Food Microbiology: Good, Bad & Ugly

Microbial association on red smear cheese

Cheese types: Mnster, Romadour, Limburger, Harzer, Vacherin Mont d`Or, Tilsiter, Livarot,and many others Bacteria:Arthrobacter nicotianum, Brevibacterium linens, Corynebacterium ammoniagenes, C. casei, C. variabile, Microbacterium gubbeenense,Rhodococcus fascians, Staphylococcus equorum, S. saprophyticus, and many others. Yeasts: Debaryomyces hansenii, Kluyveromyces marxianus, Pichia membranaefaciens Fungi: Galactomyces geotrichum

Staphylococci involved in food fermentation

S. xylosus: fermented sausages, raw ham, read smear cheese (strains forming enterotoxin E were involved in human infections) fish sauce fish sauce fish sauce, ham, fermented sausages horse skin, red smear cheese, fermented sausages, curing brines, raw ham red smear cheese had been identified in French cultures

S. piscifermentans: S. condimenti: S. carnosus: S. equorum:

S. succinus ssp casei S. warneri S. saprophyticus

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).

Probiotics - definitions
Published Definition Substances produced by microorganisms which promote the growth of other microorganisms Organisms and substances which contribute to intestinal microbial balance A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance 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 Living microorganisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition Reference Lilly and Stillwell 1965 Parker 1974 Fuller 1989 Havenaar and Huis Int Veld 1992 Schaafsma 1996 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

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

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

Schrezenmeir and de Vrese 2001 FAO/WHO report, October 2001

Probiotics requirements
Probiotics must be alive. Probiotics must be safe. Probiotics must deliver a measured physiological benefit, substantiated by studies conducted in the target host. Probiotics neednt 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 shouldnt limit the mechanism of action. Survival of gastrointestinal tract transit or impact on normal flora shouldnt be required.
Sanders 2003

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

Microorganisms in probiotic products

Lactobacilli L. acidophilus (L. casei) L. crispatus L. gallinaruma L. gasseri L. johnsonii L. paracasei L. plantarum L. reuteri L. rhamnosus Bifidobacteria B. adolescentis B. animalis B. bifidum B. breve B. infantis B. lactisb B. longum other LAB Ent. faecalisa Ent. faecium Lactoc. lactisc Leuconostoc mesenteroidesc Ped. acidilacticic Sporolactobacillus inulinusa Strep. thermophilus non-LAB Bacillus cereus (toyoi)a, d Escherichia coli (Nissle 1917)d Propionibacterium freudenreichiia, d Saccharomyces cerevisiae (boulardii)d

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

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
*Controlled fermentation

peppers onions citron beets turnips radishes chard Brussels sprouts cauliflower

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 threeblock 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

Food Spoilage Test GENERAL 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)

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

Microbial Food Spoilage Changes in Food Quality

Odor: due to production of volatile endcompounds 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 botulinum and Staphylococcus aureus Food infection - Live cells delivered by contaminated food; organism multiply once food is ingested; Salmonella

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Public health burden of major foodborne infections

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

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

The incidence of diagnosed foodborne infections 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 aureus
FOOD POISONING SYNDROME onset: recovery: major symptoms: other symptoms: 0.5 to 6 hours 24 to 72 hrs vomiting, diarrhea 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 all 5.5 h post Anchorage, steak dinner, crew 5.6 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 10C 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
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.

Prevalence of C. perfringens
Product Turkey Chicken Pork Ground pork Beef Ground beef Fish Shrimp Overall % positive 28 38 27 61 21 23 30 17 32

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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 Schwans 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 <30C 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 M 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 perinatal mortality. Encephalitis Psychosis Infectious mononucleosis Septicemia

Los Angeles Mexican Soft Cheese

- 93 Perinatal cases: 19 Still births 10 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

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

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

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They are not alive but they are very bad!

Human Intestinal Viruses with High Potential as Food Contaminants

1. Picornaviruses Polioviruses Coxsackieviruses A and B ECHO virus Enterovirus Hepatitis A 2. Reoviruses Reovirus Rotaviruses 3. Parvoviruses Human gastrointestinal viruses 4. Papovaviruses Human BK and JC viruses 5. Adenoviruses Human adenoviruses types 1 - 33

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Genomyc and Physical Characteristics

Type Nucleic Acid Description Envelope

Virus Family
Name
Herpesviridae (EBV), Poxviridae Adenoviridae, Myoviridae, Tectiviridae, Papovaviridae Parvoviridae, Circoviridae Hepadnaviridae Hepatitis B Cystoviridae Reoviridae Coronaviridae, Flaviviridae (Hepatitis C, Yellow fever) Picornaviridae (Hepatitis A), Caliciviridae (Hepatitis E, Norwalk) Retroviridae (HIV) Paramyxoviridae (Measles, Mumps), Rhabdoviridae (Rabies)

enveloped ds nonenveloped DNA ss ds/ss ds positive nonsegmented segmented nonsegmented positive ss negative
reverse transcriptase

nonenveloped enveloped enveloped nonenveloped enveloped nonenveloped enveloped enveloped

RNA

nonsegmented

http://www.mad-cow.org/~tom/prion_QuatStruc.html#Quaternary http://igs-server.cnrs-mrs.fr/Prion/prusiner.html

Definition of Prions

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 other spongiform encephalopathies of animals and humans.

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Composition of Prions
Prions are composed largely, if not entirely, of a protein designated as the scrapie isoform of the prion protein, PrPSc. A post-translational process, as yet undefined, generates PrPSc from the normal cellular isoform of the protein, designated PrPC.

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

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.

ANTIMICROBIAL CHEMICAL PRESERVATIVES

MAJOR 1. benzoates 2. parabens 3. propionates 4. sorbates OTHER 1. acidulants 2. antioxidants 3. nitrites 4. sulfites

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Mechanism of Action
RCOOpH 4.0 RCOOH RCOO+ + H+ RCOOH pH 7.0 H+

RCOOH RCOO+

pH 7.0 H+ ATP
ADP + Pi

ATPase

H+

Bacteriocins
Bacteriocins are defined as ribosomallyproduced 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 () and/or the pH gradient.

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Multiple hurdle technology in food preservation

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