"Studying multi-drug resistant Salmonella emergence and transmission in the Pacific Northwest and other WSU ZRU research activities".

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Surveillance for Salmonella subtypes shared by
human and bovine hostsMargaret A. Davis, DVM,
MPH, PhD
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Salmonellosis

• Caused by Salmonella enterica infection
• Illness includes gastroenteritis
• invasive disease in rare cases
• Overall incidence in U.S. 15/100,000
• FoodNet http//www.cdc.gov/mmwr/preview/mmwrhtml/
  
• Exposure route
• Foodborne
• ingestion of contaminated food, water
• Environmental exposures
• e.g. hand-to-mouth
• important in pediatric cases

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Jones TF, et al. Pediatrics. 2006
Dec118(6)2380-7.
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Salmonellosis

• S. enterica gt2,000 serovars
• Some serovars are host-specific
• Typhi human
• Dublin cattle
• Gallinarum poultry
• Cholerasuis swine
• Some multiple-host serovars
• Typhimurium
• Newport
• Enteritidis

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Nomenclature
Species
Genus

• Salmonella enterica
• Typhimurium
• DT104

Serovar
Phage type
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Methods for studying Salmonella molecular
epidemiology

• Serotype
• Phage type
• Antimicrobial resistance
• Plasmid profiles
• Insertion sequences
• Pulsed-field gel electrophoresis (PFGE)
• Multi-locus variable number of tandem repeats
  (VNTR) analysis (MLVA)

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Why do genotyping?
?
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Why do genotyping?
?
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TIME
X
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Country A
Country B
Transfer event 50 yrs ago
Lots of time for local genetic changes since
transfer
Gel of isolates collected over, say, 5 year span
Isolates from country A are all more closely
related to each other than to isolates from
country B and vice versa
Country
A A A A A A A B B
B B B B
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Salmonella Typhimurium epidemiology

• Periodic clonal replacement
• Regional, continental or global in scale
• Frequently multi-drug resistant
• Numerous examples
• DT10 Sens (Khakhria et al., Can J
  Microbiol198329(11)1583-8.)
• DT193 ACSSuT (Rowe et al. Vet Rec 105468, 1979)
• DT204 ACSSuT (Threlfall et al. Vet Rec 103438,
  1978)
• DT104 ACSSuT (Threlfall J Antimic Chemo 467,
  2000)

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Khakhria et al. Can J Microbiol1983
Nov29(11)1583-8.
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Threlfall EJ, Ward LR, Rowe B. Epidemic spread of
a chloramphenicol-resistant strain of Salmonella
typhimurium phage type 204 in bovine animals in
Britain. Vet Record 1978103438-440
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Threlfall et al. Br Med J 19802801210-1
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Salmonella Typhimurium DT104

• Best described and characterized
• Disseminated globally in the 1990s
• Characterized by resistance to ACSSuT
• Very clonal (PFGE profiles similar between very
  distant geographic sources)
• Successful expansion not explained by a
  resistance advantage

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Annual isolates of Salmonella Typhimurium at WADDL
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of S. Typhimurium infections that were DT104
- Helms et al. International Salmonella
Typhimurium DT104 infections, 1992-2001. EID
200511859-867
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Imbrechts et al. Vet Record 200014776-77.
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Salmonella surveillance project

• Real-time surveillance to detect emerging clones
• Doug Call Detection and characterization of
  phenotypic and genotypic traits for newly
  emergent zoonotic enteric pathogens
• Funded through NIH FWD-IRN contract

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Salmonella surveillance project

• Human-source isolates Washington State DOH PH
  Lab sends us all Salmonella isolates
• Animal-source isolates Washington Animal Disease
  Diagnostic Lab (WADDL), Phoenix Central Lab,
  occasional from DOH, and sampling for research
  studies
• All isolates were processed by the FDIU lab
• Disk-diffusion assay for resistance to 12
  antimicrobials
• Bank at -80 ºC

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Salmonella surveillance project

• Human and animal isolates were compared with
  respect to serovar and resistance pattern
• Those that matched were compared further using
  PFGE
• PFGE patterns analyzed in Bionumerics (Applied
  Maths,Sint-Martens-Latem, Belgium)

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Detection of newly emergent non-typhoid
Salmonella Typhimurium

• Distinct from DT104 by PFGE
• Resistant to 2 9 antimicrobials
• Washington PulseNet PFGE profile TYP035
• 7 of human Typhimuriums 2004-2005 in Washington

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TYP035
TYP187
TYP004
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TYP187 2004 2005 2006 2007
ACSSuTCaz 1
ACSSuTAmcCaz 21
AKSuTCaz 12 1
AKSSuT 1
AKSSuTCaz 1
AKSSuTAmcSxtCaz 1
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Owl-pellet associated outbreak, Massachusetts,
2006

• Elementary school students
• Caused by TYP035
• 46 primary (5th graders) and 12 secondary (K-4th)
  cases
• Source of owl pellets was a company in south
  central Washington State

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First detections of bovine-origin S. Typhimurium
TYP035 by location
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1999
2000
2005
2000
2001
2000
2006
2002
2000
2000
2001
2001
1999
2000
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First detections of bovine-origin S. Typhimurium
TYP187 by location
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TYP035/TYP187 in British Columbia

• From 2000 to present
• 36 TYP035
• Sixteen were from human cases
• The rest were primarily from bovine sources
• Other animal sources duck, wolf, buffalo and
  porcine.
•  1 bovine TYP187 (February, 2006)

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TYP035/TYP187 in British Columbia
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Conclusions

• TYP035/TYP187 may be a newly emerged epidemic
  clone of MDR Salmonella Typhimurium
• Currently not important in human population
• Apparently regional (Pacific Northwest)
• Primarily bovine reservoir
• Further characterization needed
• Resistance phenotypes v. genotypes

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MLVA

• Multi-locus variable number of tandem repeats
  analysis
• Based on polymorphisms at loci where tandem
  repeats occur

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Van Belkum et al. Microbiol. Mol Biol Rev
199862275-293
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Multi-locus variable number of tandem repeats
analysis (MLVA)

• Using published protocols (Lindstedt et al 2004)
• Five VNTR loci (STTR3, STTR5, STTR6, STTR9,
  STTR10pl)
• Mulltiplex PCR with dye-labeled primers followed
  by capillary electrophoresis
• Analysis of fragment size data
• Bionumerics (Applied Maths)
• STTR3
• 190/256 isolates lacked amplicon
• Passaging experiment
• 2 isolates X 95 wells X 30 passages
• At passage 30, 70/95 wells lacked STTR3
• Analysis using 4 VNTR loci

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MLVA
PFGE
Boiled lysate
PCR reaction
Capillary electrophoresis
Data
2 days
3 5 days
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MLVA using 4 loci of S Typhimurium from various
sources
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Acknowledgments

• FDIU
• Dale Hancock
• Katie Baker
• Russ McClanahan staff
• Bijay Adhikari

• ZRU
• Tom Besser
• Doug Call

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Acknowledgments

• WA DOH
• Kaye Eckmann
• Kathryn MacDonald
• David Boyle
• Cornell
• Lorin Warnick
• Martin Wiedmann
• Yesim Soyer
• BC CDC
• Linda Hoang

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Thank you!
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Other molecular characterizations of TYP035

• Plasmid Profiles
• 15/38 120 kb plasmid
• 23/38 had variable plasmid profiles
• plasmid profiling data did not correlate with
  resistance phenotype or SpeI banding variations
• Phage Typing
• Dr. Rafiq Ahmed at the National Microbiology
  Laboratory, Winnipeg, Manitoba, Canada)
• . All 31 TYP035 isolates were phage type
  untypable with standard Colindale panel of phages
• an additional set of phages were able to
  discriminate among some of them
• The resulting categories of untypable phage
  types (UT2, UT5, UT7 and UT8) were also not
  consistent with either plasmid profile or
  resistance phenotypes.