome Sequencing (WGS) 

Whole Genome Sequencing - A Collaborative Approach 

How to perform WGS - methodology (aligned methods) 

Analyze WGS data 

Interpret WGS data 

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• FSIS worked with the Food and Drug Administration, the Centers for

Disease Control and Prevention (CDC), with PulseNet partners on:

o

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• FSIS began performing WGS for Listeria monocytogenes (Lm) in FY13

(along side PFGE) and for all pathogens starting in early FY16.

• FSIS suspended PFGE analysis for Lm and started using WGS data

Jan 15, 2018.

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Whole Genome Sequencing - Benefits 

WGS benefits FSIS and its mission to protect public health: 

Detects harborage and cross-contamination of pathogens in FSIS-regulated 

facilities, 

Traceback from human illness outbreak data to regulated food products, and 

Identification of unique genes related to virulence, pathogenicity, survival, 

adaptation, and resistance to biocides (sanitizers, metal, etc.) and antimicrobials. 

Whole Genome Sequencing - Analysis 

FSIS uses different tools to analyze WGS information including: Public Sequence Typing 

Multi-locus Sequence Typing (MLST) 

Core genome analysis ( - 1800 genes for Lm) 

Phylogenetic analysis 

High-quality Single Nucleotide Polymorphisms (hqSNP) 

Whole Genome Sequencing - Single Nucleotide 

Po1ymorpnism (SNP) 

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Single Nucleotide Polymorphism (SNP)

ATGTTCCTC isolate A

ATGTTGCTC isolate B

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Whole Genome Sequencing - Sequence Typing 

Whole Genome Sequencing - Single Nucleotide 

Polymorphism (SNP) 

~ =c Sequence 

ST09 

/[ ~ 1- LMO 1.1- 37,3,2.36.1 37,3,2.11.7.13 LMO 1.1- 37,3.2.33 

Allele Code is more specific than Public Sequence Type; 

one Public Sequence Type can be inclusive of many 

Allele Codes. 

Whole Genome Sequencing - Single Nucleotide 

Polymorphism (SNP) 

/ ~ LMO - L. monocytogenes 

·rf-r'TrT Version 1.1 

Allele codes are a 

~ 1 . 1 . 2 . s . 1 

nomenclature scheme 

created by CDC a 11 field" 

Like PFGE patterns, allele codes simplify how we 

communicate about If the first four fields 

pathogen strains between two isolates 

match, the isolates may b closely related 

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Multi-locus Sequence Typing (MLST)

• MLST can generate a pattern name or designation based on differences in a pre-defined

set of genes.

• MLST Results will be Provided by FSIS as Follows:

o Public Sequence Type (“MLST ST”, “ST”, or “pubST”)

 small number of genes (i.e., 6-12)

 named using the publicly available database developed by Jolley & Maiden (2010)

(e.g., publicST09)

• Allele Code

o compares ~1,800 genes for Lm

o named by using CDC PulseNet numerical code

(e.g., LMO1.1-5.1.1.2.5.1)

Example: LMO1.1 - 5.1.1.2.5.1

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-

Whole Genome Sequencing - Allele Codes 

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Background: What does allele code tell you?

LM1.0 – 1.5.4.4.2.5

LM1.0 – 1.5.4.4.5.9

LM1.0 – 1.5.4.6.7.2

Last digit same or different

differ by 0 – 19 alleles

“closely related”

3rd to last digit different

differ by > 19 alleles

“not closely related”

LM1.0 – 1.5.4.4.2.3

Establishment-specific Datasets

Allele codes for Lm have been reported since 2019

Fields were created for Salmonella and STEC allele codes (Campylobacter in development)

Date Stamp format (allele code (space) date mm/dd/yyyy

LM1.0-23.5.6.0 04/05/2022 Retrieval of the allele code from PulseNet

Allele codes may change over time, a date-stamp supports use of the data in static reports

• Allele codes are a nomenclature scheme created by CDC.

• Like PFGE patterns, allele codes simplify how we communicate about pathogen strains.

• Allele codes can be used for trend analysis and to interpret relatedness.

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Whole Genome Sequencing - Analysis - Microbial 

Characterization Branch (MCB) - Eastern Lab, Athens, GA 

.._.,. 

_______ 

.. _,, ______ ____ . __ ,...,____________ ., ___ ...,., __ _ ,,_ ~ -- ---•---

"""'-'-•-------uo- ~ ... -•-------­ _______ ... ,_,, .. ______ _ 

Harborage and Cross-contamination 

Harborage or persistent contamination of the post-lethality environment, is suggested if WGS analysis indicates closely related Lm isolates are found in 

product, food contact, or non-food contact environmental samples that were 

collected over multiple days, weeks, months, or years. 

Cross-contamination is suggested when closely related Lm isolates are found in product, food contact, and environmental (non-food contact) samples 

collected during the same sampling event. 

If Lm Is isolated from a post-lethality exposed product sample and from a 

food contact surface sample, the food contact surface is more likely to be the source, unless under-processing of RTE product is suspected. 

Harborage and Cross-contamination 

C) 

q ..;1o1 ~·A\ • .M -i•·. !fl!! ~ 

✓ 

~ 

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SAL-2

LM-1 SAL-1 SAL-3

Harborage (likely) LM-1

different collection dates

SAL-3

Source LM-1 LM-1

Cross-contamination (possible)

same collection date Recommend corrections to food safety controls

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PulseNet Cluster Search 

f M,~~T~!iFo.~~io2),rform PHRE at cat 3 Est request search through AskFSIS 

search strategy: 

Obtain all Salmonella WGS from all raw poultry sampling projects obtained in past 52-weeks from the establishment. 

Determine if any such sequences are closely related to a recent clinical isolate associated with a Pulse Net cluster. 

PulseNet Cluster Search 

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Sample collection dates 

(PulseNet human 

cases) 

:: 

:: 

Asking for More Information 

FSIS 

sample 

matches 

recent 

PulseNet 

clinical 

isolate 

When performing a PHRE in establishments with more than one positive RTE sample, ElAOs are to: 

~S: t/;~~0-"::u~o~i:~:::~~a~v:~~~~l(~~~~::;~~crt:a~0m 

Outbreaks WGS@fsis.usda.g:ov 

The WGS analysis will indicate if there is a history of harborage or cross­ contamination in the establishment. 

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Shared Public-Private Success

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Whole Genome Sequencing - The Future 

FSIS continually works with FDA, CDC PulseNet, local & state health departments to harmonize interpretation and reporting. 

Future plans - - pathogens that will be reported by allele code: STEC and Campylobacter jejuni allele codes were released in early 2021 

Salmonella is still being finalized by PulseNet 

Whole Genome Sequencing - The Future 

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c=J □□□ I I I ---------~ 1 

Abbre,;atioa, j FDA - Food.,dDrug-olmatlo WGS - Whole genome sequencing 

Lm - Liswiamonocytogt nes NCBI - Natlonal center for Blotechnologv Information 

PFGE - Pulsed field gel electrophoresis 

NARM.S - National Antimicrobial Resistance 

~~~~=- produdngE.roll 

Summary- I 

Bacteria are either cocci, bacilli (rods) or spirochetes (cork-screw) shapedi bacilli (rods) are about 1 x 0.5 μm and are broadly characterized by their reaction to a Gram stain. 

~~~~J~(g~~~;,o~~~~:~~~~o11/i¥i~~i~lm:::i:r,~)~c, facultatively anaerobic, 

Some foodbocne Gram(+) bacteria include Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Listeria monocytogenes, Staphylococcus aureus. 

Intrinsic factors are those characteristics that are inherent to the food, 

i.e., pH, aw, nutrient content, physical structure. 

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1- FSIS began WGS 

FDA ran WGS on STEC and Lm 

on Lm for FSIS 2- Uploaded first 

Lm sequences to 

NCBI

FY14

FY13 FY15

OPHS Eastern Laboratory 

runs first sequences for 

outbreak investigations •

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1- PFGE stopped 

FSIS began WGS on for Lm

Salmonella and 2- Began WGS on 

Campylobacter NARMS cecal FSIS begins to 

for select cecal indicator obtain Salmonella 

isolates organisms serotype from WGS 

FY17 FY19

FY16 FY18 FY20

PFGE stopped on 

WGS on all Campylobacter,

Salmonella and Salmonella,

Campylobacter STEC

(product and cecal)

FSIS hosts WGS 

Public Meeting

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Summary- II 

Extrinsic factors are those characteristics that are related to the external environment of the food, i.e., temperature, relative humidity, gases in the environment (packaging), presence of other bacteria. 

The conditions for bacterial growth: Food, Acidic environment, Temperature, Time, Oxygen, Moisture (FATI'OM). 

~seil~~bY:~;;~~s }~j i~bJ'~:!~:;~~~~~~~re mesophilic bacteria are most 

:~~~~:%at~c~irre~n1!1:aB~bi!ot~c~::~i!.characteristic of water bound by food 

pH describes the acidity of a solution and is a logarithmic scale; most meats are generally between pH 5.4-6.8. 

Summary - III 

Bacillus cereus, Clostridium botulinum, and Clostridium perfringens are Gram(+) spore formers. 

CJostridium is nitrite-sensitive; C. perfringens grows quickly and controlling it generally means C. botuJinum and Bacillus cereus will also be controlled. 

Listeria monocytogenesi G{ + )i can grow at refrigerated temperatures, in high salt, and 

~~ft~;e~~::!~ab:tJ~ ~~a~efs t~~O ~~:;td!n~u:r~odi~~::. contamination by 

Salmonellai G(-); is hardyi can survive in low water activity foods for weeks; can become heat- and acid-resistant. 

STEC; G{-)j is hardy, can survive in low pH, can become heat- and acid-resistant; FSIS considers some foods containing any of the seven serovars as adulterated. 

Summary- IV 

Staphylococcus aureUSi Gram(+); human-associated and transferred by poor worker hygienej tolerates hi!U\ salt, low awj does not grow well in the presence of other bacteria; nitrite has fittle effect. 

~fe~~~~ohn~0~Wt !!J~~~:i~:[~~~~~~~1bu~¥oe~i~fJu~:i~~1o';~~-ple 

Foodborne illness is generally caused by one of three mechanisms: ingesting pathogenic bacteria, consummg food containing pre-formed toxin, or when pathogenic bacteria make toxin in the intestine. 

FSIS reports Listeria monocytogenes by an allele code; if the first four fields between two isolates match, the two isolates may be closely related (<19 alleles apart). 

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