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

Salmonella strain typing: An important Tool for

the control of food-borne illness

Sheena Chu, [email protected]

Bacteriology Section SupervisorLos Angeles County

Public Health Laboratory

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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.

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Foodborne Disease Outbreak

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.

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Role of Public Health Laboratory

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)

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What is strain typing and why is it necessary?

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….

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Salmonella

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.

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Step 1 – Salmonella ID

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.

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Step 2 -Salmonella Serotyping

Salmonella Serotype is based on the Somatic (O) and Flagellar (H) antigens.

e.g. Salmonella typhimuriumI 4,5,12 : i : 1,2I = subspecies I4,5,12=somatic antigeni = 1st Phase flagellar antigen1,2 = 2nd phase flagellar antigen

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Antigenic Formula

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Species enterica bongori

Subspecies enterica salamae

arizonae diarizonae houtenae indica bongori

Dulcitol + + - - - d +

ONPG - - + + - d +

Malonate - + + + - - -

Gelatinase - + + + + + -

Sorbitol + + + + + - +

KCN - - - - + - +

L-(+)Tartrate + - - - - - -

Galacturonate - + - + + + +

Mucate + + + -(70%) - + +

Salicin - - - - + - -

Lactose - - -(75%) +(75%) - d -

Lyse by phage -01

+ + - + - + d

Differential characters of Salmonella species and subspecies

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

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Agglutination for antigen determination

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Kauffmann-White SchemeSerotype O antigen (H)1st Phase 2ndphase

Restriction digest of Genomic DNA. Fragment analysis by Pulse-Field Gel

Electrophoresis.

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Step 3 - PFGE Molecular Subtyping

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.

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Pulse Field Gel Electrophoresis

PFGE Process

Genomic DNA extracted in agar plugs

Restriction digestion

Large size fragments separated by Pulse Field gel electrophoresis

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Gel image and data analysis

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BioNumericsEnable digital data normalization. Used to analyze the restriction fragments and upload to share the data with other Public Health agencies through PulseNet USA and internationally.

Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-100.0%]PFGE-XbaI

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PFGE-XbaI PFGE-BlnI

T46960

Z31465A

Z31470A

Z31471

Z31472

Z31474

Z31475

Z31476

Z31479

Z31480

Give

Give

Give

Give

Give

Give

Give

Give

Give

Give

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.

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PulseNet USA

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?

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Step 4- Combined Strain typing with Patient data

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”

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How is an outbreak recognized?

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

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Salmonella montevideo 2009

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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 be salmonella 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.

Salmonella Outbreak detection

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PHL serotype and PFGE

ACDC interview patients and Food

establishments

Strain typing results

reported to ACDC and

CDC

Outbreak info shared

with other local, state and federal

agencies

Food Recall, and/or

Preventive measures

Doctors reporting

cases of Salmonella cultured and

identified

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.

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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.

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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.aspx850 Marina Bay Parkway Bldg P 1st Floor, Richmond, CA 94804-6403(510) 620-3838, [email protected] (510) 231-7836, [email protected]

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Requirements for a CA Public Health Microbiologist

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Enteric Bacti & PFGE Staff

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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

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Los Angeles County Public Health Laboratory

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Acknowledgement:Los Angeles County

Public Health Laboratory

Acute Communicable Disease Control

PulseNet (CDC)