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
• Mechanism of action and resistance to
quinolones.
• Do quinolone-resistant E.coli strains
carry fewer virulence factors than their
susceptible counterparts?
COOH
BASIC STRUCTURE 4-QUINOLONE
O
O
O
F
OH
H3C
N
O
OH
N
N
N
HN
CH3
Nalidixic acid
O
Ciprofloxacin
O
O
F
F
OH
OH
N
N
N
N
HN
N
CH3
O
S
O
H3C
CH3
Norfloxacin
Levofloxacin
O
O
O
F
OH
F
OH
H3C
N
N
HN
O
O
H3C
Gatifloxacin
H
N
H
N
N
S
O
S
H
H3C
Moxifloxacin
MECHANISM OF ACTION
• Inhibition of:
–DNA gyrase.
–Topoisomerase IV.
Characteristics of the DNA gyrase.
• Structure
• A Subunit
A2B2
• gyrA gene.
• 97 kDa.
• DNA breaking and reunion.
• B Subunit:
• gyrB gene.
• 90 kDa.
• ATPase activity.
Characteristics of topoisomerase IV
• Structure
• Subunit A:
A2B2
• parC gene.
• 75 kDa.
• Subunit B:
• parE gene.
• 70 kDa.
• Associated with the inner membrane.
FUNCTIONS
Topoisomerase IV
- - - - -
+ + + +
DNA gyrase
++++
- - - - -
Organization of chromosome structure
REPLICATION
TOPO IV
GYRASE
TRANSCRIPTION
EVOLUTION OF THE RESISTANCE TO
QUINOLONES IN E.coli
% Resistance
Data from Hospital Clinic - Barcelona
40
30
28
23
24
28
24
30
25
20
10
2
5
9
11
14
1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003
Year
Evolution of the resistance to quinolones in
enterotoxigenic E. coli causing traveler’s diarrhea
% Resistance
1994-1997
NALIDIXIC ACID
6
2001-2004
22
(p<0.01)
CIPROFLOXACIN
Data from Hospital Clinic - Barcelona
1
12
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
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).
QUINOLONE RESISTANCE DETERMINING REGION
(QRDR) IN THE GENES ENCODING THE SUBUNITS OF
THE DNA GYRASE.
A subunit
67 106
NH2
HOOC
QRDR
DNA gyrase
Tyr-122
426 447
NH2
B subunit
HOOC
Substitutions of amino acids of the
subunit A of the DNA gyrase.
Position
Changes
67
81
83
84
87
106
Ala.....Ser
Gly.....Cys/Asp
Ser.....Leu/Trp/Ala
Ala.....Pro
Asp....Asn/Val/Tyr
Gln.....His/Arg
Increase in
the MIC (-fold)
4
8
32
8
16
4
Relationship between the MICs and mutations in the
gyrA gene of E.coli.
MIC (mg/ml)
Strains
CIP
Substitutions in amino acids
NAL
C20, C-13
0.007
2
C-1
0.06
4
C-11
0.06
2
C-18, C-8
0.125
2
C-5
0.25
2
C-6, C-7, C-9
0.25
2
C-15, C-4
0.25
128
C-10
1
>2.000
1327
2
512
1363*, 1289
4
>2.000
1273
8
>2.000
1331, 1574
8
>2.000
1283, 1334
16
>2.000
1360
32
>2.000
1416, 1319, 1323
64
>2.000
1383
128
>2.000
* Change of Lys-447 to Glu in the B subunit of the DNA gyrase.
GyrA
Ser-83
Ser-83
Ser-83
Ser-83
Ser-83
Ser-83
Leu-83
Leu-83
Leu-83
Leu-83
Leu-83
Leu-83
Leu-83
Leu-83
Leu-83
Leu-83
Ala-84
Ala-84
Ala-84
Ala-84
Val-84
Ala-84
Ala-84
Ala-84
Ala-84
Ala-84
Ala-84
Ala-84
Ala-84
Ala-84
Ala-84
Ala-84
Asp-87
Asp-87
Asp-87
Asp-87
Asp-87
Asp-87
Asp-87
Asp-87
Asp-87
Asp-87
Tyr-87
Asn-87
Asn-87
Tyr-87
Asn-87
Tyr-87
AAC (1994) 38: 2477
Relationship between the MICs and mutations in the
gyrA and parC genes of E.coli.
MIC (mg/ml)
Strain
C20, C-13
C-1, C-11
C-18, C-8
C-5, C-6, C-7, C-9
C-15, C-4
C-10
1327
1363*, 1289
1273
1331, 1574
1283, 1334
1360
1416, 1319, 1323
1383
CIP
NAL
0.007
0.06
0.125
0.25
0.25
1
2
4
8
8
16
32
64
128
2
4
2
2
128
>2.000
512
>2.000
>2.000
>2.000
>2.000
>2.000
>2.000
>2.000
Substitutions in amino acids
GyrA
Ser-83......Asp-87
Ser-83......Asp-87
Ser-83......Asp-87
Ser-83......Asp-87
Leu-83......Asp-87
Leu-83......Asp-87
Leu-83......Asp-87
Leu-83......Asp-87
Leu-83......Tyr-87
Leu-83......Asn-87
Leu-83......Asn-87
Leu-83......Tyr-87
Leu-83......Asn-87
Leu-83......Tyr-87
ParC
Ser-80......Glu-84
Ser-80......Glu-84
Ser-80......Glu-84
Ser-80......Glu-84
Ser-80......Glu-84
Arg-80......Glu-84
Ile-80.......Val-84
Ser-80......Lys-84
Ser-80......Lys-84
Ile-80......Glu-84
Ile-80......Glu-84
Ser-80......Lys-84
Ile-80......Glu-84
Ile-80......Lys-84
AAC (1996) 40: 491
DECREASE IN DRUG
ACCUMULATION.
• Decreased permeability.
• Increased efflux system(s).
Cell envelope of Gram-negative bacteria
OM
IM
MarA and SoxS (E.coli)
Transcriptional regulation
Direct or indirect
micF
Efflux pumps
acrAB-tolC
others?
OmpF
RESISTANCE
(Chloramphenicol, tetracycline,
quinolones)
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.
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.
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
KL16
Ec-wt Ec-Nor
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
DNA MICROARRAY
CRITERIA TO CONSIDER CHANGES IN
GENE EXPRESSION
Changes greater than or equal to 1.9-fold
in at least two of the three experiments.
GENES WITH MODIFIED EXPRESSION
• Increased expression.
– 28 genes.
• Decreased expression.
– 7 genes.
Overexpressed genes
• Transcriptional factors
Protein
Gene
Nor
Transc. Factor
Transc. Factor
soxS
marA
8.26 9.64 6.20
3.66 3.22 2.72
Putative protein
regulator
yhjB
2.84 3.55 2.75
Genes regulated by SoxS
Protein
Gene
Superoxide dismutase
Putative oxidoreductase
Ferredoxin-reductase
Repressor accum. iron
G6P dehydrogenase
Endonuclease IV
Aconitase
Nitroreductase
Fumarase
pH inducible protein
sodA
ydbK
fpr
fur
zwf
nfo
acn
mdaA/nfsA
fumC
inaA
Nor
4.04
8.24
2.56
2.30
1.96
2.80
1.57
2.35
1.21
0.50
3.31
7.14
2.97
1.99
2.12
3.64
1.76
1.20
1.64
0.74
5.12
4.72
1.81
1.64
1.90
1.67
2.58
2.29
1.31
0.86
Efflux pump acrAB-tolC
Protein
Gene
Nor
Increased expression
Efflux pump of acridine
acrA
acrB
3.07 1.82
2.27 2.83
2.89
2.29
No change in expression
tolC
1.21 0.94 1.16
RT-PCR
1
2 3 1 2 3 1 2 3 M
1 = Ec – wt
800 bp
2 = Ec – Nor
3 = DNA control
303 bp
289 bp
300 bp
50 bp
acrB
acrA
gapA
Other previously described efflux pumps
in E.coli
Protein
Gene
Nor
Increased expression
Putative transport protein ydhE
1.65 1.86 2.27
GENES POTENTIALLY RELATED TO QUINOLONE
ACCUMULATION
Protein
Gene
Nor
yceE
b1377
b1629
1.54 1.91 2.50
2.53 1.69 2.57
1.62 2.00 1.98
Increased expression
Protein transporter
Outer membrane protein
Membrane protein
Genes with decreased expression
related to quinolone-resistance
Decreased expression
Outer membrane prot.
ompF
10.37 9.93 8.03
Ec-wt Ec-Nor
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.
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
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 plasmidmediated quinolone resistance
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]
• 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]
Do quinolone-resistant E. coli strains
carry fewer virulence factors
than their susceptible counterparts?
RESISTANCE TO QUINOLONES IN E.coli
CAUSING UTI
No. (%) of strains
Syndrome
Total
Susceptible Resistant
Cystitis
10.950
8720 (80%)
2180 (20%)
p<.001
Pyelonephritis +
Prostatitis
669
615 (92%)
54 (8%)
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
VIRULENCE FACTORS OF E. coli
CAUSING UTI
• Adhesins
• Toxins
• Other potential urovirulence factors
FIMBRIAE
• Type 1 imbriae (FimH) - Glycoprotein with
mnose chains
• P Fimbria (PapG, PrsG, PrfG) - a-D-Gal-(14)-b-D-Gal
• S Fimbriae (SfaS) - a-sialyl-(2-3)-b-Gal
• F1C Fimbriae - Gal-Nac
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
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
OTHER UROVIRULENCE FACTORS
• Outer membrane protease - OmpT
• Serum resistance
– Iss protein.
– Tra protein.
• Invasion protein - IbeA
• Autotransporter protein (toxin) - Sat
To investigate the prevalence of several urovirulence
factors in uropathogenic E.coli strains and
correlate it with antimicrobial agent resistance
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.
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
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.
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
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
st
1
HYPOTHESIS
• Clonal dissemination of a nalidixic acidresistant E.coli strain which does not
have these virulence factors
CLONAL DISSEMINATION
nd
2 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
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.
EFFECT OF THE QUINOLONES ON THE LOSS OF PAIs
IN UROPATOGENIC E.coli.
Strain
Hemolysis
Gens
Nal (mg/L) CIP (mg/L)
14366wt
14366m
+
-
hly, cnf,sat,pap
pap
2
128
0.012
0.02
109wt
109m
+
-
hly, cnf, sfa, pap
pap, sfa
1
>256
0.006
>1
359wt
359m
+
-
hly, cnf, pap, sfa
pap, sfa,
1
>256
0.006
0.5
PFGE
14366
109
359
M 1 1C 1C 2 2C 2C 3 3C 3C M
sat
hlyA/cnf1
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
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)
CONCLUSION
• Quinolones induce the loss of some
pathogenicity islands in uropathogenic
E.coli throughout the induction of the
SOS system.