martin wiedmann department of food science 412 stocking hall cornell university phone: 607-254-2838...
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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”).
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
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
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
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
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**
*****
*
***
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
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