Villars-sur-Ollon (Switzerland) September 4-8, 2005

1

MECHANISMS OF RESISTANCE TO QUINOLONES
IN Escherichia coli
Jordi Vila Department of Microbiology Hospital
Clinic, Barcelona, Sccolf of Medicine,
University of Barcelon, Barcelona, Spain

• Villars-sur-Ollon (Switzerland) September 4-8,
  2005

2

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

3
COOH
BASIC STRUCTURE 4-QUINOLONE
4
Nalidixic acid
Ciprofloxacin
Norfloxacin
Levofloxacin
Moxifloxacin
Gatifloxacin
5
MECHANISM OF ACTION

• Inhibition of
• DNA gyrase.
• Topoisomerase IV.

6
Characteristics of the DNA gyrase.

• Structure A2B2
• A Subunit
• gyrA gene.
• 97 kDa.
• DNA breaking and reunion.
• B Subunit
• gyrB gene.
• 90 kDa.
• ATPase activity.

7
Characteristics of topoisomerase IV

• Structure A2B2
• Subunit A
• parC gene.
• 75 kDa.
• Subunit B
• parE gene.
• 70 kDa.
• Associated with the inner membrane.

8
FUNCTIONS
Topoisomerase IV
- - - - -
DNA gyrase
- -
- - -
9
Organization of chromosome structure
10
REPLICATION
TOPO IV
GYRASE
TRANSCRIPTION
11
EVOLUTION OF THE RESISTANCE TO QUINOLONES IN
E.coli
Resistance
Data from Hospital Clinic - Barcelona
40 30 20 10
30
28
28
25
24
24
23
14
11
9
5
2
1992 1993 1994 1995 1996 1997 1998
1999 2000 2001 2002 2003
Year
12
Evolution of the resistance to quinolones in
enterotoxigenic E. coli causing travelers
diarrhea
Resistance 1994-1997
2001-2004 NALIDIXIC ACID 6
22
(plt0.01) CIPROFLOXACIN 1
12
Data from Hospital Clinic - Barcelona
13
MECHANISMS OF RESISTANCE TO QUINOLONES IN
ENTEROBACTERIACEAE

• Changes in the protein targets.
• DNA gyrase
• Topoisomerase IV.
• Reduction in the accumulation of the quinolone.
• - Decrease in permeability.
• - Increase in active efflux system(s).
• DNA gyrase and topoisomerase IV protection -
  qnr gene

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ACQUISITION OF RESISTANCE TO QUINOLONES
- CHROMOSOMAL MUTATIONS. - Mutations in the
genes encoding the subunits of the
protein targets. - Mutations in the genes
causing the decrease in
accumulation. - PLASMID (Enterobacteriaceae).
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QUINOLONE RESISTANCE DETERMINING REGION (QRDR)
IN THE GENES ENCODING THE SUBUNITS OF THE DNA
GYRASE.
A subunit DNA gyrase B subunit
67 106
NH2
HOOC
QRDR
Tyr-122
426 447
NH2
HOOC
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Substitutions of amino acids of the subunit A of
the DNA gyrase.
Position Changes Increase in the MIC
(-fold) 67 Ala.....Ser 4 81 Gly.....Cys/Asp
8 83 Ser.....Leu/Trp/Ala 32 84 Ala.....Pro
8 87 Asp....Asn/Val/Tyr 16 106 Gln.....His/
Arg 4
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Relationship between the MICs and mutations in
the gyrA gene of E.coli.
MIC (mg/ml) Substitutions in
amino acids Strains CIP NAL
GyrA
C20, C-13 0.007 2 Ser-83 Ala-84 Asp-87 C-1 0.
06 4 Ser-83 Ala-84 Asp-87 C-11 0.06 2 Ser-83
Ala-84 Asp-87 C-18, C-8 0.125 2 Ser-83 Ala-84
Asp-87 C-5 0.25 2 Ser-83 Val-84 Asp-87 C-6,
C-7, C-9 0.25 2 Ser-83 Ala-84 Asp-87 C-15,
C-4 0.25 128 Leu-83 Ala-84 Asp-87 C-10 1 gt2.00
0 Leu-83 Ala-84 Asp-87 1327 2 512 Leu-83 Ala-
84 Asp-87 1363, 1289 4 gt2.000 Leu-83 Ala-84 As
p-87 1273 8 gt2.000 Leu-83 Ala-84 Tyr-87 1331,
1574 8 gt2.000 Leu-83 Ala-84 Asn-87 1283,
1334 16 gt2.000 Leu-83 Ala-84 Asn-87
1360 32 gt2.000 Leu-83 Ala-84 Tyr-87 1416,
1319, 1323 64 gt2.000 Leu-83 Ala-84 Asn-87
1383 128 gt2.000 Leu-83 Ala-84 Tyr-87

Change of Lys-447 to Glu in the B subunit of
the DNA gyrase.
AAC (1994) 38 2477
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Relationship between the MICs and mutations in
the gyrA and parC genes of E.coli.
MIC (mg/ml) Substitutions in
amino acids Strain CIP NAL
GyrA ParC
C20, C-13 0.007 2 Ser-83......Asp-87 Ser-80...
...Glu-84 C-1, C-11 0.06 4 Ser-83......Asp-87
Ser-80......Glu-84 C-18, C-8 0.125 2 Ser-83....
..Asp-87 Ser-80......Glu-84 C-5, C-6, C-7,
C-9 0.25 2 Ser-83......Asp-87 Ser-80......Glu-84
C-15, C-4 0.25 128 Leu-83......Asp-87 Ser-80...
...Glu-84 C-10 1 gt2.000 Leu-83......Asp-87 Arg-8
0......Glu-84 1327 2 512 Leu-83......Asp-87
Ile-80.......Val-84 1363, 1289 4 gt2.000 Leu-83.
.....Asp-87 Ser-80......Lys-84 1273 8 gt2.000 Leu
-83......Tyr-87 Ser-80......Lys-84 1331,
1574 8 gt2.000 Leu-83......Asn-87
Ile-80......Glu-84 1283, 1334 16 gt2.000 Leu-83..
....Asn-87 Ile-80......Glu-84 1360 32 gt2.000 L
eu-83......Tyr-87 Ser-80......Lys-84 1416,
1319, 1323 64 gt2.000 Leu-83......Asn-87
Ile-80......Glu-84 1383 128 gt2.000 Leu-83......T
yr-87 Ile-80......Lys-84

AAC (1996) 40 491
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DECREASE IN DRUG ACCUMULATION.

• Decreased permeability.
• Increased efflux system(s).

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Cell envelope of Gram-negative bacteria
21
MarA and SoxS (E.coli) Transcriptional
regulation Direct or indirect micF
Efflux pumps acrAB-tolC
others? OmpF RESISTANCE (Chloramphenicol,
tetracycline, 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.
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Characteristics of the strains

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

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ACCUMULATION OF NORFLOXACIN
Amount of norflo
accumulated (mg/mg
protein) Strain without CCCP with
CCCP Difference (CCCP - without
CCCP) Ec-wt 0.147 0.04 0.199 0.06
0.052 Ec-nor 0.038
0.02 0.231 0.10 0.193
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KL16 Ec-wt Ec-Nor
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RNA purification 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
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DNA MICROARRAY
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CRITERIA TO CONSIDER CHANGES IN GENE EXPRESSION
Changes greater than or equal to 1.9-fold in at
least two of the three experiments.
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GENES WITH MODIFIED EXPRESSION

• Increased expression.
• 28 genes.
• Decreased expression.
• 7 genes.

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Overexpressed genes

• Transcriptional factors

Protein Gene Nor Transc. Factor
soxS 8.26 9.64 6.20 Transc. Factor
marA 3.66 3.22 2.72 Putative
protein yhjB 2.84 3.55 2.75
regulator
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Genes regulated by SoxS
Protein Gene 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
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Efflux pump acrAB-tolC
Protein Gene Nor 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
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RT-PCR
1 2 3 1 2 3 1 2 3 M
1 Ec wt 2 Ec Nor 3 DNA control
800 bp
303 bp 289 bp
300 bp
50 bp
gapA
acrA
acrB
34
Other previously described efflux pumps in E.coli
Protein Gene Nor Increased
expression Putative transport protein ydhE 1.65
1.86 2.27
35
GENES POTENTIALLY RELATED TO QUINOLONE
ACCUMULATION
Protein Gene Nor Increased
expression Protein transporter yceE 1.54
1.91 2.50 Outer membrane protein b1377
2.53 1.69 2.57 Membrane protein
b1629 1.62 2.00 1.98
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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.
• 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
• quinolone resistance
• Other putative protein transporters and outer
  membranes proteins may be associated with
  acquisition of quinolone resistance

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PLASMID-MEDIATED RESISTANCE TO QUINOLONES IN
Escherichia coli

• First reported in a strain of K. pneumoniae
• QnrA protein 218 aa protein
• Protects DNA gyrase and topoisomerase IV from the
  inhibitory activity of quinolones
• 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

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PREVALENCE OF PLASMID-MEDIATED RESISTANCE TO
QUINOLONES IN Escherichia coli

• 1 QnrA isolates among ciprofloxacin-resistant
  E.coli from different countries AAC (2003)
  47559
• 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
• 0.4 QnrA isolates among nalidixic acid-
  resistant Escherichia coli (France) AAC (2005)
  49 3091

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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 Susceptible Resistant C
ystitis 10.950 8720 (80) 2180
(20) plt.001 Pyelonephritis 669
615 (92) 54 (8) Prostatitis
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CONCLUSIONS

• 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
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VIRULENCE FACTORS OF E. coli CAUSING UTI

• Adhesins
• Toxins
• Other potential urovirulence factors

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FIMBRIAE

• Type 1 imbriae (FimH) - Glycoprotein with
  mnose chains
• P Fimbria (PapG, PrsG, PrfG) -
  a-D-Gal-(1-4)-b-D-Gal
• S Fimbriae (SfaS) - a-sialyl-(2-3)-b-Gal
• F1C Fimbriae - Gal-Nac

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TOXINS

• a-hemolysin Cytolytic toxin (Hly)
• Produces transmembrane porus by a Ca2 dependent
  pathway in epithelial cells and leucocytes
• 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
• Siderofores
• Aerobactin (Aer).
• Yersiniobactin (Fyu).
• O-Antigen Involved in serum resistance and
  inflammation effects
• O1, O2, O4, O6, O7, O8, O14, O16, O18, O25,
  O50, O75
• Capsules Avoid phagocytosis
• K1, K5, K6, K12, K13

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OTHER UROVIRULENCE FACTORS

• Outer membrane protease - OmpT
• Serum resistance
• Iss protein.
• Tra protein.
• Invasion protein - IbeA
• Autotransporter protein (toxin) - Sat

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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
• 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
• 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
• 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

JID (2005) 191 46
52
RESULTS
FV Strains NalS-E.coli Strains NalR
-E.coli p fimA 47 (94) 42
(84) NS Type 1 fimbriae 44 (88) 30
(60) 0.014
JID (2002) 186 1039.
53
RESULTS
85 E. coli urine isolates from phylogenetic
group B2 64 nalidixic acid susceptible and 21
nalidixic acid resistant
FV Strains NalS-E.coli Strains NalR
-E.coli p hlyD 42 (66) 7
(33) 0.009 cnf 1
39 (61) 7 (33) 0.02
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
• A decrease in type 1 fimbriae expression is also
  observed in some quinolone-resistant
  uropathogenic E.coli

55
1st HYPOTHESIS

• Clonal dissemination of a nalidixic
  acid-resistant E.coli strain which does not have
  these virulence factors

56
CLONAL DISSEMINATION
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2nd HYPOTHESIS

• 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.

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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
gt256 gt 1 359wt hly, cnf, pap,
sfa 1 0.006 359m -
pap, sfa, gt256 0.5
60
hlyA/cnf1
sat
PFGE
1 1C 1C 2 2C 2C 3 3C 3C
1 1C 1C
sfa
papA
1 1N 1C 2 2C 2C 3 3C 3C
1 1N 1C 2 2C 2C 3 3C 3C
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RESULTS(Strain E. coli HC14366)
No. experiments (steps/ rate of loss) Wild-type
( - Cip) Wild-type ( Cip) RecA- ( Cip) 1
(lt10-5) 1 (1 / 1 x 10-3) 1 (lt10-5) 2
(lt10-5) 2 (2 / 2 x 10-3) 2 (lt10-5) 3 (lt10-5)
3 (1 / 5 x 10-3) 3 (lt10-5)
62
CONCLUSION

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