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Introduction
The emergence of a new phage type of
Salmonella Typhimurium in humans and
animals in New Zealand
• New Zealand is a
geographically isolated
country, similar in size to
Great Britain, with a
population of 4 million
M Dufour
AIMS NZIMLS South Pacific Congress
Gold Coast, August 2011
• The economy has a
strong agricultural and
pastoral focus
Specialist Science Solutions
Manaaki Tangata Taiao Hoki
protecting people and their environment through science
Background
© ESR 2011
NCBID
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Joint initiative from:
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foodborne disease in NZ estimated at
$86 billion, 90% attributed to the lost of
productivity due to the absence from
work
Salmonellosis is a worldwide public
health problem
National surveillance of Salmonella is
carried out at ESR’s NCBID
Wallaceville Centre
National Centre for Biosecurity and
Infectious Diseases
http://www.ncbid.govt.nz/
© ESR 2011
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MAF Biosecurity New Zealand (Ministry of Agriculture and Forestry)
- (Environment, flora, fauna, marine life, Maori resources)
AgResearch’s Infectious Disease diagnostic team (Animal Health)
ESR (People and their Environment)
AsureQuality (Food safety)
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Approximately 90 microbiologists, molecular biologists, disease modellers,
epidemiologists and researchers
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Access to the only infectious disease laboratory that provides a PC3+ level of
containment in NZ
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Increases NZ’s capability to protect human and animal health
© ESR 2011
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Salmonella
Microbiology Laboratory
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Enteric Reference Laboratory
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Provides national reference and surveillance laboratory services
Human, animal, food and environmental enteric bacterial pathogens.
Salmonella, Shigella, Yersinia, toxigenic Escherichia coli (VTEC), Vibrio and
Campylobacter.
Identification and characterisation using standard methods: Biochemical
profiling (metabolic activity), serology, phage typing, biotyping and
molecular typing (PCR and PFGE)
Reference Laboratory: Only lab in NZ with the capability to identify and type
enteric bacterial pathogens
Surveillance
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Screening and confirmatory tests for common Leptospira serovars found in
NZ
© ESR 2011
Serotyping of Salmonella
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Serotyping useful tool for surveillance
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Robust and comparable world wide
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Based on identifying the O antigen on
bacterial cell wall (lipopolysaccharide) and H
antigen on flagella (protein).
Salmonella genus has two species:
- S. enterica divided into 6 subspecies (> 2579
serovars)
ƒ Human infections predominantly caused by
subspecies I (enterica) and subspecies II
(salmae)
ƒ S. bongori
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Salmonella enterica
subspecies enterica
serovar Typhimurium
= Salmonella Typhimurium
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Salmonella Typhimurium is our most common
serotype
Incidence and distribution
Introduction of new species/strain in NZ
Outbreak detection and investigation
Leptospira Reference Laboratory
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© ESR 2011
Phage typing of Salmonella
•S. Typhi
•S. Typhimurium
•S. Enteritidis
Selective ability of
bacteriophages to infect certain
strains of Salmonella
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Subsequent bacterial lysis
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Formation of plaques
95% of human infections caused by O Groups A,
B, C, D, and E
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Based on Kauffman White scheme
S. Typhimurium (Group B)
4,[5],12 : i : 1,2
(O antigen) (H antigen)
© ESR 2011
© ESR 2011
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Molecular typing: PFGE and MLVA
Pulsed-field Gel Electrophoresis (PFGE)
• Molecular “fingerprinting” technique reflecting DNA sequence of the entire bacterial
genome (Degree of genetic diversity)
• “Gold standard” : CDC has developed and standardized protocols
(E. coli O157:H7, Salmonella, Campylobacter jejuni, Shigella, V. cholerae, Yersinia pestis, Listeria monocytogenes)
Rare-cutting enzymes are used to cut DNA
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Separated by pulsed-field electrophoresis
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Computer software to analyse and compare PFGE patterns
Stored in database for future reference (PulseNet)
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More discriminatory than phenotypic methods, accurate, reproducible, allows
comparison between laboratories
- To track spread of foodborne pathogens
- Some bacteria are highly clonal (limited number of PFGE profiles)
© ESR 2011
© ESR 2011
MLVA
Analysis of PFGE using Bionumerics software: Dendogram
Dice (Opt:0.50%) (Tol 2.5%-2.5%) (H>0.0% S>0.0%) [0.0%-98.3%]
© ESR 2011
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Discrimination between isolates is based on loci distributed throughout the
bacterial genome harboring variable numbers of short repetitive DNA sequences
(tandem repeats) (VNTR).
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The number of tandem repeats for each locus varies between isolates
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This variability was exploited by the development of MLVA (Multiple Locus
VNTR Analysis)
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MLVA is used to determine the relatedness of isolates
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Amenable to standardization and high-throughput analysis
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Methods have now been developed for large number of bacterial species and
are being used internationally
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S. Typhimurium: (Lindstedt et al. 2003) Salmonella Typhimurium DT104
- 5 VNTR (STTR3, STTR5, STTR6, STTR9, STTR10)
100
PFGE-XbaI
95
90
85
PFGE-XbaI
ERL11-0059
Human
E. coli O157:H7
ERL11-0150
Human
E. coli O157:H7
ERL11-0937
Human
E. coli O157:H7
ERL11-0807
Human
E. coli O157:H7
ERL11-0104
Human
E. coli O157:H7
ERL11-0408
Human
E. coli O157:H7
ERL11-0808
Human
E. coli O157:H7
ERL11-0223
Human
E. coli O157:H7
ERL11-0929
Human
E. coli O157:H7
ERL11-0107
Human
E. coli O157:H7
ERL11-0283
Human
E. coli O157:H7
ERL11-0021
Human
E. coli O157:H7
ERL11-0277
Human
E. coli O157:H7
ERL11-0656
Human
E. coli O157:H7
ERL11-0950
Human
E. coli O157:H7
ERL11-0145
Human
E. coli O157:H7
ERL11-0686
Human
E. coli O157:H7
ERL11-0071
Human
E. coli O157:H7
ERL11-0348
Human
E. coli O157:H7
ERL11-0390
Human
E. coli O157:H7
ERL11-0405
Human
E. coli O157:H7
ERL11-0194
Human
E. coli O157:H7
ERL11-0322
Human
E. coli O157:H7
ERL11-0858
Human
E. coli O157:H7
ERL11-0281
Human
E. coli O157:H7
ERL11-0657
Human
E. coli O157:H7
ERL11-0014
Human
E. coli O157:H7
ERL11-0233
Human
E. coli O157:H7
ERL11-0001
Human
E. coli O157:H7
ERL11-0238
Human
E. coli O157:H7
ERL11-0263
Human
E. coli O157:H7
© ESR 2011
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Salmonella Typhimurium DT RDNC-May 06
PCR amplification
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Genetic analyzer is used to determine the size of the amplicon
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Once the size is known, the number of repeat sequences is calculated
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This is repeated for multiple loci and results for all loci are represented as
a string of numbers.
© ESR 2011
Emergence of DT RDNC-May 06
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First human isolate 11th May 2006 from a 3 year old male in Auckland.
No history of overseas travel.
No source of infection determined.
Next 2 years: Auckland, Hawke’s Bay, Northland, Whanganui, Waikato
June 2008: First human case in the South Island (2Y male, Canterbury)
Dec 2008: Southern (5Y male)
Spread more in the North Island: Mid Central, Lakes, Capital Coast, Bay of Plenty
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National rate of Salmonellosis = 26.2 per
100,000 in 2010
Majority (approximately 50%) of human
Salmonella isolates are Salmonella
Typhimurium (STM)
Phage typing: To further differentiate our
STM
Phage pattern type:
- DT: Definitive type e.g. DT1
- RDNC: Isolate reacts with some
phages but the reaction pattern
does not conform to a recognised
phage type.
- Indistinguishable New Zealand
RDNC patterns are assigned a
name based upon the month and
year in which the pattern was first
identified.
- Salmonella Typhimurium DT
RDNC-May 06 was not found
in New Zealand prior to May
2006
Human Salmonella serotypes 2006 (n = 1404)
S. Brandenburg
S. Enteritidis (Phage
Type 9a ‐ 49%)
S. Infantis
S. Saintpaul
S. Typhimurium
Other serotypes
© ESR 2011
Year
North
Island
South
Island
2006
16
0
2007
51
0
2008
53
2
2009
41
2
2010
73
12
1
3
5
4
2
6
7
© ESR 2011
© ESR 2011
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Epidemiology of DT RNDC-May 06
Human S. Typhimurium DT
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2006, n=733
DT 1
DT 101
DT 156
DT 160
DT RDNC‐May 06
Other DT
© ESR 2011
Total number of cases 2006-2010 = 250 confirmed by ERL
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2010, n=594
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DT1
DT101
DT156
55% are children aged 6 years or under (50% are male)
50% are Male
Ethnicity: European (59%), Maori (13%), Asian (4%), Pacific Island (2%), Other (22%)
No deaths has been reported
12% of cases have been hospitalised
Risk factors: Food premises (16%), contact with farm animals (14%), drinking
untreated water (10%), recreational water (5%), contact with symptomatic
people (3.5%), recent overseas travel (0.4%)
DT160
DT RDNC‐May 06
Other DT
© ESR 2011
Non-human isolates of DT RDNC-May 06
Non-human isolates of DT RDNC-May 06 2006-2010 (n = 161)
Non-Human isolates (2006-2010)
• May 2006: Equine
• Aug 2006: Porcine
• September 2006: Feline, Bovine
• December 2006: Avian, Poultry feed
• Since then has been confirmed in Lapine, Canine, Food (lettuce),
Alpaca, Caprine, but NOT in Ovine
Equine
Feline
Bovine
Poultry (feed, environmental)
Year
Number of isolates
2006
9
2007
35
2008
56
2009
22
2010
39
Environmental
Others
Other: Alpaca, Food, Canine, Porcine, Avian, Caprine
© ESR 2011
© ESR 2011
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Conclusion
Molecular typing of DT RDNC-May 06
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Dice (Opt:0.50%) (Tol 2.5%-2.5%) (H>0.0% S>0.0%) [0.0%-100.0%]
100
98
PFGE-XbaI
96
94
92
PFGE-XbaI
ERL06-1751
RDNC-May 06
Human
M
3 years
AK
Not typed
ERL06-2131
RDNC-May 06
Human
F
17m
NW
Not typed
ERL06-3062
RDNC-May 06
Human
M
35Y
HB
Not typed
ERL06-3292
RDNC-May 06
Human
M
2Y
AK
Not typed
ERL06-3303
RDNC-May 06
Human
M
3Y
AK
Not typed
ERL07-0716
RDNC-May 06
Human
M
75Y
NL
2-10-5-NA-13
ERL07-0954
RDNC-May 06
Human
F
2Y
WK
2-10-5-NA-13
ERL07-1179
RDNC-May 06
Poultry
AK
2-10-5-NA-13
ERL07-2127
RDNC-May 06
Avian
AK
2-10-5-NA-13
ERL07-2317
RDNC-May 06
Human
F
50Y
WG
2-10-5-NA-13
ERL07-2670
RDNC-May 06
Human
F
47Y
WK
2-11-5-NA-13
ERL07-3089
RDNC-May 06
Bovine
WK
2-10-5-NA-13
ERL07-3168
RDNC-May 06
Human
M
2Y
AK
2-11-5-NA-13
ERL07-3818
RDNC-May 06
Human
M
2Y
WN
Not typed
ERL08-2002
RDNC-May 06
Human
M
2Y
CB
Not typed
ERL08-2648
RDNC-May 06
Human
M
14M
RO
Not typed
ERL08-2935
RDNC-May 06
Human
M
5Y
TG
Not typed
ERL08-3136
RDNC-May 06
Canine
AK
Not typed
ERL08-4647
RDNC-May 06
Human
OT
Not typed
ERL07-3287
RDNC-May 06
Laprine
AK
2-10-5-NA-13
ERL07-3878
RDNC-May 06
Equine
WK
Not typed
M
5Y
© ESR 2011
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Spread from a single location to entire country
peaked at 85 (2010) of total human Salmonella isolates (n = 1195).
% increase in 4 years: 1.1% (2006) to 7.1% (2010)
- Jan-Jun 2007: 18 human isolates
- Jan-Jun 2011: 42 human isolates
Established as a pathogen in animals, particularly cats, cattle and
horses
DT RDNC-May 06 is isolated most of the year and has the typical
spring/summer peak of Salmonella species
DT RDNC-May 06 increased at expense of other DTs and is becoming
our predominate phage type
This phage type has not yet been seen by the Australian Salmonella
Reference Centre (Dianne Davos - Personal communication)
Source of New Zealand strain remains unknown
© ESR 2011
Acknowledgements
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The staff of the Microbiology laboratory: Carolyn Nicol, David Duncan,
Karen Cullen, Hugo Strydom, Emily Motion, Mackenzie Nicol, Penelope
Hancock, Shevaun Pain.
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Dr Kristin Dyet for the MLVA typing.
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The human work was done under funding by the Ministry of Health
© ESR 2011
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