Download Mechanisms of penicillin resistance in N. Gonorrhoeae

Real-Time PCR Applications:
•
Detection of chromosomally mediated penicillin-resistant Neisseria
gonorrhoeae
• Quantitative expression of mRNA encoding pro-inflammatory and
immunomodulatory cytokines in hamsters infected with a virulent
variant of Leptospira
IPNC Juillet 2006
1
Neisseria gonorrhoeae
the etiologic agent for the sexually transmitted infection
gonorrhoea
2
•
Second most common bacterial STD in France
as in US
•
Around 75% of all reported cases in 15-29 year
old age group
•
High prevalences reported from non-genital
sites among MSM (oropharynx & rectum)
•
Usually symptomatic in males, often
asymptomatic in women
•
Can cause PID, infertility, ectopic pregnancy,
and complications in pregnancy in women
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Treatment
Depending of the national guidelines
• 1940s = Penicillin effective treatment
• 1970s = Penicillin resistance develops secondary to
acquisition of plasmid for penicillinase
• Since 1972, CDC has recommended treatment with third
generation cephalosporin or fluoroquinolones (SD)
• Increase in chromosomally acquired multi-resistance to
antibiotics
• In New Caledonia, N.gonorrhoeae were susceptible to
penicillin
•
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Benzylpenicillin, metronidazole and azithromycin
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Mechanisms of antibiotic resistance
• Alteration of the target site
• Reduction of the affinity for the antibiotic
• Access of the antibiotic to the target site
• Reduced permeability of the cell envelope
• Active export of antibiotics
• Destruction of the antibiotic
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b-lactams action mode
Antibiotics
Penicillin binding proteins
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Mechanisms of penicillin resistance
in N. Gonorrhoeae (1)
Plasmid mediated
b-lactamases
Acquired, Transferable
(e.g. TEM)
Chromosomally- mediated
alterations
• Altered penicillin binding proteins
• Porins
• Efflux pumps
is of considerable concern
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Mechanisms of penicillin resistance
in N.gonorrhoeae (2)
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Real-Time Multiplex PCR Assay
• Detection of NG from clinical samples within 2hrs
• Combined detection of penA and ponA genotypes
• ponA mutation = T to C substitution
(CMI ≥ 1 mg/ml)
• penA mutation = additional aspartic acid (Asp-345A)
codon (CMI ≥ 0.03 mg/ml)
• Correlation with Phenotypes obtained by E-test
• Status of N.gonorrhoeae strains in New Caledonia
• LightCycler: additional advantage
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Oligonucleotide sequences
Design with Probe design software 2.0 (Roche Diagnostics)
Oligonucleotides
ponA
Primers
ponA-F (Forward)
ponA-R (Reverse)
Probes
ponA-P1 (Anchor)
Sequences (5’ to 3’)
Concentrations
ponA-P2 (Sensor)
0,5 M
1 M
0,2 M
a
Positions
CGGGAACTGTATGAGAAAT
GTCTCGCCCGTACCTA
814-832
1694-1679
GGCGTTGGGCGGTGGT-fluorescein
1232-1247
b
0,2 M
LC-R705 -AAGAGCCGTTGCTGCA-phosphate
1250-1265
0,5 M
GACCTTGGCCTATGAAGAG
GGTTACGGAAACCATCAGATT
756-774
1461-1441
CGCGACGATACCCATGTTTACC-fluorescein
LC-R640c-TCTTTGGATGTGCGCGGCATTATGCA-phosphate
1057-1078
1081-1106
penA
Primers
penA-F (Forward)
penA-R (Reverse)
Probes
penA-P1 (Sensor)
penA-P2 (Anchor)
a
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GenBank accession number;
1 M
0,2 M
0,2 M
b
LC-R705, LightCycler red 705;
c
LC-R640, LightCycler red 640
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FRET based-system for the detection
of ponA mutation (1)
T
881 bp
3’
ponA-F
5’
814
C
or
ponA-P1
FL
LC705
GGCGTTGGGCGGTGGT
1232
D
5’
ponA-P2
3’
AAGAGCCGTTGCTGCA(PO4)
A
1265
hυ
ponA-R
FRET
5’
1679
5’
Forward Primer
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3’
Anchor Probe
Sensor Probe
or Detection Probe
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Reverse Primer
Tm and mutation detection
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Overlap of emission spectra from
fluorescent dyes used in the LC System
• The degree of crosstalk is strongly influenced by:
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•
Temperature
•
Coupling chemistry of the fluorescence dyes
•
properties of the optical system
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Color compensation activation
Without
compensation
With
compensation
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Experimental protocol (1)
• Chromosomal DNA Extraction Using QIA amp DNA Minikit with :
Isolated culture
Urethral or vaginal samples (stocked in transport medium)
Urines (resuspended in water after centrifugation)
• Components of PCR with LightCycler FastStart DNA Master Hybridization
Probes Kit (Roche )
Amorce penA-F (10 µM)
Amorce penA-R (10 µM)
Amorce ponA-F (10 µM)
Amorce ponA-R (10 µM)
Sonde penA-P1 (10 µM)
Sonde penA-P2 (10 µM)
Sonde ponA-P1 (10 µM)
Sonde ponA-P2 (10 µM)
Master mix 10X
MgCl2
eau pcr
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1 tube (µl)
1
2
1
2
0,4
0,4
0,4
0,4
2
2,4
6
18 µl mix et 2 µl ADN extrait
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Final
0,5 µM
1 µM
0,5 µM
1 µM
0,2 µM
0,2 µM
0,2 µM
0,2 µM
1X
4 mM
Experimental protocol (2)
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Partial sequence alignment
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ponAWT
066213
275183
GTT CAA GAG CCG TTG CTG CAG GGG GCT TTG GTT TCG
GTT CAA GAG CCG TTG CTG CAG GGG GCT TTG GTT TCG
GTT CAA GAG CCG TTG CTG CAG GGG GCT TTG GTT TCG
V Q
E
P
L
A
L
V
S
L Q G
ponAmUT
356390
GTT CAA GAG CCG TTG CCG CAG GGG GCT TTG GTT TCG
GTT CAA GAG CCG TTG CCG CAG GGG GCT TTG GTT TCG
V Q E
P
L
Q
G
A
L
V
S
P
penAWT
037350
CCG TCT CCC GTG CGC … GAT ACC CAT GTT TAC CCC
CCG TCT CCC GTG CGC … GAT ACC CAT GTT TAC CCC
P S P V R
D T H V Y P
penAmUT
066213
CCG TCT CCC GTG CGC GAC GAT ACC CAT GTT TAC CCC
CCG TCT CCC GTG CGC GAC GAT ACC CAT GTT TAC CCC
P S P V R
D T H V Y P
D
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Characteristics of N. gonorrhoeae
strains used in the study
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MIC of
Penicillin
(g/ml)
0.016 et inf
9
0.032
S
Asp-345A: 3/9
ponA mutant: 0/8
12
0.047
S
Asp-345A: 9/12
ponA mutant: 0/12
8
0.064
I
Asp-345A: 3/8
ponA mutant: 1/8
7
0.094
I
Asp-345A: 7/7
ponA mutant: 0/6
3
0.19
I
Asp-345A: 3/3
ponA mutant: 1/3
8
0.125
I
Asp-345A: 8/8
ponA mutant: 1/8
6
0.25
I
Asp-345A: 6/6
ponA mutant: 2/6
7
0.38
I
Asp-345A: 6/7
ponA mutant: 4/7
2
0.5
I
Asp-345A: 2/2
ponA mutant: 0/2
2
0.75
I
Asp-345A: 2/2
ponA mutant: 2/2
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1 et sup
R
Asp-345A: 18/18
ponA mutant: 18/18
Number of
strains
Phenotypic
status
penA genotype
ponA genotype
S
Asp-345A: 2/38
ponA mutant: 0/38
120 NG strains
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Gene expression at mRNA level
Quantitative expression of mRNA encoding pro-inflammatory and
immunomodulatory cytokines in hamsters infected with a virulent variant of
Leptospira
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Cytokines and immunity
•
Regulatory proteins
•
Inflammatory responses (TNFa vs IL-10)
•
Immune and pathological pathways
 Why quantifying cytokine expression?
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•
Specific immune mechanisms
•
Mechanisms of anti-Leptospira immunity
•
Th1-Th2 profile
•
Pathogenesis of infection
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Syrian hamster
(Mesocricetus auratus)
• Experimental model
• Highly susceptible to Leptospira
• Nucleotide sequence of cytokine genes (Melby et al., 1998)
Quantification of specific cDNA targets
• Absolute quantification
• Relative quantification
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Definition of used terms
Definitions of Frequently Used Terms
Term
Definition
Sample
Material of interest (tissue, cells, blood etc.)
Target
Nucleic acid of interest (specific RNA or DNA sequence)
Reference
Nucleic acid that is found at a constant copy number in all samples (=
endogenous control) The reference is used for normalization of sampleto-sample differences (nucleic acid quality and quantity).
Housekeeping gene
For mRNA quantification in gene expression
studies.
A gene that is expressed constitutively on an identical level in all samples to
be analyzed.
Calibrator
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A sample that is used for normalization of the final results. The calibrator is
positive for target and reference, and must have a constant expression
ratio of target to reference.
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Absolute quantification
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Relative quantification
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Normalization
Concentration of target
Calibrator
=
Normalized Ratio
Concentration of reference
Concentration of target
Concentration of reference
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(sample)
(calibrator)
Standard Curve
Generated with serial dilutions of a standard
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From blood to curves
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•
1 to 2 ml of blood
•
PBMC purification and in vitro culture
•
Stimulation of PBMC (LPS, PMA-ionomycin)
•
Total RNA extraction (High Pure RNA isolation kit, ROCHE)
•
cDNA preparation (Transcriptor First Strand cDNA synthesis kit, ROCHE)
•
LightCycler protocol (FastStart SYBR Green)
•
Specificity : melting curves, gel electrophoresis
•
cDNA dilutions (2 to 4x107 copies/µl)
•
Standard curves
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Standard curves
E= 1,903
E= 1,899
E= 1,891
Ainsi que IL-1, IL-2, IL-4, IL-6, IL-10, IL-12p40
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Set of target and reference genes
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Cytokine
Size
Hyb. Temp. Tm
IL-2
349 bp
61°C
85.2°C
IL-4
342 bp
61°C
87.8°C
IL-10
308 bp
61°C
87.8°C
IL12p40
308 bp
61°C
89.9°C
IFNg
226 bp
60°C
84.6°C
TGFb
245 bp
60°C
90.0°C
TNFa
278 bp
60°C
89.6°C
HPRT
242 bp
60/61°C
82.4/82.9°C
b actin
357 bp
60/61°C
90.0/90.4°C
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Expression profile of TNF-alpha
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Expression profile of IFN-gamma
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Ratio
Expression profile of IL-12p40
2500
2000
1500
1000
500
0
1
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2
4
6
8
10 18 22 D1 D2 D3 D4
Hours p.i.
Days p.i.
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4
2500
2000
3
+?
1500
2
1000
1
500
0
0
1
2
4
6
8
10
18
22
Hours p.i.
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D1
D2
D3
Days p.i.
D4
Ratio IL-12
Ratio IFN
Expression profiles of g IFN and IL-12
From lymphocyte T to Th1
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