1. Villars-sur-Ollon (Switzerland) September 4-8, 2005 MECHANISMS OF RESISTANCE TO QUINOLONES IN Escherichia coli Jordi Vila Department of Microbiology Hospital Clinic, Barcelona, Sccolf of Medicine, University of Barcelon, Barcelona, Spain

2. Mechanism of action and resistance to quinolones. Mechanism of action and resistance to quinolones. Do quinolone-resistant E.coli strains carry fewer virulence factors than their susceptible counterparts? Do quinolone-resistant E.coli strains carry fewer virulence factors than their susceptible counterparts?

3. BASIC STRUCTURE 4-QUINOLONE COOH

4. Gatifloxacin Ciprofloxacin Nalidixic acid Norfloxacin Levofloxacin Moxifloxacin

5. MECHANISM OF ACTION Inhibition of: Inhibition of: – DNA gyrase. – Topoisomerase IV.

6. Characteristics of the DNA gyrase. Structure A 2 B 2 Structure A 2 B 2 A Subunit A Subunit gyrA gene. gyrA gene. 97 kDa. 97 kDa. DNA breaking and reunion. DNA breaking and reunion. B Subunit: B Subunit: gyrB gene. gyrB gene. 90 kDa. 90 kDa. ATPase activity. ATPase activity.

7. Characteristics of topoisomerase IV Structure A 2 B 2 Structure A 2 B 2 Subunit A: Subunit A: parC gene. parC gene. 75 kDa. 75 kDa. Subunit B: Subunit B: parE gene. parE gene. 70 kDa. 70 kDa. Associated with the inner membrane. Associated with the inner membrane.

8. - - - - - + + + + + + + + - - - - - DNA gyrase DNA gyrase Topoisomerase IV FUNCTIONS

9. Organization of chromosome structure Organization of chromosome structure

10. REPLICATION TRANSCRIPTION TOPO IV GYRASE

11. EVOLUTION OF THE RESISTANCE TO QUINOLONES IN E.coli % Resistance Year 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 40302010 2 5 9 11 14 23 24 28 24 25 28 30 Data from Hospital Clinic - Barcelona

12. Evolution of the resistance to quinolones in enterotoxigenic E. coli causing traveler’s diarrhea % Resistance % Resistance 1994-1997 2001-2004 1994-1997 2001-2004 NALIDIXIC ACID 6 22 (p<0.01) (p<0.01) CIPROFLOXACIN 1 12 Data from Hospital Clinic - Barcelona

13. MECHANISMS OF RESISTANCE TO QUINOLONES IN ENTEROBACTERIACEAE Changes in the protein targets. Changes in the protein targets. DNA gyrase DNA gyrase Topoisomerase IV. Topoisomerase IV. Reduction in the accumulation of the quinolone. Reduction in the accumulation of the quinolone. - Decrease in permeability. - Decrease in permeability. - Increase in active efflux system(s). DNA gyrase and topoisomerase IV protection - qnr gene DNA gyrase and topoisomerase IV protection - qnr gene

14. ACQUISITION OF RESISTANCE TO QUINOLONES - CHROMOSOMAL MUTATIONS. - Mutations in the genes encoding the subunits of the protein targets. the subunits of the protein targets. - Mutations in the genes causing the decrease in accumulation. the decrease in accumulation. - PLASMID (Enterobacteriaceae).

15. QUINOLONE RESISTANCE DETERMINING REGION (QRDR) IN THE GENES ENCODING THE SUBUNITS OF THE DNA GYRASE. QUINOLONE RESISTANCE DETERMINING REGION (QRDR) IN THE GENES ENCODING THE SUBUNITS OF THE DNA GYRASE. A subunit DNA gyrase DNA gyrase B subunit B subunit HOOC NH 2 QRDR QRDR Tyr-122 HOOC NH 2 67 106 67 106 426 447

16. Substitutions of amino acids of the subunit A of the DNA gyrase. PositionChangesIncrease in the MIC (-fold) 67Ala.....Ser4 81Gly.....Cys/Asp8 83Ser.....Leu/Trp/Ala32 84Ala.....Pro8 87Asp....Asn/Val/Tyr16 106Gln.....His/Arg4

17. MIC (  g/ml) Substitutions in amino acids MIC (  g/ml) Substitutions in amino acids Strains CIP NAL GyrA C20, C-130.0072Ser-83Ala-84Asp-87 C-10.064Ser-83Ala-84Asp-87 C-110.062Ser-83Ala-84Asp-87 C-18, C-80.1252Ser-83Ala-84Asp-87 C-50.252Ser-83Val-84Asp-87 C-6, C-7, C-90.252Ser-83Ala-84Asp-87 C-15, C-40.25128Leu-83Ala-84Asp-87 C-101>2.000Leu-83Ala-84Asp-87 13272512Leu-83Ala-84Asp-87 1363*, 12894>2.000Leu-83Ala-84Asp-87 12738>2.000Leu-83Ala-84Tyr-87 1331, 15748>2.000Leu-83Ala-84Asn-87 1283, 133416>2.000Leu-83Ala-84Asn-87 136032>2.000 Leu-83Ala-84Tyr-87 1416, 1319, 132364>2.000 Leu-83Ala-84Asn-87 1383128>2.000 Leu-83Ala-84Tyr-87 Relationship between the MICs and mutations in the gyrA gene of E.coli. * Change of Lys-447 to Glu in the B subunit of the DNA gyrase. AAC (1994) 38: 2477

18. MIC (  g/ml) Substitutions in amino acids MIC (  g/ml) Substitutions in amino acids Strain CIP NAL GyrA ParC C20, C-130.0072Ser-83......Asp-87Ser-80......Glu-84 C-1, C-110.064Ser-83......Asp-87 Ser-80......Glu-84 C-18, C-80.1252Ser-83......Asp-87Ser-80......Glu-84 C-5, C-6, C-7, C-90.252Ser-83......Asp-87Ser-80......Glu-84 C-15, C-40.25128Leu-83......Asp-87Ser-80......Glu-84 C-101>2.000Leu-83......Asp-87Arg-80......Glu-84 13272512Leu-83......Asp-87 Ile-80.......Val-84 1363*, 12894>2.000Leu-83......Asp-87Ser-80......Lys-84 12738>2.000Leu-83......Tyr-87Ser-80......Lys-84 1331, 15748>2.000Leu-83......Asn-87 Ile-80......Glu-84 1283, 133416>2.000Leu-83...... Asn-87 Ile-80......Glu-84 136032>2.000Leu-83...... Tyr-87 Ser-80......Lys-84 1416, 1319, 132364>2.000Leu-83...... Asn-87 Ile-80......Glu-84 1383128>2.000Leu-83...... Tyr-87 Ile-80......Lys-84 Relationship between the MICs and mutations in the gyrA and parC genes of E.coli. AAC (1996) 40: 491

19. DECREASE IN DRUG ACCUMULATION. Decreased permeability. Decreased permeability. Increased efflux system(s). Increased efflux system(s).

20. OM IM IM Cell envelope of Gram-negative bacteria

21. MarA and SoxS (E.coli) Transcriptional regulation Direct or indirect Direct or indirect micF Efflux pumps acrAB-tolC acrAB-tolC others? others?OmpFRESISTANCE (Chloramphenicol, tetracycline, quinolones) quinolones)

22. OBJECTIVE To investigate changes in the expression of outer membrane protein(s) and efflux system(s) associated with quinolone resistance in an E.coli quinolone-resistant mutant using DNA microarrays containing the whole genome.

23. Characteristics of the strains E.coli (Ec-wt) (Clinical isolate). E.coli (Ec-wt) (Clinical isolate). – MIC of norflo of 0.5  g/ml. – Mutation Ser-83 to Leu. (GyrA) E.coli (Ec-Nor mutant). E.coli (Ec-Nor mutant). – MIC of norflo of 32  g/ml. – Mutation Ser-83 to Leu. (GyrA). No changes in the gyrB, parC and parE genes.

24. ACCUMULATION OF NORFLOXACIN Amount of norflo accumulated (  g/mg protein) (  g/mg protein) Strainwithout CCCP with CCCPDifference (CCCP - without CCCP) (CCCP - without CCCP) Ec-wt0.147 + 0.04 0.199 + 0.06 0.052 Ec-nor0.038 + 0.02 0.231+ 0.10 0.193

25. KL16 Ec-wt Ec-Nor

26. RNApurification of the Ec-wt and Ec-Nor strains. cDNA preparation (Reverse transcription and fluorescent labelling) Cy3 (Ec-wt) // Cy5 (Ec-Nor) Combine equal quantity Hybridize with the microarray Scanning

27. DNA MICROARRAY

28. CRITERIA TO CONSIDER CHANGES IN GENE EXPRESSION Changes greater than or equal to 1.9-fold in at least two of the three experiments.

29. GENES WITH MODIFIED EXPRESSION Increased expression. Increased expression. – 28 genes. Decreased expression. Decreased expression. – 7 genes.

30. Overexpressed genes Transcriptional factors Transcriptional factors ProteinGene Nor Transc. Factor soxS 8.26 9.64 6.20 Transc. Factor marA 3.663.222.72 Putative protein yhjB 2.84 3.55 2.75 regulator

31. Genes regulated by SoxS ProteinGene Nor Superoxide dismutase sodA 4.04 3.31 5.12 Putative oxidoreductase ydbK 8.24 7.14 4.72 Ferredoxin-reductase fpr 2.56 2.97 1.81 Repressor accum. iron fur 2.30 1.99 1.64 G6P dehydrogenase zwf 1.96 2.12 1.90 Endonuclease IV nfo 2.80 3.64 1.67 Aconitase acn 1.57 1.76 2.58 Nitroreductase mdaA/nfsA 2.35 1.20 2.29 Fumarase fumC 1.21 1.64 1.31 pH inducible protein inaA 0.50 0.74 0.86

32. Efflux pump acrAB-tolC ProteinGeneNor Increased expression Efflux pump of acridine acrA 3.07 1.82 2.89 acrB 2.27 2.83 2.29 No change in expression tolC 1.21 0.94 1.16

33. gapAacrAacrB 300 bp 50 bp 800 bp 1 2 3 1 2 3 1 2 3 M 1 = Ec – wt 2 = Ec – Nor 3 = DNA control 303 bp 289 bp RT-PCR

34. Other previously described efflux pumps in E.coli ProteinGeneNor Increased expression Putative transport protein ydhE 1.65 1.86 2.27

35. GENES POTENTIALLY RELATED TO QUINOLONE ACCUMULATION ProteinGeneNor Increased expression Protein transporter yceE 1.54 1.91 2.50 Outer membrane proteinb1377 2.53 1.69 2.57 Membrane protein b16291.62 2.00 1.98

36. Genes with decreased expression related to quinolone-resistance Decreased expression Outer membrane prot. ompF 10.37 9.93 8.03 Ec-wt Ec-Nor

37. CONCLUSIONS The DNA microarray is a powerful tool to study the expression of genes associated with quinolone resistance. The DNA microarray is a powerful tool to study the expression of genes associated with quinolone resistance. MarA and SoxS can both be overexpressed in a quinolone-resistant strain. MarA and SoxS can both be overexpressed in a quinolone-resistant strain.

38. CONCLUSIONS AcrAB and likely ydhE(NorM) and yceE (pmrA) may play a role in the acquisition of AcrAB and likely ydhE(NorM) and yceE (pmrA) may play a role in the acquisition of quinolone resistance quinolone resistance Other putative protein transporters and outer membranes proteins may be associated with acquisition of quinolone resistance Other putative protein transporters and outer membranes proteins may be associated with acquisition of quinolone resistance

39. PLASMID-MEDIATED RESISTANCE TO QUINOLONES IN Escherichia coli First reported in a strain of K. pneumoniae First reported in a strain of K. pneumoniae QnrA protein – 218 aa protein QnrA protein – 218 aa protein Protects DNA gyrase and topoisomerase IV from the inhibitory activity of quinolones Protects DNA gyrase and topoisomerase IV from the inhibitory activity of quinolones Qnr proteins Qnr proteins – QnrA2 – K. oxytoca (China) – QnrB - E. coli, K. pneumoniae, E. cloacae, C. koseri (USA and India) - 40% aa identity with QnrA – QnrS – S. flexneri (Japan) - 59% aa identity with QnrA The presence of other mechanisms of resistance may increase plasmid- mediated quinolone resistance The presence of other mechanisms of resistance may increase plasmid- mediated quinolone resistance

40. PREVALENCE OF PLASMID-MEDIATED RESISTANCE TO QUINOLONES IN Escherichia coli 1% QnrA+ isolates among ciprofloxacin-resistant E.coli from different countries [AAC (2003) 47:559] 1% QnrA+ isolates among ciprofloxacin-resistant E.coli from different countries [AAC (2003) 47:559] 11% QnrA+ isolates among ciprofloxacin-resistant K. pneumoniae and 0% in E.coli from USA [AAC (2004) 48: 1295] 11% QnrA+ isolates among ciprofloxacin-resistant K. pneumoniae and 0% in E.coli from USA [AAC (2004) 48: 1295] 7.7% QnrA+ isolates among ciprofloxacin-resistant E. coli in Shanghai (China) [AAC (2003) 47: 2242] 7.7% QnrA+ isolates among ciprofloxacin-resistant E. coli in Shanghai (China) [AAC (2003) 47: 2242] 0.4% QnrA+ isolates among nalidixic acid- resistant Escherichia coli (France) [AAC (2005) 49: 3091] 0.4% QnrA+ isolates among nalidixic acid- resistant Escherichia coli (France) [AAC (2005) 49: 3091]

41. Do quinolone-resistant E. coli strains carry fewer virulence factors than their susceptible counterparts?

42. RESISTANCE TO QUINOLONES IN E.coli CAUSING UTI No. (%) of strains Syndrome Total SusceptibleResistant Cystitis 10.950 8720 (80%)2180 (20%) p<.001 Pyelonephritis + 669 615 (92%) 54 (8%) Prostatitis

43. CONCLUSIONS Quinolone-resistant E.coli strains are less able to cause invasive urinary tract infection such as (pyelonephritis or prostatitis) than quinolone-susceptible strains Quinolone-resistant E.coli strains are less able to cause invasive urinary tract infection such as (pyelonephritis or prostatitis) than quinolone-susceptible strains CID (2001) 33: 1682

44. VIRULENCE FACTORS OF E. coli CAUSING UTI Adhesins Adhesins Toxins Toxins Other potential urovirulence factors Other potential urovirulence factors

45. FIMBRIAE Type 1 imbriae (FimH) - Glycoprotein with mnose chains Type 1 imbriae (FimH) - Glycoprotein with mnose chains P Fimbria (PapG, PrsG, PrfG) -  -D-Gal-(1- 4)-  -D-Gal P Fimbria (PapG, PrsG, PrfG) -  -D-Gal-(1- 4)-  -D-Gal S Fimbriae (SfaS) -  -sialyl-(2-3)-  -Gal S Fimbriae (SfaS) -  -sialyl-(2-3)-  -Gal F1C Fimbriae - Gal-Nac F1C Fimbriae - Gal-Nac

46. TOXINS  -hemolysin – Cytolytic toxin (Hly) – Produces transmembrane porus by a Ca 2+ dependent pathway in epithelial cells and leucocytes Cytotoxic necrotizing factor-1 (Cnf-1 ) Cytotoxic necrotizing factor-1 (Cnf-1 ) – Modification of Rho (GTP binding protein) which acts in the rearrangement of actin

47. OTHER UROVIRULENCE FACTORS Iron uptake Iron uptake Siderofores: Siderofores: – Aerobactin (Aer). – Yersiniobactin (Fyu). O-Antigen Involved in serum resistance and inflammation effects O-Antigen Involved in serum resistance and inflammation effects O1, O2, O4, O6, O7, O8, O14, O16, O18, O25, O50, O75 Capsules Avoid phagocytosis Capsules Avoid phagocytosis K1, K5, K6, K12, K13 K1, K5, K6, K12, K13

48. OTHER UROVIRULENCE FACTORS Outer membrane protease - OmpT Outer membrane protease - OmpT Serum resistance Serum resistance – Iss protein. – Tra protein. Invasion protein - IbeA Invasion protein - IbeA Autotransporter protein (toxin) - Sat Autotransporter protein (toxin) - Sat

49. To investigate the prevalence of several urovirulence factors in uropathogenic E.coli strains and correlate it with antimicrobial agent resistance

50. METHODOLOGY 100 E.coli clinical isolates causing cystitis or pyelonephritis 100 E.coli clinical isolates causing cystitis or pyelonephritis PCR detection of the following urovirulence factors PCR detection of the following urovirulence factors – Pap, Sfa, Foc, Fim, Afa, Bma, Gaf, Hly, Cnf, Fyu, Aer, Iron, Iss, Tra, Ibe, OmpT, Sat Determination of type 1 frimbriae expression by Saccharomyces cerevisae agglutination Determination of type 1 frimbriae expression by Saccharomyces cerevisae agglutination Analysis of the mutations in the gyrA and parC genes as mechanisms of quinolone resistance. Analysis of the mutations in the gyrA and parC genes as mechanisms of quinolone resistance.

51. RESULTS No relationship was detected between susceptibility to ampicillin, gentamicin, cotrimoxazol or chloramphenicol and the presence or absence of UVF No relationship was detected between susceptibility to ampicillin, gentamicin, cotrimoxazol or chloramphenicol and the presence or absence of UVF The resistance to nalidixic acid or ciprofloxacin was associated with the absence to several VF The resistance to nalidixic acid or ciprofloxacin was associated with the absence to several VF PapG, Sfa, Foc, Afa, Bma, Gaf, Fim, Hly, Cnf, Fyu, Aer, Iron, Iss, Tra, Ibe, OmpT, Sat PapG, Sfa, Foc, Afa, Bma, Gaf, Fim, Hly, Cnf, Fyu, Aer, Iron, Iss, Tra, Ibe, OmpT, Sat JID (2005) 191: 46

52. RESULTS FV Strains Nal S -E.coli Strains Nal R -E.coli p fimA 47 (94%)42 (84%)NS Type 1 fimbriae 44 (88%)30 (60%) 0.014 JID (2002) 186: 1039.

53. RESULTS FV Strains Nal S -E.coli Strains Nal R -E.coli p hlyD 42 (66%)7 (33%) 0.009 cnf 1 39 (61%)7 (33%) 0.02 85 E. coli urine isolates from phylogenetic group B2 64 nalidixic acid susceptible and 21 nalidixic acid resistant JCM (2005) 43: 2962

54. CONCLUSIONS Quinolone-resistant uropathogenic E.coli strains possess urovirulence factors such as Pap, Hly, Cnf o Sat with a lower frequency Quinolone-resistant uropathogenic E.coli strains possess urovirulence factors such as Pap, Hly, Cnf o Sat with a lower frequency A decrease in type 1 fimbriae expression is also observed in some quinolone-resistant uropathogenic E.coli A decrease in type 1 fimbriae expression is also observed in some quinolone-resistant uropathogenic E.coli

55. 1 st HYPOTHESIS Clonal dissemination of a nalidixic acid- resistant E.coli strain which does not have these virulence factors Clonal dissemination of a nalidixic acid- resistant E.coli strain which does not have these virulence factors

56. CLONAL DISSEMINATION

57. 2 nd HYPOTHESIS Quinolones can induce the loss of these urovirulence factors. Quinolones can induce the loss of these urovirulence factors. – Hly, Cnf, Pap and Sat genes are encoded in pathogenicity islands (PAI) - PAI similar to bacteriophages - Quinolones facilitate phage elimination through SOS activation – Quinolones may favor the loss of PAIs in the same way

58. EFFECT OF THE QUINOLONES ON THE LOSS OF PAIs IN UROPATHOGENIC E.coli. Three uropathogenic E. coli strains with different PAIs containing Hly, Cnf, Pap, Sat were submitted to subinhibitory concentrations of quinolones, analyzing the loss of hemolysin production. Three uropathogenic E. coli strains with different PAIs containing Hly, Cnf, Pap, Sat were submitted to subinhibitory concentrations of quinolones, analyzing the loss of hemolysin production.

59. EFFECT OF THE QUINOLONES ON THE LOSS OF PAIs IN UROPATOGENIC E.coli. Strain Hemolysis Gens Nal (mg/L) CIP (mg/L) 14366wt+ hly, cnf,sat,pap 2 0.012 14366m - pap 128 0.02 109wt+ hly, cnf, sfa, pap 1 0.006 109m- pap, sfa >256 > 1 359wt+ hly, cnf, pap, sfa 1 0.006 359m- pap, sfa, >256 0.5

60. sfa papA 1 1N 1C 2 2C 2C 3 3C 3C PFGE M 1 1C 1C 2 2C 2C 3 3C 3C M 14366 109 359 hlyA/cnf1 3C 3C 1 1C 1C 2 2C 2C 3 3C 3C 1 1C 1C sat

61. RESULTS (Strain E. coli HC14366) No. experiments (steps/ rate of loss) Wild-type ( - Cip)Wild-type ( + Cip)RecA - (+ Cip) 1 (<10 -5 )1 (1 / 1 x 10 -3 )1 (<10 -5 ) 2 (<10 -5 )2 (2 / 2 x 10 -3 )2 (<10 -5 ) 3 (<10 -5 ) 3 (1 / 5 x 10 -3 )3 (<10 -5 )

62. CONCLUSION Quinolones induce the loss of some pathogenicity islands in uropathogenic E.coli throughout the induction of the SOS system. Quinolones induce the loss of some pathogenicity islands in uropathogenic E.coli throughout the induction of the SOS system.