Discusses the different types of carbapenamases in gram negative pathogens and overall classification of beta lactamases.

1. ACQUIRED
CARBAPENEMASES IN
GRAM-NEGATIVE
PATHOGENS

2. Carbapenems
Penams
Cephems
Monobactams
Oxacephems Carbacephems
N
COO
-
O
R2
OH
R1
N
O
SNH
COO
-
R
O
N
O
NH
R1
O
S
COO
-
R2
N
O SO3
-
NH
O
R
R
N
O
NH
R1
O
COO
-
R2
N
O
NH
R1
O
O
COO
-
R2
N
COO
-
O
R
OH
S
Penems
An exceptionally broad antimicrobial spectrum due to:
• High stability to b-lactamases
• High affinity to PBPs
• Good penetration across the OM of Gram-negatives
• Poor substrates for efflux systems

3. Carbapenems: an exceptionally broad spectrum
Staphylococci (exc. MRS)
Pneumococci (incl. PRP)
Other streptococci
Listeria
E. faecalis (Imipenem)
Bacteroides
other gram-negative anaerobes
most gram-positive anaerobes
Neisseria
Moraxella
(incl. b-lactamase+)
Haemophilus
(incl. b-lactamase+)
Enterobacteriaceae
(incl. AmpC+ & ESBL+)
Pseudomonas (exc. Ertapenem)
(incl. AmpC+ & ESBL+)
Acinetobacter (exc. Ertapenem)
Recommended among the drugs of choice for empiric
treatment of several types of serious infections
(e. g. FN, HAP/VAP, IAIs etc.)

4. Classification of β-lactamases
ESBL
Pen-Cephs-Inh-S
β-lactamases
(Plasmid) (Chromosomal) (Plasmid) (Chromosomal)
AmpC
Cephs-Inh-R
MbL (IMP/VIM)
Carbapenems
Inh-R
OXA
Pens, esp Oxa
Inhib-R/S
Serine enzymes Metallo-enzymes
Class A
enzymes
Class C
enzymes
Class D
enzymes
Class B
enzymes
Bush. Rev Inf Dis 1987;10:681; Bush et al. Antimicrob Agents Chemother 1995;39:1211–1233
Bush. Curr Opin Investig Drugs 2002;3:1284–1290

5. • Impermeability/efflux
• Carbapenemases
• Carbapenemases
• Impermeability/efflux
• Modified target?
• Carbapenemases
• Impermeability +
ESBL/AmpC
Pseudomonas aeruginosa ++
Acinetobacter
Enterobacteriaceae
++
+/- (++*)
* in some geograhical areas
Acquired resistance to
carbapenems, an issue for:

6. Carbapenemases β-
lactamases:

7. Acquired carbapenemases in Gram-
negative pathogens
Enzyme family Class Pathogens
Serine-β-lact. A Enterobacteriaceae
P. aeruginosa
Serine-β-lact. D Acinetobacter
Enterobacteriaceae
Metallo-β-lact. B
Pseudomonas
Acinetobacter
Enterobacteriaceae

8. Klebsiella Pneumoniae
Carbapenemase
• KPC is a class A b-lactamase
– Confers resistance to all b-lactams including
extended-spectrum cephalosporins and
carbapenems
• Occurs in Enterobacteriaceae
– Most commonly in Klebsiella pneumoniae
– Also reported in: K. oxytoca, Citrobacter freundii,
Enterobacter spp., Escherichia coli, Salmonella
spp., Serratia spp.,
• Also reported in Pseudomonas aeruginosa
(Columbia)

9. KPC-type
SME-type
NMC/IMI-type
Class A serine-carbapenemases in
Enterobacteriaceae

10. Frequent Occurrence
Sporadic Isolate(s)
Geographical Distribution of KPC-
Producers

11. Yigit et al. AAC 2003
MIC (μg/ml)
KPC-2
KPC carbapenemases: a very broad
spectrum

12. 0
10
20
30
40
50
1999 2001 2006
%carbapenem-resistant
Years
22% of isolates resistant to:
- Aminoglycosides
- Fluoroquonolones
- 3rd 4th gener. Cephems
- Carbapenems
JAC 2007
Klebsiella pneumoniae
XDR phenotype
Susceptibility only to:
- Colistin
- Tigecycline
Due to spread of KPC
carbapenemases
Also in E. coli, C. freundii, and
E. cloacae from the same area
Jones et al – DMID 2008

13. 0
10
20
30
40
50
2004 2005 2006
No.ofcases
Years
No KPC
KPC-2
KPC-3
Proportion of carbapenem-resistant isolates:
0.4% 0.1% 3.1%
75% clonal transmission (KPC-3)
25% multiple clones
The major clone susceptible only
to Ciprofloxacin, Amikacin,
Colistin and Tigecycline
Leavitt et al. AAC 2007
Emergence of carbapenem-resistant K. pneumoniae
producing KPC serine carbapenemases in a Israeli hospital

14. Giakkoupi et al. 10th β-lact. meeting 2008
Emergence of carbapenem-resistant K. pneumoniae
producing KPC serine carbapenemases in Greece
 26 KPC-2-producing K. pneumoniae
in 6 different hospitals from 3 cities
(incuding one in Crete island)
 Mostly clonal spread
 Often but not always resistant to
carbapenems
Cuzon et al. AAC 2008
Crete island

15. Class A serine-carbapenemases in
Enterobacteriaceae
KPC-type
SME-type
NMC/IMI-type
KPC-2 in
P. aeruginosa

16. Three isolates from Medellin (Colombia) producing KPC-2
High-level carbapenem resistance:
MICs of IMI and MER ≥256 mg/L
Susceptible only to amikacin and colistin

17. In vivo transfer of KPC carbapenemase genes
NYH Medical Center – Queens
Patient admitted in late February for gastric perforation
March April May
LRT
IMI-S P. aeruginosa
IMI-R (KPC-2+) P. aeruginosa (S to COL GEN AMK only)
IMI-R (KPC-2+) K. pneumoniae
Urine
Decubitus
Urban et al – unpublished

18. Weak class A serine-carbapenemases
Agent
MIC (mg/L) for P. aeruginosa PU21
WT PU21 (GES-2)
Imipenem 2 16
Meropenem 0.5 2
kcat for IMI = 0.004 sec-1
KM for IMI = 0.45 μM

19. Acquired carbapenemases in Gram-
negative pathogens
Enzyme family Class Pathogens
Serine-β-lact. A Enterobacteriaceae
P. aeruginosa
Serine-β-lact. D Acinetobacter
Enterobacteriaceae
Metallo-β-lact. B
Pseudomonas
Acinetobacter
Enterobacteriaceae

20. Class D Oxacillinase —
Carbapenemases
• Class D enzymes
• OXA-23, -24, -25, -26, -27, -28, -40, -49, -58, ….
• Highly mobile (integron, plasmid)
• Multi-drug resistance (penicillins and 3rd & 4th
generation cephalosporins, BL/BL-inhibitors,
aminoglycosides, SXT,…)
• Variable resistance levels to imipenem and
meropenem (4–>256 mg/mL)

21. OXA-58-type
OXA-23-type
OXA-24-type
OXA-48
A. baumannii
P. mirabilis
A. baumannii
A. baumannii
K. pneumoniae
Acquired class D serine-
carbapenemases

22. Contribution of class D serine-carbapenemases to β-
lactam resistance in A. baumannii
Strain
MIC (mg/ml)
TIC IPM MEM
A. baumannii MAD (OXA-58+) >256 32 >64
A. baumannii FER (OXA-23+) >256 >32 >32
A. baumannii CLA-1 (OXA-24+) >256 >32 >32
A. baumannii CIP 7010T
4 0.25 0.25
A. baumannii CIP 7010T (OXA-58+) >256 0.5 0.5
A. baumannii CIP 7010T (OXA-23+) >256 4 4
A. baumannii CIP 7010T (OXA-24+) >256 4 4
Heritier et al. – AAC 2005

23. Gales et al. CMI 2006
0%
20%
40%
60%
80%
100%
%Susceptibility
Italy, 2002 – 2003; J Chemother 2006
Acinetobacter: wordlwide surveillance data (2001-
2004)
(n = 2621 isolates)

24. Acquired carbapenemases in Gram-
negative pathogens
Enzyme family Class Pathogens
Serine-β-lact. A Enterobacteriaceae
P. aeruginosa
Serine-β-lact. D Acinetobacter
Enterobacteriaceae
Metallo-β-lact. B
Pseudomonas
Acinetobacter
Enterobacteriaceae

25. Penicillins
Cephalosporins (all)
Carbapenems
Monobactams
Suscept. to inhibitors
(clavulanate & sulphones) -
activity
good
no
Acquired metallo-b-lactamases
(MBLs): functional features

26. Class B (Metallo)-Carbapenemases
• Hydrolyzing virtually all b-lactams
• Mediate broad spectrum b-lactam resistance
• No clinical inhibitor available
• Present on large plasmids and integrons
• Genes are continuously spreading
• Associated (80%) with aminoglycoside
resistance
Still rare but increasing, especially in non-fermenters

27. In-vitro activity (%) of various antimicrobials
against MBL+ve and MBL–ve P. aeruginosa
strains
MBL –ve (n=1006)
RESORT study 2002–2004
46.2
56.8
28.6
41.2
54.6
43.2
61.3
63.8
36.6
35.3
26.4
60.7
Piperacillin
Piper.-Tazo.
Cefoperazone
Cefoper.-Sulb.
Ceftazidime
Cefepime
Meropenem
Imipenem
Ciprofloxacin
Levofloxacin
Gentamicin
Amikacin
Polymyxin B 93.8
MBL +ve (n=47)
100.0
8.5
Dekhnich, et al. ICAAC 2006

28. VIM
SPM
IMP
GIM
SIM
MBLs and acquired MBLs in gram-negative pathogens
FEZ
GOB
L1
CphA
Sfh
CVI
B3
B2
THIN-B
NOV
CAU
BJP
Bc-II
IND/CGB
JOHN
BlaB
EBR
CcrA
TUS
MUS
SLB/SFB
B1
B4
AIM
CAR
POC
KHM
ILM

29. Acinetobacter
P. aeruginosa Acinetobacter oth. GNNFs Enterics Aeromonas
P. aeruginosa Acinetobacter oth. GNNFs Enterics Aeromonas
P. aeruginosa
P. aeruginosa
SPM-1
GIM-1
VIM (>20 variants)
SIM-1
IMP (>20 variants)
AIM-1 P. aeruginosa
KHM-1 Enterics
Distribution of acquired MBLs

30. Acquired MBLs in Gram-negative nonfermenters
P. aeruginosa and Acinetobacter are
the most common hosts, but
occasional detections also in other
species (P. putida, Achromobacter)
Wordlwide distribution (P. aeruginosa)
Overall low prevalence, but notable
regional differences and possibility of
even large outbreaks

31. GM
COL
AK
TOB
FEP IPM
MEMCAZTZP
ATM
CIP
PRL
Lauretti et al. – AAC 1999
Cornaglia et al. - CID, 2000
MBL (VIM-1)-producing P. aeruginosa index strain Verona,
Italy, 1997
(now a major Italian clone – ser. O11; ST227; BG11)
Very large outbreak ongoing since 2000;
Multiward, even LTCFs and outpatients;
Significant increase of carbapenem resistance rates
Lagatolla et al. - EID 2004
Riccio et al. – AAC 2005
Giske et al. – JCM 2006

32. P. aeruginosa Producing Acquired MBLs:
MDR / XDR Resistant Phenotypes
Strain Enz. CAZ FEP P/T IMI MEM ATM CIP AK GM TOB COL
VR-143/97 VIM-1 R R R R R R R I R R S
PPV-97 VIM-1 R R R R R S R R R R S
TS-832347 VIM-2 R R R R R R R S R R S
MB397 VIM-4 R R R R R I R R R R I
MNA1455 IMP-1 R ND ND R R R R R R R ND
CGH IMP-7 R R S R R I R I R R ND
AV-78 IMP-13 R R R R R R R S R R S
PA-Cl.1 SPM-1 R R R R R S R R ND ND S
73-15553 GIM-1 R R R R R I R S R R I
Castanheira et al. AAC 2004; 48:4654
Zavascki et al. JAC 2005; 56:1148
Riccio et al. AAC 2005; 49:104
Libisch et al. AAC 2006; 50:4220
Senda et al., AAC 1996; 40: 349
Gibb et al., AAC 2002; 46: 255
Giuliani et al., AAC 2005; 49: 1973
Pagani et al., JCM 2005; 43: 3824

33. 0.50
1
2
4
8
16
32
64
128
S
I
R
MIC(mg/ml)
IMI MER IMI MER
Carbapenem MICs of MBL
producers

34. IPM
IPM + EDTA (750 mg)
Jong et al - JCM 2002
Combo disk test
Double-disk tests
Arakawa et al - JCM 2000
Lee et al – JCM 2003
Etest MBL
IPM
IPM + EDTA
Confirmation by
molecular testing
Phenotypic tests for MBL detection

35. Acquired metallo-β-lactamases in Enterobacteriaceae
 Sporadic reports since the mid 1990s (Far East, Europe,
Austraila)
 Recent worrysome evolution in the mediterranean area
K. pneumoniae:
carbapenem resistance
surveillance in Europe
(EARSS 2006 report)
VIM-type
metallo-β-lactamases
Vatopoulos et al. - Eurosurveillance 200833%

36. Klebsiella pneumoniae isolated from BSIs (2004-06):
- 38% MBL-positive (VIM-1)
- 25% MBL- positive (VIM-1) and ESBL-positive (SHV-5)
- Multiple clones
ESBL+/MBL+ strains usually exhibit XDR phenotype
(susceptible only to colistin and tigecycline)
JAC 2008

37. Carboxy-pen. R
Ureido-pen. R
BLICs R
Cefepime R
Ceftazidime R
Cefotaxime R
Imipenem S
Meropenem S
Cagnacci et al. – JAC 2008
MICs, 1-4 mg/L*
* >8 mg/L in a single isolate which had also lost k36 porin
Resistance profile of ESBL+/MBL+
K. pneumoniae

38. ESCMID Expert Meeting
on Metallo-β-Lactamases
November 14-15, 2005,
Siena, Certosa di Pontignano
MBL-producing Enterobacteriaceae
may appear still susceptible to
carbapenems according to current
breakpoints, although showing
carbapenem MICs higher than
modal values for the species

39. CLSI
2008
Diagnostic issue
Therapeutic issue

40. IPM
IPM + EDTA
IPM
EDTA
Combo-disk Double-disk
Etest
IPM
IPM + EDTA
Phantom zone
Phenotypic tests for detection of
MBLs in Enterobacteriaceae

41. Carbapenems for MBL producers with low
MICs?
• No clinical evidence, except for a single report
and a study in an animal model
• Strong inoculum-size effect
• Infections by carbapenem-susceptible MBL
producers observed under empirically started
carbapenem therapy
• Carbapenems should be avoided or considered
with caution for confirmed MBL producers that
are appaently susceptible to carbapenems

42. 14 cases of BSIs (4 CVC-related) and 3 cases of VAP,
caused by VIM-1 MBL-producing Enterobacteriaceae (15
Klebsiella, 2 Enterobacter)
5 cases occurred under carbapenem-based regimens (in
one case the strain was carbapenem susceptible)
Carbapenem MICs: 1 - >32 mg/L (7/17 susceptible)
Success with imipenem in only one case (MIC, 8 mg/L)

43. CLSI 2008

45. b-lactamases: Summary
Broad Spectrum Expanded Spectrum AmpC Carbapenemase
TEM/ SHV OXA TEM/SHV CTX-M OXA KPC MBL OXA
Class A D A A D C A B D
Inhibition by
Clavulanic Acid
Penicillins
Oxacilin
Narrow Spectrum
Cephalosporins
Cephamycins
Oxyimino-
cephalosporins
Cefepime
Monobactam
Carbapenems
Polymyxin E