Clostridium botulinumStandardized PFGE Protocol

Development

Clostridium botulinumStandardized PFGE Protocol

Development

Elise B. Smith B.S., MT(ASCP)Commonwealth of Virginia

Division of Consolidated Laboratory Services

Clostridium botulinumGram Positive Rod Obligate AnaerobeSubterminal SporeCapable of Producing NeurotoxinsUbiquitous in Soils World Wide Causative agent of food poisoningBacteria and associated toxins are designated as potential biological warfare agentswww.healthofchildren.com

http://www.neuro.wustl.edu/neuromuscular/nother/bot.htm#proteinhttp://chip.med.nyu.edu

Clostridium botulinumToxin Types

Toxin Type G isolated from soil in Argentina Possible cause of sudden, unexpected death Plasmid encoded

Toxin Types A, B, E, and F Cause disease in humans Chromosomally encoded

Toxin Types C, and D Cause disease in animals Bacteriophage encoded

A Kornberg

wildlife1.usask.ca

http://www.neuro.wustl.edu/neuromuscular/nother/bot.htm#protein www.american.edu

CDC Project Goals for C. botulinum PFGE Method

Development

To determine the PFGE pattern diversity of neurotoxin producing Clostridia species in theUnited States

To establish a national database for use by state health laboratories and CDC during natural and intentional outbreaks of botulism

Pitfalls to PFGE Fingerprinting of Clostridium species

Clumping of Clostridium spp. in cell suspensionOrganism often requires extended anaerobic incubation time to obtain sufficient growthC. botulinum spores are extremely resistant to PFGE cell lysis conditionsDNA degradation results in unresolved or faint PFGE patterns for certain strain types

Previous Studies PFGE of Clostridium perfringens

Previous Studies PFGE of Clostridium perfringens

After:PFGE procedure modifications resulted in distinct reproducible DNA fingerprint patterns from previously untypeable strains

PFGE yielded faint DNA fingerprint patterns

Before:PFGE did NOT yield DNA fingerprint patterns

Split a single isolated colony to Chopped Meat Glucose (CMG) broth and a Blood Agar Plate (BAP)

Suspend growth from BAP in Cell Suspension Buffer and adjust turbidity using a MicroScan Turbidity Meter

Centrifuge 1ml of cell suspension at 5000 rpm for 5 minutes.

Suspend pellet in 0.5 ml of Cell Suspension Buffer. Mix gently and

flood Egg Yolk Agar plate with suspended cells.

Modified Culturing Techniques for PFGE of Clostridium perfringens

Modified Culturing Techniques for PFGE of Clostridium perfringens

Incubate at 37oC overnight

anaerobically

Flood Egg Yolk agar plate with 0.5 ml of

CMG broth after incubation

BEFORE AFTER

PFGE analysis of outbreak-related Clostridium perfringens isolates

BEFOREBEFORE AFTER AFTER

Modification using Cell Suspension Buffer Modification using Cell Suspension Buffer to inoculate the Egg Yolk Agar plates and to inoculate the Egg Yolk Agar plates and

the addition of the addition of Thiourea to gel to gel electrophoresis electrophoresis

Isolates grown in CMG broth prior Isolates grown in CMG broth prior to inoculating Egg Yolk Agar to inoculating Egg Yolk Agar

platesplates

DCLS Objectives

To develop a PFGE protocol in collaboration with CDC to subtype proteolytic and non-proteolytic straintypes of Clostridium botulinum Type A

To initiate the development of a BioNumerics PFGEdatabase of C. botulinum Type A using ~ 200fingerprint patterns from local and CDC prototype strains

To develop techniques for safe and secure PFGE handling of Select Agents to facilitate rapid subtyping as a result of a natural or intentional contamination event

HHS and USDA Select Agents

Botulinum neurotoxin-producing species of

Clostridium

Select Agent ProgramThe "Public Health Security and Bioterrorism Preparedness and Response Act of 2002 and the Agricultural Protection Act of 2002" (the Acts) require laboratories to register with the U.S. Department of Health and Human Services (HHS) or the USDA if they possess,use, or transfer biological agents or toxins (i.e. select agents and toxins) that could pose a severe threat to public health and safety; to animal or plant health; or animal or plant products.

In addition to ensuring that laboratories safely handle these select agents and toxins, the Acts also require increased safeguards and security measures for these agents, including controlling access, screening laboratories and personnel (i.e., security risk assessments) and establishing a comprehensive and detailed national database of registered entities.

The Act also imposes criminal and civil penalties for the inappropriate use of select agents and toxins.

http://www.cdc.gov/od/sap/faq.htm

Select Agent ProgramPFGE Considerations

Laboratory facility and all select agent working spaces must be inspected and approved for the handling of Select Agents

All personnel MUST undergo fingerprint and FBI background security clearance and a security risk assessment process to receive authorization to handle select agents such as Clostridium botulinumDetailed and timely process to obtain approval to ship or receive select agents

Form 2

SAP authorization required

Agents MUST be verified and inventoried at all times

Documentation required for intra-facility (room to room) transfer and destruction of select agents

Laboratories and all equipment used to store or incubate agents MUST be secured (ie. locked)

The Public Health Security and Bioterrorism Preparedness and Response Act of 2002

C. botulinum ProtocolCell Suspension

Prepare bacterial cell suspension Pellet 1ml of Cell Suspension at 5000 rpm for 5 minutesSuspend pellet in pre-warmed 2X Cell Lysis Buffer

Treat with Lysozyme at 37oC for 10 minTreat with Mutanolysin and Proteinase K at 37oC for 10 min

Cast plug using 400 μl of 1.2% SeaKem Gold Agarose without SDS

Cell LysisLyse plugs in ES Buffer containing Proteinase K for 2 hours at 54oC

Plug Washing2X sterile water; 6X sterile TE at 50˚C for 15 min at 70 rpm

C. botulinum Protocol

Restriction DigestionPre-digestion, incubate plug in 1X Restriction Buffer 4 for 15 min at RTDigest with Sma I for 4 hours at 25oCPost-digestion, incubate plug in 200 μl of 0.5x TBE for 5 min

Gel ElectrophoresisElectrophorese using 1% SeaKem Gold Agarose Gel Running conditions:

Running Buffer temperature: 14oC Running buffer containing ThioureaRun Time Duration: 19 hours Kb range: 30 kb to 600 kbInitial and Final Switch times: 0.5 seconds and 40.0 seconds

Stain gel with EtBr and destain using 500 ml Milli Q water

Representative PFGE GelClostridium botulinum Strains

Representative PFGE GelClostridium botulinum Strains

Project Status

Completed PFGE testing of over 200 isolates of Clostridium botulinum Type A

Tested a limited panel of Clostridium botulinum Type B, E and F isolates using newly developed PFGE method

Established a BioNumerics database normalized to the Salmonella ser. Braenderup, H9812 global standard; however all gels also contain three Clostridium control strains (CPERF1)

Received first panel of 50 CDC isolates of Clostridium botulinum Type B (Year 2)

Evaluated numerous parameters to render organisms non-viable –all parameters unsuccessful; studies ongoing

Viability StudiesViability Studies

Standard PFGE method post cell lysis, cell washing, and restriction digestion

Standard PFGE cell lysis conditions including increased concentrations and incubation periods with:

Lysozyme Mutanolysin Proteinase K

Post Cell Lysis Heat Treatment (100oC for 35 minutes)

Treatment of cell suspensions with:PIV buffer with 10% formalin

Serial dilutions of cell suspension (1:2 to 1:16) followed by standard method cell lysis

The following conditions FAILED to render C. botulinumisolates non-viable (non-Select Agents): The following conditions FAILED to render C. botulinumisolates non-viable (non-Select Agents):

Future Studies

Determine the ability of the Clostridium botulinum method to assess the diversity of other neurotoxin producing Clostridium species

Work with CDC Database Team to establish an official CDC database for Clostridium botulinumAssess the robustness of the developed PFGE method through a multi-laboratory validation

Provide the standardized method to other laboratories interested in testing strains isolated during local investigations of botulism

Continue to evaluate testing parameters to render organisms non-viable

AcknowledgementsDCLS PFGE Team

Kelly TomsonFrancis TannorCrystal JohnstonMatthew York

DCLS BT and Training TeamSonya CauseyJody LowmanJennifer SharpEmily HopkinsEllen BasingerJoAnn JellisonSusan Murphy

DCLS AdministrativeChristina Lambert-MosleyAnn Munson Dr. Denise ToneyDr. Thomas YorkDr. James Pearson

Dr. Susan Maslanka and CDC Botulism team