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Plasmid-mediated quinolone resistance.

Plasmid-mediated quinolone resistance. Dr. Jose Manuel RODRÍGUEZ-MARTÍNEZ. Department of Microbiology , Faculty of Medicine , University of Seville E-mail : jmrodriguez@us.es. EPIDEMIOLOGY OF THE RESISTANCE. E. coli & FQ-R en 2009 (Europe).

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Plasmid-mediated quinolone resistance.

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  1. Plasmid-mediated quinolone resistance. Dr. Jose Manuel RODRÍGUEZ-MARTÍNEZ Department of Microbiology, Faculty of Medicine, University of SevilleE-mail : jmrodriguez@us.es

  2. EPIDEMIOLOGY OF THE RESISTANCE E. coli & FQ-R en 2009 (Europe) Resistance to FQ in E. coli (hemocultures) in Europe (EARSS, www.earss.rivm.nl)

  3. 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 (+++)

  4. MECHANISM OF ACTION (2) First and second targets (1) • Gram negative Bacteria : First Target = DNA gyrase • Gram positive Bacteria : First Target = Topoisomerase IV

  5. MECHANISM OF ACTION (3) Complexs of DNA-Topoisomerase-Quinolone Inhibition of religation (Bacteriostatic) (Covalent Fixation) Fragmentation of chromosome (Bactericidal) Drlica & Zhao, MMBR1997

  6. IN VITRO ACTIVITY (2) FQ & Gram negative bacteria 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)

  7. Wild-type population ECOFF • www.eucast.org

  8. Low level resistance? Decreased susceptibility? High level resistance ECOFF R S • www.eucast.org

  9. Consequences of low level antimicrobial resistance • Selection of lowlevelresistancesubpopulations. • Increasedprobability of acquiringhighresistancelevels. • Lack of homogeneous response toantimicrobials • Potentialclinicalfailure at standard dosis • Increasedresistancetounrelatedcompounds. • Growthadvantages, increasedadherence and virulence……….

  10. 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 • 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 www.scq.ubc.ca NB : All the mechanisms can be associated +++

  11. 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 Resistance-Determining 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: CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

  12. QRDR of GyrA in E. coli (1) GyrA (875 AA) 67 106 C N QRDR (Yoshida et al., AAC 1990) Weigel et al., AAC 1998 CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

  13. Association of mutations and resistance to quinolones in E. coli WT Vila et al., AAC 1994 et 1996 CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

  14. Phenotype of resistance in E. coli (1) NAL NAL PEF CIP PEF CIP WT 1 mutation into GyrA L’antibiogramme 2006 CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

  15. Phenotype of resistance in E. coli (2) PEF NAL CIP CIP NAL PEF 1 mutation into GyrA + 1 into ParC 2 mutations into GyrA + 1 into ParC L’antibiogramme 2006 CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

  16. At leastthreemutations, two of whichmustbe in gyrA, wererequiredtoexceedthe CLSI breakpointforfluoroquinoloneresistance

  17. Interplaysbetweenresistance mechanisms in GNB Target modifications Outer membrane permeability Active efflux CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

  18. Target protection: plasmid mediated quinolone resistance.

  19. 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. • …………………..

  20. Gram +: potential reservoir of Qnr-like proteins.

  21. 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.

  22. PLASMID-MEDIATED QUINOLONE RESISTANCE QnrA QnrB QnrS QnrC Prevalence = 1-5% QnrD

  23. Epidemiology of Qnr • Mainly described among Enterobacteriaceae: Prevalence: qnrA ~ 1.5% qnrB ~ 4.5% qnrS ~ 2.5% • 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)

  24. 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

  25. In the presence of qnrA1, mutations in gyrA y parC are easily induced causing high level fluoroquinolone resistance.

  26. Effect of breakpoints (CLSI Vs EUCAST) in Ciprofloxacin susceptibility.

  27. Changes in qnr prevalence and fluoroquinolone resistance. Strahilkevitz et al. AAC 2007

  28. 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

  29. PK/PD parameters of fluorquinolones related to in vivo activity (AUC)/MIC: ≥ 25-30 (immunocompetent) (AUC)/MIC: ≥ 100-125 (immunodeppressed) Cmax/MIC: >8

  30. Effect of qnrA, qnrB and qnrS on the in vivo activity of fluoroquinolones. Dominguez-Herrera et al. ECCMID 2010 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.

  31. Clinical consequences of Qnr? • MPC > peak serum mutant selection Therapeutic failure? • Effect of breakpoint guidelines. • Increases clinical failures in experimental model.

  32. QepA (qepA1 y qepA2) OqxAB (Chromosome of K. pneumoniae!) Moderate increase in MIC values Plasmid-Mediated Active efflux Yamane et al., AAC 2007 ; Périchon et al., AAC 2007 Hansen LH, AAC 2004; Kim HB, AAC 2009

  33. 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

  34. aac-(6´)-Ib-cr prevalence and fluorquinolone resistance Warburg et al. AAC 2009

  35. 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

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