Introduction The emergence of a new phage type of Salmonella Typhimurium in humans and animals in New Zealand M Dufour AIMS NZIMLS South Pacific Congress Gold Coast, August 2011 New Zealand is a geographically isolated country, similar in size to Great Britain, with a population of 4 million The economy has a strong agricultural and pastoral focus Specialist Science Solutions Manaaki Tangata Taiao Hoki protecting people and their environment through science Background 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/ NCBID Joint initiative from: - 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 1
Microbiology Laboratory Enteric Reference Laboratory - Provides national reference and surveillance laboratory services -, 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 Incidence and distribution Introduction of new species/strain in NZ Outbreak detection and investigation Leptospira Reference Laboratory - Screening and confirmatory tests for common Leptospira serovars found in NZ Salmonella Salmonella genus has two species: - S. enterica divided into 6 subspecies (> 2579 serovars) 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 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). 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) 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 2
Dice (Opt:0.50%) (Tol 2.5%-2.5%) (H>0.0% S>0.0%) [0.0%-98.3%] 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 (, 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) Analysis of PFGE using Bionumerics software: Dendogram MLVA Discrimination between isolates is based on loci distributed throughout the bacterial genome harboring variable numbers of short repetitive DNA sequences (tandem repeats) (VNTR). 85 90 95 100 ERL11-0059 ERL11-0150 ERL11-0937 The number of tandem repeats for each locus varies between isolates ERL11-0807 ERL11-0104 ERL11-0408 ERL11-0808 ERL11-0223 ERL11-0929 This variability was exploited by the development of MLVA (Multiple Locus VNTR Analysis) ERL11-0107 ERL11-0283 ERL11-0021 ERL11-0277 MLVA is used to determine the relatedness of isolates ERL11-0656 ERL11-0950 ERL11-0145 ERL11-0686 ERL11-0071 Amenable to standardization and high-throughput analysis ERL11-0348 ERL11-0390 ERL11-0405 ERL11-0194 ERL11-0322 ERL11-0858 Methods have now been developed for large number of bacterial species and are being used internationally ERL11-0281 ERL11-0657 ERL11-0014 ERL11-0233 ERL11-0001 ERL11-0238 S. Typhimurium: (Lindstedt et al. 2003) Salmonella Typhimurium DT104-5 VNTR (STTR3, STTR5, STTR6, STTR9, STTR10) ERL11-0263 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. 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 Salmonella serotypes 2006 (n = 1404) S. Brandenburg S. Enteritidis (Phage Type 9a 49%) S. Infantis S. Saintpaul S. Typhimurium Other serotypes Emergence of DT RDNC-May 06 First human isolate 11 th 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 Year North Island South Island 2006 16 0 2007 51 0 2008 53 2 2009 41 2 2010 73 12 3 4 1 5 2 6 7 4
S. Typhimurium DT 2006, n=733 2010, n=594 DT 1 DT 101 DT 156 DT 160 DT RDNC May 06 Other DT DT1 DT101 DT156 DT160 DT RDNC May 06 Other DT Epidemiology of DT RNDC-May 06 Total number of cases 2006-2010 = 250 confirmed by ERL - 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%) Non-human isolates of DT RDNC-May 06 Non-human isolates of DT RDNC-May 06 2006-2010 (n = 161) Non- 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 Year Number of isolates 2006 9 2007 35 2008 56 2009 22 2010 39 Other: Alpaca, Food, Canine, Porcine, Avian, Caprine Equine Feline Bovine Poultry (feed, environmental) Environmental Others 5
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%] 92 94 96 98 100 ERL06-1751 RDNC-May 06 M 3 years ERL06-2131 RDNC-May 06 F 17m NW ERL06-3062 RDNC-May 06 M 35Y HB ERL06-3292 RDNC-May 06 M 2Y ERL06-3303 RDNC-May 06 M 3Y ERL07-0716 RDNC-May 06 M 75Y NL ERL07-0954 RDNC-May 06 F 2Y WK ERL07-1179 RDNC-May 06 Poultry ERL07-2127 RDNC-May 06 Avian ERL07-2317 RDNC-May 06 F 50Y WG ERL07-2670 RDNC-May 06 F 47Y WK ERL07-3089 RDNC-May 06 Bovine WK ERL07-3168 RDNC-May 06 M 2Y ERL07-3818 RDNC-May 06 M 2Y WN ERL08-2002 RDNC-May 06 M 2Y CB ERL08-2648 RDNC-May 06 M 14M RO ERL08-2935 RDNC-May 06 M 5Y TG ERL08-3136 RDNC-May 06 Canine ERL08-4647 RDNC-May 06 M 5Y OT ERL07-3287 RDNC-May 06 Laprine ERL07-3878 RDNC-May 06 Equine WK 2-11-5-NA-13 2-11-5-NA-13 Conclusion 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 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 6