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Food Microbiology:Good, Bad & Ugly

Microbial association on red smear cheese

• Cheese types: Münster, Romadour, Limburger, Harzer, Vacherin Mont d`Or, Tilsiter, Livarot,andmany others

• Bacteria:Arthrobacter nicotianum, Brevibacterium linens, Corynebacteriumammoniagenes, C. casei, C. variabile, Microbacterium gubbeenense,Rhodococcusfascians, Staphylococcus equorum, S. saprophyticus, and many others.

• Yeasts: Debaryomyces hansenii, Kluyveromycesmarxianus, Pichia membranaefaciens

• Fungi: Galactomyces geotrichum

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S. xylosus: fermented sausages, raw ham, read smear cheese (strains forming enterotoxin E were involved in human infections)

S. piscifermentans: fish sauce

S. condimenti: fish sauce

S. carnosus: fish sauce, ham, fermented sausages

S. equorum: horse skin, red smear cheese, fermented sausages, curing brines, raw ham

S. succinus ssp casei red smear cheese

S. warneri had been identified in French culturesS. saprophyticus

Staphylococci involved in food fermentation

The safe tradition in food fermentation of strains of certain Staphylococcus species confirms that within a taxonomically related group of organisms safe strains or even species exist. The same applies e.g. to enterococci, streptococci (S. thermophilus),Aspergillus oryzae ( used for koji production and closely related to A. flavus).

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FAO/WHO report, October 2001

Live microorganisms which when administered in adequate amounts confer a health benefit on the host.

Schrezenmeir and de Vrese 2001

A preparation of or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora (by implantation or colonization) in a compartment of the host and by that exert beneficial health effects in this host

Naidu et al. 1999 A microbial dietary adjuvant that beneficially affects the host physiology by modulating mucosal and systemic immunity, as well as improving nutritional and microbial balance in the intestinal tract

Schaafsma 1996 Living microorganisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition

Havenaar and Huis In’t Veld 1992

A viable mono- or mixed-culture of microorganisms which applied to animal or man, beneficially affects the host by improving the properties of the indigenous microcflora

Fuller 1989 A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance

Parker 1974 Organisms and substances which contribute to intestinal microbial balance

Lilly and Stillwell 1965

Substances produced by microorganisms which promote the growth of other microorganisms

Reference Published Definition

Live microorganisms which when administered in adequate amounts confer a health benefit on the host.

Probiotics - definitions

• Probiotics must be alive. • Probiotics must be safe.• Probiotics must deliver a measured physiological

benefit, substantiated by studies conducted in the target host.

• Probiotics needn’t be restricted to food applications or oral delivery.

• Probiotics used as pharmaceuticals or as topical agents are not excluded from this definition.

• A definition of probiotics shouldn’t limit the mechanism of action.

• Survival of gastrointestinal tract transit or impact on normal flora shouldn’t be required.

Probiotics – requirements

Sanders 2003

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Expected Benefits Associated with Consumption of Probiotics

•Increased tolerance to infections•Control of diarrhea•Reduction of blood pressure•Cholesterol reduction•Allergy control•Immunomodulation•Cancer reduction

Human probiotics

• Gastro-intestinal probiotics• Skin probiotics• Scalp probiotics• Probiotics of the oral cavity• Probiotics of the underarm and feet• Urogenital (including vaginal) probiotics

Human probiotics can be grouped according to the ecological niches of the human body they occupy (or the part of the body they deliver their health-promoting action to), such as:

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Lactobacilli Bifidobacteria other LAB non-LAB

L. acidophilus(L. casei)L. crispatusL. gallinaruma

L. gasseriL. johnsoniiL. paracaseiL. plantarumL. reuteriL. rhamnosus

B. adolescentisB. animalisB. bifidumB. breveB. infantisB. lactisb

B. longum

Ent. faecalisa

Ent. faeciumLactoc. lactisc

Leuconostoc mesenteroidesc

Ped. acidilacticic

Sporolactobacillusinulinusa

Strep. thermophilus

Bacillus cereus (‘toyoi‘)a, d

Escherichia coli (‘Nissle 1917‘)d

Propionibacteriumfreudenreichiia, d

Saccharomycescerevisiae(‘boulardii‘)d

Microorganisms in probiotic products

The Bulgarian farmer's longevity and healthy life are the result of the consumption of fermented dairy products.

E. Metchnikoff

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If the science of probiotics holds up in clinical trials,...

...yogurt could replace many pharmaceutical drugs.

http://www.usprobiotics.org/news/healing_power.htm

Fermented Vegetables

•sauerkraut •olives •cucumbers*•carrots •celery •beans •peas •corn •tomatoes

•peppers •onions •citron •beets •turnips •radishes •chard •Brussels sprouts•cauliflower

*Controlled fermentation

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Food Spoilage Microorganisms

• It is important to be able to distinguish food poisoning from food spoilage

• Food poisoning is when food is eaten which looks normal, smells normal and tastes normal: you eat enough to make you ill from the ingested pathogens or toxins

• Spoiled food does not normally cause food poisoning because it is rejected by the consumer before ingestion

Food Spoilage Test

• EGGS: When something starts pecking its way out of the shell, the egg is probably past its prime

• MAYONNAISE: If it makes you violently ill after you eat it, the mayonnaise is spoiled

• FROZEN FOODS: Frozen foods that have become an integral part of the defrosting problem in your freezer compartment will probably be spoiled -- (or wrecked anyway) by the time you pry them out with a kitchen knife

• MEAT: If opening the refrigerator door causes stray animals from a three-block radius to congregate outside your house, the meat is spoiled

• BREAD: Sesame seeds and Poppy seeds are the only officially acceptable"spots" that should be seen on the surface of any loaf of bread. Fuzzy and hairy looking white or green growth areas are a good indication your bread has turned into a pharmaceutical laboratory experiment

• FLOUR: Flour is spoiled when it wiggles• CANNED GOODS: Any canned goods that have become the size or shape

of a softball should be disposed of• POTATOES: Fresh potatoes do not have roots, branches, or dense, leafy

undergrowth• CHIP DIP: If you can take it out of its container and bounce it on the floor, it

has gone bad

http://science.ntu.ac.uk/external/foodmicrobiol.htm

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Food Spoilage TestGENERAL RULE OF THUMB

• Most food cannot be kept longer than the average life span of a goldfish.

• Keep a goldfish nearby your refrigerator to gauge this.

http://science.ntu.ac.uk/external/foodmicrobiol.htm

Microbial Food Spoilage

•Microbial growth in a food - faster•Release of enzymes - slower

•extracellular•intracellular (lysis)

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Microbial food spoilage: favorable conditions

• Transfer of the microorganisms into the food• Optimal environment: • AW, nutrients, atmosphere• “Proper” abuse of temperature• Sufficient time of storage

Microbial food spoilage: “first come – first serve”

•Bacteria•Yeast•Molds

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Microbial Food SpoilageChanges in Food Quality

• Odor: due to production of volatile end-compounds

• Color: pigment production or oxidation• Texture: softening due to the breakdown of

pectin in vegetables or the tissues by proteinases

• Accumulation of gas: CO2, H2 or H2S2

• Slime formation: due to the production of dextrans and/or amount of microorganisms

Foodborne diseases

• Food poisoning - Caused by preformed toxin in the food; organism may or may not be alive and growing; Clostridium botulinumand Staphylococcus aureus

• Food infection - Live cells delivered by contaminated food; organism multiply once food is ingested; Salmonella

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Pathogen Cases Hospitalizations Deaths Salmonella 1,350,000 16,000 560 Campylobacter 2,000,000 10,500 100 Shiga-toxin E. coli 110,000 950 26 Listeria 2,500 2,300 500 Norovirus 9,200,000 20,000 124 Toxoplasma 112,500 2,500 375

Public health burden of major foodborne infections

Mead et al., EID 5:707-25, 1999

The incidence of diagnosed foodborneinfections is a small fraction of total

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Staphylococcus aureus

• Gr+, non-motile, asporogenous cocci

• “grape-like” clusters• enterotoxin• effective at 1ug/kg• protein of 239 amino

acids• serological types: A, B, C,

D, F

Staphylococcus aureusFOOD POISONING SYNDROME

• onset: 0.5 to 6 hours• recovery: 24 to 72 hrs• major symptoms: vomiting, diarrhea• other symptoms: nausea, salivation,

cramps, retching,prostration

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Staphylococcus aureus case study

• Flight from Tokyo to Copenhagen via Anchorage• 196 of 343 passengers, 1 of 20 crew• food eaten:

1 h post Anchorage, snack, served to all5.5 h post Anchorage, steak dinner, crew5.6 h post Anchorage, cheese omelet w/ ham -passengers only

• onset: 0.5 to 5.5 h after breakfast, average 2.5 h

• symptoms:diarrhea (88%), vomiting (82%)cramps (74%), nausea (68%)

Staphylococcus aureus investigation

• Isolated SED-producing S. aureus from fecal swabs of 5 patients and from left over omelet and ham.

• Isolated SED from omelet and ham.• Breakfast prepared day before flight, held at

room temperature for 6 h during preparation• Held 14.5 h at 10°C prior to the flight.• Held at room temperature during the flight, then

heated.• Isolated SED-producing S. aureus from inflamed

lesion on the hand of the cook who had made the breakfast.

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Botulism (C. botulinum)

• The most potent toxin known; few cases but high mortality (25%); destroyed by 10 min in 80 oC

• Flacid paralysis of muscles (acetylcholine release is blocked)

• Common in soil and water• Improper canning -> spore germination -> toxin

production -> canned food used without cooking ->disease

• Infant botulism: consumption of honey that is contaminated by spores (0 - 2 months)

• Treatment: antitoxin and ventilation

Clostridium botulinum toxin

• 1 mld = 0.1ng• Dose of 3,000 mld (0.3 ug) will kill a 104 kg

person.• Cultures easily produce 105 to 106 mld per ml. • Very heat sensitive

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Botulism (C. botulinum)

Botulism and Temperature Abused Pot Pie(Morbidity and Mortality Weekly Report, Vol. 32, # 3, January 1983).

• Patient: 56 year old diabetic woman• Symptoms: diplopia, weakness, difficulty

breathing, chest pain, respiratory arrest.• Scenario: Son prepared pot pie for mother.

Father brought home take-out hamburgers. Mother left pot pie out on shelf (in California, in August), and ate it two and one-half days later without reheating.

• Illness: next day. Type A botulinum toxin was isolated from the left-over food and patient's serum.

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C. perfringens produces cpe gene product as paracrystalline inclusion body released during sporulation. Also makes beta-toxin (necrotic).14 different toxins and genes in species, but given strain carries a few.

cpe action - 35 kDa protein (tissue damage and permeability change):

1) receptor mediated binding w/claudin + 50 kDa protein; 2) forms larger complexes; 3) channel formation.

C. perfringens

32Overall

% positiveProduct

17Shrimp30Fish23Ground beef21Beef61Ground pork27Pork38Chicken28Turkey

Prevalence of C. perfringens

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Salmonellosis

• Gram-negative enteric bacterium; all strains are pathogenic; transmission is from sources (eggs, meats) and by food handlers

• Colonization of intestinal epithelium• Two diseases:

– Enterocolitis (most commonly by S. typhimurium): 105 - 108 viable cells; disease onset within 8 - 48 hrs; headaches, chills, vomiting, diarrhea and fever (2-3 days); continuous shading of organism for months/years; treatment - none

– Typhoid fever (Salmonella enterica serovar Typhi ): Septicemia leading to high fever that can last for several weeks; mortality is 15% if untreated; antibiotics

• Prevention: Cooked food (70 ◦C for 10 min); monitor for carrier state among food handlers

Salmonella Enteriditis: ice cream outbreak

• Nation-wide outbreak• 224,000 people developed gastroentiritis after

eating Schwan’s ice cream• Salmonella was isolated from 8 of 266 ice cream

products (3%)• Trailers were transporting non-pateurized eggs

immediately before carrying ice cream premix

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Escherichia coli• Gram-negative rod• non-sporeformer• Flagellated• facultative anaerobe• generally harmless, • pH: 6.0-8.0• Temp.:37oC• Aw: 0.94-0.97

Pathogenic E. coli• Some strains of E. coli; diarrhea and urinary tract

infection; classification of pathogens is based on toxin and diseases

• Enterohemorrhagic (O157:H7) - colonization of the small intestine and verotxin production -> hemorrhagic diarrhea and kidney infection; uncooked and undercooked ground meat; occasional epidemics

• Enterotoxigenic (Travelers diarrhea) - heat labile toxin; water and produce in developing countries; immunity

• Enteropathogenic - diarrhea that afflicts young children • Enteroinvasive - invasive colon infection; bloody

diarrhea; survival in phagosomes; in developing countries• Treatment and prevention: diseases are self-contained

but antibiotics help.

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E. coli O157:H7

• Japan, >9,000 cases, 7 deaths• Linked to school lunches• Radish sprouts?• Beef products of US origin?• Sick people discriminated against• School official blamed for outbreak

committed suicide

Campylobacter jejuni

• Characteristics– High morbidity, low mortality– Sensitive to freezing– Survives in refrigerated foods– will not grow at <30◦C– Microaerophilic– Fragile organism, sensitive to drying, heat,

acidic conditions, and disinfectants- So why is it such a problem?

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Campylobacter jejuni

• Disease– Onset 48-82 h following

ingestion– Profuse diarrhea– Invasive– Survives phagocytosis

Listeria monocytogenes

• Gram+, small, non-sporeforming rod• 1-2 X 0.5 μM Long.• Can Have "Y", "V", or even streptococcal

forms.• Motile at 20-25 °C, but Not at 37 °C.• Serotype Based on O & H Antigens.• Produces haemolysin.• Infective dose may be as low as 8 CFU/G.

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L. monocytogenes- disease syndromes

• Low grade "flu-like" infection - not serious, except in pregnant woman (who abort).

• Up to 16% women carry L. monocytogenes without illness. (Larmont and Postlethwaite, 1986, J. Infection 13:187-193.)

• Listeric meningitis- headache, drowsiness, coma. 50% fatality rate. If very young and old are excluded, this drops to 30%.

• Perinatal infection - 0.15% to 2.0% of all perinatalmortality.

• Encephalitis• Psychosis• Infectious mononucleosis• Septicemia

Los Angeles Mexican Soft Cheese

- 93 Perinatal cases:19 Still births10 Postnatal deaths

- 49 Adult cases:18 Deaths

- Linked with Mexican soft cheese- Same phage type isolated from cases and processing

plant- Plant's milk throughput exceeded capacity of

pasteurizer- Cheese was alkaline phosphate positive

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Incidence of reported cases and outbreaks of listeriosis in the United States, 1986-2004

April 15, 2005 / 54(14);352-356

Since 1996-98, significant decreases in infections with: Salmonella - 8% Campylobacter- 31% Listeria - 40% E. coli O157 - 42%

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They are not alivebut they are very bad!

Human Intestinal Viruses with High Potential as Food Contaminants

1. PicornavirusesPoliovirusesCoxsackieviruses A and BECHO virusEnterovirusHepatitis A

2. ReovirusesReovirusRotaviruses

3. ParvovirusesHuman gastrointestinal viruses

4. PapovavirusesHuman BK and JC viruses

5. AdenovirusesHuman adenoviruses types 1 - 33

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Genomyc and Physical Characteristics

Virus Family

Type

Nucleic Acid Description

Envelope

Name

enveloped Herpesviridae (EBV), Poxviridae

ds

nonenveloped Adenoviridae, Myoviridae, Tectiviridae, Papovaviridae

ss

nonenveloped Parvoviridae, Circoviridae

DNA

ds/ss

enveloped Hepadnaviridae – Hepatitis B

nonsegmented

enveloped Cystoviridae

ds

positive segmented nonenveloped Reoviridae

enveloped Coronaviridae, Flaviviridae (Hepatitis C, Yellow fever)

nonsegmented

nonenvelopedPicornaviridae (Hepatitis A), Caliciviridae (Hepatitis E, Norwalk)

positive reverse transcriptase enveloped Retroviridae (HIV)

RNA

ss

negative

nonsegmented

enveloped Paramyxoviridae (Measles, Mumps), Rhabdoviridae (Rabies)

Prions are small, proteinaceous infectious particles that resist inactivation by procedures which affect nucleic acids. To date, no detectable nucleic acids of any kind and no virus-like particles have been assoc iated with prions. Prions cause scrapie and otherspongiform encephalopathies of animals and humans.

Definition of Prions

http://www.mad-cow.org/~tom/prion_QuatStruc.html#Quaternary

http://igs-server.cnrs-mrs.fr/Prion/prusiner.html

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Prions are composed largely, if not entirely, of a protein designated as the scrapie isoform of the prionprotein, PrPSc.

A post-translational process, as yet undefined, generates PrPSc from the normal cellular isoform of the protein, designated PrPC.

Composition of Prions

Antibodies protect against prions in vivo

http://www.hfsp.org/pubs/Awards_articles/heppner2.htm

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Antibodies protect against prions in vivo

FDC = follicular dendritic cells

http://www.hfsp.org/pubs/Awards_articles/heppner2.htm

New pathogen x food combinations identified in outbreaks

• E. coli O157 and ground beef, apple cider, sprouts• Salmonella Enteritidis and eggs, broilers, almonds• Salmonella Poona and cantaloupe• MDR Salmonella Newport and ground beef, raw milk

cheese• Several Salmonellaserotypes and tomatoes• Listeria monocytogenes and sliced deli turkey, hot dogs,

Mexican queso fresco• Norovirus and raw oysters• Cyclospora and raspberries, basil• Hepatitis A and strawberries, green onions

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The new foodborne outbreak scenario

Old focal scenario:• Acute large local

outbreak• High dose, high attack

rate• Detected by group

themselves• Local investigation • A local food handling

error, often egregious• Local solution

New diffuse scenario:• Diffuse widespread

outbreak• Low dose, low attack rate• Increase in "sporadic"

cases• Detected by lab-based

subtype surveillance• Complex multistate

investigation• "Industrial contamination

event“earlier in food chain• Industry-wide implications

Why do "new" pathogens emerge?(Lynton Cox, Food Technol. 43(12)52-59, 1989)

• CHANGES IN EATING HABITS: fresh, organic• CHANGES IN AWARENESS: computer databases• CHANGES IN DEMOGRAPHICS: larger sensitive

populations• CHANGES IN PRIMARY FOOD PRODUCTION: scale

of operation, global production• CHANGES IN FOOD PROCESSING TECHNOLOGY:

chill, vacuum packaging• CHANGES IN HANDLING AND PREPARATION:home

refrigeration, microwave• CHANGES IN THE MICROORGANISMS:plasmid

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Physical, Chemical, and Biological Preservation Methods

IRRADIATED FOOD PRODUCTS

• Spices (EC Banned Ethylene Oxide in 1981. Irradiated Spices Do Not Require Specialized Labeling in the U.S.)

• Frog Legs in France• Fruit and Vegetables in South Africa• Strawberries in U.S (Consumers Prefer 20:1)• Nation's Pride (U.S.) Raw Chicken

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“Any chemical that when added to food, tends to prevent or retard deterioration, but does not include common salt, sugars, vinegars, spices, or oils extracted from spices, substances added to food by direct exposure to wood smoke, or chemicals applied for their insecticidal or herbicidal properties.”

FDA DEFINITION OF A CHEMICAL PRESERVATIVE FDA-CFR TITLE 21, p. 101.22 (A)

ANTIMICROBIAL CHEMICAL PRESERVATIVES

MAJOR

1. benzoates2. parabens3. propionates4. sorbates

OTHER

1. acidulants2. antioxidants3. nitrites4. sulfites

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Mechanism of ActionRCOO- + H+

RCOOH RCOO- + H+

RCOOH

pH 4.0 pH 7.0

RCOOH

RCOO- + H+

pH 7.0

H+

ATP

ADP + PiATPase

Bacteriocins are defined as ribosomally-produced proteinaceous substances of bacterial origin that exhibit antimicrobial activity.

They kill sensitive cells by forming pores in the membrane causing the leakage of cellular materials, and the depletion of transmembrane potential (Δψ) and/or the pH gradient.

Bacteriocins

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Multiple hurdle technology in food preservation