National Zoonoses Conference (NZC), O Reilly Hall, University College Dublin, Wednesday-8 th June, 2011.

Transcription

1 National Zoonoses Conference (NZC), O Reilly Hall, University College Dublin, Wednesday-8 th June, Séamus Fanning, UCD Centre for Food Safety School of Public Health, Physiotherapy & Population Science, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland.

2 Salmonella the bacterium General Characteristics!! Member of the Enterobacteriaceae family!! Gram-negative bacilli, non-spore forming!! Facultative anaerobes!! Normally motile with peritrichous flagella!! Often inhabit gastrointestinal tract and also found in the environment Mode of infection!! Ingestion of contaminated food/water!! Passage through digestive system and invasion of intestinal epithelia!! Subsequent passage to vasculature system can lead to systemic infection

3 !! rarely lactose fermenting, there are 2,500 serotypes of Salmonella!! Salmonella serotypes occur worldwide, infecting mammals, birds and reptiles and are excreted in their faeces!! Enteritidis and Typhimurium are the serotypes often reported!! diagnostic protocols aid in elucidating the epidemiology of these pathogens

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5 Traditional Method ISO 6579:2002 DAY 0 25 g food sample in 225 ml Buffered Peptone Water (37 o C, 18 h) DAY 1 DAY 2 DAY 3 DAY ml culture in 10 ml RVS broth (41.5 o C, 24 h) XLD & BGA (37 o C, 24 h) TSA (37 o C, 24 h) 1 ml culture in 10 ml MKTTn broth (37 o C, 24 h) Biochemical & Serological Testing!! standardised and validated protocols are in place to facilitate the recovery of Salmonella from a food matrix!! present in food in low numbers!! Salmonella are usually stressed and must be allowed to recover prior to isolation on selective culture media!! the strategy can facilitate the culture of Salmonella!! for epidemiological purposes additional tests must be performed to improve the definition of the isolate Salmonella genus

6 Why sub-type bacterial isolates?!! for quality control purposes, where bacteria are added to foods!! to define the ecology of a food production site and identify persistent Salmonella Agona Salmonella Agona Sal. Typhimurium Do these isolates have the same phenotype? isolates that have adapted and to locate contamination hot-spots!! to protect public health

7 Stomach (ph 2) Salmonella in contaminated food (ingestion) Intestine (ph 9) Attachment to epithelial cells Host cells Injection of effector proteins Host cells Invasion & Production of chemotactic factors Lamina propria (ph ) Phagocytosis (macrophages) and/or Infiltration of PMN s Macrophage death and/or Survival inside these cells Migration of PMN s (through epithelial cells) Systemic infection Detachment of epithelial cells Fluid secretion (diarrhoea)

8 !"#$%&'($ Stress increases Bacterial stress challenges- -! ph food matrix -! hygiene measures (biocides) -! moisture (a w ) -! heat -! antibacterial measures CCP Step )&*+$%&'($,-.+"*/0$/'1-2/$

9 It is not the strongest of the species that survives, nor the most intelligent that survives. It is the one that is the most adaptable to change Charles Darwin

10 !!!"# $% "&'()*"+,"%-./.%"0123"4156"'"!"&'()*"+,"7/8 % 9"!! :*'5";*<1<5'2=*"1<">;*'5(?"12=;*'<*@"12"(+4"' 4 "'2@"12",++@<"4156"'"61>6",'5"=+25*25"!! 9'2"<);&1&*",+;"4**A<B"0+256<"'2@"?*';<"12"(+4"0+1<5)5*",++@<"C4156"'2"' 4 ")D"5+"%-EF"!!!2"' 4 "+,"%-G7";*H)1;*@",+;">;+456" The infectious dose of Salmonella is dependent on-!! degree of host (human/animal) resistance!! age extremes!! immuno-supressive illness!! bacterial numbers in the contaminated food!! nature of the food matrix and!! physiological status of bacterial cells Food matrix Serotype Infective dose (CFU) Cheese Typhimurium 1-10 Chocolate Eastborne Naploi Typhimurium < Maize snack Agona 2-45 Paprika-flavoured patato chips Saint Paul; Javiana; Rubislaw < 45 Peanut butter Mbandaka

11 Outbreaks linked to Salmonella Agona Food matrix Cereal (puffed) Cereal (toasted) Tea (aniseed) Snack (savory) Year Pathogen No. affected (country/ies) Details 2008 S. Agona 28 Puffed rice and puffed wheat cereals implicated in the outbreak were manufactured at the same plant that manufactured toasted oat cereal implicated in a 1998 outbreak of S. Agona infections. S. Agona was isolated from the plant and from bags of puffed rice cereals 1998 S. Agona 209 An opened box of cereal yielded a S. Agona isolate with a PFGE pattern indistinguishable from the predominant PFGE pattern among outbreakassociated clinical isolates. Cereal from unopened boxes was also positive for S. Agona 2003 S. Agona 42 (Germany) 1994 S. Agona PT15 >2,200 (Israel; UK & USA) S. Agona was isolated from six brands of tea containing aniseed. Various serotypes were isolated from 61 (11%) of 575 tea and other products containing aniseed. S. Agona survived upon exposure to hot water during tea-making Snacks were manufactured on at least seven separate dates over a 4-month period. Levels were estimated to be 2-45 CFU/25 g packet of peanut flavoured snack common feature in all of these outbreaks low moisture food

12 Collection of isolates [pre-, post-outbreak] Sub-typing by PFGE Genetic characterisation [genotype] Physiological characterisation [phenotype] Growth curves Optical mapping Acid tolerance Genome sequencing gap closure annotation Tolerance to biocides Motlity Biofilm formation Ex vivo cell culture studies

13 Malt-o-Meal outbreak: Outbreak strain: Sump strain: R0102/09

14 Genomic DNA is captured as single DNA molecules DNA is digested with a restriction endonuclease Whole genome restriction map Fluorescent intensity is measured to determine fragment sizes Order is maintained

15 !! sub-typing supports epidemiological investigation, though these methods can also be applied in a broader context!! though isolates may have indistinguishable DNA fingerprints, there phenotypes may differ, reflecting adaptation to a stress condition!! failure in a critical CCP may lead to the escape of an adapted pathogen (following preselection at one or more stages)!! food safety management systems must be integrated with an environmental management system!! a structured collaboration between stakeholders can facilitate a better understanding of these issues

16 Acknowledgements Marta Martens Matthew McCusker Sarah Finn Karen Power Evonne McCabe Denis O Leary Kaye Burgess Geraldine Duffy Alan Reilly Wayne Anderson Food Institutional Research Measure (FIRM)- Network & Team Building Initiative grant no.: 06/TNIUCD10