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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 • Joint initiative from: • • • • 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 - 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) • Approximately 90 microbiologists, molecular biologists, disease modellers, epidemiologists and researchers • Access to the only infectious disease laboratory that provides a PC3+ level of containment in NZ • Increases NZ’s capability to protect human and animal health © ESR 2011 1 Salmonella Microbiology Laboratory • Enteric Reference Laboratory - • 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 • • • Screening and confirmatory tests for common Leptospira serovars found in NZ © ESR 2011 Serotyping of Salmonella • Serotyping useful tool for surveillance • Robust and comparable world wide • 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) Human infections predominantly caused by subspecies I (enterica) and subspecies II (salmae) S. bongori • Salmonella enterica subspecies enterica serovar Typhimurium = Salmonella Typhimurium • Salmonella Typhimurium is our most common serotype Incidence and distribution Introduction of new species/strain in NZ Outbreak detection and investigation Leptospira Reference Laboratory - • © ESR 2011 Phage typing of Salmonella •S. Typhi •S. Typhimurium •S. Enteritidis Selective ability of bacteriophages to infect certain strains of Salmonella ⇓ Subsequent bacterial lysis ⇓ Formation of plaques 95% of human infections caused by O Groups A, B, C, D, and E • Based on Kauffman White scheme S. Typhimurium (Group B) 4,[5],12 : i : 1,2 (O antigen) (H antigen) © ESR 2011 © ESR 2011 2 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 ⇓ Separated by pulsed-field electrophoresis ⇓ Computer software to analyse and compare PFGE patterns Stored in database for future reference (PulseNet) • 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 • Discrimination between isolates is based on loci distributed throughout the bacterial genome harboring variable numbers of short repetitive DNA sequences (tandem repeats) (VNTR). • The number of tandem repeats for each locus varies between isolates • This variability was exploited by the development of MLVA (Multiple Locus VNTR Analysis) • MLVA is used to determine the relatedness of isolates • Amenable to standardization and high-throughput analysis • Methods have now been developed for large number of bacterial species and are being used internationally • 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 3 Salmonella Typhimurium DT RDNC-May 06 PCR amplification ⇓ Genetic analyzer is used to determine the size of the amplicon ⇓ Once the size is known, the number of repeat sequences is calculated ⇓ 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 • • • • • • • 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 • • • • 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 4 Epidemiology of DT RNDC-May 06 Human S. Typhimurium DT • 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 - 2010, n=594 • • • 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 5 Conclusion Molecular typing of DT RDNC-May 06 • • • 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 • • • • • 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 • The staff of the Microbiology laboratory: Carolyn Nicol, David Duncan, Karen Cullen, Hugo Strydom, Emily Motion, Mackenzie Nicol, Penelope Hancock, Shevaun Pain. • Dr Kristin Dyet for the MLVA typing. • The human work was done under funding by the Ministry of Health © ESR 2011 6