Biologia molecular: Una necessitat de la
microbiologia clàssica
Jordi Vila
Hospital Clinic
DIAGNOSTIC TECHNOLOGIES IN
CLINICAL MICROBIOLOGY
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Direct examination Microscopy
Culture
Detection of antibodies (serology)
Detection of microbial antigens
Detection of nucleic acids
Main features for an optimal test
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Detection and identification of several
microorganisms related to a specific
syndrome in a single test
High sensitivity and specificity
Rapid, in minutes better than hours
Automatized
Cheap
Rapid diagnostic methods
• Adventages
– To implement adequate antimicrobial therapy
– To favor the control of the emergence of
antimicrobial resistance
– Decrease the cost to the hospital
EVOLUTION OF THESE MOLECULAR TOOLS
• Hybridization of nucleic acids
• DNA or RNA amplification
• Real time PCR
• DNA microarrays
TYPES OF NUCLEIC ACID AMPLIFICATION
• Target amplification system
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PCR
Ligase chain reaction (LCR)
Self-sustaining sequence amplification (3SR)
Nucleic acid sequence-based amplification (NASBA)
Transcription-mediated amplification (TMA)
Strand displacement amplification (SDA)
• Signal amplification system
– Q-b-replicase
– Branched DNA technologies (bDNA)
– Cleavage-based signal amplification
VARIATIONS OF THE PCR
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Multiplex PCR
Nested PCR
RT-PCR
Broad-range PCR
Real-time PCR
Arbitrarily primed PCR
Broad-range PCR
P1 P2
16S
ITS
23S
798 bp
Consensus Regions
Variable Regions
ITS = Internal transcribed spacer
5S
APPLICATIONS 0F 16S rRNA
• From colony:
– Bacteria with difficult phenotypic identification
• Species of Acinetobacter, Corynebacterium, some anaerobes
– Fastidious bacteria, due to nutritional requirements
• Eikenella corrodens, Nocardia spp., etc.
– Slow growth bacteria
• Non-tuberculosis Mycobacteria
– Bacteria with no phenotypic match with known bacteria
• From clinical samples:
– Previously treated with antibiotics (CSF, amniotic fluid, joint fluid, cardiac
valves)
• When culture is negative
– Non-cultured bacteria
• Rochalimae (Bartonella) quintana
VARIATIONS OF THE PCR
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Multiplex PCR
Nested PCR
RT-PCR
Broad-range PCR
Real-time PCR
Arbitrarily primed PCR
Real time PCR (specific probes)
A
Probe
B
A
Primer
B
Primer
3’
5’
3’
5’
Molecular beacons
3’
Taq
TaqMan probes
A = Reporter dye
B = Quenching dye
A
5’
B
Primer
3’
5’
FRET Probes
Real time PCR
• Advantages:
– Speed (1-3 hours)
– Closed system decreasing the risk of contamination
– Quantitation of the initial
nucleic acid
APPLICATIONS
Detection of toxins
• Detection of C. difficile toxin
• Detection of PVL in S. aureus
• Detection of virulence factors in diarrhoeagenic
E.coli (Multiplex PCR directly from a colony identified as E.coli)
– Enterotoxigenic E.coli
– Enteroaggregative E.coli
– Enteropathogenic E.coli
APPLICATIONS
Detection of specific microorganisms
• Grow slowly, or for which the cultivation methods
are not widely available or do not exist.
– Examples:
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Bartonella spp.
Borrelia burgdorferi
Ricketssias, Ehrlichia and Coxiella spp.
Mycoplasma pneumoniae
Chlamydophila pneumoniae
Bordetella pertussis
Tropheryma whipplei
APPLICATIONS
Detection of specific virus
Detection
Viral load
– Parainfluenza virus 1,2,3 and 4
– HIV
– Influenza virus A, B and C
– HBV
– Adenovirus
– Respiratory syncytial virus A and B
– Coronavirus
– Herpes simple virus
– Human papillomavirus
– Enterovirus
– HCV
– CMV
– EBV
APPLICATIONS
• Where reliable and rapid detection of infected or
colonized patients and health care workers is used for
minimizing spread of antimicrobial resistant bacteria in
health care institution. Ex. MRSA
• Detection of S. agalactiae in vaginal swabs.
Detection and identification of several
microorganisms in a single test
• Respiratory infections
• Gastrointestinal infections
• Sexually transmited diseases infection
Current alternatives to detect
respiratory virus
• Detection of specific microorganisms.
– Virus
5’
– Parainfluenza virus 1,2,3 and 4
– Influenza virus A, B and C
5’
5’
– Adenovirus
5’
– Respiratory syncytial virus A and B
– Coronavirus
N IVB
ADV
IVB
RSV
IVA RSV + ADV
– Enterovirus
– Metapneumovirus
Nested-RT-PCR
Current alternatives to detect
respiratory virus
• Detection of specific microorganisms.
– Filmarray
– Magicplex-RV (Seegene)
– Abbot
– Luminex
– Resplex (Qiagen)
– Most of them are detecting the most prevalent respiratory virus some of
them can also detect atypical bacteria causing pneumonia
– Overall good sensitivity and specificity
Blood
Classical
Blood culture
Gram stain
Culture
Semi-molecular
Blood culture
Gram stain
Identification/Resistance
ID/Antibiogram
- DNA Miroarrays
- Filmarray
Microarrays de ADN
120 sondas para identificar:
Pseudomonas aeruginosa
Staphylococcus aureus
Escherichia coli
Genes:
Housekeeping
Virulencia
Resistencia
gen aac-aphD
gen cat
gen dfrS1
gen ermA
gen mecA
Tiempo total 8h.
JCM (2006) 44:2389
Blood
Classical
Semi-molecular
Blood culture
Blood culture
Gram stain
Culture
ID/Antibiogram
Gram Stain
Culture
ID/Anti
Identification/Resistance
MS
DNA Miroarrays
Filmarray
Matrix-assisted laser desorption/ionization
mass spectrometry (MALDI-TOF MS)
Acceleration
Matrix-embedded
Analyte on
Microtitreplate Target
Drift
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Electrodes
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Detector
Laser Desorption/Ionization
Time-of-Flight
Intensity
m/z
MALDI-TOF MS
Identified species
Bacillus globigii
BioProfiler
Data interpretation
Generate MALDI-TOF
profile spectrum
Profiling results from different Bacillus strains
a.i.
6000
4000
Smear a thin-layer onto
a MALDI target plate
2000
B. globigii
0
Select a colony
-2000
-4000
Unknown
microrganism
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-6000
B. licheniformis
B. subtilis
B. thuringiensis
Direct testing of positive blood cultures by
MALDI-TOF
WHEN
POSITIVE
Sampling
Incubation of blood
culture bottles
Comparison with
a database
Acquisition of the
proteic profile
Preparation of a
bacterial pellet
Deposition of
bacterial pellet on
MALDI microplate
Identification of bacteria growth in
blood cultures
%
97
93
97
70
Juiz et al. EJCM (2011) “in press”
Burckhardt and Zimmerman: Using matrix-assisted laser
desoprtion ionization-time of flight mass spectrometry to
detect carbapenem resistance within 1 to 2.5 hours
Journal of Clinical Microbiology 2011; 49: 3321
• 10 mcl loopful of bacteria to 1 ml of 0.45% NaCl with or without
0.5 g/liter ertapenem. Incubation 2.5 h at 36ºC
This methods works for strains
carrying NDM-1, VIM-1, VIM-2,
KPC-2 and different IMP enzymes
Blood
Classical
Semi-molecular
Blood culture
Blood culture
Gram stain
Gram Stain
Culture
ID/Antibiograma
Identification/Resistance
MS
Miroarrays de ADN
Filmarray
Genexpert (MRSA)
Parta M et al. Identification of methicillin-resistant or methicillinsusceptible Staphylococcus aureus in blood cultures and
wound swabs by GeneXpert.
J Clin Microbiol 2009;47:1609
• 223 blood cultures – 68 positive to S. aureus
– 47 MRSA and 21 MSSA
– PCR 46/47 (98%) MRSA and 21/21 MSSA (100%)
Blood
Clasical
Semi-molecular
Blood culture
Molecular
Blood culture
Direct detection
- Septifast
- Magicplex-sepsis
- SeptiTest
Gram Stain
Culture
ID/Antibiogram
Gram Stain
Culture
ID/Anti
Identification/Resistance
MS
Miroarrays de ADN
Filmarray
Genexpert (MRSA)
CONCLUSIONS
Advantages of these rapid tests compared to BC:
- Patients receiving antibiotic
- Detection of fungemia caused mainly by Aspergillus
- More rapid identification than the BC.
Advantages of the BC compared to these rapid tests:
- Possibility to determine antimicrobial susceptibility
- Detect microorganisms not included in the rapid test
PCR and Mass Spectrometry
(Detection of nucleic acids)
• SEQUENOM
– Amplification / Transcription / Cleavage / Detection of
restriction fragment by MALDI-TOF
• ESI Mass Spectrometry (PLEX-ID)
ESI-TOF Mass Spectrometry
Reflectron
Q- Separation
CID
TOF-MS
Sprayer
Dry Gas
Heater
Glass
Capillary
API Spray Chamber
Dual Ion Funnel
Flight Tube
Collision
Gas Supply
Hexapole
Analytical
Quadrupole
Collision
Cell
Orthogonal
Accelerator
Detector
PCR/ESI Mass Spectrometry
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First, broad-range primers, targeting sites that are highly conserved
in all members of a microbe family, are used to amplify PCR products
from groupings of microbes rather than single species.
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These primers are coupled with species- or strain-specific primers
for the identification of specific pathogens or antibiotic targets.
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Second, PCR conditions are, by design, permissive and thus tolerant
of mismatches, so that even sequences from novel strains can be
amplified.
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Third, inosine and other “wild-card” nucleotides are used in primers
to facilitate PCR analysis of mispaired sequences.
PCR/ESI Mass Spectrometry
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Fourth, because MS simply measures the mass-to-charge ratio (m/z),
the sequence of the amplicon need not be known in order to detect it.
The technology offers advantages over routine single-target and
multiplex PCR in that it is a full bioinformatics sequence analysis
system.
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After amplification, MS is used to rapidly determine the precise
mass-to-charge ratio for the amplified nucleic acid fragments
present, and the A, C, T, and G contents (i.e., the base composition)
of each amplicon are determined using proprietary software that
creates a signature to allow organism identification and genotyping.
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This novel MS technology enables the rapid, sensitive, cost-effective,
and simultaneous detection of a wide range of typical pathogenic
organisms.
PCR/ESI Mass Spectrometry
• Broad amplification of all microorganisms
– Bacteria, virus, fungi and protozoos
• Rapid detection-identification (6-8 hours)
• High throughput
• Able to detect co-infections
• Quantitative
• Detect new pathogens
Next generation DNA sequencing
• Full genome sequence
• 1-2 weeks
• All genomic information for a specific
microorganism
Next generation DNA sequencing
• Intrahost variability of HIV or HBV
– Tropism
– Antiviral resistance
• Compartmentalization of viral quasispecies
• Viral dynamics
– During natural history
– After therapeutic intervention
Do you think that molecular tools will fully
replace conventional bacteriology?
MB
I DO NOT THINK SO