Salmonella Enteritidis for Genetic Similarity Using Multiple Locus Variable Number Tandem Repeat Analysis MLVA Andrew Chang Public Health Internship Program School of Biological Sciences University of Texas at Austin ID: 675306
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Examining Past Isolates of Salmonella Enteritidis for Genetic Similarity Using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA)
Andrew ChangPublic Health Internship ProgramSchool of Biological SciencesUniversity of Texas at Austin
Mentors: Aaron Benfield, Ph.D and Tori A. PonsonMolecular Biology DepartmentTexas Department of State Health ServicesSlide2
INTRODUCTIONSlide3
SalmonellaFamily: Enterobacteriaceae
Gram-negative, rod-shaped bacilli, facultative anaerobesNon-spore formingPeritrichous flagella ChemoorganotrophsSlide4
Species and Subspecies
SpeciesS. bongori S. entericaSubspecies of S. enterica
S. enterica (I)S. salamae (II)S. arizonae (IIIa
)
S.
diarizonae
(
IIIb
)
S. houtenae (IV)S. indica (VI)Over 2300 serotypes of Salmonella Slide5
Gastroenteritis (Salmonellosis)
SymptomsGI tract inflammation, bloody diarrhea, fever, vomiting, abdominal cramps 12-72 hours after infection, severe dehydrationIllness lasts 4-7 daysPathogenesisReaches GI tract, enters enterocytes and M-cells, replicates in the lamina propria
Can disseminates via the lymphatic system septicemia Slide6
Modes of Transmission Fecal-oral route
Consumption of contaminated foodsShell eggs, peanut butter, tomatoes, spinach, alfalfa sproutsExposure to animalChicken, reptiles, amphibians, cats, dogsSlide7
Modes of TransmissionSlide8
Bioterrorism1984 use of Salmonella Typhimurium on local restaurants in Oregon
Rajneeshee cult group attempt to incapacitate voters in upcoming electionOver 750 people infectedSlide9
Salmonella Foodborne Illnesses2
nd most common food borne illness Estimated > 1 million cases each yearOnly 40,000 reported due to milder symptomsEach year20,000 cases result in hospitalization350-500 deathsSlide10
2010Shell eggs-
S. Enteritidis2009Peanut butter- S. Typhimurium2008Tomatoes, jalapeno and serrano peppers- S.
Saintpaul2007Pot pies- Salmonella 4,[5], 12:i-:
Food Related Outbreaks Slide11
2010 Salmonella Egg Outbreak
Number of
Salmonella
Enteritidis
Cases Matching PFGE Pattern JEGX01.0004 Reported to
PulseNet
, United States, 2010Slide12
Outbreak Source Epidemiological investigations
Wright County Egg Hillandale FarmS. Enteritidis isolated from: egg water wash, chicken feed, manure, and walkwaysSlide13
Outbreak Aftermath Voluntary recall of ~500 million eggs
May-November: 3,578 illnesses reported1,639 illnesses expected based on past 5 year averagesEstimated 1,939 illnesses associated with outbreakSlide14
Outbreak Impact in Texas “Here’s the issue: in terms of Salmonella, it’s multiplied fourfold in Texas – a large number of increases” – Zachary Thompson Dallas County Health and Human Services Director
More than 150 S. Enteritidis infections in over 40 different countiesSlide15
TDSHS Molecular Biology Dept. Response
Pure isolates received
Isolates processed
PFGE performed
PFGE pattern assigned
Clusters reported to epidemiologistsSlide16
Molecular Subtyping of Outbreak StrainsTwo common techniques
Pulse field gel electrophoresis (PFGE)Multiple locus variable number tandem repeat analysis (MLVA)Slide17
Pulsed-Field Gel Electrophoresis (PFGE)Slide18
Multiple Locus Variable Number Tandem Repeat
Analysis (MLVA)Locus 1`Strain A: VNTR 4x3atgggtaatccgtcgACgCACgC
ACgCgccaatcgatacgatStrain B: VNTR 4x5atgggtaatccgtcgACgCACgCACgCACgC
ACgC
gccaatcgatacgat
Trees, Eija. PulseNet MLVA protocols – General overviewSlide19
PFGE vs MLVA
Pulsed-Field Gel Electrophoresis
Multiple Locus Variable Number Tandem Repeat Analysis
Large fragments (2-10 Mb) can be resolved
Current CDC “gold standard” for molecular subtyping
Limitations: requires precise standardization, discriminatory power lacking against highly clonal organisms
Differentiates based on repeat sequences in the DNA –superior discriminatory power
Not widely used
Limitations: requires knowledge of organism DNA sequence; results not always reproducible between labs using difference DNA sequencersSlide20
PurposeThe purpose of this project was two-fold:
to analyze patient demographics and isolate characteristics to analyze genetic similarity of past Salmonella Enteritidis isolates sent to TDSHS from 12/09 – 12/10 using multiple locus variable number tandem repeat analysis (MLVA)Slide21
METHODSSlide22
Overview of Methods
Cultured
Salmonella
Enteritidis isolates
Extracted DNA from isolates
Amplified purified DNA via multiplex-PCR
Performed MLVA on amplified DNA
Raw data imported into BioNumerics software
Calculated dendogram showing genetic similarities Slide23
Preparation of IsolatesSamples kept in freezer @ -70O
CSamples grown on BHI platesInoculated into BHI brothIncubated @ 37OCSlide24
Extraction MethodsManual
Blood & Tissue Kit by QiagenAutomatedMagNA Pure Compact DNA Isolation Kits by RocheSlide25
Multiplex-PCR AmplificationSlide26
MLVALoaded dilute amplified DNA product onto 96-well plates
Sequenced samples using the Beckman Coulter CEQ 8000 sequencerExamined fragment sizesSlide27
DataSlide28
Data Processing Imported peak file into
BioNumerics SEMLVA databaseEstablished phylogenetic relationships between isolates based on VNTR copy numbers Slide29
RESULTSSlide30
Patient DemographicsSlide31
Patient Gender Distribution
N=224Slide32
Age Distribution of PatientsSlide33
Isolate InformationSlide34
Salmonella Enteritidis Isolates
224
Isolates
51
Egg-outbreak related
173
Egg-outbreak
unrelated
3
NC-outbreak relatedSlide35
Source of Salmonella Enteritidis Isolates Sent to TDSHS, 12/09-12/10Slide36
Distribution of Specimens by County, 12/09-12/10
Counties with < 4 isolatessentCounties with > 4 isolates sentSlide37
Top 10 Counties Submitting > 4 Salmonella Enteritidis Isolates to TDSHS, 12/09- 12/10
CountyNo. (%) of specimens submittedDallas81 (42%) Travis52 (27%)
Bexar12 (6%)Bell11 (5.7%)Tarrant10 (5%)Lubbock9 (4.7%)
Williamson
6 (3.1%)
Collin
4 (2%)
Nueces
4 (2%)
Nacogdoches
4 (2%)Total193 (100%)Slide38
Distribution of Isolates
N=224
Number of
Salmonella
Enteritidis
Cases Matching PFGE Pattern JEGX01.0004 Reported to
PulseNet
, United States, 2010
Isolation Dates of
Salmonella
Enteritidis
Isolates Sent to TDSHS 12/09-12/10Slide39
PFGE and MLVA ResultsSlide40
Salmonella Enteritidis Isolates
224
Isolates
51
Egg-outbreak related
173
Egg-outbreak
unrelated
3
NC-outbreak relatedSlide41
Genetic Similarity Between S. Enteritidis Isolates with Outbreak Code 1006TXJEG-1 Using PFGESlide42
Genetic Similarity Between S. Enteritidis Isolates with Outbreak Code 1006TXJEG-1 Using MLVASlide43
Salmonella Enteritidis Isolates
224
Isolates
51
Egg-outbreak related
173
Egg-outbreak
unrelated
3
NC-outbreak relatedSlide44
Genetic Similarity Between S. Enteritidis Isolates with Outbreak Code 1005NCJEG-1c Using MLVASlide45
Salmonella Enteritidis Isolates
224
Isolates
51
Egg-outbreak related
173
Egg-outbreak
unrelated
3
NC-outbreak relatedSlide46
Genetic Similarity Between S. Enteritidis Isolates with Outbreak Code 1005NCJEG-1c and Non-outbreak Associated Isolates Using MLVASlide47
Salmonella Enteritidis Isolates
224
Isolates
51
Egg-outbreak related
173
Egg-outbreak
unrelated
3
NC-outbreak relatedSlide48
Genetic Similarity Between
S. Enteritidis Isolates with Outbreak Code 1005NCJEG-1c and Outbreak Code 1006TXJEG-1Slide49
CONCLUSIONSSlide50
Conclusions: Patient DemographicsPatient gender
No gender preferencePatient age groups0-20 = 33%0-10 = 18% 21-50 = 33%51-70+ = 28%Slide51
Conclusions: Isolate InformationCounties
3 counties in the Dallas/Ft. Worth area- 49%2 counties in the Austin area- 30%Isolation date45% isolated in June; corresponded with peak number of cases reported nationally Isolate sourceStool samples – 91%Slide52
Conclusions: MLVA ResultsMLVA-
greater power of differentiation than PFGEIsolates associated with same outbreakGenetically identical by PFGEFurther differentiated by MLVA into 3 distinct clustersMLVA useful to demonstrate genetic relatedness of isolates associated with different outbreaks in NC and TXSlide53
LimitationsSubjectivity of MLVA
Replication of dataTimeCostly reagents/equipmentSlide54
Future Studies
Furthur analyze isolates not assigned outbreak codeWere they part of 2010 egg outbreak in TX?Compare MLVA results with other known MLVA results egg outbreak-related isolatesSlide55
AcknowledgementsTDSHS
Tori PonsonAaron Benfield, Ph. DUT AustinLeanne Field, Ph. DDiane Kneeland, Ph. DNancy Elder, MLS
THANK YOU!Slide56
THANK YOU!Funding generously provided by an Association of Schools for Public Health/Association of Public Health Laboratories “Pathways to Public Health Careers and Internships Grant” From The Centers for Disease Control and Prevention