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

2. EPIDEMIOLOGY OF THE RESISTANCE Resistance to FQ in E. coli (hemocultures) in Europe (EARSS, www.earss.rivm.nl) E. coli FQs I + R 2002 E. coli FQs I + R 2012

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 Drlica & Zhao, MMBR 1997 Inhibition of religation (Bacteriostatic) Fragmentation of chromosome (Bactericidal) (Covalent Fixation)

6. 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 IN VITRO ACTIVITY (2) FQ & Gram negative bacteria Specie MIC50 (µg/ml) : OFXNORCIPLEVMOXGATTRO E. coli Others enterobacterial Aeromonas spp. Acinetobacter spp. P. aeruginosa S. maltophilia H. influenzae Neisseria spp. C. jejuni B. fragilis group Prevotella spp. 0,060,03-0,12<0,010,25120,03<0,010,52-0,060,06-0,12<0,0140,510,06<0,010,5--0,010,01-0,03<0,010,250,252-40,01<0,010,254-840,030,03-0,06<0,010,250,510,01<0,010,25210,060,03-0,12<0,010,031-20,50,01<0,010,1210,50,030,01-0,12<0,010,1210,5-1<0,01<0,010,120,50,5<0,01<0,01-0,25<0,010,030,50,5<0,01<0,01-0,50,5 Cip-S if MIC  1 mg/L (according to CLSI)

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

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

9.  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 MECHANISMS OF RESISTANCE NB : All the mechanisms can be associated +++

10. CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE Target Modification (1)  Mutations in the genes coding for type II topoisomerases:. DNA gyrase (gyrA, gyrB). Topoisomerase IV (parC, parE) QRDR  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: 1st Mutant2nd Mutant3rd Mutant Gram - BacteriagyrAparCgyrA Gram + BacteriaparCgyrAparC

11. GyrA (875 AA) N C 67106 Weigel et al., AAC 1998 QRDR (Yoshida et al., AAC 1990) QRDR of GyrA in E. coli (1) CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

12. Association of mutations and resistance to quinolones in E. coli GyrAGyrBParCMIC Nal (µg/ml) MIC CIP (µg/ml) 83874478084 Ser Leu Asp - Tyr Asn Tyr Asn Tyr Lys - Glu - Ser - Arg Ile Arg - Ile Arg Ile - Ile Glu - Val - Lys - Lys - Val Lys 2-4 128-256 >2000 512 >2000 0,007-0,25 0,25 1 2 4 8 16 32 64 128 WT Vila et al., AAC 1994 et 1996 CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

13. Phenotype of resistance in E. coli (1) WT1 mutation into GyrA L’antibiogramme 2006 NAL PEF CIP NAL PEF CIP CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

14. 2 mutations into GyrA + 1 into ParC1 mutation into GyrA + 1 into ParC L’antibiogramme 2006 NAL PEF CIP NAL PEF CIP Phenotype of resistance in E. coli (2) CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

16. Interplays between resistance mechanisms in GNB Active efflux Outer membrane permeability Target modifications CHROMOSOMAL-MEDIATED QUINOLONE RESISTANCE

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

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

24. 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 PLASMID-MEDIATED QUINOLONE RESISTANCE

27. Resistencia y Fitness bacteriano

28. Sin PMQR QnrA1QnrB1QnrCQnrD1QnrS1QepA2 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 Puntos de corte CLSI: Se requieren 4 mecanismos de resistencia para obtener cepas resistentes: 3 cromosómicos y 1 plasmídico. Puntos de corte CLSI: Se requieren 4 mecanismos de resistencia para obtener cepas resistentes: 3 cromosómicos y 1 plasmídico.

29. Sin PMQR QnrA1QnrB1QnrCQnrD1QnrS1QepA2 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 Puntos de corte EUCAST: Se requieren 3 mecanismos de resistencia para obtener cepas resistentes. Puntos de corte EUCAST: Se requieren 3 mecanismos de resistencia para obtener cepas resistentes. 0,016 0,03

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

32. Coste Biológico de la Resistencia qnrA1 qnrS1

33. Correlación entre fitness in vitro e in vivo

34. Resistencia Coste biológico

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

37. 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

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

39. Effect of qnrA, qnrB and qnrS on the in vivo activity of fluoroquinolones. Dominguez-Herrera et al. ECCMID 2010 StrainGroup Log 10 CFU / g of lung (Mean ± SD) Mortality(%) Positive blood cultures (%) E. coli ATCC 25922 (pBK-CMV) Control 9.37 ± 0.48 100100 CPX 1.87 a ± 2.07 53.33 a 0a0a0a0a LVX 1.67 a ± 2.50 28.57 a 0a0a0a0a E. coli ATCC 25922 (pBK-Qnr A) Control 9.40 ± 0.25 10092.86 CPX 5.71 ab ± 0.86 53.33 a 26.67 a LVX 5.71 ac ± 0.75 71.43 a 21.40 a E. coli ATCC 25922 (pBK-Qnr B) Control 8.84 ± 0.75 100100 CPX 4.95 ab ± 1.71 57.14 a 35.71 ab LVX 4.54 ac ± 1.49 50 a 28.57 ac E. coli ATCC 25922 (pBK-Qnr S) Control 8.36 ± 1.26 93.3393.33 CPX 5.38 ab ± 1.75 42.86 a 28.57 ab LVX 4.99 ac ± 1.63 35.71 a 21.43 a 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.

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

41. 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

42. 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 Enzimatic inactivation: AAC(6’)-Ib-cr Robicsek A et al, Nature Med 2005

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

45. 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 Low Level Antimicrobial Resistance Conclusions

46. Bases Moleculares de la Resistencia a Antimicrobianos y Fijación en Poblaciones BacterianasBases Moleculares de la Resistencia a Antimicrobianos y Fijación en Poblaciones Bacterianas Sensibilización de Bacterias Resistentes Mediante Búsqueda de Nuevas Dianas TerapéuticasSensibilización de Bacterias Resistentes Mediante Búsqueda de Nuevas Dianas Terapéuticas Líneas de Investigación