Aminoglycosides Macrolides Fluoroquinolones Paul M Tulkens MD PhD Cellular and Molecular Pharmacology Louvain Drug Research Institute Université catholique de Louvain Brussels Belgium ID: 816141
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Slide1
The Major Mechanisms of Resistance to Aminoglycosides, Macrolides, Fluoroquinolones
Paul M. Tulkens, MD, PhDCellular and Molecular PharmacologyLouvain Drug Research InstituteUniversité catholique de LouvainBrussels, Belgium
2014 ISF Stephen F. Lowry
Colloquium Intersection between Virulence and Antimicrobial Resistance in the ICUParis, France -- 1-3 December 2014
2/12/2014
2014 ISF Stephen F. Lowry Colloquium
1
Slide2Disclosures and slides availabilityResearch grants Theravance, Astellas
, Targanta, Cerexa/Forest, AstraZeneca, Bayer, GSK, Trius, Rib-X, EumedicaBelgian Science Foundation (F.R.S.-FNRS), Ministry of Health (
SPF), and Walloon and Brussels Regions Speaking fees
Bayer, GSK, Sanofi, Johnson & Johnson, OM-Pharma, CubistDecision-making and consultation bodiesGeneral Assembly and steering committee (2008-2010) of EUCASTEuropean Medicines Agency (external expert)US National Institutes of Health (grant reviewer)
Slides: http://www.facm.ucl.ac.be
Lectures
2/12/2014
2014 ISF Stephen F. Lowry Colloquium
2
Slide3The aminoglycosides in shortThe first class of wide-spectrum antibiotics(as the result of a first systematic screening of natural sources) Concentration-dependent and highly bactericidal
Active of most aerobic Gram-negative bacteria including P. aeruginosaFor some of them, also active against Mycobacterium tuberculosis and other Mycobacteriae
Synergy with cell-wall acting agentsResistance variable between regions and settings but usually low … because of limited use
2/12/20142014 ISF Stephen F. Lowry Colloquium3
Slide4Discovery by screening2/12/20142014 ISF Stephen F. Lowry Colloquium4
From the point of view of human benefit, never was a Nobel prize so justifiably awarded as was the award to Selman Waksman for the discovery of streptomycin and other antibiotics produced from
Streptomyces spp. Waksman and his talented team (many of whom went on to make important antibiotic discoveries in their own right) developed the concept of
systematic screening of microbial culture products for biological activity, a technology which has provided the foundation of the antibiotic industry, and for this alone his name should rank high in any pantheon of microbiology.J. Davies: In Praise of Antibiotics, ASM Newshttp://www.asm.org/memonly/asmnews/may99/feature6.html
Waksman and Fleming …
streptomyces
grisaeus
Slide5The aminoglycosides in shortThe first class of wide-spectrum antibiotics(as the result of a first systematic screening of natural sources) Concentration-dependent and highly bactericidal
Active of most aerobic Gram-negative bacteria including P. aeruginosaFor some of them, also active against Mycobacterium tuberculosis and other Mycobacteriae
Synergy with cell-wall acting agentsResistance variable between regions and settings but usually low … because of limited use
2/12/20142014 ISF Stephen F. Lowry Colloquium5
Slide6Concentration-dependence2/12/20142014 ISF Stephen F. Lowry Colloquium6
Craig & Ebert SC. Killing and regrowth of bacteria in vitro: A review. Scand J Infect Dis. 1990;74:63-70.
Slide7The aminoglycosides in shortThe first class of wide-spectrum antibiotics(as the result of a first systematic screening of natural sources)
Concentration-dependent and highly bactericidalActive of most aerobic Gram-negative bacteria including P. aeruginosaFor some of them, also active against Mycobacterium tuberculosis and other MycobacteriaeSynergy with cell-wall acting agents
Resistance variable between regions and settings but usually low … because of limited use2/12/2014
2014 ISF Stephen F. Lowry Colloquium7
Slide8Resistance of P. aeruginosa to antibiotics in ICU (VAP patients; Belgian hospitals; 2007-2009) 2/12/20142014 ISF Stephen F. Lowry Colloquium
8
EUCAST bkpt > R
CLSI bkpt
≥ R
16
1
8
16
8
8
Slide9Aminoglycosides: main mechanisms of resistanceIntrinsic resistanceAll anaerobic bacteria (no accumulation in bacteria)Enterococci (facultative anaerobics)Bacteria with mutations at 16S rRNA
(resistant M. tuberculosis)(includ. point mutations: M. abcessens, M. chloanae)Acquired (or resulting from overexpression) resistanceReduced entry and effluxEnzymatic modificationMethylation of targetSmall colony variantsBiofilms
2/12/20142014 ISF Stephen F. Lowry Colloquium9
Slide10Aminoglycosides: main mechanisms of resistanceIntrinsic resistanceAll anaerobic bacteria (no accumulation in bacteria)Enterococci (facultative anaerobics)Bacteria with mutations at 16S rRNA
(resistant M. tuberculosis)(includ. point mutations: M. abcessens, M. chloanae)Acquired (or resulting from overexpression) resistanceReduced entry and effluxEnzymatic modificationMethylation of targetSmall colony variants
Biofilms 2/12/20142014 ISF Stephen F. Lowry Colloquium
10
Slide11Aminoglycosides: effluxMostly related to the presence tripartite pumps (MexXY-OprM in P. aeruginosa; AcrA/B-T
olC in Enterobacteriaceae; AmrAB in Burkholderia…)Recognition may be after association/binding to phopsholipids (amphiphilic complex) but may also involve specific recognition sitesProbably responsible forDecreased susceptibility during treatment and/or exposure to sub-inhibitory concentrations (overexpression)
Adaptative resistance ( susceptibility after high peak)2/12/20142014 ISF Stephen F. Lowry Colloquium
11
Slide12Asymmetric trimer model of MexY (homology modeling on the crystal structure of E. coli AcrB)2/12/2014
2014 ISF Stephen F. Lowry Colloquium12
Lau et al.
MBio. 2014 Apr 22;5(2):e01068.
General structure
of
Mex
pumps
Eda
S et al. J. Biol. Chem. 2003;278:2085-2088
Slide13Prevalence of efflux pumps in 62 pairs of Pseudomonas aeruginosa collected from ICU patients2/12/201413
DAY 0
DAY X
mexA
and
mexX
(basal expression in
wild-type strains)
treatment
mexA
+
mexX
+
mexA
-
mexX
+
mexA
+
mexX
-
mexA
-
mexX
-
mexA
+
mexX
+
mexA
-
mexX
+
mexA
+
mexX
-
mexA
-
mexX
-
The prevalence of
mexA
+
and
mexX
+
was already high in first isolates.
For all genes tested, the number of isolates with overexpression increased during treatment
(P < 0.05, Fisher's Exact Test 2-sided ).
Riou
et al.
20
th
ECCMID, 2010, Poster
780
Riou
et al
., in preparation
2014 ISF Stephen F. Lowry Colloquium
Slide14Aminoglycosides: enzymatic modification2/12/20142014 ISF Stephen F. Lowry Colloquium14
O
O
O
H
2
N
N
H
2
H
2
N
O
H
H
O
H
2
N
O
N
H
2
O
H
O
H
kanamycin B
O
H
2'-acetylation
6'-acetylation
3-acetylation
2"-adenylation
2"-phosphorylation
3'-phosphorylation
4'-adenylation
O
O
O
C
H
H
2
N
N
H
2
O
H
H
N
H
3
C
C
H
3
O
H
H
O
H
2
N
g
e
n
t
a
m
i
c
i
n
C
1
:
R
1
=
C
H
3
;
R
2
=
C
H
3
g
e
n
t
a
m
i
c
i
n
C
1
a
:
R
1
=
H
;
R
2
=
H
g
e
n
t
a
m
i
c
i
n
C
2
:
R
1
=
C
H
3
;
R
2
=
H
O
H
N
R
1
R
2
O
O
O
H
O
H
2
H
2
N
O
H
H
O
H
2
N
O
N
H
2
O
H
O
H
kanamycin A
O
H
N
Slide15Enzymatic modifications: the situation in the late 90’s2/12/20142014 ISF Stephen F. Lowry Colloquium15
Slide16Aminoglycosides: the family (based on susceptibility to enzymatic resistance mechanisms)2/12/20142014 ISF Stephen F. Lowry Colloquium
16grouptypical approved or in development antibiotics
most
susceptibleless susceptiblelesser susceptible
least susceptible
Streptomycins
streptomycin
Neomycins
Kanamycins
kanamycin A
amikacin
kanamycin B
tobramycin
dibekacin
Gentamicin
“gentamicin” *
gentamicin
B
isepamicin
Sisomicin
sisomicin
netilmicin
plazomycin
Spectinomycin
* Mixture of
gentamicins
C1, C1a, C2 and C2a
Slide17Plazomycin… 2/12/20142014 ISF Stephen F. Lowry Colloquium17
plazomicin
sisomicin
still susceptible
to AAC(2’)
(but rare enzyme)
Slide18Plazomycin…2/12/20142014 ISF Stephen F. Lowry Colloquium18
Slide19Aminoglycosides: main mechanisms of resistanceIntrinsic resistanceAll anaerobic bacteria (no accumulation in bacteria)Enterococci
(facultative anaerobics)Bacteria with mutations at 16S rRNA (resistant M. tuberculosis)(includ
. point mutations: M. abcessens, M. chloanae
)Acquired (or resulting from overexpression) resistanceReduced entry and effluxEnzymatic modificationMethylation of targetSmall colony variantsBiofilms 2/12/2014
2014 ISF Stephen F. Lowry Colloquium
19
Exogenously acquired 16S
rRNA
methyltransferase
(16S-RMTase) genes responsible for a very high level of resistance against various aminoglycosides have been widely distributed among Enterobacteriaceae and glucose-nonfermentative microbes recovered from human and animal. Wachino & Y. Arakawa, Drug Resist. Updat. 15 (2012) 133–148.
Slide20Methylases may be part of the normal bacterial “equipment” 2/12/20142014 ISF Stephen F. Lowry Colloquium20
Slide21Methylases may be part of the normal bacterial “equipment” 2/12/20142014 ISF Stephen F. Lowry Colloquium21
Slide22Macrolides2/12/20142014 ISF Stephen F. Lowry Colloquium22
ErythromycinRoxithromycinClarithromycin
Erythromycylamine
SpiramycinJosamycinMiocamycinRokitamycin
X
O
H
3
C
OH
OH
O
R
CH
3
H
3
CH
2
C
O
O
CH
3
CH
3
H
3
C
O
O
O
HO
(H
3
C)
2
N
CH
3
OH
H
3
C
CH
3
H
3
CO
H
3
C
X
C=O
C=O
C-NH
2
R
H
H
CH
3
H
O
O
H
3
C
O
O
R
1
H
3
CO
OHC
CH
3
O
R
2
O
HO
(H
3
C)
2
N
O
O
CH
3
O
R
3
CH
3
O
R
4
CH
3
R
1
H
COCH
3
COCH
2
CH
3
H
R
2
Forosamine
H
COCH
3
H
R
3
H
H
COCH
3
COCH
2
CH
3
R
4
H
COCH
2
CH(CH
3
)
2
COCH
2
CH
3
CO(CH
2
)
2
CH
3
O
H
3
C
OH
OH
OH
CH
3
H
3
CH
2
C
O
O
CH
3
CH
3
H
3
C
O
O
O
HO
(H
3
C)
2
N
CH
3
OH
N
CH
3
CH
3
H
3
C
H
3
C
H
3
CO
14-membered
15-membered
16-membered
C=N-O-CH
2
-O-CH
2
-CH
2
-O-CH
3
« OLD »
MACROLIDES
Van
Bambeke
F. In Fundamentals off
Antimicrobial Pharmacokinetics and
Pharmacodynamics (201,
p 257-278
Springer, ISBN 978-0-387-75612-7 - ISBN 978-0-387-75613-4 (eBook)
Slide23Macrolides main mechanisms of resistanceIntrinsic resistanceGram-negative bacteria: impermeability (with exceptions and possible modulation by the medium)AcquiredEfflux
msrA in Staphylococci (MSB phenotype)mefA in Streptococci and Enterococci(dissociation between clindamycin and macrolides)Target mutationsdimethylation of the A2058 residue within a conserved region of domain V of the 23S rRNA(several genes and cross-resistance with clindamycin)
Drug inactivation (phosphotransferases, esterases…)2/12/2014
2014 ISF Stephen F. Lowry Colloquium23Most frequent
in Europe
Slide24Can macrolides be active against P. aeruginosa ?2/12/20142014 ISF Stephen F. Lowry Colloquium24
Clinical Infectious Diseases (2012) 55:534-542
Slide25Can macrolides be active against P. aeruginosa ?2/12/20142014 ISF Stephen F. Lowry Colloquium25
Clinical Infectious Diseases (2012) 55:534-542
Slide26“Old” macrolides are no longer usable for “common” respiratory infections due to S. pneumoniae2/12/20142014 ISF Stephen F. Lowry Colloquium26
Community-acquired pneumonia 2006-2009Lismond et al.
Int J Antimicrob Agents (2012) 39:208– 216
Chronic obstructive pulmonary disease 2010-2013 (bacterial exacerbations)Vandevelde et al. Int J Antimicrob Agents (2014) 44:209–217
EUCAST
breakpoints
EUCAST
breakpoints
Slide27Macrolide resistance: mechanisms…2/12/20142014 ISF Stephen F. Lowry Colloquium27
Most of the resistant isolatesin Europe are “high level” and “methylase” positive
But clinical isolates may have both mechanisms… or additional ones
Lismond et al. Int J Antimicrob Agents (2012) 39:208– 216
Slide28A possible future for macrolides…2/12/20142014 ISF Stephen F. Lowry Colloquium28
KETOLIDES
Cethromycin
Solithromycin
Modithromycin
11-N-ketolides
6-O-ketolides
bridged bicyclic
ketolides
fluoroketolides
Slide29Solithromycin and resistance to “old” macrolides …2/12/20142014 ISF Stephen F. Lowry Colloquium29
S. pneumoniae isolates fromBelgium and Germany(n=741)submitted
Slope = 0.17
Slide30FluoroquinolonesThe first major class of antibiotics of synthetic origin (no obvious equivalent in natural products) resistance was though to be unlikely…
But…spontaneously occurring mutations in chromosomal genes that alter the target enzymes (DNA gyrase and topoisomerase IV) high frequency: 10-7 / 10-8
!!Efflux-mediated reduction of intrabacterial concentration in both Gram-positive and Gram-negative bacteria, even if not pre-exposed to the same molecule
favours the selection of resistant mutantsAcquisition of plasmid-encoded qnr genes protecting DNA gyrase and topoisomerase IV from quinolone action
spreading…
modification of a plasmid-encoded aminoglycoside
acetylating enzyme (AAC(6′)-Ib-cr) that
cetylates
the C7
aminofunction
of piperazinyl-substituted fluoroquinolones (ciprofloxacin, norfloxacin). 2/12/20142014 ISF Stephen F. Lowry Colloquium30
Most frequent
Slide31Fluoroquinolones: prevalence of resistance2/12/20142014 ISF Stephen F. Lowry Colloquium31
Jacoby G. Clin Infect Dis. 2005 Jul 15;41 Suppl 2:S120-6
Slide32Fluoroquinolones: Mutant Prevention Concentration (MPC)2/12/20142014 ISF Stephen F. Lowry Colloquium32
0.01
0.10
1.00
10.00
10
-2
1
concentration
10
-4
10
-6
10
-8
10
-10
MPC
10
=
9
Dong et al: AAC 1999; 43:1756-1758
"Classic" bactericidal effect
Elimination
of resistant organisms
Surviving bacteria
MIC
99
=
0.8 mg/L
(in this example)
poorly
susceptible
organisms…
Slide33Fluoroquinolones: Mutant Prevention Concentration (MPC)2/12/20142014 ISF Stephen F. Lowry Colloquium33
0.01
0.10
1.00
10.00
10
-2
1
concentration
10
-4
10
-6
10
-8
10
-10
MPC
10
=
9
Dong et al; AAC 43:1756-1758
Surviving bacteria
MIC
99
=
0.8
Concentration
that
inhibits
the majority
of the organisms
Concentration needed to prevent the
selection
of
resistant organisms
(about 10 x the MIC)
Slide34Fluoroquinolones: a high peak may be necessary2/12/20142014 ISF Stephen F. Lowry Colloquium34
Slide35Fluoroquinolones: a high peak may be necessary2/12/20142014 ISF Stephen F. Lowry Colloquium35
Delivering the same fAUC/MIC over short durations of exposure (i.e. 1, 4 or 10h) achieved more rapid killing with no or very limited emergence of resistance, whereas longer
durations of exposure over 16 and 24h led to a dramatic (5 log10) increase in the concentration of resistant bacteria.Clinical
ciprofloxacin regimens with high intensity, short-exposure durations may provide extensive and rapid bacterial killing with no or limited resistance.
Slide36Fluoroquinolones: dissociated resistance and low MICs2/12/20142014 ISF Stephen F. Lowry Colloquium36
Van Bambeke et al. Clinical Microbiology and Infection (2005) 11:256-280
Van Bambeke F Annals of Medicine (2014) 46(7):512-29.Lemaire et al. Antimicrobial Agents and Chemotherapy (2011) 55:649-58.
Slide37Fluoroquinolones: preexisting efflux(in S. pneumonia [Belgian CAP isolates])2/12/20142014 ISF Stephen F. Lowry Colloquium
37
control
+ reserpineLismond et al. J
Antimicrob Chemother (2011
) 66:948-951
Not recommended
Never used in Europe
Slide38Efflux and selection of resistance 2/12/20142014 ISF Stephen F. Lowry Colloquium38
Change in MICs of Levofloxacin
inPseudomonas aeruginosa if deleting the efflux pump operons
Pump status LVX MIC Frequency of LVX- resistant mutants WT 0.25 2 × 107 - 4 × 107 mexAB-oprM
0.015
mexCD-oprJ 0.25
mexEF-oprN 0.25
mexAB-oprM
; mexEF-oprN 0.015 mexCD-oprJ; mexEF-oprN 0.25 mexAB-oprM; mexCD-oprJ 0.015 mexAB-oprM;
mexCD-oprJ; 0.015
mexEF-oprN
Lomovskaya
et al,
AAC (
1999:
43:1340-1346
The MIC falls to low values ...
Slide39Efflux and selection of resistance 2/12/20142014 ISF Stephen F. Lowry Colloquium39
Frequency of Levofloxacin-resistant mutants
inPseudomonas aeruginosa if deleting the efflux pump operons
Pump status LVX MIC Frequency of LVX- resistant mutants WT 0.25 2 × 107 - 4 × 107 mexAB-oprM
0.015 2 × 107 - 4 × 10
7
mexCD-oprJ 0.25 2 × 107
- 4 × 10
7
mexEF-oprN 0.25 2 × 107 - 4 × 107 mexAB-oprM; mexEF-oprN 0.015 2 × 107 - 107 mexCD-oprJ; mexEF-oprN 0.25 2 × 106
mexAB-oprM;
mexCD-oprJ
0.015 1 × 10
9
mexAB-oprM
;
mexCD-oprJ
; 0.015
<1 × 10
11
mexEF-oprN
AND the selection of mutants in FQ target becomes
undetectable when ALL pumps are disrupted
Lomovskaya
et al,
AAC (1999) 43:1340-1346
Slide40Thank you for your attention!2/12/20142014 ISF Stephen F. Lowry Colloquium40
And ask questions