fsct326: exam 2

CDC 2016 List of Notifiable Foodborne Diseases, Bacterial

• Botulism (All forms including foodborne, infant)

• Brucellosis (Undulant Fever)

• Campylobacteriosis

• Cholera

• Listeriosis

• Q-Fever (Coxiella burnetii)

• Salmonellosis

• Shiga toxin-producing E. coli (STEC) disease, acute/chronic

• Shigellosis

• Vibriosis

• Yersiniosis (Not CDC, but is mandatory in Texas)

foodborne disease outbreak

• typically occurring with 2 or more persons consume shared food item (contaminated) within localized timeline and/or geographic area

  • shared food vehicle → not alive carrier of disease

  • vector → living that carries disease to you

• exception: C. botilinum disease → only 1 case required to define as outbreak

• sporadic disease: typically single patients/cases, not affiliated with other persons or food items (not outbreak related)

• outbreaks can range from very small and localized to very large (multi-state, -national, -continent)

Bacillus (Latin = “small staff“)

• belongs to the family Bacillaceae

• aerobic spore-forming rods (obligate aerobes), bearing psychrotolerant, mesophilic, and thermophilic spp.

  • some capable of anaerobic fermentation of glucose

  • proteolytic and non-proteolytics

• spores do not distend (expand, disshapen) the sporangium (mother cell)

• environmental reservoirs (ecological niche in which foodborne pathogens gains access to food-producing environment/processing) exist (soils, grasses, crop production, environments)

• Bacillus cereus group: B. cereus, B. anthracis, B. thuringiensis, and B. weihenstephanensis → plasmid-borne pathogenesis elements

B. cereus and human disease

• isolates capable of producing 1 or 2 key toxins

  • emetic toxin (intoxication)

    • intoxication → bacteria/toxin formed prior to consumption

    • emesis = throwing up, vomiting

  • enterotoxin/diarrheal toxin (toxico-infection)

    • toxico-infection → must consume bacterium, survives stomach, gets in lower intestinal tract, secretes enterotoxin and disrupts stomach balance)

    • entero- = gut in Latin

• commonly associated with starchy foods and/or high proteins foods (milk, fish, meats)

  • contamination of animal or milk

  • spores survive pasteurization

  • some psychrotolerant strains identified- milk safety during refrigeration (growth at > 4°C)

B. cereus foodborne disease

characteristic	enterotoxin	emetic toxin
# of cells needed for illness	10^5 - 10^7 CFU total	10^5 -10^9 CFU/g food
toxin production?	small intestine	pre-formed in food
incubation period	8 -16 hr (typical)	0.5 - 6 hr
duration	12- 24 hr (or longer)	6 - 24 hr
symptoms	abdominal pain, watery diarrhea, nausea	nausea, vomitting

emetic toxin

• a form of cereulide (amino-acid based)

• enzymatically synthesized, plasmid-encoded

• tolerant to large pH range (pH 2-11) and to heating/cooking (90 min at 121 °C)

  • longer than mandatory decontamination for some bio-waste in micro labs…

• similar process tolerance as S. aureus enterotoxin

• no impact of protease treatment

toxin production conditions

a_w, pH, O2 content, temperature, nutrients, [NaCl] → growth of microbes, respiration → toxin production

other Bacillus spp.

• dairy spoilage

  • B. weihenstephanensis

  • B. wiedmanii (2016 discovered)

• other genera derived, reclassified from Bacillus

  • Paenibacillus (“Like Bacillus): P. polymyxa, P. macerans

  • Geobacillus: G. stearothermophilus - Thermophilic flat
    sour spoilage LACF

Clostridium botilinum

• family Clostridiaceae (Greek = spindle, small spindle) (type species = C. butyricum)

• C. botulinum first described in connection to consumption of raw undercooked blood sausages (Botulus: Latiin- sausage)

• anaerobic regularly-shaped spore-forming rod (spore distends sporangium)

• psychrotrophic, mesophilic, and thermophilic species

• C. botulinum possess both psychotropic and mesophilic strains

C. botulinum physiology

• cells may be pleomorphic in shape

• motile by flagella (peritrichous)

• may ferment various acids and can produce gas from sugars

• typically catalase negative

C. botulinum classifications

• 8 toxin types: A. B, C_1, C_2, D, E, F, G

  • human disease : A, B, E, and F

• groups:

  • 1 (toxin A and proteolytic strains) → associated with large outbreaks and meat/poultry dishes

  • 2 (non-proteolytics, type E)

  • 3 (toxin types C and D)

  • 4 (toxin type G)

foodborne botulism

• adult, infant, wound, and indeterminate form

• flaccid paralysis → blocking of acetylcholine release from muscle/nerve junction

• adult botulism → foodborne intoxication

• food vehicles: home-canned vegetables/foods (low-acid), vacuum-packed smoked fish, under-cooked canned foods, honey (infants), baked potatoes wrapped in foil, warmed-over extended holding foods

• intoxicating dose: 0.4 ng (10^-9 g) per kg of body weight

symptoms of disease

• incubation 12-36 hr post-consumption

• symptoms

  • nausea, abdominal pain, vomiting

  • descending paralysis

    • droopy eyelids

    • slurred speech, loss of motor control in face

  • can have respiratory failure, inability to breathe (leading cause of botulism-derived fatality)

• abdominal pain follows, constipation (typically infant botulism)

disease therapies

• providing antiserum to bind and remove toxin

• enema to expel toxin from GI tract rapidly

• stomach pump, or induced vomiting

• ventilator to assist, maintain breathing

• most critical to proper treatment- proper diagnosis!

botulism in the US

• increasingly associated with home-preserved foods

  • home-canning of vegetables, meats

  • low-acid foods

• often a result of insufficient heating in jars (spores survive and germinate post-cooling)

• proper cooking in home requires use of pressure cooker

• cooking lowers E_h supporting growth

botulism cases, US, 2009-2018

• about 80 cases of disease in US, 5 fatalities

• most cases arise from consumption of contaminated fish, seal meat, grains, and canned veggies

recent foodborne botulism outbreaks in US

• 2017: nacho cheese from convenience stores (CA): 10 cases

• 2015: potato salad (home-canned potatoes)- OH (29 cases confirmed, suspected) [Described as largest botulism outbreak in 40 years in US]

• 2014: pesto (canned)- OH/CA (2 cases of adult females with disease)

• 2012: home-fermented tofu- NY (2 cases)

botulinal neurotoxin

• two components: light and heavy chains

• chains must separate to exact final toxin activity

• Zn-metalloprotease: blocks acetyl-choline release

• thermally labile (sensitive):

• cooking 80 °C, 10 min (or boiling)

best practices of home canning (US CDC)

• use recommended pressure canner holding at least four 1 qt. jars upright

• ensure pressure cooker gauge accuracy

• clean lid gaskets and components via manufacturer recommendations

• vent canner before pressuring and follow recommended cooling procedures

• use current process time/temperature combinations for specific food items of interest, jar size, method of food packaging in jar (with brine, without, salted, unsalted)

Clostridium perfringens

• similar to other Clostridium species: rod-shaped, sporeformer, mesophilic pathogen

• originally named C. welchii

• some identify as facultative anaerobe due to plating capacity, but typically behaves as obligate anaerobe

• non-motile by flagella

• encapsulated organisms

  • bacterial capsules: excreted polysaccharides bonded to outer layers of cell wall, teichoic acids in Gram-positives

  • useful for attaching to solid surfaces, protecting against dehydration

• capable of very rapid doubling times (10 min) under optimal conditions

• optimal growth: 40-45 °C

differentiating C. perfringens strains

foods associated with C. perfringens disease

• common food vehicles are high protein-containing

• stews, roasts, gravies, meat + gravy, seafood, poultry

• 13 essential amino acids cannot be synthesized (source effectively in high protein foods)

• often foods undercooked or are properly cooked but cooled too slowly

  • vegetative cells destroyed; spores survive

  • re-heating shocks spores into germination and replication

preventing disease in the home

• cooks foods thoroughly

  • use meat thermometer; check to ensure proper cooking tp recommended internal temperatures

  • consider size of meat dish in cooling plans

  • vegetative cells possess elevated heat tolerance

• chill rapidly

  • use the refrigerator to chill leftovers- that’s why it was purchased; don’t use counter-top to “cool“

preventing C. perfrigens growth in food processing

• very similar to consumer recommendations: proper cooking to destroy vegetative cells, rapid chilling to prevent spores from germinating/replicating

• USDA-FSIS 1999: appendix B- stabilization

  • demands cooling of fully cooked roasts, meat, poultry products

  • increase in C. perfringens counts: no more than 1.0 log_10-cycle allowed during cooling

  • 130 to 80 °F in 5 hr

  • addition of curing agents increases the amount of time required for cooling by preventing spores from germinating

processing hurdles to C. perfringens survival/growth

• heat (thermal processing) and cold (refrigerated storage at/or below 6 °C)

• reduction in pH by acidification; isolates don’t tend to grow below pH 5.0

• reduction in a_w: 0.93 lower limit to growth

• maintenance of elevated E_h (not always an option and there are tradeoffs)

foodborne perfringenosis

• diarrheal disease (toxico-infection): commonly associated with type A toxin-producing C. perfringens (sometimes also type E)

  • acute (explosive) diarrheal with cramping (12-24 hr symptoms) → symptoms painful but short-lived

  • incubation period 8-12 hr

• necrotic enteritis (pigbel; Darmbrand)

  • uncommon in US (observed in Papua New Guinea and surrounding regions today in non-industrialized countries

  • observed with C. perfringens producing the β-toxin (typically type C)

  • characterized by necrosis of jejunum and ileum; can be fatal if not diagnosed quickly and treated aggressively

  • also identified to occur in livestock animals via toxigenic C. perfringens

• CDC estimates as 3rd leading cause of bacterial foodborne human disease!

characteristic of disease

• typically requires moderate consumed dose (10^6 - 10^7 CFU/g); 8-16 hr incubation

• fever is uncommon

• passage through stomach in meaty protects during stomach passage

• toxin released in small intestine as consequence of sporulation (accumulates in cell prior to mother cell lysis)

• toxin causes damage to villus tips in intestinal cells

• diarrheal symptoms continues to flush out excess toxin particles

Listeria monocytogenes

• gram-positive cocco-bacillus facultatively anaerobic psychrotrophs

• tumblee motility by flagella

• over 15 species and sub-species (most identified within the last 5 years!)

• genus shares similarities to Clostridium, Brochothrix, and other gram-positive genera

• only L. monocytogenes (Lm) and L. ivanovii are identified pathogens (L. ivanovii is animal pathogen)

• described as being environmental ubiquitous

Lm physiology

• psychrotrophic growth (1-45 °C)

  • t_d (hr) = 43 @ 4 °C, 1.1 hr @ 37 °C)

• can grow at low pH (4.3 - 4.4) but most rapid at neutral pH

• growth at > 0.93 a_w

• not very strong competitor in mixed microbial ecologies

• ferments D-xylose, L-rhamnose, D-mannitol

Listeriosis disease syndrome

• foodborne infectious infectious, intracellular pathogen

• syndomes

  • adult onset: symptoms of meningitis, septicemia with fatality of 1/5 to ¼ (immuno-compromised adults at highest risk)

  • pregnant mother: flu-like symptoms with possible long-term shedding, other complications

  • late-onset neonatal: exposure during vaginal birth, post-delivery nosocomial from shedding adults

  • early-onset neonatal: exposure to fetus during gestation, potentially resulting in stillbirth, spontaneous abortion, or systemic infection post-natal

  • feverish gastroenteritis: those with no other predispositions; often requires high consumption for infection to occur with incubation periods 12-72 hr

foodborne listeriosis: generalities

• higher fatality rate versus other viral, bacterial pathogens (maybe 3rd highest amongst foodborne pathogens)

• observed in multiple types of fresh and processed foodstuffs

  • pathogen (Lm) can perisist in numerous of food process systems

  • can form biofilms, resist sanitizer attack, and tolerate acid, moderate heat exposure

• incubation period can be very long! (up to 70 days)

• infectious dose may be low (as few as 1 CFU/gram of product in some outbreaks)

• intracellular pathogen (invades, moves within human GI cells escaping immune response)

• pathogenic Lm differentiated into 13 serotypes: ½a, ½b, and 4b are most commonly identified in human disease

• often associated with ready-to-eat (RTE) food products: cheeses, processed meats, produced foods (whole and sliced)

environmental transmission to foods

foods of concern: raw milk and dairy

• raw milk: cross-contamination in milk collection equipment, holding tank, on farm/cow teats

• outbreaks of disease associated with raw milk-made cheeses occurring all over US

  • 1985: Los Angeles, CA → 142 cases (93 associated with pregnant women, fetuses)

  • 2001: Winston-Salem, NC → 12 cases (10 pregnant women, 5 stillborns, 3 premature, 2 post-delivery infected)

  • 2015: Blue Bell…

RTE meat, poultry, seafood

• pathogen may infiltrate post-lethality environment, causing contamination risk on processed product

• some products are more supportive of growth than others

  • fermented sausages, seafood less supportive at low pH

  • salted foods/meats, dried meats do not support growth of pathogen

• opportunity exists to cross-contaminated post-lethality exposed product from food contact surfaces, adjacent surfaces, walls, floors, drains, and other niches

  • process facility sanitation systems are key yo Lm control on foods

  • isolates (strain) shown to have tolerance development to sanitizers (rotate sanitizers to inhibit this) → dubbed persistent Lm

Lm pathogenesis

• internalins: help pathogen to enter human GI epithalials (escape immune response, competitors, environmental stressors)

• Listeriolysin O (LLO): aids escape of internalized (vacuolar) cells via destruction of vacuolar/phagosome membrane

  • hemolysin: Lm and β-hemolytic (escape phagosomes)

• actin mobilization: assists movement through cell and translocation to adjacent GI cells

US regulations surrounding Lm

• zero-tolerance for RTE foods

  • fully cooked meat, poultry, seafood

  • dairy products, produce

  • non-detectable in 1 or 2 identical 25 g food samples

• USDA-FSIS: 9 CFR 430 (i.e., “The Listeria Rule“)

• 3 alternatives for processors to handle Lm in PLE foods with respect to processing and sampling

1) use of post-lethality treatment (1.0 log_10 reduction in Lm counts) and antimicrobial agent/process (< 2.0log_10 in growth over product shelf life)

2a, b) use of post-lethality treatment (2a) or microbial agent (2b)

3) sanitation-based prevention of contamination

• provides additional processing requirements for manufacturers of deli products

Straphylococcus aureus (Greek: grape-like coccoid)

• family Straphylococcaceae (S. aureus is type species)

• facultatively anaerobic non-sporulating coccoid

• cause foodborne intoxication: pre-formed toxin consumption

  • pathogen not required for disease

  • toxin not process sensitive

• may also cause toxic shock syndrome (TSS), wound infections, and animal disease (mastitis)

S. aureus physiology, dissemination

• catalase +, non-motile, environmentally ubiquitous

• coagulase-variable; thermostable DNAse-variable

• human reservoir; 1 in 2 to 1 in 3 carry at any time

  • skin, hair

  • nasal mucosa

  • food preparers often linked to pathogen contamination (duh…)

• mesophilic facultative anaerobe (optimal growth: 35 °C)

• exhibits growth to a_w > 0.85 (aerobic) and 0.9 (anaerobic); toxin production typically halts at a_w 0.9

• describes as poor competitor: does not effectively sequester/control nutrients, water in mixed microbial ecology

the pathogen in foods and processing

• meat processing equipment contamination sites

• inadequate refrigeration or temperature abuse allows growth, toxin production

• inadequate cooking, heating of foods

Staphylococcus environmental stress tolerance

• most low a_w-tolerant bacterial pathogen!

  • accumulation of compatible solutes

  • reduction of cytoplasmic a_w to neat equal exterior environment → prevents water movement out of pathogen cell

• may escape host immune systems via phagocytosis

• ubiquity: being everywhere in production environment increases difficulty of control

• cooperative in biofilm development, maintenance

foodborne Staphylococcal disease

• characterized by short-lived, self-limiting disease with low fatality rate

• CDC estimates ~ 241K cases per year in US with ~6 fatalities (highly under-estimated incidence)

• infectious dose of enterotoxin: <1.0 CFU/g to achieve toxic doses of SE

• incubation: 0.5-7 hr (dose consumed, sensitivity); duration is short (hrs - 1 day)

• symptoms: nausea, heavy/violent vomiting (emesis), diarrhea (pathogen and/or toxin can be recovered from stools), dehydration

foods associated with Staphylococcal disease

• meats, poultry/eggs, dishes requiring and/or human handling allowing for S. aureus contamination

• confections, bakery items, meat/starchy salads (tuna, chicken, potato)

  • products with fully cooked ingredients still requiring human mixing/handling

• some milk/dairy products (cheeses)

• any foods where background microbes have been eliminated and food is kept under temperature abuse for extended period can be transmission vehicle for S. aureus

Staphylococcal enterotoxins

• SEs are proteins (single chains) anti-genetically distinct from one another: A, B, C, D, E, G, H, J (now known over 20 distinct toxin types)

• some cross-reaction with toxin form-specific antibodies

• SEA is most common disease-causing form, but SEB is most severe form (symptoms)

• SEA, B are super-antigens (bind to major histo-compatibility class II (MHC II) molecules expressed as antigen-presenting cells (e.g., macrophages)

• classically used coagulase test to predict SE producing S. aureus strains (coagulase + staphylococci)

• single peptides (25-28 kDa)

SEs are food process resistant

• tolerant to protease attack (pepsin, trypsin) (pH dependent; may be degraded under conditions of severely low pH)

• thermo-tolerant

  • can withstand autoclaving conditions, short term canning

  • microbe inactivated much more easily than toxin

  • heating tolerance best at low acidity or under sight alkaline condition

Enterococcus spp.

• capable of growth at environmental temperatures ranging 10-45 °C, in up to 6.5% NaCl

• may grow in conditions of pH 9.5-9.6, and can hydrolyze esculin in conditions of high bile salts concentration

• will appear as cocci ~2 µm in diameter in pairs or chains (similar to Streptococci)

• broken out into five distinct groupings based on biochemical capabilities

• typical residents of GI tracts of both warm- and cold-blooded animals: can reside in human tract GI tract also

  • some utility as fecal residues indicator, but typically overlooked in US for this purpose

  • growth in these environments predicated on multiple systems of environmental tolerance, metabolism of various nutrients, and capacity to resist antimicrobials, reactive species, etc

Enterococcus diarrheal disease: humans

• may possess low infectious dose, but estimates vary

• typically acute, and self-limiting, but can become severe with possibly ½ chance of succumbing

• symptoms can mirror those of staphylococcal food disease

• E. faecium and sometimes E. faecalis

• many isolates difficult to treat due to multi-drug resistance (e.g., vancomycin-resistant enterococci [VRE])

Enterococci in livestock species disease

• E. durans: diarrheal disease reported in calves, piglets, foals

• E. avium, E. faecalis, E. gallinarum: septicemia in bords

• E. porcinus, E. villorum: piglet diarrhea/neonatal disease

GRAM-NEGATIVE bacterial foodborne pathogens

Brucella abortus

• non-sporulating facultatively anaerobic coccobacilli; 11 species in genus

  • reference organism: B. melitensis

  • some species known animal pathogens (B. abortus, B. canis)

• family Brucellaceae

• zoonotic pathogen: can be spread between animals and humans

• reservoirs: sheep, goats, bison, elk

human brucellosis (undulant fever)

• uncommon in US; FDA/CDC estimates 120-840 cases/year (higher incidence in other countries with lower food safety demands, animal vaccination programs versus US)

  • infectious dose: ~500 cells (possibly fewer)

  • fatality rate: <2%

• symptoms: chills, sweating, muscle weakness, headache, joint/muscle pain (acute)

• chronic conditions: endocarditis, liver/spleen swelling, chronic fatigue, arthritis, recurring fever

  • animal farmers

  • meat industry employees (slaughter workers and QA laboratory)

  • vets

• consumers of raw milk, fresh-made cheese

• pregnant women

bovine brucellosis (bang’s disease)

• primarily impacting cattle, bison, and some cervids (deer)

• pathogens: B. abortus, B. melitensis, B. suis (swine)

• typically no clinical symptoms appear: abortions or weakness in calves, fawns, piglets

• pathogen eradication program present in US- significant cost-savings in production/meat processing

  • vaccination

  • surveillance

  • herd/animal quarantine

Campylobacter spp.

• family Campylobacteraceae (greek: curved rod); type species: C. fetus (previously named Vibrio fetus)

  • over 30 species with genus

  • key human foodborne pathogenic species: C. jejuni subsp. jejuni, C. coli

  • some species known to cause disease/abortion in livestock: C. fetus, C. jejuni

• spiral rods with one or more twists to body (0.2 -0.9 µm length)

• organisms are microaerophiles (optimal: 3-5% O2, 10% CO2); high oxygen tension can be lethal

• motile by flagella (mono- or amphi-trichous with one unsheathed flagellum at one, both poles)

• optimal growth: 37-42 °C

• nutritionally fastidious; unable to utilize/oxidize CHO (yield energy from amino breakdown) (does contain cytochrome oxidase [electron carrier])

Campylobacter jejuni

• human foodborne disease (infectious)

• thermo-tolerant Campylobacter (good growth: 37-42 °C; no proliferation <30 ° C)

• some isolates shown to continue respiration at 4 ° C

• does not generally catabolize CHO, but contains some genetics necessary for limited sugar (fucose) use in some strains

  • lactate. pyruvate, acetate, etc

  • glutamate, aspartate, serine, proline

• colonization sites in animal, human GI tract with sufficient O2 availability

• multiple respiratory components sustain growth in gut

Campylobacteriosis epidemiology

• globally reported #1 cause of bacterial foodborne disease

• CDC estimates between 1st to 3rd most common human foodborne disease

  • ~1.5 million incidence cases/year estimated

• reservoirs for pathogen identified in multiple meat animals, companion animals, and humans

• outbreaks in animal-derived foods, produce, water, human-human cross-contacts

• children mist likely impacted, along with adolescents and young adults

  • immune-compromised

  • HIV+

• higher disease, outbreak frequency in warmer months/seasons

campylobacter in foods

• principally associated with animal-derived foods not properly cooked, cross-contaminated

  • poultry, swine meat

  • non-pasteurized milks

• fecal shedding leads to water, produce cross-contamination

• full cooking of meat, poultry/pasteurization of milk destroys organism

human camoylicater enteritis

• typically self-limiting/acute disease: up to 2-10 days of clinical symptoms

  • clinical symptoms resolve w/o medical treatment (self-limiting)

  • fever, diarrhea (heavy, frequent, contain undetected blood [occult]), abdominal pain, vomiting (violent, repeated)

• infectious dose is low: <500 CFU (generally around 10^4)

• incubation period: 2-5 days post-exposure

• low frequency of chronic sequelae (post-acute disease) onset

  • bacteremia: bacteria in blood stream

  • inflammation: meningitis, pancreatitis, endocarditis

  • miscarriage of fetus, neonatal sepsis (C. fetus subap. fetus)

  • auto-immune disorders: Guillain-Barre Syndrome (GBS), reactive arthritis

pathogens in humans

factors aiding Campylobacter disease, human GI cell invasion

• adhesions for GI cells with subsequent invasion

• flagellar motility: critical for colonization of gut

• GI Mucin (O2 tension; fucose secretion)- chemo-attractants

• cytolethal distending toxin (Cdt)

  • leads to cell death eventually

  • damages host DNA

• thermo-tolerance: greater survival- higher numbers consumed

• LPS component: sialic acid (mirrors components of human gangliosides)

  • antibodies attack human nerve cell endings

  • autoimmune condition onset

control in food handling

• use of sanitary/potable drinking water for animals

• poultry/meat/food products: proper cooking

• prompt chilling, full reheating leftovers

• WASH YOUR HANDS!

  • frequently

  • working with food animals, pets, exotics

helicobacter pylori

• previously Campylobacter pylori (pyloric; stomach)

• spiral-shaped Gram-negative motile rod capable of motility and colonization or GI mucosum

• widespread throughout world (50-66% infection/carriage rate); likely transmission/exposure through contaminated drinking water

  • fecal/oral; oral/oral routes of spread

  • causative of gastric ulcers; contributor to gastric cancers

  • may provide some protection against esophageal cancer

helicobacter pathogenesis

• capable of survival in acidic conditions

  • motility: locate b/w tight cell junctions in stomach epithelium

  • urease secretion

• chronic infection by organisms leads to inflammation, exacerbated by acid → ulcer

• older adults; senior citizens mostly impacted

• toxins

  • vacuolating toxin: disrupts GI tight cell junction

  • phospholipase

  • cytotoxic elements

vibrio spp.

• family vibrionaceae: facultatively anaerobic irregularly shaped rods (curved rods), halophilic (require Na+ for survival and successful growth)

• > 90 species in genus, 8 are foodborne pathogens

• Infectious disease spp: V. cholerae, V.

  parahaemolyticus, V. vulnificus, V. mimicus, V.

  alginolyticus

• foodborne disease most commonly associated with contaminated marine (salt-water) food animal meat

  • raw shellfish, sushi

  • cross-contaminated post-cooking

• sensitive to cold storage; no growth at refrigeration/freezing conditions

• can enter VNC state: shrink, coccoidal cells

foodborne disease trends

• CDC estimate: ~80 K cases per year (~52K are foodborne)

• overall increases in US vibriosis cases since 1996

• CDC indicated higher disease rates in warmer months/waters (higher pathogen loads in harvest waters)

V. cholerae

• foodborne GI disease and/or cholera (severe: cholera gravis)

• can enter VNC states

• cholera strains belong to serotypes O1 and O139 (encapsulated)

• non-O1 and non-O139 V. cholerae can produce GI disease; not cholera-associated generally

• reservoirs in marine and fresh waters (drinking water contaminated with fecal sewage common cause of disease)

cholera

• estimated ~3 million cases globally per year, with 95K fatalities occurring

• can take mild to heavy/severe forms; partially mediated by toxin expression capacity of isolate (cholera enterotoxin (CT))

• CT induces export of ions and loss of transmembrane ion regulation ability- high ion content leads to flooding of water into colon

• high infectious dose (~1 million cells), with incubation up to 3 days post consumption

• mortality is high without treatment but can be successfully treated with antibiotics and rehydration therapy

• severe diarrhea, pain, cramps, heart racing, muscle aches, rice water-stools (progressed)

• small numbers of cases still occurring in US; most cases occur in under-developed countries, Africa

V. parahaemolyticus

• optimal growth in 2% NaCl in medium; doubling time as little as -10 min at 37 ° C

• produces thermostable direct hemolysin (TDH), AKA Kanagawa hemolysin (beta-hemolytic on Wagatsuma Agar)

• infectious pathogen: 2% mortality common (elevated when patient (pt) is septic)

• capable of very rapid replication numbers necessary for disease

• infectious dose 10^5-10^7 cells; incubation period typically 24 hr

• reservoirs: estuarine waters, seafood animals, sediments

• symptoms typically 2-6 days (acute): diarrhea, abdominal pain, cramps. blood in stool, possible septicemia

V. vulnificus

• highest fatality rate of pathogens discussed (35-60%), but very low incidence (~90 cases annually)

• infections leads to septicemia

• wound infections occur in addition to foodborne infections (20-25% fatality)

• estuarine envirnoment reservoirs

• similar to other vibrio in cold sensitivity, acid sensitivity

• incubation period can be very long (up to 21 days); infectious dose estimated at 1,000 cells

• typical symptoms of GI disease are not common in cases (diarrhea, vomiting)

• highest disease incidence: US, Japan, Mexico, Taiwan, S. Korea

• chronic hepatic disease predisposes to diseases(males more frequent pt)

• syndromes:

  • primary septicemia

  • primary gastroenteritis

  • primary wound infection

Cronobacter sakazakii

• formerly enterobacter sakazakii (family enterobacteriaceae) (genus change suggested in 2008)

• rod-shaped mesophilic facultative anaerobe

• encapsulated organism

  • enhanced biofilm participation

  • enhanced dehydration tolerance

• non-pasteurization tolerant: contaminates post-process

human cronobacter disease

• human infectious disease agent; CDC estimates 4-6 cases per year

• processed powdered infant formula (PIF)

• noenates (<2 months) at risk for disease, death

  • infection can cause bowel damage- septicemia

  • spread to brain- fatality (10-90%)

  • infectious dose estimated at 10 CFU

• incubation is 2-5 days

• symptoms: jaundice, persisting irritability, poor feedings, seizure, fluctuations in body temperature, etc

• treatment often requires antibiotics, but some isolates are drug-resistant

• FDA dictates zero-tolerance in cronobacter and salmonella in infant formulas

escheria coli and the EHEC

• member of family enterobacteriaceae

• gram-neg, oxidase-negative facultative anaerobic rod, motile by peritrichious flagella

• mesophilic growth, with capacity to grow at pH 4.0-4.5 in some systems

  • acid tolerance response system (rpoS)

  • glutamate- and arginine-dependent systems

• pathogenic and non-pathogenic E. coli

• other pathogenic Escheria spp.: E. albertii, E. fergusonii

E. coli pathgroups

• disease syndromes differ between patho-groups, as well as high-risk populations targeted

  • entero-toxigenic E. coli (ETEC): toxico-infection

  • entero-pathogenic E. coli (EPEC): infection/invasion

  • entero-invasive E. coli (EIEC): infection/invasive

  • diffuse-adhering E. coli (DAEC): infection/invasive

  • entero-aggregative E.coli (EAEC): infection

  • Shiga toxin-producing E. coli (STEC) and the entero-hemorrhagic E. coli (EHEC): infection/invasion; toxico-infection

• strains are differentiated into subgroups by three antigens on cell: somatic (LPS; O), flagellar (H), and capsule (K) antigens

EHEC, STEC

• over 100 serotypes of STEC, with some EHEC-type organisms within STEC group

• EHEC are differentiated by characteristic bloody (frank) diarrhea

• STEC and Shiga toxin (verotoxin)

  • nearly identical to shillega dysenteria stx toxin (phage encoded toxin genetics)

  • leads to post-acute disease sequelae HUS and TPP

  • O157 and non-O157 STEC: E. coli O157: H7, O103, O111, O45, O145, O121

  • these 7 STEC serogroups treated as adulterants in fresh/raw non-intact beef products (ground beef, gyro, beef, tenderized, or enhance beef products)

other characteristics

• capable of growth at higher temperatures (> 44.5 °C)

• enzyme glucuronidase is made

• can grow in presence of bile and some other detergents

• inability to use some CHO versus other E. coli

• high acid tolerance (pH 4.0-4.5)- growth

• may survive even lower pH conditions in foods

• no more heat tolerant than other E. coli and Salmonella

sources of STEC in foods

• animal-derived foods: E. coli maintain reservoirs in bovine GI tract (commensal)

  • beef, dairy breeds

  • calves have higher carriage rates

  • dairy breeds of greater concern for carriage, likely conversion of carcass meat into ground beef

• recovered from game animal carcasses, feces

• fecal matter cross-contamination of produce-irrigating waters, produce production systems

• human-human; fecal/oral transmission

foodborne disease outbreaks and beef

• 1992: outbreak which identifies E. coli O157 as foodborne pathogen

• 1993: Jack-in-the-Box (Pacific NW): 179 cases, fatalities- leads to mandatory adulterant status by USDA and pushes HACCP forward for USDA-inspected products in 1999

• 2007: Topp’s ground beef: 40 cases, over 1 M lbs recalled

• 2013: Coco Loco’s (5 confirmed cases, 5 non-confirmed cases): under-cooked ground beef, E. coli O157:H7

• 2014: Wolverine Meats: 12 cases, 1.8 M lbs recalled

• 2015: Chipotle Restaurants (2 outbreaks, E. coli O26)

other outbreak food vehicles

• dairy: non-pasteurized milk

• produce: leafy greens (spinach, lettuce), walnuts, cucumbers, raw apple ciders, sprouts

• drinking water

• further processed foods: pizza rolls with pepperoni, cookie dough, flour (GM, E. coli O121, O26)

• recent outbreaks in US: romaine lettuce (2019, 2018, 2017): lettuce grown western US (AZ, CA)

another way to be exposed

• direct contact with fecal matter, contaminated dust, bedding- fairs and petting zoos

• multiple outbreaks in US surrounding

• NC (2011): cases of disease at state fair (2 mo- 62 years old in pt)

• wash hands thoroughly after handling livestock, adults, and children

hemorrhagic colitis, STEC disease

• mild diarrhea (non-bloody) to severe bloody diarrhea and post-acute syndromes also

• incubation period: 2-12 days (3-4 is most common): colonization of gut occurs

• abdominal pain, cramps is common; symptoms resolve typically within 7-10 days

• hemolytic uremic syndrome (HUS): most common in young children but can affect teens, young and older adults

• thrombotic thrombocytopenia (TPP): adults

  • low incidence; low disease risk

  • similar symptoms pf HUS but with bleeding in brain and other organs in body

• disease dose: ~10 CFU has been reported (generally thought <100 CFU)

• fatality is rare, but can occur in severe cases: estimated rate 1%

pathogenic elements, EHEC

• intimin and tir: allows for formation of pedestal-type intimate attachment of producing E. coli and other enterics

  • encoded genetically on locus of Enterocyte Effacement (LEE)

  • attaching/effacing (A/E) lesions: Effacement (removal) of GI microvilli

• 60 MDa plasmid (pO157): enterohemolysin, catalase

shiga toxins

• the STEC and EHEC produce stx1 and/or stx2

• either chromosome or phage-encoded

• multiple variant forms of stx2 spread throughout EHEC, EPEC, Citrobacter spp., and other Escherichia spp.'

  • human and livestock disease

  • 2e: pig edema

• holotoxin (protein): one A unit and 5 B-units

• binds to specific renal cell surface lipid receptor and is then imported

• induces failure of intoxicated cells to complete translation (protein synthesis) by halting tRNA successful binding in ribosomes- leads to cell death

pathogenic E. coli control

• proper cooking of foods (where applicable)

  • fully cooked meats, pasteurized dairy

  • ground, non-intact beef: 160 °F internal temperature (USDA recommended)

• other validated antomicrobial interventions to reduce, prevent growth

• wash produce with chlorinated water in home, restaurant

• good personal hygiene

• sanitation in process environment, restaurant

Salmonella enterica

• facultatively anaerobic regular rods, oxidase negative, mesophilic infectious/invasive pathogen

• catalase-negative, urease-negative, produce lysine decarboxylase, reduce H2S and precipitate sulfur in presence of iron to FeS

• can ferment glucose, but not lactose or sucrose; can use citrate as carbon source

• infectious/invasive pathogen of humans and animals

  • typhoidal salmonellae: S. Typhi, paratyphi

  • non-typhoidal salmonella

• some salmonella flagellated (peritrichous), but some non-flagellated (S. Gallinarum, S. Pullorum)

• adulterant in RTE foods, fully cooked meat/poultry (zero-tolerance)

• identified by CDC as #1 cause of human foodborne bacterial disease

disease incidence, outbreaks

• estimated ~1.4 million cases per year

• animal- and plant-derived foods

• numerous serovars involved in differing outbreaks

• globally spread but differing serovars tend to dominate in differing regions

• fresh and further-processed foods

Salmonella taxonomy

• two species with genus: S. enterica, S. bongori

• within S. enterica, six sub-species:

  • I: enterica

  • II: salamae

  • IIIa: arizonae

  • IIIb: diarizonae

  • IV: houtenae

  • VI: indica <3

• separated by likenesses in biochemical and genomic attributes

• serovars identified and differentiated (O, H, K antigens)

Salmonella serovars

• serological identification rests on differentiation of somatic, capsular, and flagellar antigens (Kaufmann-White scheme)

  • capsule: Typhi, Paratyphi, Dublin

  • flagellar: most salmonellae, but some non-flagellated

  • somatic: all (major, minor LPS components)

• serovars were often used as species names prior to revision in serotyping and nomenclature

• >2500 known serovars (and counting…)

reservoirs of salmonella

• livestock (meat, milk), poultry animals (poultry meat, eggs)

• identification of multiple other transmission vehicles/exposure sources:

  • irrigation water, drinking water

  • plant-derived foods

  • companion, livestock animals (feces, urine)

salmonella growth

• can grow optimally at 32-38 °C, bur reports of low and high-temperature growth also exist

  • prior cold, heat exposure can lead to adaptation of cell

  • no growth, but long-term survival in frozen foods reported

• can remain viabke in dehydration/low a_w foods for long time periods, resisting heat application (also can remain infectious)

• tolerance to acidity at pH 4.5 and alkali up to pH 9.5 (optimal at neutral pH)

  • acetic acid may be more functional vs. lactic acid

  • three genetically encoded acid tolerance systems allows increasing acid tolerance as lower pH environments encountered

    • mild pH reduction: ATP consuming homeostasis, log-phase ATR

    • moderate (pH 5.5-4.3): stationary phase inducible ATR

    • hard (pH 5.0-4.3): rpoS inducible system

• mild acid shock result in cross-protection to subsequent heat exposure (changes in membrane lipids, metabolic outputs, chaperone proteins activated to protect DNA, RNA, other critical systems)

foodborne Salmonellosis

• typically caused by non-typhoidal salmonellae: invasive pathogen

• incubation period ranges from 8-72 hr, with symptoms running 2-7 days (typically self-limiting)

• fever, abdominal pain, frequent diarrhea, vomiting, headache

• acute disease is more common, but fatalities occur, or post-acute systemic/chronic sequelae are reported

  • Reiter’s syndrome, reactive arthritis

  • septicemia or bacteremia may also occur

• antibiotics are not typically prescribed, and most cases require fluids, rest

typhoid fever (enteric fever)

• S. Typhi, Paratyphi A

• incubation period runs 1 week to 2 months; severe diarrhea and constipation can occur

• rash may occur; septicemia can occur

• asymptomatic carrier state can occur following decline of clinical symptoms (gallbladder infection sub-clinical)

  • Mary Mallon (Typhoid Mary)

pathogenesis, non-typhoidal Salmonella

• attachment of Salmonella to GI cells allows subsequent uptake into the GI cell (invasion)

• GI cell membrane ruffles, resulting in endocytosis (uptake/invasion) of Salmonella into GI epithelials

• may be followed by GI programmed cell death (Salmonella-directed)

• pathogen can also survive in phagocyte acidic phagosome (membrane-bound small compartment entrapping Salmonella)

• some serovars contain virulence plasmid(s)

• siderophores (Fe-acquisition from host); vaccines for animals have targeted these surface-located proteins

controlling Salmonella

• cooking: pathogen not excessively heat-tolerant

  • heat tolerance increased in low a_w environments

  • cooking of some produce items (e.g., nuts)

  • high-fat foods provide enhanced Salmonella protection (e.g., chocolate, peanut butter)

• sanitation, pathogen-reducing interventions on food animal carcasses, produce

• pre-harvest interventions: competitive exclusion bacteria, vaccinations, animal handling practices

• pathogen source tracking in live animal, food process environment

Shigella spp.

• regular rods, facultatively anaerobic mesophiles, oxidase-negative, non-motile

• unlike other enterics, not able to use lactose

• genus contains 4 species: S. dysenteriae, S. boydii, S. flexneri, and S. sonnei

• nearly identical to E. coli genetically

  • capacity of stx-carrying phages to infect E. coli from Shigella

  • difficulty human infectious disease; human are only reservoir of organism

  • cause of foodborne shigellosis (mild self-limiting disease) ranging to bloody acute diarrhea and bacillary dysentery (more severe, possibility fatal)

foodborne disease

• transmission via fecal/oral route following poor personal hygiene practices and spread of contaminated feces

• disease symptoms vary greatly between cases but generally include severe diarrhea (species-specific) and potential for heavy blood, mucosal stools (bacillary dysentery)

  • watery diarrhea + pathogen colonization of colon leads to mucous cells sloughing off

  • fatality rate varies with severity of disease; in dysentery fatality rate ranges 10-15%

• infective dose approximately 10-200 cells

• pathogen can survive for long periods in frozen food, allowing for distribution and retention of possible infectivity

Shigellosis

• incubation: 8-50 hr, with duration lasting 5-7 days in acute cases

• disease occurs most frequently in younger children (<6 years old)

• Shiga toxin produced by isolates; can produce HUS and other chronic sequelae

• fever occurs drives extensive GI colonization

• estimated as 6th most frequent cause of human foodborne disease (500,000 cases annually)

pathogenesis of Shigella

• enterotoxin production by bacterium

• invasion capacity, similar to pathogenic E. coli

• Shiga toxin production

• incubation of organism at physiological temperature induces invasion capabilities

prevention/control

• proper cooking of foods, refrigeration to prevent growth

• good personal hygiene of process facility employees

• FDA Food Code (2017 edt.) lists Shigella as reportable for restauranteurs and recommends restriction from food handling duties for at least 3 days post-exposure

Yersinia spp.

• family Enterobacteriaceae; infectious zoonotic pathogen; oxidase-negative regular rod that can oxidize/ferment glucose (facultative anaerobe)

• type species: Y. pestis (cause if bubonic, pneumonic plague) (named for A. Yersin who discovered agent as plague cause, 1894)

• 18 species and multiple subspecies

• foodborne species of concern in food safety: Y. enterocolitica, Y. pseudotuberculosis

• Y. enterocolitica exhibits psychotropics capacity with growth at 4 °C and survival in cooked foods enhanced over raw foods

  • cooking enhances nutrient utility to organism

  • fewer competitors

  • does not survive proper cooking so fresh and post-process contaminated foods at highest risk (pork and other animal-derived foods including shellfish)

• capable of growth from pH 4-10, with alkali tolerance high

Yersinia enterocolitica physiology, typing

• most isolates are Voges-Proskauer+ (indicates capacity to produce and metabolize acids into non-acidic metabolites)

• many isolates use D-xylose, trehalose but most cannot produce beta-D-glucosidase (releases glucose from oligomers)

• typing based on O-antigen (e.g., 4/O:3) and H-antigen profiles

• urease+

Yersiniosis

• typically acute diarrheal disease with higher incidence in young children (<5 years)

• symptoms in young children include diarrhea, vomiting, fever and abdominal pain

• older children may present with symptoms mirroring appendicitis

• infectious dose estimated 10^4-10^6 cells; incubation period ranges from 1-11 days post-consumption

• clinical symptoms may persist for few days up to 3 weeks

• chronic sequelae can occur, with reactive arthritis most commonly reported (other include hyperthyroidism, Graves’ disease, goiter, Hashimoto’s thyroiditis)

• bacteremia can lead to higher mortality rate (30-60%) post-acute disease

• CDC reported 1% of yersiniosis-identified outbreaks in US were caused by Y. pseudotuberculosis from 1996-2007

symptoms, diseases

• enterocolitis, pharyngitis

• autoimmune: arthritis, uveitis, myocarditis, Hashimoto’s, Grave’s Disease

• septicemia, pneumonia, endocarditis, peritonitis, meningitis, eye infections

sources of pathogenic Yersiniae

• CDC estimates ~117K cases per year with about 35-40 deaths

• disease fits definition of zoonoses (animal- human transmission)

• animal-derived foods (meat, milk, byproducts)- undercooked or raw

  • swine tonsils: carriage site for serotype 4/O:3 Y. enterocolitica

  • tripe, chitterlings, tongue may also be contaminated

• proper sanitation in processing, home can reduce contamination, spread to consumers

pathogenesis elements

• invasive pathogen (similar to Salmonella)- modulated by invasin protein and host cell for internalization

• enterotoxin production - role in disease unclear

• flagella- not produced in lab medium at physiological temperature but recovered from patients

• phospholipase production- hemolysis of blood cells

• urease production: acid protection

• pYV: plasmid-encoded protection factors against immunity and phagocytosis