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Next Generation PulseNet

PulseNet USA strategy to meet the challenge of Culture Independent Diagnostic Methods

PulseNet must keep pace with new testing methods if it is to preserve its ability to quickly detect state and multi-state foodborne outbreaks caused by Listeria, E. coli, Salmonella and other dangerous foodborne bacteria.

PulseNet relies on pure bacterial cultures to do DNA “fingerprinting” (or subtyping) to find groups (or clusters) of sickness that might be outbreaks. These cultures come from patient samples, such as stool samples, requested by doctors. The information from patient samples helps scientists track the “who, what, when, where, and why” of foodborne illness, and can help find outbreaks.

The basic process for identifying the type of bacteria making a patient sick is:

  1. The doctor requests a sample from the sick patient, such as a stool sample.
  2. A hospital, clinic, or private lab tests the sample for certain kinds of bacteria. This step can take up to three days.
  3. If the suspected bacteria is found, or cultured, it is sent to a local or state public health lab that will find the bacteria’s DNA “fingerprint” using Pulsed-field Gel Electrophoresis (PFGE) and/or Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) and submit it to the National PulseNet database at CDC.
  4. The lab may also send the culture to CDC, PulseNet headquarters, for more information about the bacteria making patient sick.

Culture Independent Diagnostic Testing

Graphic: Test for Foodborne Illnesses

Similarities and differences between culture-dependent and culture-independent tests. Click for larger view.

The scientist takes bacterial cells from an agar plate

The scientist takes bacterial cells from an agar plate.

Culture independent diagnostic testing (CIDT) can identify the bacteria that makes a person sick in patient samples without using pure cultures. That means that doctors can find out what is making a patient sick without waiting up to three days for the bacteria culture to grow in a lab (step 2 in the list above).

 The advantages of CIDT, such as fast results, mean that many labs will stop doing pure cultures for patients with foodborne illness. As this happens, PulseNet will need to develop new “fingerprinting” techniques that do not depend on bacterial cultures.

Meeting the Challenge

PulseNet’s strategy to meet the challenge of culture independent diagnostic testing includes preserving cultures, conducting whole genome sequencing, and working with meta-genomics. The list below outlines the details of each strategy element.

Preserve cultures

  • Continue to ask labs to submit cultures or patient samples
  • Use current methods (PFGE and MLVA) for subtyping
  • Preserve, or save, the "fingerprints" of the samples to help find future outbreaks

Whole genomic sequencing

  • Requires pure cultures of bacteria
  • Use whole genome sequencing which by itself is a very powerful DNA fingerprinting method to help identifying and investigating outbreaks
  • Identify genetic targets for surveillance
  • Build reference database for metagenomics

Metagenomics

  • No pure culture of bacteria required
  • Sequence select genetic targets or all bacteria in a patient sample (stool, blood, etc.)
  • Identify bacteria making a patient sick and subtype at the same time

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Next Generation Sequencing

Next generation sequencing (NGS) is a fast, cheap way to do whole genome sequencing. NGS offers many ways to improve PulseNet’s surveillance and use resources more efficiently.

Efficient use of resources

With NGS the laboratory finds information about the species, serovar, and subtype of bacteria in just one test. Currently, those three important pieces of information are generated by at least three separate tests by two or more scientists.

To explore these opportunities, PulseNet is actively involved in validating NGS machines and evaluating the tools for analyzing the data from NGS machines. The potential advantages and comparison to the current PulseNet gold standard subtyping technique, PFGE, are shown in the table below.

Key Factors Next Generation Sequencing (NGS) Current "Gold Standard" (PFGE)
Time to get data 1-2 days Days
Time to analyze data Hours to days Hour or less
Cost per bacteria isolate (for reagents only) ~$150-300
(depends on platform)
~$10-15
Ability to differentiate and cluster isolates To be determined but likely very good Well-established for bacteria tracked by PulseNet, few exceptions
Potential for automation Yes No
Intended objective Genetic serotyping, virulence, antimicrobial resistance profiling, and subtyping possibly based on SNPs or other variant regions Only subtyping

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Projects and collaborations

PulseNet's evaluation of NGS as a potential tool for surveillance and outbreak investigations has many projects and collaborators, shown below.

Projects Collaborations
Sequence strains from recent and historical outbreaks as well as strains not associated with outbreaks both in house and with collaborators CDC internal partners, FDA, device manufacturers, academic institutions such as UC Davis in the 100K Foodborne Pathogens Project
Evaluate molecular subtyping schemes based on whole genome sequencing that provide the best clustering Partners from universities, PulseNet participants in the states, PulseNet International and other public health laboratories abroad
Determine what genetic cues (sequences) from clinical samples such as stool can be used to identify and cluster bacteria causing foodborne illness
Develop standardized laboratory and data analysis workflows International partners for developing standards for whole genome sequencing and analysis, such as the Global Microbial Identifier initiative and bioinformatics software manufacturing partners

Many groups are producing genome sequences, including CDC’s Enteric Diseases Laboratory Branch, the CDC Biotechnology Core Facility, collaborative projects with device manufacturers, and collaborative projects with federal agencies and academic institutions. 

The future of “DNA fingerprinting”

Evaluating the different approaches to genome sequencing will show if the future of subtyping (or “fingerprinting”) will be based on sequencing whole genomes or just targeted regions of DNA, and if the strategy can be used with clinical samples as well as bacterial isolates. These projects may lead to new ways to make identifying and “fingerprinting” foodborne bacteria easier, improving our surveillance.

We are confident that CDC’s work with our collaborators will allow scientists to develop and implement strategies to enhance PulseNet’s ability to detect clusters of foodborne illness faster and more accurately, so we can decrease illness and save lives.

Related Links

Articles about culture independent diagnostic testing

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