Sheena Chu, M.S. [email protected] Bacteriology Section
Supervisor Los Angeles County Public Health Laboratory 1
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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. 2
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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. 3
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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) 4
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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. 5
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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. 6
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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. 7
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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 = 1 st Phase flagellar
antigen 1,2 = 2 nd phase flagellar antigen 8
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9 Species enterica bongori Subspecies
entericasalamaearizonaediarizonaehoutenaeindica 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
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Slide agglutination for the somatic (O) antigen (LPS) Tube
agglutination for the 1 st phase H antigen Phase Reversal to find
the 2 nd phase H antigen 10
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11 Serotype O antigen (H)1 st Phase 2 nd phase
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Restriction digest of Genomic DNA. Fragment analysis by
Pulse-Field Gel Electrophoresis. 12
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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. 13
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Genomic DNA extracted in agar plugs Restriction digestion Large
size fragments separated by Pulse Field gel electrophoresis 14
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16 Enable 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.
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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. 17
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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? 18
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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 19
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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 20
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21 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.
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22 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
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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. 23
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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. 24
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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, [email protected]@cdph.ca.gov
(510) 231-7836, [email protected]@cdph.ca.gov
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Bacteriology, Molecular Diagnostics, Mycobacteriology,
Mycology, Parasitology, Serology, Virology, Water and Sanitation,
All Hazard response Unit Pandemic Flu Response. 12750 Erickson
Avenue Downey, CA 90242 Telephone: (562) 658-1300 Fax: (562)
401-5999 28
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29 Los Angeles County Public Health Laboratory Acute
Communicable Disease Control PulseNet (CDC)
