Transcript Document

Plasmid-mediated quinolone resistance.
Dr. Jose Manuel RODRÍGUEZ-MARTÍNEZ
Department of Microbiology,
Faculty of Medicine, University of Seville
E-mail : [email protected]
EPIDEMIOLOGY OF THE RESISTANCE
E. coli FQs I + R
2002
E. coli FQs I + R
2012
Resistance to FQ in E. coli (hemocultures) in Europe (EARSS, www.earss.rivm.nl)
MECHANISM OF ACTION (1)
Functions of Type II Topoisomerases
DNA gyrase :
• Replication :
. Initiation of replication
. Progression of fork of replication
. Decatenation (+)
• Transcription :
. Progression of RNA polimerase
• Recombination and reparation of DNA
Topoisomerase IV :
• Replication :
. Decatenation (+++)
REPLICATION
TOPO IV
GYRASE
TRANSCRIPTION
MECHANISM OF ACTION (2)
First and second targets (1)
Gram negative Bacteria : First Target = DNA gyrase
 Gram positive Bacteria : First Target = Topoisomerase IV
MECHANISM OF ACTION (3)
Complexs of DNA-Topoisomerase-Quinolone
Inhibition of religation
(Bacteriostatic)
(Covalent Fixation)
Fragmentation of chromosome
(Bactericidal)
Drlica & Zhao, MMBR 1997
IN VITRO ACTIVITY (2)
FQ & Gram negative bacteria
Specie
E. coli
Others enterobacterial
Aeromonas spp.
Acinetobacter spp.
P. aeruginosa
S. maltophilia
H. influenzae
Neisseria spp.
C. jejuni
B. fragilis group
Prevotella spp.
MIC50 (µg/ml) :
OFX
NOR
CIP
LEV
MOX
GAT
TRO
0,06
0,03-0,12
<0,01
0,25
1
2
0,03
<0,01
0,5
2
-
0,06
0,06-0,12
<0,01
4
0,5
1
0,06
<0,01
0,5
-
0,01
0,01-0,03
<0,01
0,25
0,25
2-4
0,01
<0,01
0,25
4-8
4
0,03
0,03-0,06
<0,01
0,25
0,5
1
0,01
<0,01
0,25
2
1
0,06
0,03-0,12
<0,01
0,03
1-2
0,5
0,01
<0,01
0,12
1
0,5
0,03
0,01-0,12
<0,01
0,12
1
0,5-1
<0,01
<0,01
0,12
0,5
0,5
<0,01
<0,01-0,25
<0,01
0,03
0,5
0,5
<0,01
<0,01
0,5
0,5
Le Noc et al., JAC 1993 ; Cunha et al., JAC 1997 ; Fernandez-Roblas et al., JAC 2000 ; Milatovic et al., AAC 2000 ;
McCloskey et al., JAC 2000 ; Fung-Tomc et al., JAC 2000 ; Barry et al., AAC 2001 ; Sheng et al., JMII 2001 ;
Hoban et al., DMID 2001 ; Christiansen et al., AAC 2004
Cip-S if MIC  1 mg/L (according to CLSI)
Wild-type population
ECOFF
www.eucast.org
Low level resistance?
Decreased susceptibility?
High level
resistance
ECOFF
S
R
www.eucast.org
MECHANISMS OF RESISTANCE
 Chromosomal-mediated Resistance: MAIN
• Decreasement of affinity on the target by
modification of intracellular targets (DNA gyrase,
Topo IV)
• Decreasement of intracellular accumulation of
FQ by deficient penetration and/or efflux pumps
www.scq.ubc.ca
 Plasmid-mediated Resistance: EMERGENT
• Protection of the target (proteins Qnr, PRP),
1998
• Enzymatic
Inactivation
(acétyltransferase
AAC(6’)-Ib-cr), 2005
• Efflux pumps (QepA, OqxAB), 2007
NB : All the mechanisms can be associated +++
Target Modification (1)
 Mutations in the genes coding for type II topoisomerases:
. DNA gyrase (gyrA, gyrB)
. Topoisomerase IV (parC, parE)
 Substitutions at short conserved region named « Quinolone ResistanceDetermining Regions » (QRDR)
 Resistance multi-step (first level mutants, second level mutants…) with:
. 1 mutation facilitating a second and etc
.  level of resistance with the number of mutations
. First level mutation in the main target:
1st Mutant
2nd Mutant
3rd Mutant
Gram - Bacteria
gyrA
parC
gyrA
Gram + Bacteria
parC
gyrA
parC
CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE
QRDR of GyrA in E. coli (1)
67
106
GyrA (875 AA)
C
N
QRDR
(Yoshida et al., AAC 1990)
Weigel et al., AAC 1998
CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE
Association of mutations and
resistance to quinolones in E. coli
GyrA
WT
GyrB
ParC
83
87
447
80
84
Ser
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Asp
Tyr
Asn
Asn
Asn
Asn
Tyr
Asn
Asn
Asn
Tyr
Lys
Glu
-
Ser
Arg
Ile
Arg
Ile
Arg
Ile
Ile
Ile
Ile
Ile
Glu
Val
Lys
Lys
Lys
Lys
Val
Lys
MIC Nal
(µg/ml)
MIC CIP
(µg/ml)
2-4
128-256
>2000
512
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
0,007-0,25
0,25
1
2
4
4
8
8
8
16
16
32
32
64
64
128
Vila et al., AAC 1994 et 1996
CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE
Phenotype of resistance in E. coli (1)
NAL
PEF
CIP
NAL
WT
PEF
CIP
1 mutation into GyrA
L’antibiogramme 2006
CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE
Phenotype of resistance in E. coli (2)
NAL
PEF
NAL
CIP
1 mutation into GyrA + 1 into ParC
PEF
CIP
2 mutations into GyrA + 1 into ParC
L’antibiogramme 2006
CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE
Interplays between resistance
mechanisms in GNB
Outer membrane
permeability
Target modifications
Active efflux
CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE
Target protection:
plasmid mediated quinolone
resistance.
Types of qnr
• qnrA in K. pneumoniae. 1998.
– qnrA1, A2, A3, etc
• qnrS (homology 59%) in S. flexneri. 2005.
– qnrS1, qnrS2, qnrS3, etc
• qnrB (40%) in K. pneumoniae. 2006.
– qnrB1, B2, B3, etc
•
•
•
•
qnrC in P. mirabilis. 2008.
qnrD (48%) in S. enterica. 2008.
qnrVC in V. cholerae. 2008.
…………………..
Gram +: potential reservoir of Qnr-like proteins.
QNR Proteins
•
•
•
•
QnrA, QnrB, QnrC, QnrD, QnrS, QnrVC…
Pentapeptide repeat proteins
Expressed by different bacteria
Protect DNA-gyrase ad topoisomerase IV
from quinolone attack.
• Origin: chromosome of different
environmental and aquatic bacteria.
PLASMID-MEDIATED QUINOLONE
RESISTANCE
QnrA
QnrB
QnrS
QnrC
QnrD
Prevalence = 1-5%
Epidemiology of Qnr
 Mainly described among Enterobacteriaceae:
Bacterial species
E. coli
K. pneumoniae
K. oxytoca
E. cloacae
E. aerogenes
E. sakazakii
C. freundii
C. koseri
C. werkmanii
S. marcescens
P. mirabilis
M .morganii
S. enterica
Shigella spp.
Aeromomnas spp.
Pseudomonas spp.
Campylobacter spp.
a
Prevalence of qnr-like genesa:
qnrA
qnrB
qnrS
+++
++
++
++++
++++
+++
+
+
++++
++++
+++
+
+
+
+
++
+
+
+
+
+
+
+
++
++
++++
+
+
+
-
No. of published qnr-like positive isolates: -, 0; +, 1-10;
++, 10-50; +++, 50-100; ++++, >100.
Prevalence:
qnrA ~ 1.5%
qnrB ~ 4.5%
qnrS ~ 2.5%
Cattoir & Nordmann, CMC 2009
 qnrC1 identified from a single Proteus mirabilis isolate in China (Wang et al., AAC 2009)
 qnrD1 identified from four Salmonella isolates in China (Cavaco et al., AAC 2009)
PLASMID-MEDIATED QUINOLONE
RESISTANCE
Association with -lactamases
 Frequent association with ESBLs:
- qnrA : SHV-2/7/12/92, CTX-M-1/9/14/15/24, VEB-1, PER-1
- qnrB : TEM-52, SHV-12/30, CTX-M-3/12/14/15/24, VEB-1
- qnrS : TEM-52, SHV-2/5/12, CTX-M-1/9/14/15/24
 Association with several plasmid-mediated cephalosporinases (AmpC):
- qnrA : FOX-5 (pMG252), CMY-2
- qnrB : CMY-1, DHA-1 (qnrB4)
 Worrying association with carbapenemases (class A and B):
- qnrA : IMP-4, KPC-3
- qnrB : IMP-8, KPC-2, KPC-3
- qnrS : IMP-8, VIM-1
Resistencia y Fitness bacteriano
Puntos de corte CLSI:
Se requieren 4 mecanismos de resistencia para obtener cepas resistentes:
3 cromosómicos y 1 plasmídico.
Sin
PMQR
ATCC 25922
ATCC ΔmarR
ATCC S83L
ATCC S83L ΔmarR
ATCC S83L S80R
ATCC S83L S80R ΔmarR
ATCC S83L D87N
ATCC S83L D87N ΔmarR
ATCC S83L D87N S80R
ATCC S83L D87N S80R ΔmarR
QnrA1
QnrB1
QnrC
QnrD1
QnrS1
QepA2
Puntos de corte EUCAST:
Se requieren 3 mecanismos de resistencia para obtener cepas resistentes.
Sin
PMQR
ATCC S83L
ATCC S83L ΔmarR
ATCC S83L S80R
ATCC S83L S80R ΔmarR
ATCC S83L D87N
ATCC S83L D87N ΔmarR
ATCC S83L D87N S80R
ATCC S83L D87N S80R ΔmarR
QnrB1
QnrC
QnrD1
0,03
ATCC 25922
ATCC ΔmarR
QnrA1
0,016
QnrS1
QepA2
0,016
Serum or tissue drug concentration
Cmax
MPC
Mutant
selection
window
MIC
Time post-administration
In the presence of
qnrA1, mutations
in gyrA y parC are
easily induced
causing high level
fluoroquinolone
resistance.
qnrA1
Coste Biológico
de la Resistencia
qnrS1
Correlación entre fitness in vitro e in vivo
Resistencia
Coste biológico
Changes in qnr prevalence and
fluoroquinolone resistance.
Strahilkevitz et al. AAC 2007
The presence of qnrA1 in E. coli from a patient with a UTI
(treated with norfloxacin) preceeds the emergence of changes
in Ser83Leu and Asp87Asn, (GyrA gyrase subunit) and Ser80Ile
(ParC topoisomerase IV subunit)
MICs of ciprofloxacin increased from 0.5 to >32 mg/l
PK/PD parameters of fluorquinolones
related to in vivo activity
(AUC)/MIC: ≥ 25-30 (immunocompetent)
(AUC)/MIC: ≥ 100-125 (immunodeppressed)
Cmax/MIC: >8
Effect of qnrA, qnrB and qnrS on the in vivo activity of fluoroquinolones.
Dominguez-Herrera et al. ECCMID 2010
Strain
E. coli
ATCC 25922
(pBK-CMV)
E. coli
ATCC 25922
(pBK-Qnr A)
E. coli
ATCC 25922
(pBK-Qnr B)
E. coli
ATCC 25922
(pBK-Qnr S)
Log10 CFU / g of lung
Mortality
Positive blood
(Mean ± SD)
(%)
cultures (%)
Control
9.37 ± 0.48
100
100
CPX
1.87a ± 2.07
53.33a
0a
LVX
1.67a ± 2.50
28.57a
0a
Control
9.40 ± 0.25
100
92.86
CPX
5.71ab ± 0.86
53.33a
26.67a
LVX
5.71ac ± 0.75
71.43a
21.40a
Control
8.84 ± 0.75
100
100
CPX
4.95ab ± 1.71
57.14a
35.71ab
LVX
4.54ac ± 1.49
50a
28.57ac
Control
8.36 ± 1.26
93.33
93.33
CPX
5.38ab ± 1.75
42.86a
28.57ab
LVX
4.99ac ± 1.63
35.71a
21.43a
Group
Conclusion
The presence of the qnrA, qnrB or qnrS genes in Escherichia coli strains reduces the therapeutic
efficacy of ciprofloxacin and levofloxacin in a murine experimental pneumonia model.
Clinical consequences of Qnr?
• MPC > peak serum
mutant selection
Therapeutic failure?
• Effect of breakpoint guidelines.
• Increases clinical failures in
experimental model.
Plasmid-Mediated Active efflux
• QepA (qepA1 y qepA2)
• OqxAB (Chromosome of K. pneumoniae!)
• Moderate increase in MIC values
Yamane et al., AAC 2007 ; Périchon et al., AAC 2007
Hansen LH, AAC 2004; Kim HB, AAC 2009
Enzimatic inactivation: AAC(6’)-Ib-cr
Two substitutions at the aac(6’)-Ib gene: Trp102Arg and Asp179Tyr
N-acetylation at the amino radical of the piperacynil group
(It does not compromise activty against aminoglycosides)
It affects ciprofloxacin, norfloxacin,… (but not other quinolones)
Moderate increase in MIC values
It favors the emergence of more resistant mutants
Robicsek A et al, Nature Med 2005
aac-(6´)-Ib-cr prevalence and
fluorquinolone resistance
Warburg et al. AAC 2009
Low Level Antimicrobial Resistance
Conclusions
•
Caused by multiple mechanism encoded by
chromosomal or plasmid genes. Phenotypic methods are
not reliable for detecting many of these mechanisms
•
LLR increases the level of resistance due to high-level
resistance mechanisms, and ensures bacterial viability
to allow acqusition of additional resistance mechanisms
•
Overexpression of LLR or coexpression of several LLR
mechanisms may translate into clinical resistance, as
defined by usual breakpoints
•
PK/PD data indicate that even the moderate changes in
MIC caused by LLR affect the clinical efficacy of
quinolones
Líneas de Investigación
•
Bases Moleculares de la Resistencia a Antimicrobianos y
Fijación en Poblaciones Bacterianas
•
Sensibilización de Bacterias Resistentes Mediante
Búsqueda de Nuevas Dianas Terapéuticas