Title: "Studying multi-drug resistant Salmonella emergence and transmission in the Pacific Northwest and other WSU ZRU research activities".
1Surveillance for Salmonella subtypes shared by
human and bovine hostsMargaret A. Davis, DVM,
MPH, PhD
2Salmonellosis
- 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
3Jones TF, et al. Pediatrics. 2006
Dec118(6)2380-7.
4Salmonellosis
- 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
5Nomenclature
Species
Genus
- Salmonella enterica
- Typhimurium
- DT104
Serovar
Phage type
6Methods 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)
7Why do genotyping?
?
8Why do genotyping?
?
9TIME
X
10Country 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
11Salmonella 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)
12Khakhria et al. Can J Microbiol1983
Nov29(11)1583-8.
13Threlfall 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
14Threlfall et al. Br Med J 19802801210-1
15Salmonella 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
16Annual isolates of Salmonella Typhimurium at WADDL
17 of S. Typhimurium infections that were DT104
- Helms et al. International Salmonella
Typhimurium DT104 infections, 1992-2001. EID
200511859-867
18Imbrechts et al. Vet Record 200014776-77.
19Salmonella 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
20Salmonella 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
21Salmonella 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)
22(No Transcript)
23(No Transcript)
24Detection 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
25TYP035
TYP187
TYP004
26(No Transcript)
27(No Transcript)
28(No Transcript)
29(No Transcript)
30(No Transcript)
31TYP187 2004 2005 2006 2007
ACSSuTCaz 1
ACSSuTAmcCaz 21
AKSuTCaz 12 1
AKSSuT 1
AKSSuTCaz 1
AKSSuTAmcSxtCaz 1
32Owl-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
33First detections of bovine-origin S. Typhimurium
TYP035 by location
341999
2000
2005
2000
2001
2000
2006
2002
2000
2000
2001
2001
1999
2000
35First detections of bovine-origin S. Typhimurium
TYP187 by location
36(No Transcript)
37TYP035/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)
38TYP035/TYP187 in British Columbia
39Conclusions
- 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
40MLVA
- Multi-locus variable number of tandem repeats
analysis - Based on polymorphisms at loci where tandem
repeats occur
41Van Belkum et al. Microbiol. Mol Biol Rev
199862275-293
42Multi-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
43MLVA
PFGE
Boiled lysate
PCR reaction
Capillary electrophoresis
Data
2 days
3 5 days
44MLVA using 4 loci of S Typhimurium from various
sources
45(No Transcript)
46(No Transcript)
47(No Transcript)
48Acknowledgments
- FDIU
- Dale Hancock
- Katie Baker
- Russ McClanahan staff
- Bijay Adhikari
49Acknowledgments
- WA DOH
- Kaye Eckmann
- Kathryn MacDonald
- David Boyle
- Cornell
- Lorin Warnick
- Martin Wiedmann
- Yesim Soyer
- BC CDC
- Linda Hoang
50Thank you!
51Other 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.