Salmonella strain typing : An important Tool for the control of food-borne illness

1. Salmonella strain typing: An important Tool forthe control of food-borne illness Sheena Chu, M.S. shchu@ph.lacounty.gov Bacteriology Section Supervisor Los Angeles County Public Health Laboratory

2. Foodborne Disease Outbreak • Defined as 2 or more cases of similar illness resulting from the ingestion of a common food. • Magnitude may range from 2 local cases to thousands cases in a multi-national level.

3. Role of Public Health Laboratory • Culture and isolate Pathogen involved in the outbreak from human, environment, and/or food/water source. • Strain type the bacterial isolates • Report to other Public Health agencies and compare to other isolates.

4. What is strain typing and why is it necessary? • Phenotypic or genomic characteristics that helps discriminate the relatedness between isolates of the same species. • Examples of phenotypes: Biotypes, Phage types, Serotypes • Examples of Genotypes: Pulese-Field gel electrophoresis (PFGE), Restriction Fragments Length Polymorphism (RFLP), Multi-loci variable tandem repeats (MLVA)

5. Salmonella • Causes 1.4 million illnesses and 600 deaths in U.S. • Biochemical tests used for Identification of the genus and subspecies. • Serotyping further differentiate isolate based on the O (somatic) antigen and H (flagellar) antigen • Serotypes are further subtyped by Pulse-Field Gel Electrophoresis. • Source: Chicken, Beef, Pork, Produce, Spices, Chocolate, Dirty hands….

6. Step 1 – Salmonella ID • Title 17, CCR, Section 2505 requires that an isolate of salmonella must be submitted to the local public health laboratory for definitive identification and serotype. • LA PHL still identify all isolates using conventional culture plates and biochemical tests.

7. Step 2 -Salmonella Serotyping • 2 Salmonella species (Enterica and bongori) • Salmonella enterica is separated Biochemically into 6 subspecies • >99.5% of salmonella isolates belong to enterica ssp. enterica • >2,500 serotypes (antigenic formula) • >2,000 serotypes performed annually by LA PHL.

8. Antigenic Formula Salmonella Serotype is based on the Somatic (O) and Flagellar (H) antigens. e.g. Salmonella typhimurium I 4,5,12 : i : 1,2 I = subspecies I 4,5,12=somatic antigen i = 1st Phase flagellar antigen 1,2 = 2nd phase flagellar antigen

9. Differential characters of Salmonella species and subspecies

10. Agglutination for antigen determination • Slide agglutination for the somatic (O) antigen (LPS) • Tube agglutination for the 1st phase H antigen • Phase Reversal to find the 2nd phase H antigen

11. Kauffmann-White Scheme Serotype O antigen (H)1st Phase 2ndphase

12. Step 3 - PFGE Molecular Subtyping • Restriction digest of Genomic DNA. • Fragment analysis by Pulse-Field Gel Electrophoresis.

13. Pulse Field Gel Electrophoresis • In 1984, Schwartz and Cantor described pulsed field gel electrophoresis (PFGE), introducing a new way to separate DNA. In particular, PFGE resolved extremely large DNA for the first time, raising the upper size limit of DNA separation in agarose from 30-50 kb to well over 10 Mb (10,000 kb). • PFGE is essentially the comparison of large genomic DNA fragments after digestion with a restriction enzyme. Since the bacterial chromosome is typically a circular molecule, this digestion yields several linear molecules of DNA. The basic concept of interpretation of this experiment is the following: if one is comparing two strains that are clonal (i.e. the same strain), the sites at which the restriction enzymes act on the DNA and the length between these sites would be identical. Therefore, after digestion of the DNA and electrophoresis through an agarose gel, if the DNA banding patterns between any two isolates is identical, then these isolates are considered the same strain. Conversely, if two isolates are not the same strain, then the sites at which the restriction enzymes act on the DNA and the length between these sites would be different; thus their DNA banding patterns will be different.

14. PFGE Process

15. Gel image and data analysis

16. BioNumerics Enable digital data normalization. Used to analyze the restriction fragments and upload to share the data with other Public Health agencies through PulseNetUSA and internationally.

17. PulseNet USA • PulseNet is part of CDC and it uses the PFGE data from PulseNet participating labs to identify clusters across state and sometimes across countries. State/Local public health labs, Federal agencies (FDA, USDA, and CDC), and 67 countries all participate and submit their PFGE patterns to identify food borne disease case clusters by PFGE.

18. Step 4- Combined Strain typing with Patient data • Clusters of similar PFGE patterns • Food History • Exposure to Reptile • Other risk factor? • Cluster comparison to national data base • Communicate with CDC and other state agencies • Link to Possible Food / Animal source?

19. How is an outbreak recognized? • Victims recognize the common nature in an acute local outbreak and report to the local Public Health. • Diffuse Outbreaks – laboratory based public health surveillance systems –When data shows a surge of a particular serotype or PFGE pattern that is more frequent than “Baseline”

20. Salmonella montevideo2009 • 44 States impacted!, >250 cases Reported • Salmonella serotype Montevideo: (I 6,7: g,m,s:1,2) • Cluster detected by PulseNet and posted on Aug. 23, 2009. • Recall issues! WASHINGTON, January 23, 2010 - Daniele International Inc., is recalling approximately 1,263,754* pounds of ready-to-eat (RT.E) varieties of Italian sausage products, including salami/salame • LA County : 6 laboratory confirm case, serotype and PFGE type all matched to the national cluster. • Locate Food samples from local distributor and samples sent to PHL. • Salmonella isolates from Daniele Salame but NOT serotype montevideo!? Senftenberg No patient identified..PulseNet alerted and search continues… • FDA looking into red and black pepper as possible source

21. LAC Salmonella Give outbreak • Salmonella serotype Give : Formula Salmonella enterica I 3,10:l,v:1,7 • 2 parties with 41 ill reported eating food purchases from a meat market on 2/21/2010. • 8 patient specimens were culture positive for salmonella by clinical lab. • LA PHL serotyped all 8 to besalmonella serotype give • 56 interviewed and all 41 patients (100%) reported eating canitas, compared to 45% for the control group (5/11). • Carnitas samples were sent to the LA county PHL and salmonella give was cultured and isolated. • PFGE confirms the genotype to be identical from the patient isolates.

22. Salmonella Outbreak detection x

23. Public Health Microbiologist Public health microbiologists are a select group of professional laboratorians whose comprehensive approach to the identification and characterization of microorganisms of public health significance contributes to the control and prevention of disease.The microbiologists perform a variety of complex laboratory techniques from traditional isolation methods and fluorescent  microscopy to flow cytometry and molecular technology. In testing specimens from human, animal, food, water and  dairy products and environmental sources.

24. Public Health Microbiologist Public health microbiologists are active in training laboratory personnel for public health and clinical laboratories. They develop improved laboratory procedures and practices. They provide consultation services to other laboratories  and the medical community.

25. Requirements for a CA Public Health Microbiologist 1. B.S. Degree 2. completion of 24 semester units (or equivalent quarter  units) of the following: microbiology, bacteriology, immunology, virology, parasitology, hematology, cellular biology,  biochemistry, clinical chemistry, genetics, microtechnique, instrumentation, epidemiology, other related courses. The above units must include at least 6 semester units (or equivalent quarter units) of medical or pathogenic microbiology/bacteriology. A course in immunology or serology may be accepted as a substitute for three or less of the medical microbiology units. 3. 6 month training in a approved CA Public Health Laboratory. Laboratory Field Services - Public Health Laboratory Program http://www.cdph.ca.gov/programs/lfs/Pages/PublicHealth.aspx 850 Marina Bay Parkway Bldg P 1st Floor, Richmond, CA 94804-6403 (510) 620-3838, kathy.williams@cdph.ca.gov (510) 231-7836, genie.tang@cdph.ca.gov

26. Enteric Bacti & PFGE Staff

27. Los Angeles County Public Health Laboratory

28. Los Angeles County Public Health Laboratory • Bacteriology, Molecular Diagnostics, Mycobacteriology, Mycology, Parasitology, Serology, Virology, Water and Sanitation, All Hazard response Unit Pandemic Flu Response. • 12750 Erickson AvenueDowney, CA 90242 • Telephone: (562) 658-1300Fax: (562) 401-5999

29. Acknowledgement: Los Angeles County Public Health Laboratory Acute Communicable Disease Control PulseNet (CDC)