Transcript Dias nummer 1
Quinolones: mechanisms of resistance
Niels Frimodt Møller
National Center of Antimicrobials and Infection Control Statens Serum Institut Copenhagen, Denmark
Quinolone mechanisms of resistance 1) Change in receptor
Mutations in genes for gyrases/topoisomerases
2)
Change in penetration
Efflux-mechanisms: Proton-pumps with active transport of quinolone out of cell 3)
Enzymatic degradation
Always (almost) chromosomal (plasmid carried transfer found in Klebsiella, mechanism?)
Gram negativ bakterie kinolonresistensmekanismer.
DNA + gyrasekomplex Porer Effluxpumper
DNA
Structure of the Topo I/DNA complex
. During replication, the unwinding of DNA may cause the formation of tangling structures, such as supercoils or catenanes . The major role of topoisomerases is to prevent DNA tangling.
The structure of supercoils.
(a)
Positive supercoils - the front segment of a DNA molecule cross over the back segment from left to right.
(b)
Negative supercoils.
bacteria during DNA replication.
(c)
The positive supercoil in
There are two types of topoisomerases
:
type I
produces transient
single-strand
breaks in DNA:
The topo I
of both prokaryotes and eukaryotes and
types II
produces transient
double-strand
breaks: The eukaryotic topo II,
bacterial gyrase
, and
bacterial topo IV
belong to the type II The
gyrase
has two functions: (1) to remove the positive supercoils during DNA replication, (2) to introduce negative supercoils (one supercoil for 15-20 turns of the DNA helix) so that the DNA molecule can be packed into the cell. During replication, these negative supercoils are removed by topo I.
Malfunction in topoiomerases causes cell death.
The function of topo II: (a)
To remove supercoils. This involves a double-strand break (indicated by a short line), allowing the tangled segment to pass through. The break is then resealed.
(b)
To remove catenanes. The topo II makes a double-strand break in one DNA molecule (the blue one), allowing the other molecule to pass through. The break is then resealed.
Quinolone resistance mechanism
Topoiso merase II Gyrase
(catalyses ATP-dep. negative supercoiling of DNA-molecule)
Topo IV
(Effect on chromo somal segregation) Subunit A (
gyrA
) Subunit B (
gyrB
) Subunit C (
parC
) Subunit E (parE)
Quinolone resistance mechanism
Topoiso merase II
Gyrase
(catalyses ATP-dep. negative supercoiling of DNA-molecule)
Topo IV
(Effect on chromo somal segregation)
Subunit A (gyrA ) Subunit B (gyrB) Subunit C (parC) Subunit E (parE) Mutations in gyrA QRDR resistance in Gram-negatives Mutations in parC QRDR resistance in Gram-positives
Gram negativ bakterie kinolonresistensmekanismer.
DNA + gyrasekomplex Porer Effluxpumper
Accumulation of moxifloxacin in
P. aeruginosa
+/- CCCP (efflux inhibitor)
Ng mox./mg dry cell
90 80 70 60 50 40 30 20 10 0 5 10 15 20 alone + CCCP
Minutes
Accumulation of ciprofloxacin and lomefloxacin in fluoroquinolone-resistant strains of Escherichia coli
XIA Peiyuan et al. Chin Med J 2002; 115:31-5.
Accumulation of LMLX in E. coli strains. Each curve indicates the accumulated concentration of LMLX in one strain at diffe rent time point.JF701 and JF703: control strains ; Ecs: susceptible strain. R 2 and R256: the in vitro selected resistant strains;
R5 and R6: the clinical res istant strains.
Involvement of Topoisomerase IV and Gyrase as Ciprofloxacin targets in
S. pneumoniae
Strain
D5 B10 D11 E4
Mutations in QRDR of: ParC GyrA GyrB ParE Cipro MIC
1 Ser-79 Phe Ser-79 Phe Arg-95 Cys Glu-87 > Lys 4 64 4 Pan et al. Antimicrob Ag Chemother 1996, 40: 2321-6
Quinolone resistance types in
E. coli
Strain Mutation in gyrA Mutation in parC Redu ced accum.
ATCC25922 WT-4 MI WT-3 WT-3-M4 MII MIII S83 S83+D87 S83+D87 S83 S83+D87 S80I S80I S80I + + Cipro MIC 0.008
0.008
0.5
1 64 2 256 Moxiflox MIC 0.03
0.25
1 2 32 4 128 Schedletzky et al. JAC, 1999, 43 suppl.B: 31-7
Effect of fluoroquinolone concentration on the recovery of single-step, resistant mutants.
Moxifloxacin
(open circles) or
levofloxacin
(solid circles). Unlabeled arrows indicate MIC 99 . The triangle indicates no colony recove red at that drug concentration and bac terial load. The dashed line indicates one colony recovered per 10 10 cells tested. Three strains were tested: wild-type strain ATCC 49619 (
A
); strain KD2138, a ParC (Ser-79 to Tyr) variant (
B
); and strain KD2139, a GyrA (Ser-81 to Phe) variant (
C
).
Two double mutants were recovered from each point indicated by an arrow .
Li et al. Antimicrob Ag Chemother 2002, 46: 522-524.
Changes in the susceptibility of S. aureus 201 during and after 3-day treatments with four fluoroquinolones at different AUC 24 /MIC ratios.
Firsov et al. Antimicrob Ag Chemother 2003, 47: 1604-1613
Mutant Prevention Concentrations of Fluoroquinolones for Clinical Isolates of Streptococcus pneumoniae
Blondeau et al., Antimicrob Ag Chemother 2001, 45: 433-438 Relationship of pharmacokinetics and MPC Fluoroquinolone Moxifloxacin Gatifloxacin Trovafloxacin Grepafloxacin Levofloxacin MPC pr90 (µg/ml) 2 4 4 8 8 Dose (mg) 400 400 200 600 500
C
max (µg/ml) 4.5
4.2
3.1
<2.7
5.7
t 1/2 (h) 12 8 12 14 8
Thomas JK, et al. Antimicrob Agents Chemother 1998; 42: 521-7.
Pharmacodynamic evaluation of factors associated with the development of bacterial resistance in acutely ill patients during therapy.
•
• Five different regimes in ICU could be avaluated for resistance developments in different bacterial species (total of 128 patogenes in 107 ptt.).
– cirofloxacin alone – ciprofloxacin + piperacillin – ceftazidime alone – ceftazidime +tobramycin – cefmenoxime alone
The overall predictor for development of resistance was AUC/MIC < 100.
• Combination therapy resulted in lower rates of resistance.
Thomas JK et al. Antimicrob Ag Chemother 1998; 42: 521-7.
AUC/MIC > 100
• Resistance developed in lactamase-type-I Gram-neg. rods even when AUC/MIC > 100 after -lactam monotherapy.
• Median time to resistance: 6 days if AUC/MIC < 100.
AUC/MIC < 100
Quinolone resistance: Summary
1. Mutations in genes for topoisomerases (gyr, par): MIC increases with no. of mutations; rate ~ 1 in 10-7 2. Changes in efflux mechanisms: Pumps out drug; NB: inhibitors; Can be first step in development of resistance 3. Both 1 and 2 can be present in same strain – leads to high MIC ´s