RECENT ADVANCES IN RAPID MICROBIAL IDENTIFICATION AND CHARACTERIZATION TECHNIQUES Mackenzie Slifierz...

RECENT ADVANCES IN RAPID MICROBIAL

IDENTIFICATION AND CHARACTERIZATION

TECHNIQUES

Mackenzie SlifierzHiu Ching LaiJames Feiner

PUBLIC HEALTH EMERGENCIES

Hurricane Katrina, 2005 At least five level 3

biosafety labs within hurricane zone.

PUBLIC HEALTH EMERGENCIES

Hurricane Ike, 2008 Galveston Nation Labs, a level 4 biosafety lab working with

potential bioterrorism pathogens including hantavirus, anthrax, and ebola virus.

PRESENTATION OVERVIEW

1. Traditional Detection Methods

2. Advanced Rapid Detection Methods:i) PCR-based Methodsii) Nanotechnologyiii) Immunoassaysiv) Other Rapid Microbial Methods

3. General advantages and limitations

4. Conclusion

TRADITIONAL DETECTION METHODS

Culturing bacteria: colony morphology, colour, and size.

Staining: Gram stain, spore stain, flagella, cell morphology.

Biochemical analysis: carbohydrate utilization and fermentation.

Inhibition: Bile-salts, antibiotic resistance, dye tolerance.

Traditional Detection Methods

RAPID DETECTION TECHNIQUES

What is a rapid detection technique? A rapid microbial method (RMM) is any technique

which can identify or characterize a microorganism in hours or minutes as opposed to days or weeks.

Some techniques that can be used for rapid analysis: PCR-based Methods Nanotechnology Immunoassays Spectroscopy Chromatography

PCR-based Methods

Rapid Identification and Characterization Techniques

PCR: POLYMERASE CHAIN REACTION

Amplification of microbial DNA.

Detection only requires very little DNA.

Sample is directly from food/clinical sources.

No Culturing.

Advantages:Low cost (PCR machine: $500)Quick assay (3.5 hrs)

Real-Time PCR (RT-PCR) estimates the quantity of microorganisms in the sample:

Designing Primers

Primers can be designed from broad-range/highly conserved bacterial 16S ribosomal genes.

This allows detection of most bacteria but also species-level identification.

Primers can target genes that encode proteins that differentiate known bacteria.

Further detection from the amplified DNA.

DIRECT METHOD

Design specific fluorescently labeled oligonucleotide probes to differentiate between microorganisms in the sample:

Specific Species (eg E. coli)Gram DifferentiationSpore formation

Combine with microarray: allows for many PCR reactions in a single

rapid procedure. Quick Efficient Automated

HRMA: HIGH-RESOLUTION MELTING ANALYSIS

Design three conserved primers for three hypervariable regions (V1, V3, and V6) within the 16S rRNA gene.

Double stranded DNA is “melted” and then binds to a fluorescent dye.

Compare combinations of V1, V3, V6:

EIMS:ELECTROSPRAY IONIZATION MASS SPECTROMETRY

Primers designed for conserved bacterial 16S ribosomal genes. DNA is amplified by PCR.

Ionize the amplified DNA for mass spectrometry analysis.

Detect the mass of amplified DNA and compare to a database.

DGGE:DENATURING GRADIENT GEL ELECTROPHORESIS Use specific primers to target a region of the bacterial

genome and amplify with PCR.

Amplified DNA is run through a denaturing gel.

Different bacterial DNA has different DGGE patterns due to nucleotide content and the secondary structures formed during partial denaturing.

Can determine genus of unknown microorganisms by comparing known DGGE profile.(eg. Nitrosomonas which oxidizes NH3)

Lactobacillus Bacteroides

Nanotechnology

Rapid Identification and Characterization Techniques

NANOTECHNOLOGY

Nanotechnology is the creation and manipulation of

matter at the nanoscale.

Nano-constructs capable of self-assembly and specific activity:

imitation of natural molecular structures.

Extreme sensitivity reduces

needed sample size.

Self-propelled microtubes:

capture-and-release bacteria. 8 um long; 0.5 um diameter.

300 body-lengths/second.

Microengine: platinum-catalyzed oxidation of H2O2 fuel.

Lectin receptor selectively bind bacterial polysaccharides.

Lectin-bacterial bond broken by low-pH gylcine solution.

Mass production: membrane template electrodepostion. Nanorockets are inexpensive and reusable.

NANOROCKETS

Rapid capture and isolation of pathogenic

bacteria in complex media.

Duel action: magnetic attachment and release of

therapeutic molecules.

H2O2 fuel makes bacteria non-cultural but they

remain viable and thus testable.

Biochips: nanorockets scour samples for

pathogens in microfluidic channels.

NANOROCKETS

SURFACE-ENHANCED-RAMAN-SCATTERING

Raman spectroscopy:

Viruses bound to Ag substrate.

Exposed to an infrared laser.

Laser scattered by viral particles.

Spectrograph records signal.

Virus spectral barcodes made.

Silver nanorod substrate:

Enhances spectra cross section by orders of magnitude.

Oblique angle deposition: cheap and simple procedure.

Deposits nanorods in random arrays at an uniform angle.

1300 1200 1100 1000 900 800 700 600 500

Raman Shift (cm-1)

Three strains of the influenza

virus (flu): Each share conserved spectra due

to close relation. Highlighted: non-conserved

spectra correlate to variable

nucleic acids and surface proteins.

Fast method for detection and

classification of viruses, bacteria

or toxins.

Monitoring of pollutants.

SURFACE-ENHANCED-RAMAN-SCATTERING

Bent Cantilever

Antibody

Biomarker Proteins

CANTILEVERS

Cantilevers are horizontal

beams anchored on one end: Mass bends the cantilever downwards.

A counterbalancing force can restore the original position, resulting in a certain

resonance frequency.

The resonance frequency is determined electronically.

Cantilevers can be manufactured at the nanoscale: Made in dimensions as small as: 5 um x 2 um x 30 nm.

Antibodies attached to cantilevers can detect the binding of single viral or

bacterial particles.

Ideal for airborne virus detection or microfluidic biochips.

Photodiode Laser

Cantilever

Sample

Piezoelectric scanner

Tip Cantilevers and Atomic

Force Microscopy (AFM):A nano-pin attached to a cantilever traces cell surfaces.

Pin-cantilever movement is detected by laser deflection.

Produces detailed high resolution topological images of cells and

profiles of molecular bond strength.

Rapid microbial detection applications:Imaging completed in minutes: location of cell-surface markers.

Capable of analyses in aqueous solutions and in vivo.

CANTILEVERS

OmpF: a porin protein of E. coli’s outer membrane: Forms channels composed of β – strands (circled). Left: X-ray crystallography image. Right: AFM image made with constant force microscopy. Scale bar: 50 Å.

CANTILEVERS

AFM image of a Saccharomyces cerevisiae yeast cell trapped

in a microporous membrane, scale: 1 um.

CANTILEVERS

Force spectrometry on a typical bacterial cell:Left: a cantilever attached to a membrane polysaccharide encounters

resistance as it pulls away from the molecule.Right: corresponding force-extension curve shows the growing molecular

force offered by the polysaccharide as it is stretched.

200 300 400 500

Extension (nm)Fo

rce (

pN

)

600

500

400

300

200

100

0

CANTILEVERS

IMMUNOASSAYS

Rapid Identification and Characterization Techniques

IMMUNOASSAYS:RAPID QUANTIFICATION OF FOODBORNE PATHOGENS

Multiplex immunoflourescent technique:Antibody-Antigen interaction provides specificity and the conjugated flourescent quantum dots allow for rapid detection of multiple pathogens.

QUANTUM DOTS

Multiplex ImmunoflourescentAssay

RESULTS

IMMUNOFLOURESCENT ASSAY

Can quantify the number of microorganisms

Relatively quick (< 2 hrs) Simultaneous detection

of multiple microorganisms

Does not require culturing

Sensitive and specific

Expensive Current methods require

technical expertise Requires lab equipment,

but there is a potential for portable equipment

Advantages Disadvantages

IMMUNOASSAYS:DIPSTICK ASSAY FOR HIV-1 AND HIV-2

SD Bioline HIV-1/2 3.0 Test One step, rapid, immunochromatographic test that can

distinguish between HIV-1 and HIV-2, and can detect all isoforms of each!

Sample: human serum/plasma (10 microliter sample) or whole blood (20 microliter sample).

Evaluated by WHO: 99.3% specificity, 100% sensitivity Time to run test: 5-20 minutes Price per test: $0.85 – 1.10 USD

DIRECT SANDWICH ELISA

RESULTS

DIPSTICK IMMUNOASSAYS

Relatively cheap Rapid results Requires very little sample Very specific and sensitive Stable over broad

temperature range Easy to transport Does not require technical

expertise to use

Qualitative only Invasive procedure Disposal Accidental infections

Advantages Disadvantages

IMMUNOASSAYS:USING CHEWING GUM TO DETECT MALARIA Researchers at UCLA have received funding from the Bill

and Melinda Gates Foundation to develop an innovative way to detect malaria using chewing gum (MALiVA).

The technique: chewing gum containing antibodies detects malaria-specific antigens in the saliva. The gum is then blotted onto a paper to give a visual result.

This method will be ideal for undeveloped countries where electrical lab equipment and technical expertise is not readily available.

Other Rapid Microbial Methods

Rapid Identification and Characterization Techniques

Computational Analysis and Databases The use of computational analysis provides high-

throughput results in a short amount of time.

Data can be shared in online databases.

MALDI – TOF Mass SpectrometryMatrix-Assisted Laser Desorption Ionisation Time-of-

Flight Species-specific mass spectra of peptides, protein, or other

organic molecules by mass spectrometry.

MALDI – TOF Mass Spectrometry

Very common in clinical biology laboratory:Efficient

(multiple samples per run)Cost-effective

($1.80 USD/sample)Rapid

(<10mins)

FAME:FATTY ACID METHYL ESTER

Cellular fatty acid analysis by Gas Chromatography.

MIDI Sherlock Microbial Identification System.

Comparing fatty acid components to database.

Conclusion

Rapid Identification and Characterization Techniques

Overall Advantages and Disadvantages of Rapid Microbial Methods:

Results are rapid! Ease-of-use testing. Tests are made to be very

sensitive and specific. The tests can be portable. Reduces workload on

medical laboratories. Can test for microorganisms

that cannot be cultured. Allows for earlier detection

and faster treatment.

Expensive investment in new lab equipment.

May require additional technical expertise.

Potential for abuse: may be used for bioterrorism.

Censorship of scientific data can hinder new developments in this field.

Advantages Disadvantages

THE FUTURE! Phasing out of culture-based microscopy:

Can’t compete with superior techniques and lower cost methods. Unable to rectify its own flaws.

Take-home diagnosis and treatment kits: Prepackaged reagents and protocols:

available at your local superstore! Rapidly identify disease-causing

pathogens and recommend or provide the appropriate treatment.

Cheap and mass produced, affordable to the developing nations: improvement in global health.

Biology Museum

Bacterial Cultures

QUESTIONS?