1. Quinolones: mechanisms of resistance
Niels Frimodt-Møller
National Center of Antimicrobials and Infection Control
Statens Serum Institut
Copenhagen, Denmark

2. Quinolone mechanisms of resistance
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?)

3. Gram negativ bakterie kinolonresistensmekanismer.
DNA + gyrasekomplex
Effluxpumper
Porer

4. 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.

5. The structure of supercoils
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.  (c) The positive supercoil in bacteria during DNA replication.

6. 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 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.  .

7. The function of topo II: (a) To remove supercoils
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.

8. Quinolone resistance mechanism

9. Quinolone resistance mechanism
Mutations in gyrA QRDR resistance in Gram-negatives
Mutations in parC QRDR
resistance in Gram-positives

10. Gram negativ bakterie kinolonresistensmekanismer.
DNA + gyrasekomplex
Effluxpumper
Porer

11. Accumulation of moxifloxacin in P
Accumulation of moxifloxacin in P. aeruginosa +/- CCCP (efflux inhibitor)
Ng mox./mg dry cell
Minutes

12. 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.

13. Involvement of Topoisomerase IV and Gyrase as Ciprofloxacin targets in S. pneumoniae
Strain
Mutations in QRDR of:
ParC GyrA GyrB ParE
Cipro
MIC D5 1
B10
Ser-79
Phe 4
D11
Glu-87
> Lys 64 E4
Arg-95
Cys
Pan et al. Antimicrob Ag Chemother 1996, 40:

14. Quinolone resistance types in E. coli
Strain
Mutation in gyrA
Mutation in parC
Redu-
ced accum.
Cipro
MIC
Moxiflox
ATCC25922
0.008
0.03
WT-4
S80I
0.25 MI
S83
0.5 1
WT-3
S83+D87 2
WT-3-M4 64 32
MII + 4
MIII
256
128
Schedletzky et al. JAC, 1999, 43 suppl.B: 31-7

15. Effect of fluoroquinolone concentration on the recovery of single-step, resistant mutants Moxifloxacin (open circles) or levofloxacin (solid circles) Unlabeled arrows indicate MIC The triangle indicates no colony recove-red at that drug concentration and bac-terial load. The dashed line indicates one colony recovered per 1010 cells tested. Three strains were tested: wild-type strain ATCC (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:

16. Changes in the susceptibility of S
Changes in the susceptibility of S. aureus 201 during and after 3-day treatments with four fluoroquinolones at different AUC24/MIC ratios.
Firsov et al. Antimicrob Ag Chemother 2003, 47:

17. Relationship of pharmacokinetics and MPC
Mutant Prevention Concentrations of Fluoroquinolones for Clinical Isolates of Streptococcus pneumoniae
Blondeau et al., Antimicrob Ag Chemother 2001, 45:
Relationship of pharmacokinetics and MPC
Fluoroquinolone
MPCpr90 (µg/ml)
Dose (mg)
Cmax (µg/ml)
t1/2 (h)
Moxifloxacin 2
400
4.5 12
Gatifloxacin 4
4.2 8
Trovafloxacin
200
3.1
Grepafloxacin
600
<2.7 14
Levofloxacin
500
5.7

18. Combination therapy resulted in lower rates of resistance.
Thomas JK, et al. Antimicrob Agents Chemother 1998; 42: 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.

19. Thomas JK et al. Antimicrob Ag Chemother 1998; 42: 521-7.
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
AUC/MIC < 100

20. Quinolone resistance: Summary
Mutations in genes for topoisomerases (gyr, par): MIC increases with no. of mutations; rate ~ 1 in 10-7
Changes in efflux mechanisms: Pumps out drug; NB: inhibitors; Can be first step in development of resistance
Both 1 and 2 can be present in same strain – leads to high MIC´s
Majority of R-genes chromosomal – plasmid transported gene reported