1. Development of Resistance mechanisms in Pseudomonas aeruginosa
Luis Martínez Martínez
Service of Microbiology
University Hospital Marqués de Valdecilla
Santander, Spain
5th ESCMID School, Santander June 2006

2. Targets Antimicrobial agent LPS Outer Memb. Peripl. space Inner Memb.
Porina
Phospholípids
Peripl. space
Peptidoglycan
ß-lactamase
PBP
Inner Memb.
Phospholípids
Targets
Cytoplasm

3. Mechanisms of Resistance to ß-lactams

4. Natural Resistance
CXM
AMP
CFZ
AMC
PIP
CAZ
CTX
FOX
OXA
PTZ

5. Mechanisms of natural resistance to ß-lactams
AmpC production
Low permeability of outer membrane
Expression of efflux pumps
Altered PBPs?

6. AmpC: Basal production
Cell Wall
ampR
ampC
ampD
PG Recycling
AmpC: Low Level
Hydrolysis

7. AmpC: Induction AmpC: High Level ß-lactam PG Recycling
Cell Wall
ampR
ampC
ampD
PG Recycling
AmpC: High Level
[Partial hydrolysis]

8. AmpC: Derepression AmpC: Very high level [ß-lactam] PG Recycling
Cell Wall
ampC
ampD
ampR
PG Recycling
AmpC: Very high level

9. AmpC: Resistance phenotypes
TIC
PIP
CAZ
CPM
CBPM
INDUCIBLE
PRODUCTION S
S/r
PARTIAL DEREP. I
TOTAL DEREP. R
S/R

10. Deficiente en AmpC Inducible AmpC CAZ CAZ IPM IPM FEP FEP MPM MPM PIP

11. AmpC Derepression CMI Interpr. Act. Ticarcilina Piperacilina
CAZ
Ticarcilina
Piperacilina
Piper/tazob
Ceftazidima
Cefepime
Aztreonam
Imipenem
Meropenem
>64 64 16 32 1 2
CMI R I S
Interpr.
Act.
FEP
IPM
PIP
MPM

12. Secondary ß-lactamases
B-J-M Group
PROPERTIES
EXAMPLES 2a
PenSe/CLV (S)
NPS-1 2b
BroadEsp/CLV (S)
TEM 1/2
2be
ExtEsp/CLV (S)
PER-1; TEM-42 2c
CARBse/CLV (S)
PSE-n, CARB-n 2d
OXAse
OXA-n, LCR-1 3
MetEnz/EDTA (S)
IMP,VIM, SPM,...

13. Secondary ß-lactamases
ANTIM.
WILD TYPE
PSE1
TEM1
OXA3
OXA11
PER1
IMP1
TIC 16
1024
2048
512
256
TIC-CLV 32 64
128
PIP 4 8
CAZ 1
PIM 2 ND
AZT
[4]
IMP
16(var)

14. ß-lactamasa OXA-31 CMI Interpr. Real Ticarcillin Piperacilin
Piper/tazob
Ceftazidime
Cefepime
Aztreonam 64
16
 1 16
CMI S
I/R
Interpr. I
Real
FEP
ATM
CAZ

15. Metallo-ß-lactamases
IMP
VIM
SPM
GIM
SIM

16. VIM METALLO--LACTAMASES Kcat/Km (M.s) VALUES
IMP
1.3x 105
3.8x 106
MPM
2.6x 105
2.5x 106
Docquier et al. J-D, JAC 2003

17. VIM METALLO--LACTAMASES MIC VALUES
IMP
MPM
P. aeruginosa (VIM-1)
>128
128->128
E. coli (VIM-1) 8 2
E. cloacae (VIM-2) 4
Miriagou V et al, AAC 2003; Jeong SH et al, JAC 2003, Cornaglia G et al, CID 2000

18. MBL Production CMI Real Intepr. Ticarcillin >64 R R Piperacillin
CAZ
Ticarcillin
Piperacillin
Piper/tazob
Ceftazidime
Cefepime
Aztreonam
Imipenem
Meropenem
>64 64 32 4
>16
CMI
R R
S S
Real Intepr.
FEP
PIP
ATM
IPM
MPM

19. MBL Detection ¿Using Etest?
IP: imipenem
IPI: imipenem + EDTA

20. OprF P. aeruginosa

21. OmpA E. coli

22. OmpF E. coli

24. OprD EXPRESSION AND CARBAPENEM RESISTANCE IN P. aeruginosa
Dib et al. EJCMID 1995

25. AmpC/OprD IN P. aeruginosa

26. OprD Loss CMI Real Interpr. Inducible AmpC Ticarcillin <16 S
FEP
CAZ
PIP
IPM
MPM
ATM
Ticarcillin
Piperacillin
Piper/tazob
Ceftazidime
Cefepime
Aztreonam
Imipenem
Meropenem
<16 1 2 4 16
CMI S R
Real I
Interpr.
Inducible AmpC

27. OprD loss in clinical isolates of P. aeruginosa producing VIM-1
Cornaglia et al, CID 2000

28. Loss of OprD porin in P. aeruginosa
RNA expression of oprD
IS within oprD
PCR amplification of oprD
Walters et al, FEMS ML 2004

29. ZINC DECREASES OprD LEVELS IN P. aeruginosa: RESISTANCE TO IMIPENEM
66.2
45.0
31.0
OprD
OprD3 MH
SLUC
Zn2+ MH
SLUC
Zn2+
P. aeruginosa PAO1
P. aeruginosa PAO1 D-
Conejo MC et al, AAC 2003

30. EFFLUX SYSTEMS MAJOR FAMILIES
1. MFS: Major Facilitator Superfamily
2. RND: Resistance Nodulation-Division
3. SMR: Small Multidrug Resistance
4. ABC: ATP-Binding Cassette
5. MATE: Multidrug And Toxic Extrusion

32. mexA-mexB-OprM mexE-mexF-OprN mexR mexA mexB oprM nalB mexT mexE mexF
Nakae, Microbiología SEM, 1997 (Mod.)

33. EFFLUX PROTEINS Located in Cytoplasmic Membrane
Multiprotein Systems (PUMP+MFP+OMF)
Multiple variants in the same host
Broad/Narrow substrate profile

34. OprM P. aeruginosa

35. MULTIDRUG EEFLUX PUMPS IN P. aeruginosa

36. BASAL EXPRESSION OF EFFLUX SYSTEMS
Constitutively Expressed: MexAB-OprM, AcrAB-TolC,…
Inducible systems: MexXY-OprM,…
Silent in wild type strains: MexCD-OprJ, MexEF-OprN,…
INTRINSIC RESISTANCE CONTRIBUTED BY CONSTITUTIVE AND INDUCIBLE SYSTEMS

37. EXPRESSION OF ACTIVE EFFLUX
BASAL EXPRESSION
OVEREXPRESSION
Active efflux is present in both (clinically) susceptible and resistant organisms

38. CONSTITUTIVE EXPRESSION OF EFFLUX PUMP(S) IN P. aeruginosa
Antimicrobial Agent mexAB-OprM DmexAB-oprM
Nalidixic Acid
Ciprofloxacin
Tetracycline
Chloramphenicol
Amoxicillin
Piperacillin
Carbenicillin
Ceftazidime
Cefepime
Meropenem 19

39. EFFLUX PUMPS MexAB/OprM S R MexCD/OprJ SS MexEF/OprN* S/SS S/R nfxB-B
SISTEMA
MUTACION
IMP
MPM
PEN/CEF/IBL
CF4G
TET/CLO/TMP
ERY
FLQ
MexAB/OprM
nalB S R
MexCD/OprJ
nfxB-A SS
nfxB-B
MexEF/OprN*
nfxC
S/SS
S/R
*OprD(-)

40. OprM/OprD IN P. aeruginosa

41. mexE-mexF-OprN (-) (+) (-) (+) czcCBA mexT mexE mexF oprN oprD czcC
czcB
czcA
oprD
czcS
czcR
(-)
(+)
czcCBA
Perron et al, JBC (Mod.)

42. ZINC AND RESISTANCE TO IMIPENEM. ROLE OF THE CzcCBA SYSTEM
Sublethal [Zn] induce resistance to Zn and to IMP
Imipenem may also select for Zn-R
Lethal [Zn] select stable mutants resistant to Zn and to IMP, because of mutation (V194L) in CzcS
Expression of mutated CzcS causes increased expression of czcC and decreased expression of oprD.

43. Expression of OprN and OprD
OprM
OprN
OprD
PT75
PAO1
Köhler et al. Mol. Microbiol. 1997

44. MICs of Imipenem against P. aeruginosa hyperproducing MexCD-OprJ
Wolter et al, AAC 2005

45. Mechanisms of Resistance to other antimicrobials

46. Natural Resistance
DOX
TET
CHL
RIF
SXT

47. Resistance to Aminoglycosides
Altered permeability
Modifying enzymes
Efflux pumps (MexXY-OprM)

48. Resistance to Aminoglycosides
Interpretative reading: Difficult to perform
>10 compounds to be tested)
>80 different Mechanisms (but...5 of them represent 75% of cases)

49. Aminoglycosides S R Nt AAC(3)-I APH(3’)-VI AAC(6’)-I
AAC(6’)-II, AAC(3)-II,IV,V
ANT(2”)-I
Enzyme AK
TOB GM

50. Resistance to AminoglycosidesAK
TOB GM
TOB GM AK
ANT(2”)-I
AAC(6’)-II
Combined mechanisms

51. Resistance to Fluoroquinolones: Wild-type phenotype
CIP
NAL
LEV
OFL
NOR

52. Resistance to Fluoroquinolones
Efflux pumps
Target modification (Gyrase, Topo IV)
Altered permeability: LPS

53. Resistance to Fluoroquinolones
CIP
OFX
Ciprofloxacin
Ofloxacin 1 4
CMI I
Interpr. S
Real
Mex AB-OprM

54. EXPRESSION OF EFFLUX PUMPS IN P. aeruginosa
Lee A., J Bacteriol 2000

55. EFFECT OF PAN ON MIC OF LEVOFLOXACIN AGAINST P. aeruginosa
Strain
PAN
MIC of Levofloxacin+[PAN]
0 mg/L
10 mg/L
20 mg/L WT
>512
0,125
0,015
0,008
nalB 2
0,03
nfxB
nfxC 4
0,25
0,06
Mex(*) 32
Lomovskaya O et al, AAC 2001