Martin Wiedmann

Department of Food Science

412 Stocking Hall

Cornell University

Phone: 607-254-2838

E-mail: mw16@cornell.edu

Microbial food safety issues – real Microbial food safety issues – real world problems that get undergrads world problems that get undergrads

excited about microbiologyexcited about microbiology

Foodborne diseasesFoodborne diseases

• Latest CDC estimates indicate the following annual burdens due to foodborne diseases in the US per year:• 325,000 serious illnesses resulting in

hospitalizations• 76 million cases of gastrointestinal illnesses• 5,000 deaths

Transmission of foodborne diseasesTransmission of foodborne diseases

Manure

Plant derived raw products

Humans

Animal feed/environment/protozoans

Food animals

Animal derived food products

Food Processing Plants

RTE Foods

SalmonellaSalmonella• About 1.4 million cases annually in the US resulting in

approx. 550 deaths• Mostly gastrointestinal syndrome, but also invasive

disease• Bioterrorism agent• Many different subtypes, e.g., Salmonella Typhi,

Salmonella Dublin, Salmonella Typhimurium• While cases caused by many foodborne pathogens

(Listeria, Campylobacter, E. coli O157:H7) have decreased over the years, Salmonella case numbers have not

• Apparent increase in Salmonella resistant to one or more antibiotics has been observed

Antibiotic resistant Antibiotic resistant SalmonellaSalmonella• In 2002, 22% of Salmonella Newport, 27% of

Salmonella Typhimurium isolates, and 8% of Salmonella Heidelberg displayed multi-drug resistance (MDR)• MDR Salmonella Typhimurium isolates

commonly shows two resistance type: (i) resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline; or (ii) resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline

Salmonella as bioterrorism agentSalmonella as bioterrorism agent

Torok TJ, Tauxe RV, Wise RP, Livengood JR, Sokolow R, Mauvais S, Birkness KA, Skeels MR, Horan JM, Foster LR. A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars.JAMA. 1997 Aug 6;278(5):389-95.

Listeria monocytogenesListeria monocytogenes

• Causes septicemia, abortion and encephalitis in humans and in animals

• Incubation period 7 - 60 days• Human listeriosis occurs in both epidemic and sporadic

cases• Affects predominantly elderly and immuno-

compromised people, pregnant women and newborns.• Approx. 2,500 human cases/year in the U.S., resulting in

350 - 450 deaths/year• Responsible for majority of microbial food recalls

E. coliE. coli O157:H7/Enterohemorhagic O157:H7/Enterohemorhagic E. coliE. coli (EHEC) (EHEC)

• Severe human disease transmitted through many foods, including undercooked meat, raw milk, apple cider, raw vegetables, etc.• Also transmitted by other means including drinking

water, direct animal - human transmission • Approx. 60,000 human cases and 50 deaths annually

in US• Symptoms include bloody diarrhea, hemolytic

uremic syndrome (kidney damage)• Children are particularly at risk

NorovirusesNoroviruses• Noroviruses are most common cause of acute gastroenteritis in

the United States and are believed to be the most common cause of food borne illnesses (23 million total cases annually, 9.2 million foodborne)• Often associated with infections/outbreaks in “crowding

situations” (cruise ships, homes for elderly etc.)• Noroviruses cannot be grown in the laboratory

• Studies on heat resistance and disinfection strategies are difficult to perform

• Detection in foods and infected humans requires electron microscopy or molecular biology approaches

• Sources are most likely humans with infection that prepare or handle foods• Virus appears to be able to survive on surfaces for days

Norwalk-like viruses: When the runs Norwalk-like viruses: When the runs can slow you down can slow you down

• In January 1999, an outbreak of viral gastroenteritis affected more than 300 people who attended a metropolitan concert hall over a 5-day period.

• The first case was a concert attendee who vomited in the auditorium and adjacent male toilet• Gastrointestinal illness occurred among members of 8/15 school

parties who attended the following day. • Children who sat on the same level of the auditorium as the first

case were much more likely to be ill than those seated elsewhere • Transmission most likely occurred through direct contact with

contaminated fomites (“any inanimate object or substance capable of absorbing, retaining, and transporting contagious or infectious organisms from one individual to another”).

Case study – human listeriosis Case study – human listeriosis outbreakoutbreak

Human listeriosis cases in NYS Human listeriosis cases in NYS 1/97-10/981/97-10/98

0

1

2

3

4

5

6

7

8

Jan

Mar

May Ju

lSep Nov Ja

nM

ar Jun

Aug Oct

Ribotyping results - November 8, 9 pm

Ribotyping results - November 8, 12 pm

Epidemic curve for 1/97 - 2/99 in NYSEpidemic curve for 1/97 - 2/99 in NYS

0

1

2

3

4

5

6

7

8

Jan

MarMay Ju

lSep Nov Ja

nMar Ju

nAug Oct

Dec Feb

1044A

Other Ribotypes

Follow-Up investigations by Follow-Up investigations by NYSDOH, Cornell University, CDC NYSDOH, Cornell University, CDC and other state health departmentsand other state health departments

Time Total isolates DUP-1044A

Connecticut 8/98-1/99 14 1Ohio 8/98-1/99 35 20NY City DOH 8/98-1/99 21 3South Carolina 8/98-12/98 3 1Michigan 8/98–12/98 2 2Massachusetts 8/98-1/99 10 5Oregon 8/98-12/98 6 1Vermont 8/98-12/98 2 2Arizona -1/98 11 8

Subtyping of human isolates - Subtyping of human isolates - summarysummary

• More than 100 human listeriosis cases and 21 deaths in more than 15 states; all caused by the identical DNA subtype

The outbreak source?The outbreak source?

• Patients included in this outbreak were significantly more likely to have eaten hot dogs then controls

• November: matching subtypes found in opened packages of deli meats collected in retail (including deli meats produced by Sara Lee) by NYSAGM

• Middle of December: L. monocytogenes isolated from opened package of hot dogs from affected patient; subsequent tests reveal L. monocytogenes DUP-1044A contamination of unopened packages

• December 22, 1998: Sara Lee announces recall of hot dogs and other packaged meats produced at its Bil Mar Foods unit in Zeeland, MI

DNA fingerprinting methodsDNA fingerprinting methods

Molecular characterization and Molecular characterization and taxonomytaxonomy

AA

AC

CC

TT

TG

GG

AAACCCTTTGGG

AAACCCTTTGGG

700 kb

600 kb

1200 kb

AA

AT

CC

TT

TA

GG

AAACCCTTTGGG

AAACCCTTTGGG

1300 kb1200 kb

Gel Electrophoresis (separates DNA pieces by size)

Bacterium #1 Bacterium #2

Bact #1

Bact #2

PCR-RFLP characterization of PCR-RFLP characterization of hlyhly

• PCR amplification of complete hly ORF, followed by restriction enzyme digest using HhaI and Hpa II

• Differentiates 8 different hlyA alleles

M 1 2 3 4 5 6 7 8 9 10

Uneven lanes:HhaIEven lanes: HpaII

Examples of differentExamples of different L. monocytogenesL. monocytogenes ribotypes ribotypes

PFGE TypesPFGE TypesPFGE TypesPFGE Types

CCCC CCCC CCCC

DNA sequencing-DNA sequencing-based subtypingbased subtyping

Isolate 1 AACATGCAGACTGACGATTCGACGTAGGCTAGACGTTGACTGIsolate 2 AACATGCAGACTGACGATTCGTCGTAGGCTAGACGTTGACTGIsolate 3 AACATGCAGACTGACGATTCGACGTAGGCTAGACGTTGACTGIsolate 4 AACATGCATACTGACGATTCGACGAAGGCTAGACGTTGACTG

2289

j2-045

j1-038

L99

j2-068

j2-003

10403S

j1-047

c2-006

n1-064

c2-008

dd680

c2-011

n1-067

n1-079

92

92

92

88

75

92

92

92

75

91

89

Use of “DNA fingerprinting” to Use of “DNA fingerprinting” to control Listeria in food processing control Listeria in food processing

plantsplants

Sample Source

*

VISIT 2

VISIT 3

VISIT 1

****

**

*****

*

***

Sample Ribotype Sample Source RiboPrint® Pattern

1039C (E) Floor drain, raw materials area1039C (E) Floor drain, hallway to finished area1039C (IP) Troll Red King Salmon, in brine, head area1039C (IP) Troll Red King Salmon, in brine, belly area1039C (IP) Brine, Troll Red King Salmon1039C (IP) Faroe Island Salmon, in brine, head area1039C (F) Smoked Sable1039C (F) Cold-Smoked Norwegian Salmon1044A (E) Floor drain, brining cold room 11044A (R) Raw Troll Red King Salmon, head area1044A (IP) Brine, Faroe Island Salmon1045 (R) Raw Troll Red King Salmon, belly area1045 (IP) Faroe Island Salmon, in brine, head area1053 (IP) Norwegian Salmon, in brine1062 (E) Floor drain #1, raw materials preparation1039C (E) Floor drain #1, raw materials preparation1039C (E) Floor drain, brining cold room 11039C (E) Floor drain #2, raw materials preparation1039C (E) Floor drain #2, raw materials receiving1039C (E) Floor drain, finished product area1039C (E) Floor drain, hallway to finished area1039C (IP) Brine, Troll Red King Salmon1039C (F) Smoked Sable1044A (IP) Sable, in brine1044A (IP) Brine, Faroe Island Salmon1062 (IP) Brine, Norwegian Salmon

Subtyping Results – seafood Subtyping Results – seafood processing plantprocessing plant

VISIT 4

VISIT 5

*

*

*

*

**

*

Sample Ribotype Sample Source RiboPrint® Pattern

1039C (E) Floor drain #1, raw materials preparation

1039C (E) Floor drain #1, raw materials receiving

1039C (IP) Brine, Atlantic Salmon

1039C (F) Cold-smoked Salmon trimmings

1062 (E) Floor drain #2, raw materials receiving

1044A (IP) Troll Red King Salmon, in brine

1048 (E) Floor drain #2, raw materials preparation

1052 (F) Smoked Sable

1053 (R) Raw Atlantic Salmon, in spawn

1053 (IP) Atlantic Salmon, in brine, head area

1053 (IP) Atlantic Salmon, in brine, belly area

1062 (E) Floor drain, brining cold room

1039C (E) Floor drain #2, raw materials preparation

1039C (E) Floor drain #2, raw materials receiving

1039C (F) Smoked Sea Bass

1042B (E) Floor drain #1, raw materials preparation

1042C (IP) Salmon-Trout, in brine

1044A (F) Smoked Sable

1062 (E) Floor drain #2, finished product area

1062 (E) Floor, finished product freezer

1062 (E) Floor drain #1, raw materials preparation

Subtyping Results - cont.Subtyping Results - cont.

L. monocytogenesL. monocytogenes persistence in plants persistence in plants

Samples

Plant B

n=129

Plant C

n=173

Plant D

n=229

P-value

Ribotype % Prevalence

1039C 0.0 0.0 10.0 0.0000

1042B 0.8 1.2 0.4 0.8221

1042C 6.2 0.6 0.4 0.0003

1044A 0.0 2.3 3.1 0.1494

1045 5.4 0.0 0.9 0.0006

1046B 0.0 2.3 0.0 0.0144

1053 0.0 0.6 1.7 0.2686

1062 0.8 0.6 2.6 0.1822

2000 US outbreak - Environmental 2000 US outbreak - Environmental persistence of persistence of L. monocytogenes? L. monocytogenes?

• 1988: one human listeriosis case linked to hot dogs produced by plant X

• 2000: 29 human listeriosis cases linked to sliced turkey meats from plant X

Ideas for food micro activities for Ideas for food micro activities for undergradsundergrads

• Brainstorm about (1) what microorganisms cause foodborne illness (and how much), (2) what foods cause foodborne illness (and why), and (3) how can you reduce your personal risk of foodborne illness

• Use current examples (ProMed; FSNet)• Lab activities (stage outbreak investigation):

• Can include simple (detection with selective media) to fairly complex (DNA sequencing with bioinformatics) lab activities

Food Micro resourcesFood Micro resources

• FSNet: http://www.foodsafety.ksu.edu• ProMed: http://www.promedmail.org• The PathogenTracker game:

http://scicentr1.tc.cornell.edu/pathogentrackergame/Intro/introduction/welcome.htm

• FightBac: http://www.fightbac.org/• Encyclopedia of Food Microbiology (can be made

available on-line through institutional libraries)• Food Microbiology: an Introduction (ASM Press)• Food Microbiology: Fundamentals and Frontiers –

more advanced (ASM Press)

• Modern Food Microbiology (CHIPS)

• Food Microbiology Laboratory (Taylor & Francis Ltd)

Summary and conclusionsSummary and conclusions

• Foodborne disease affects everyone and foodborne disease burdens in US and internationally are not trivial• Foodborne disease is a highly suitable topic for

introducing undergrads to many/all concepts in microbiology

• Undergrad food micro education can be as simple and as complex as you wish