The Wonders of Living and
Teaching in the Third Golden
Age of Microbiology
Thanks to the Organizers (especially Dr. Patrick Cummings)
of this 18th Annual American Society of Microbiology
Conference for Undergraduate Educators at Johns Hopkins
Amy J. Horneman,
Horneman, PhD, SM(ASCP)
Dircctor,
Dircctor, Microbiology and Molecular Diagnostics
VA Maryland Health Care System
Baltimore,Maryland
University Homewood Campus
June 2-5, 2011
Classical Golden Age of Microbiology
(1857(1857-1914)
• Microbiology as a discipline “arrived”
arrived” with the
validation of the existence and importance of
microbes
• Scientists discovered the main bacterial
etiological agents of disease in humans and
animals
• Field of immunology was developed leading to
vaccine development and serological testing
1
Sir Joseph Lister
“Father of Antiseptic
Surgery” who as an
English surgeon in 1867
advocated the use of
“carbolic acid” or
“phenol” to sterilize
surgical instruments and
to clean wounds
Ignaz Philipp Semmelweis (1818-65)
He dared to advocate “handwashing”
before Surgery to stop the spread of
“puerperal sepsis” or “childbirth fever”
in 1850 in Vienna, Austria
(1827-1912)
http://www.nndb.com/people/597/000091324/joseph-lister-1-sized.jpg
Robert Koch (1843-1910)
1876 – First Proof of Germ Theory
of disease with discovery of
Bacillus anthracis
Dr. Louis Pasteur, a great French chemist and bacteriologist (1822-1895)
1881 – Growth of Bacteria on Solid
Media
1861- Disproved “spontaneous generation”
1882 – Discovery of the cause of
Tuberculosis
1862 – Supported “germ theory” of disease
1885 – Developed first rabies vaccine
http://www.bio.miami.edu/dana/pix/pasteur.jpg
Google Images
1884 – Outlined Koch’s Postulates
2
Paul Ehrlich
(1854-1915)
More Key Players in First Golden Age
• 1884 – Christian Gram developed Gram Stain
1882 – Developed the
“acid-fast” stain
• 1887 – R.J. Petri invented the Petri Dish
• 1892 – Dmitri Ivanovski discovered “viruses”
viruses”
• 1900 – Walter Reed proved that mosquitoes
carried the “yellow fever”
fever” agent
Google Images
1910 – Discovered the
“magic cure” of
Salvarsan (606) for the
treatment of syphilis
(with help of Hata, a
Japanese Graduate
student)
Results from Discoveries of the First
“Golden Age of Microbiology”
• Pasteurization
• Antibiotics
• Germ Theory
• Vaccination
• Fermentation
Dr. Albert Neisser, a famous German bacteriologist (1855-1916), in the lab
where he discovered the gonococcus (Note the presence of his
graduate student in the background!!)
ASM Archives
3
Second Golden Age of Microbiology
(1943(1943-1970)
• This began with the “rediscovery”
rediscovery” of penicillin
by Florey and Chaney (originally observed by
Sir Alexander Fleming in 1928)
• 1944 – Avery, McLeod, and McCarty
determine that DNA is the “genetic material”
material”
Second Golden Age of Microbiology
(1943(1943-1970)
• 1953 – Watson and Crick at Cambridage
determine the structure of DNA is a
“double helix”
helix” (with extensive help from
Rosalind Franklin – a graduate student of
Maurice Wilkins at Kings College)
• Accompanied by the birth of
“molecular genetics”
genetics”
Google Images
Second Golden Age of Microbiology
(1943(1943-1970)
• 1960 – Jacob and Monod develop the concept
of an “operon”
operon”
• 1966 – Nirenberg and Khorana discover
Sadly, as antibiotic usage becomes widespread,
the study of pathogens begins to lose importance.
1967 - the U.S. Surgeon General William Stewart said
that it was “time to close the books on infectious
diseases, declare the war against pestilence won,
and shift national resources to such chronic
problems as cancer and heart disease.” *
the genetic code
• 1969 – Temin, Baltimore and Dulbecco discover
retroviruses/reverse transcriptase
1970’s – President Nixon declares “War on Cancer” **
• 1970 – Hamilton Smith reveals the specificity of
of the action of restriction enzymes
*I was reading Paul de Kruif’s 1926 book Microbe Hunters for the first time.
** I was entering Virginia Tech as a Biology Major on my way to becoming a
Clinical Microbiologist.
4
Third Golden Age of Microbiology
(Late 1970’
1970’s –Present Tme)
Tme)
• 1977 – Both Gilbert and Sanger developed methods
to sequence DNA
• 1983–
1983– Kary Mullis discovers PCR (polymerase
In this Third Golden Age of
Microbiology, we begin to really see the
“marriage”
marriage” of classical microbiology to
molecular biology which leads to the:
chain reaction)
• 1995 – Craig Venter and Hamilton Smith complete
•
the first genome sequence of a bacterium,
Haemophilus influenzae
2003 – Human Genome Sequencing Project
Completed
Birth of Molecular
Diagnostics
Methods of Bacterial Identification
• Biochemical assays for phenospecies
• DNA/DNA Hybridization for
genospecies
• PFGE and 16S rDNA Ribotyping
• MLST (Multi(Multi-Locus Sequence Typing)
Gram stain of Klebsiella pneumoniae
ASM
5
Growth at 35-37o Celsius for 24-48 Hours
ASM
Motility using
Semi-Solid Agar
Flagella Stains
6
MicroScan (Siemens)
Vitek (BioMerieux, Inc)
1980s to Present
Automated ID and AST
API-20E Strip for Identification of Enterobacteriaceae
1970”s
ASM
Phoenix (BDMS)
Google Images
Methods of Bacterial Identification
MOLECULAR TYPING
• Biochemical assays for phenospecies
• DNA/DNA Hybridization for
• Based on genetic material
• More accurate in differentiating strains
• Analyzed on flexible and complex
genospecies
• PFGE and 16S rDNA Ribotyping
• MLST (Multi(Multi-Locus Sequence Typing)
computercomputer-based programs
• Easily accessible in clinical settings
• Ideal for epidemiological investigations
• PFGE – was the “gold standard”
standard”
7
Pitfalls of PFGE
Salmonellois associated with marijuana:
a multistate outbreak traced by plasmid
fingerprinting
Taylor et al., 1981, New
England Journal of Medicine
Taxonomic Note (IJSEM March 2002)
„
„
• Complex interpretation
of results
• Time consuming
process
• Data is not portable
MLST(Multi LocusSequence Typing)
Report of the Ad Hoc Committee for the ReReEvaluation of the Species Definition in
Bacteriology
Acknowledge methods of promise to augment “gold
standard”
standard” of DNA/DNA hybridization for species
delineation
„
„
„
Sequencing of housekeeping or other genes (MLST)
DNA profiling
DNA arrays
8
Concatenated
MULTLOCUS SEQUENCE TYPING
Neighbor joining:
Concatenated Tree
NJ
A.bestiarum ATCC51108 AA8
A.hydro&sal&best&dhak JB28E
A.hydro&sal&best&dhak JB49E
A.hydro&sal&best&dhak JB17E
A.hydro&sal&best&dhak JB7E
A.hydro&sal&best&dhak JB15E
A.bestiarum AA9
A.hydro&sal&best&dhak JB3E
A.hydro&sal&best&dhak JB27E
A.hydro&sal&best&dhak JB1E
A.hydro&sal&best&dhak JB13E
A.hydro&sal&best&dhak JB14E
A.hydro&sal&best&dhak JB8E
A.popoffi LMG17541 AA31
atypical Aeromonas JB40E
A.veron.sobri&jand&schub&trot JB39E
A.salmonicida CDC0434.84 AA10E
A.salmonicida AA11
A.hydro&sal&best&dhak JB33E
A.hydro&sal&best&dhak AA23
AH dhakensis 80
AH dhakensis 127
AH dhakensis 104
AH dhakensis 84
AH dhakensis 79
AH dhakensis 10
AH dhakensis 133
AH dhakensis 56
AH dhakensis 14
A.hydro&sal&best&dhak AA19
A.hydro&sal&best&dhak AA24
AH dhakensis 106
AH dhakensis 64
AH dhakensis LMG19562 AA29
A.hydro&sal&best&dhak JB11E
AH dhakensis 136E
AH dhakensis 81
AH dhakensis 82
A.hydro&sal&best&dhak AA21
A.veron.sobr&jand&schu&tro AA20
AH dhakensis 126
A.hydro&sal&best&dhak AA18
A.hydro&sal&best&dhak JB4E
A.hydro&sal&best&dhak JB5E
A.hydro&sal&best&dhak JB26E
A.hydrophila ATCC7966 AA1
A.hydro&sal&best&dhak JB41E
A.hydro&sal&best&dhak AA26
A.hydro&sal&best&dhak JB38E
A.hydrophila MB4
A.hydro&sal&best&dhak NM16a
A.hydro&sal&best&dhak NM42E
A.hydro&sal&best&dhak AA25
A.hydrophila MB5
AH ranae LMG19707 AA30
A.hydro&sal&best&dhak JB19E
A.hydro&sal&best&dhak JB20E
A.hydro&sal&best&dhak JB21E
A.hydro&sal&best&dhak JB35E
atypical Aeromonas MB9
A.hydro&sal&best&dhak AA28
A.hydro&sal&best&dhak AA27
A.hydro&sal&best&dhak JB12E
A.hydro&sal&best&dhak NM7
A.hydro&sal&best&dhak NM41
A.caviae&media&eucreno AA13E
A.caviae&media&eucreno JB18E
A.caviae&media&eucreno AA14
A.caviae&media&eucreno AA16E
A.caviae&media&eucreno AA15
A.caviae MB7
A.caviae MB10
A.caviae MB8
A.caviae MB1
A.caviae&media&eucreno JB45E
A.caviae MB3
A.caviae&media&eucreno NM22
A.caviae ATCC15468 AA3
A.caviae MB14
atypical Aeromonas MB6
A.caviae MB12
A.caviae MB16
A.caviae&media&eucreno NM2
A.caviae&media&eucreno NM3
A.caviae MB13
A.caviae&media&eucreno NM4
A.caviae&media&eucreno NM14
A.caviae&media&eucreno JB37E
A.caviae&media&eucreno JB16E
A.trota ATCC49657 AA7
A.schubertii ATCC43700 AA6
A.jandaei ATCC49568 AA4
A.hydro&sal&best&dhak AA22
A.veronii.sobria ATCC9071 AA2
A.veron.sobr&jand&schu&trot NM74E
A.veron.sobr&jand&schu&trot NM75E
A.veron.sobr&jand&schu&trot JB22E
A.veron.sobr&jand&schu&trot JB23E
A.veronii.veronii ATCC35624 AA5
A.sobria CIP7433 AA17
A. bestiarum
A. popoffii
A. salmonicida
AH subsp dhakensis
• Four Gene Loci
• 17 species of Aeromonas
• Maximum Discriminatory
Power
A. hydrophila
A. caviae/media
Amy J. Horneman, PhD
PAUP
A. veronii
0.005 substitutions/site
Field of Molecular Diagnostics
What’
What’s New in the World of
Microbial ID:
Genomics and Proteomics
• Infectious Diseases Molecular Testing
• Molecular Oncology
• Identity Testing or DNA Fingerprinting
• HLA Testing or Immunogenetics
• Pharmacogenetics
Courtesy of: Patrick R. Murray, PhD
Director, Microbiology at NIH
Medscape, 2010
9
Genomics
• Molecular Probes
– DNA Probes
– PNA Probes
• Gene Amplification
– Sanger Sequencing
– Pyrosequencing
• Next Generation
Sequencing
Molecular Probes
Proteomics
• MALDIMALDI-TOF Mass
Molecular Probes
• DNA Probes (e.g., GenProbe AccuProbe)
AccuProbe)
– SingleSingle-stranded DNA probe with a
chemiluminescent label (acridinium
(acridinium ester)
complementary to ribosomal RNA target.
Spectophotometry
– Bacterial identification
– Mycobacterial
Identification
– Yeast Identification
– Used for identification of cultured organisms; not
sensitive enough for detection of organisms in
clinical specimens
– Used to identify selected bacteria,
bacteria, mycobacteria,
mycobacteria,
and dimorphic fungi
Peptide Nucleic Acid
Fluorescent In Situ Hybridization (PNA FISH)
10
AdvanDx PNA FISH Assays
FDAFDA-Approved Nucleic Acid Amplification
Tests:
Microbial Detection Assays
Amplification Method
Company
Polymerase chain
reaction (PCR)
Roche, Cepheid N. gonorrhoeae,
gonorrhoeae, C. trachomatis,
trachomatis,
M. tuberculosis, grp.
grp. B
Streptococcus, MRSA, VRE, C.
difficile,
difficile, Influenza virus, HIV, HBV,
HCV, Enterovirus,
Enterovirus, respiratory
viruses
GenProbe
N.
gonorrhoeae,
gonorrhoeae, C. trachomatis,
trachomatis,
Transcription mediated
amplification (TMA)
FDAFDA-Approved Tests
M. tuberculosis, HIV, HBV, HCV
Strand displacement
amplification (SDA)
Becton
Dickinson
N. gonorrhoeae,
gonorrhoeae, C. trachomatis,
trachomatis,
grp.
grp. B Streptococcus, MRSA
Nucleic acid sequence
based amplification
(NASBA)
bioMerieux
CMV, HIV
Branched DNA signal
amplification (bDNA
(bDNA))
Siemens
HIV, HBV, HCV
Molecular Probes
Nucleic Acid Amplification Tests
• Nucleic acid amplification + probes
– Cepheid Xpert assays (e.g., MRSA, VRE,
C. difficile,
difficile, Flu A)
– Multiplex assays (e.g., respiratory
viruses)
• Nucleic acid amplification + sequencing
– Sanger sequencing
– Pyrosequencing
11
Cepheid Xpert MRSA Assay
Cepheid Xpert Assay
Cepheid Xpert VRE Assay
Cepheid Xpert Clostridium difficile Assay
• Previous assays for C. difficile toxins (i.e.,
cytotoxicity,
cytotoxicity, culture, EIA for glutamate
dehydrogenase or toxins A/B) were insensitive
and/or nonspecific.
• In contrast with the data for the MRSA and VRE
assays, the C. difficile assay has been reported to
be highly accurate,
accurate, as well as rapid and costcosteffective.
• Because previous C. difficile toxin assays were
insensitive, repeat testing to confirm a negative
result was commonplace. This is not necessary
with this C. difficile assay.
12
New Assay Additions – C.diff/epi
Current, FDA Approved Menu
•MRSA Nasal
•SA Nasal Complete (MRSA and MSSA)
•MRSA/SA BC
•MRSA/SA SSTI
•vanA for VRE
•FLU *Requires 66-color Modules
•C.diff (Toxin B only)
•C.diff/epi (Toxin B and BI/NAP1/027) *Requires 4.3
New Assay Additions - FLU
A Better Way to Platform Design
GeneXpert
Infinity-48
GX-16
GeneXpert®
Module
GX-1
GX-4
software
•Factor II and V
•Group B Strep
•Enterovirus
13
Detection of rifampin-resistant
TB strains (Boehme et al., 2010,
Multiplex Genomic Assays
• A variety of commercial multiplex assays have been
develop for detection of respiratory pathogens including –
New England Journal Medicine)
– Luminex xTAG
– EraGen Biosciences MultiCodePLx
– Qiagen ResPlex II Panel
• A variety of pathogens can be detected including
•
influenza A and B viruses, adenovirus, parainfluenza
viruses, respiratory syncytial virus, metapneumovirus,
metapneumovirus,
rhinovirus, coronaviruses,
coronaviruses, coxsackie and ECHO viruses,
and bocavirus.
bocavirus.
These assays are consistently more sensitive than
conventional cultures and stains although some of the
current technologies are labor intensive and costly.
Cepheid
QIAGEN – Leader in
Sample & Assay Technologies
QIAGEN – Leader in
Sample & Assay Technologies
ResPlex II v2.0
Technology overview
Single-Plex Tests
Technology Overview
March, 2011
May, 2011
Preparing the Future
Preparing the Future
14
From Sample To Clinical Results
Combination Of FrontFront-End Solutions With artus Products
Patient
sample
Sample technologies
Assay technologies
Detection
QIAamp
CMV
Urine
1-12
Swabs
EBV
QIAcube
Swabs
• Molecular Differential Detection MDD
– Amplification of multiple targets from single sample in one tube
– Proprietary QIAplex technology
• Detection
Sample
– Luminex IS 200 Workstation
– BeadBead-array technology
Nucleic acid purification
(QIAamp)
– Target specific probes coupled to beads
– Hybridization to specific amplicons
1-6
CSF
MultiMulti-Plex Technology
QIAplex™
QIAplex™ for Molecular Differential Detection
HSV
PCR
(ABI9700)
1-6
Blood
EZ1 Advanced
BKV
Plasma
Sample Type
Hybridization
(Target specific beads)
VZV
1-96
QIAsymphony
Detection & Analysis
(Luminex IS 200 Workstation)
Integrated
Integrated solution
solution from
from sample
sample preparation
preparation to
to pathogen
pathogen detection
detection
QIAplex – ResPlex II v2.0 Panel
Summary
ResPlex II v2.0 Panel composition
ResPlex II v2.0
– Amplification and detection of 18 respiratory viruses in one tube
tube
– TwoTwo-box concept
• Amplification includes all reagents needed for PCR setup
• Detection includes all reagents needed for hybridization
– Workflow
• 4.5 hours from sample to results
• 1 – 1.5 hours hands on time
• No multiple openings of amplified products
– Internal control for process verification
– Sample control for verification of sample quality
– QIAplex MDD Software and MDD Plate for analysis, result
interpretation and calibration
ResPlex™ is for research use only.
Not for use in diagnostic procedures.
– Respiratory Syncytial
Virus A
– Respiratory Syncytial
Virus B
– Influenza A
– Influenza B
– Parainfluenza 1
– Parainfluenza 2
– Parainfluenza 3
– Parainfluenza 4
– Human Metapneumovirus
(A, B)
– Enterovirus (Coxsackie,
Echo)
–
–
–
–
–
–
–
–
–
–
Coronavirus 229E
Coronavirus OC43
Coronavirus NL63
Coronavirus HKU1
Bocavirus
Adenovirus B (3, 7, 21)
Adenovirus E (4)
IC (internal control)
IDS (sample control)
Rhinovirus
15
4 Steps from prep to detection
Nucleic acid purification to
amplification, hybridization and
detection
Nucleic Acid Amplification Tests
Fw Super primer
Fo
• Nucleic acid amplification + probes
Fi
Forward Pri
One-step NA extraction
(40 min)
Virus or other
pathogens
Ri
One-Step QIAplex PCR
(210 min)
Extracted
nucleic acid (NA)
Ro
Rev Super primer
“
Amplification and labeling
– Cepheid Xpert assays (e.g., MRSA, VRE,
C. difficile)
difficile)
– Multiplex assays (e.g., respiratory
viruses)
• Nucleic acid amplification + sequencing
– Sanger sequencing
– 454 Sequencing or Pyrosequencing
– IBIS and Illumina
Hybridization
(20 min)
Hybridization
Detection and read out
Sanger Method of DNA Sequencing
Genomic Approach
Gene Amplification/Sequencing
16
16S rRNA as a target for
molecular phylogenetic analysis
Genomic Approach
Gene Amplification/Sequencing
• Small subunit of ribosomal
RNA
• Present in all bacteria
• Large sequence database
•
for identification closest
related species
Broad primers (E(E-TIGER)
or group/species specific
primer sets available
Bacterial Identification by Gene Sequencing
Abiotrophia defectiva
Capnocytophaga sputigena
Helicobacter cinaedi
Achromobacter
xylosoxidans
Brevundimonas diminuta
Cardiobacterium hominis
Corynebacterium accolens
Herbaspirillum
huttiense
Kingella denitrificans
Dysgonomonas
capnocytophagoides
Haemophilus aphrophilus
Tsukamurella pulmonis
Campylobacter fetus
Campylobacter upsaliensis
Molecular Identification of Fungi
• Large subunit rRNA (28S)
• Internal transcribed spacers (ITS)
- Universal
- Very large database including environmental fungi
- ~500 bp region sequenced
Kytococcus schroeteri
17
Clinical Case
• 48 year old male with an undefined immunodeficiency
• Infection developed at his nares and spread to his cheek, sinuses, palates and skin Identification of the Mold
Culture –
28S rRNA Sequencing –
Sporulation after 9 weeks; • Failed to identify the mold identified as Corynespora
(sequence not in database)
ITS Sequencing –
• Identified as Corynespora
cassiicola (“cucumber” mold)
• Patient required extensive surgical debridement
Detection and Identification of Mycobacteria
SecA1 Gene Sequencing
Clinical Case
18
IMPORTANT NOTICE
Intended Use
High Throughput DNA
Sequencing &
the Revolution in Life
Science Research
Unless explicitly stated otherwise, all Roche Applied
Science and 454 Sequencing products and services
referenced in this presentation are intended for the following
use:
For Life Science Research Only.
Not for Use in Diagnostic Procedures.
Christopher McLeod
President & CEO, 454 Life Sciences, A
Roche Company
The DNA Sequencing Revolution
Impact on nearly every field of
biological research
www.454.com
Proven Technology: Customer Success
Enabling breakthrough genomic discoveries
90
80
70
60
50
40
30
20
10
0
Publications by Quarter
>800 peer-reviewed
publications
Q1'05 Q3'05 Q1'06 Q3'06 Q1'07 Q3'07 Q1'08 Q4'08 Q2'09 Q4'09
Publications by Research Area
"Evolution & ecology
Human Genetics
& Genomics
Plants &
Agriculture
Microbes, Viruses
& Infectious
Diseases
8%
Environmental
Genomics
6%
7%
"Human genetics & genomics
"Metagenomics & microbial diversity
13%
21%
"Microbes, viruses & infectious diseases
"Cancer research
15%
5%
"Plants & agricultural biotechnology
25%
"Model & non-model organisms, systems biology
"Technology & informatics
www.454.com
www.454.com
19
Sequencing James Watson’
Watson’s Genome
The first of the rest of us
• First whole human genome to be
sequenced with nextnext-generation technology
• 24.5 Billion bases of genomic DNA
sequence generated at the 454 Sequencing
Center
• 3.6 Million variants detected, including
several disease susceptibility gene
associations
Jim Watson
Complete Southern African
Genomes & Exomes
Sequencing the Kalahari
Bushman
Schuster et al. Nature 2010
Human Genome
Project
454 Life Sciences,
A Roche Company
Sanger
2 months,
3 instruments
1010-13 years
<$1 million
$250,000 with Titanium
$100 million - $2.7
billion
7.4x coverage
7.5x coverage
250 bp read length
400bp with Titanium
500500-800 bp read length
• Complete genome sequence of an indigenous hunter-gatherer from the
Kalahari desert & a Bantu from southern Africa (Desmond Tutu)
• Three whole exome sequences of Kalahari hunter-gatherers using NimbleGen
Sequence Capture arrays
www.454.com
Schuster SC et al. Nature. (2010)
Human Gut Metagenomics
Characterizing the communities within each of us
Turnbaugh et al. Science Translational Medicine 2009
• The human body harbors trillions of microbial organisms
which collectively make-up the human “microbiome”
• We are dependent on these organisms for known functions
such as digestion and immune defense
• Sequencing studies to characterize the human gut
microbiome, transplanting human microbes into germ-free
mice models
• Two groups of mice with the same transplanted human gut microbial
community
– One group on new high-fat diet, one on same low-fat diet as before
transplant
– Types of bacteria changed rapidly and dramatically with high-fat, highsugar diet
• What we eat has a significant impact on our gut microbial communities!!
This has significant implications for research on human nutrition, obesity
www.454.com
and famine
† Turnbaugh P.J. et al. Nature 444:1027-1031 (2006), Turnbaugh P.J. et al. Science Translational Medicine 1:6 (2009).
Environmental Metagenomics
Characterizing earth’
earth’s extreme environments
VegaVega-Thurber et al. PNAS 2008
• Metagenomics-- Sequencing a mixed sample to identify
the diversity of organisms present and their function
• Metagenomics sequencing study to explore the role of
viral pathogens in declining coral health*
• Sequence coral samples under varying environmental
•
•
•
stressorsstressors- reduced pH, elevated nutrients, increased
temperaturetemperature- to mimic current ecological changes
Study found high levels of a herpesherpes-like virus in
stressed coral samples
Virus was not detected in healthy, unstressed coral
Study sheds light into one of many factors which
explain the death of coral reefs as ocean temps rise
and pollution increases
www.454.com
* Vega-Thurber R.L. et al. PNAS 105:18413-8 (2008).
20
Pyrosequencing (454 Life Science)
Microbes, Viruses & Infectious Disease
Sequencing to identify drugdrug-resistance in HIV
Simen et al. JID 2009
• HIV drug resistance is attributed to minority viral variants
which can lead to regimen failure
• Current methodologies, based on Sanger technology, can
only detect rare variants present at >20% frequency
• Research study used 454 Sequencing Systems to detect
rare drugdrug-resistance variants in a little as 1% of the viral
population†
HIV virus
– Low-frequency mutations had significant impact on clinical outcomes, i.e.
early antiretroviral treatment failure
– The fraction of patients harboring drug-resistant variants was twice as high
as previously thought
• Barcoded primer sets allow for sequencing of multiple libraries in one run
• 10 / 100 libraries can be sequenced at a depth of 40K / 4K sequences
• FDA guidance now requires a viral population profiling test prior to, during and
after antiretroviral therapy during drug trials to identify drug-resistance
New bioinformatic tools required to handle such large data sets
www.454.com
† Simen B. et al. Journal of Infectious Disease 199:1275-85. (2009).
Advantages of 454 Sequencing Systems
Read length, quality and speed
Long Reads
400 bp average read lengths enable complete
coverage of genomic regions and haplotyping
Quick Time to Result
Fast 10-hour sequencing run time
5%
High Quality
99% accuracy at the 400th base position and
higher for preceding bases (Q20 read length of
400 bp)
4%
% Error
Pyrosequencing
3%
2%
1%
0%
0
100
200
300
400
500
600
Base Position (bp)
21
Pyrosequencing
• Pyrosequencing has a relatively rapid turnaround time,
Pyrosequencing for Microbial Identification
is more cost effective than Sanger sequencing and can
be used to identify a broad range of organisms.
The next big thing in sequencing is
small Perfectly suited for medical research
applications
• Perfectly sized for labs that require:
– Targeted sequencing of genomic
regions associated with disease,
e.g. diabetes, cancer
– Genotyping research, e.g. HLA
typing
– Whole microbial genome
sequencing
– Metagenomics
– Novel pathogen detection
GS Junior Bench Top System
• Tailored to the needs of individual labs
www.454.com
22
23
24
Genomics – Future
• Three applications of microbial sequencing
– Target gene sequencing – e.g., 16S rRNA gene for
identification of cultured organisms; SecA gene for
direct detection and identification of mycobacteria in
clinical specimens
– Whole genome sequencing – e.g., used for strainstrain-totostrain comparisons (typing), detection of virulence
factors or antibiotic resistance markers
– Gene sequencing directly in clinical specimens – e.g.,
“Next Generation”
Generation” or “Deep”
Deep” sequencing
MALDIMALDI-TOF MS
MatrixMatrix-Assisted Laser Desorption/Ionization –
Time of Flight Mass Spectrometry
Proteomics
Courtesy of: Patrick R. Murray, PhD
Director, Microbiology at NIH
25
MALDIMALDI-TOF Systems
Mass Spectrometry
• 2 commercial MALDIMALDI-TOF
instruments are currently
available for microbiology
labs:
– microFlex – Bruker
Daltonics
– Vitek MS - bioMerieux
• Bruker developed the
software and database used
in the microFlex.
microFlex.
Po we r
Io n is in g
Va cu u m
N e g ative
• Shimadzu developed the
instrument and software for
the Vitek MS, and uses a
database developed by
AnagnosTec.
AnagnosTec.
Mass Spectrometry
Shimadzu Axima – bioMerieux Vitek MS
AXIMA Assurance
Computer with
LaunchPad software
Computer with
SARAMIS database
• Samples mixed with a matrix
– Composed of small acid molecules that absorb ions in range of
laser wavelength
– Composition selected for sample molecules of interest
• α-cyanocyano-4-hydroxycinnamic acid (CHCA)
• Sinapinic acid (SA)
• Ferulic acid (FA)
• 2,52,5-dihydroxybenzoic acid (DHB)
– CHCA, SA, and FA used to detect proteins; DHB used for
glycopeptides and glycoproteins
– CHCA and DHB are optimum for detection of low mass proteins
• Samples can be pretreated with a strong organic acid or
mixed directly with the matrix and analyzed
– Only bacteria can be reliably analyzed without pretreatment
– Better extraction (higher identification scores) are obtained with
with
pretreatment.
26
Biomarkers
MALDI Matrix
• Both the Bruker microFlex and ShimadzuShimadzu-bioMerieux
Vitek MS systems use CHCA in acetonitrile and
trifluoroacetic acid; Shimadzu also has the option of
using DHB in waterwater-ethanolethanol-acetonitrile.
acetonitrile.
• Although both systems optimally detect small mass
proteins.
• If the matrix is changed, then the spectral matching
software may not be able to match the new organism
spectra with the existing databases.
Mass Spectrometry – Profile
Reproducibility
MS Databases and Software
• The BioTyper reference library for the microFlex MS
system contains reference spectra for >3200 strains;
each reference spectrum generated from 20 measured
spectra.
• The AnagnosTec SARAMIS database consists of 2700
“supersuper-spectra”
spectra” (consensus spectra from multiple
spectra of individual reference strains); corresponds to
>900 species.
• Reliability of identifications dependent on reference
database and software algorithm used for spectral
comparisons.
27
MALDIMALDI-TOF MS
Intens. [a.u.]
Unique Profiles for Each Organism 3000
Aspergillus fumigatus
2000
Intens. [a.u.]
1000
0
8000
Bacillus subtilis
6000
4000
0
5380
0.6
0.4
0.2
0.0
7870
100
0
Escherichia coli
2500
8368
200
0
Candida albicans
0.8
6410
5096
400
0
300
0
1.0
7157
7273
500
0
6315 6254
Intens.
[a.u.]
4364
2000
2000
1500
1000
0
4000
500
5000
6000
7000
8000
m/z
0
3000
4000
5000
6000
7000
8000
9000
10000
m/z
MALDIMALDI-TOF Identification
Bacterial Clinical Isolates
MALDIMALDI-TOF Identification
Protocol for Processing Blood Culture Broths
Stevenson et al: J Clin Microbiol 2010
28
MALDIMALDI-TOF Identification
Blood Culture Isolates
Stevenson et al: J Clin Microbiol 2010
MALDIMALDI-TOF Identification
Mycobacterial Isolates
Sample Preparation Protocol (1 hour)
• 212 positive blood cultures were analyzed:
– 42 (20%) with no identification
– 170 (95.3%) of the remaining organisms were identified
correctly
– 8 organisms were misidentified (all Streptococcus mitis
isolates were misidentified as S. pneumoniae)
pneumoniae)
• The most common organisms with no ID were
Propionibacterium and coagulasecoagulase-negative
staphylococci
• Spectral scores for bacteria in blood culture broths
were generally lower than scores for bacteria tested
from isolated colonies
MALDIMALDI-TOF Identification
Database of Reference Mycobacterial Strains
MALDIMALDI-TOF Identification
Identification of 73 Clinical Isolates
Organism (No. Tested)
M. tuberculosis complex (30)
Other slowslow-growers (43)
M. asiaticum (2), M. avium (7), M.
gordonae (5), M. intracellulare (6), M.
kansasii (15), M. marinum (5), M. szulgai
(2), M. xenopi (1)
RapidRapid-growers (33)
M. abscessus (6), M. chelonae (5), M.
fortuitum (6), M. massiliense (6), M.
mucogenicum (5), M. peregrinum (4), M.
smegmatis (1)
No.
Identifie
d
30
43
33
29
MALDIMALDI-TOF Identification
MALDIMALDI-TOF Identification
Yeast Clinical Isolates
Yeast Reference Database
Stevenson et al: J Clin Microbiol,
Microbiol, 2010
Stevenson et al: J Clin Microbiol,
Microbiol, 2010
Genus
Candida
Cryptococcus
Geotrichum
7
2
No. of
Isolates
58
22
4
Kodamaea
Malassezia
Pichia
1
2
1
1
2
1
Rhodotorula
Trichosporon
2
9
2
19
TOTALS
44
109
Summary
Species
20
Summary (cont.)
• Identification of organisms by gene sequencing is the
gold standard; however, multiple gene targets may be
required. Sanger sequencing and pyrosequencing are both
useful, but at the present time are associated with higher
reagent and personnel costs.
• MALDIMALDI-TOF mass spectrometry is a powerful tool that
can identify most bacteria, mycobacteria and fungi at the
species level. The test is rapid, consumable and personnel
costs low, but associated with high equipment costs.
• Metabolomics is in an early stage of development but may
be the next generation system for organism detection and
identification, strain typing, and assessing response to
antimicrobials.
30
Metabolomics
• Newborn “cousin”
cousin” to genomics and proteomics
• Involves rapid, high throughput characterization
•
•
of the small molecule metabolites found in an
organism
Metabolome is closely tied to the genotype of an
organism
Human Metabolome Project (HMP) is ongoing at
the University of Alberta, Canada
The 21st Century will be the era of the microbe
“We live now in the ‘Age of Bacteria’. Our planet has always
been in the ‘Age of Bacteria,’ ever since the first fossils,–
bacteria, of course, were entombed in rocks more than 3 billion
years ago. On any possible, reasonable or fair criterion,
bacteria are-and always have been-the dominant forms of life
on Earth. Our failure to grasp this most evident of biological
facts arises in part from the blindness of our arrogance but
also, in large measure, as an effect of scale. We are so
accustomed to viewing phenomena of our scale-sizes measures in
feet and ages in decades - as typical of nature.”
-Stephen Jay Gould (1996) in Planet of the Bacteria
Reflections - Integrative Microbiology The Third Golden Age*
• Advances in Microbiol Ecology
– Biofilm communities and Quorum Sensing
– Types II-VI Secretion Systems
• Evolution – evidence for lateral gene transfer
• Microbial Cell Biology
– Bacteria are not just “bags of enzymes”
enzymes”
– “Within 1 cubic micron or so, there is a sophisticated and unexpected
unexpected
compartmentalization.”
compartmentalization.”
• Eucaryotic Cell Biology – studying bacteriabacteria-host interactions
• Threat of Bioterrorism – detection and treatment
• Industrial and Pharmaceutical Applications
*Author -Moselio Schaechter in a Perspectives Article from the Journal of Biosciences,
Vol. 28, pp. 149-154, 2003
Reflections - Integrative Microbiology The Third Golden Age*
• “Microbiology is becoming more unified.”
unified.”
• “Microbiologists speak ever more a common
language.”
language.”
• “Microbiology has become an integrative
•
science, one that thrives to combine and
coordinate diverse elements into a biological
whole.”
whole.”
“This is the new culture of microbiology.”
microbiology.”
*Author -Moselio Schaechter in a Perspectives Article from the Journal of Biosciences,
Vol. 28, pp. 149-154, 2003
31
Acknowledgements
• ASMCUE
• Patrick Murray, PhD
• Cepheid
• 454 Life Sciences (Roche)
• Qiagen
• BioMerieux,
BioMerieux, Inc.
• IBIS
• (Abbott)
• Illumina
32