Detection of b-LactamaseMediated Resistance
David M Livermore,
Specialist & Reference
Microbiology Division
Action of a b-lactamase
S
H2O
N
O
Inactive penicilloate
COOH
S
Active penicillin
HN
O
OH
COOH
b-Lactamase families
A
Staph penicillinase; TEM & SHV; chromosomal
b-lactamases of Proteus, Klebsiella & Bacteroides
B
Zinc types
C
Chromosomal AmpC b-lactamases of most
enterobacteria
D
OXA-class plasmid b-lactamases
Established problems
• Staphylococcal penicillinase
• G-ve bacteria with penicillinases, TEM etc.
• G-ve bacteria with AmpC enzymes
• G-ve bacteria with TEM & SHV ESBLs
Other types emerging...
Emerging problems
• Plasmid AmpC enzymes
• ESBLs not derived from TEM & SHV
• Carbapenemases
• Resistance to inhibitor combinations
Spread of TEM plasmid
b-lactamases
• 1963 Ampicillin; 1st broad spectrum penicillin
• 1965 TEM b-lactamases in E. coli
• 1969 TEM b -lactamase in P. aeruginosa
• 1974 TEM in H. influenzae & N. gonorrhoeae
• Now TEM in 30-60% E. coli & enterobacteria
& in 5-20% of H. influenzae & gonococci
b-Lactamase stable cephs
a-methoxy, stability to TEM, SHV,
ESBLs & Bacteroides enzymes
H2 N
S
OCH3
N
C
S
CONH
N
OR
O
Oxyimino-aminothiazolyl
stability to classical TEM/SHV
R
COOH
AmpC b-lactamases
Derepressed
Amt b-lactamase
Basal in:
• E. coli & shigellae
Inducible in:
• Enterobacter spp.
• C. freundii
• M. morganii
• Serratia spp.
• P. aeruginosa
2nd, 3rd gen cephs:
• Labile, but weak
inducers, select
derepressed mutants
Inducible
[b -lactam]
AmpC b-lactamases
• Cephalosporins select derepressed mutants from
inducible populations
• Selection c. 20% in Enterobacter bacteraemia
• 30-40% of all Enterobacter and C. freundii now
derepressed at first isolation
• Resistant to inhibitors; escaping to plasmids
Extended-spectrum b-lactamases
Activity vs.
3rd gen cephs
TEM-1
1965
TEM-2
1970
TEM-3
1987
and now up to TEM-126…, also SHV-48
MICs (mg/L) for ESBL +ve E. coli
R-
TEM-1+ TEM-3+ TEM-10+
Ampicillin
2
1024
256
1024
Piperacillin
1
128
64
64
1
2
1
Pip + 4 mg/L taz 0.5
Ceftriaxone
0.03
0.03
64
2
Ceftazidime
0.12
0.12
32
128
Cefoxitin
4
4
8
4
Imipenem
0.12
0.12
0.12
0.12
Meropenem
0.03
0.03
0.03
0.03
Exotic ESBLs, not derived
from TEM & SHV
• CTX-M- 29 variants, some derived from
chromosomal b-lactamase of K. ascorbata
– PER- PER-1 in Turkey; PER-2 in Argentina
– ESBL OXA-2 & -10 mutants- mostly P.
aeruginosa Turkey
CTX-M b-lactamases
• Escaped from the chromosomes of Kluyvera spp.
• More active vs. cefotaxime than ceftazidime
– But mutation can confer ceftazidimase activity
• Predominant ESBLs in Argentina since 1990
• Disseminating rapidly in Asia & Europe
– 2003 ICAAC full of poster on ‘First CTX-M from ....’
Activity of CTX-M2
Vmax Km
(%) (mM)
Cephaloridine
MIC MIC
+
R
R
(mg/L) (mg/L)
-
100
-
Piperacillin
-
-
1
1024
Cefotaxime
12.5
70
0.06
16
Ceftazidime
0.02
203
0.13
2
0
-
0.03
0.03
Imipenem
Bauernfeind et al., 1992 Infection 20, 158
CTX-M in the UK
• 2000- First producers
– K. oxytoca, Leeds,CTX-M-9
• 2001/2- First hospital outbreak
– B’ham, 33 patients, K. pneumoniae, CTX-M-25
• 2003- Community E. coli from UTIs
– Diverse strains & locales, 2 CTX-M variants
Brenwald JAC 2003, 51, 195; Alobwede JAC 2003, 51, 470: HPA data on file
Acquired carbapenemases rare
• BUT….
• IMP & VIM metallo-enzymes increasing,
• OXA carbapenemases in Acinetobacter spp.;
• Few class A types…. KPC, IMI, SME• Few major outbreaks of producers
VIM & IMP metallo-blactamases
• IMP, 16 types; VIM, 12 types; SPM-1
– 15% variation in families; 70% between them
• Hydrolyse b-lactams except monobactams; inhibited by
EDTA, not clav or sulphones
• Mostly Far East & S. Europe- few UK isolates
– Mostly P. aeruginosa; 2 Acinetobacter; 2 Klebsiella
• Not all gene +ve isolates are obviously R
MICs (mg/L) for E. cloacae
with metallo-b-lactamases
MEM IMP AZT CTX CTZ CFM
R947
Y580
T524
N947
C. freundii
IMP-8
TEM-1
IMP-8
TEM-1
IMP-8
TEM-1
VIM-2
1
2
0.03 >256 >256
32
0.5
2
0.02
16
128
32
1
4
0.03
32
>256
32
0.5
1
0.06
32
64
16
Yan et al., JAC 2002, 50, 503
b-Lactamase detection
• Nitrocefin- very sensitive, expensive, good for
fastidious GNB & Moraxella, not staph
• Acidimetric -sensitive, care with controls to avoid
false +ve
• Iodometric -sensitive, fiddly, care with controls to
avoid false +ve
• Microbiological -v. sensitive, slow
Challenges for the diagnostic lab
• Detection…. Haemophilus, Neisseria etc.
• Predicting b-lactamase types. Have GNB got ?:
 ESBL,
AmpC
Metallo types, VIM, IMP etc…
• Spotting unusual patterns; knowing what to refer
Some useful knowledge
TEM
ESBL
CTX-M
K1
Ceftazidime
AmpC
Hi-level
R
R
v
S
Cefotaxime
R
v
R
S
Cefoxitin
R
S
S
S
Aztreonam
R
v
v
R
Synergy + clav
No
+++
+++
No
Know the species
ESBLs: times a’ changing with CTX-M
• Old advice- test ceftazidime; ESBL test if R
• New advice- test ceftazidime & cefotaxime;
ESBL test if R to either
• Alternative- test cefpodoxime; ESBL test if R
• Still true- Only testing cefuroxime is inadequate
ESBL detection
• Double disc synergy
• Combination discs
• E-test
– Test potentiation of ceftazidime, cefotaxime or
cefpodoxime by clavulanate……
– Use whichever suggested ESBL production
Detection of ESBLs with
combination discs (MAST)
+ve result, zone enlarged 50%
Discs (30+10 mg)
% Detected (n =100)
Ceftazidime +/- clav
88
Cefotaxime +/- clav
66
Both
93
M’Zali et al. 2000, JAC, 45, 881
Zone differences (mm), Klebsiella & E. coli
c’pod/clav 10+1 mg minus c’pod 10 mg
60
Control
AmpC
K1
ESBL
CTX-M
50
40
30
20
10
0
-3
-1
1
3
5
7
9
11
13
15
17
19
21
23
25
Etest for ESBLs
Cefotaxime
Cefotaxime
+
clavulanate
Etest for ESBLs
Cefotaxime
Cefotaxime
+
clavulanate
Difficulties in ESBL detection
• Ceph R Enterobacter etc. most likely AmpC
derepressed ….. But may have ESBL
• Clav induces AmpC; hides ESBL inhibition
– Try to read anyway
– Suspect ESBL if pip/taz or cefotetan S
– Synergy test between cefepime & clav
Bacteria not to test for ESBLs
• Acinetobacters
– Acinetobacters often S to clavulanate alone
• S. maltophilia
– You get +ve results via inhibition of L-2
chromosomal b-lactamase, which is
ubiquitous in the species
AmpC inducibility- when to look
• Rarely!!!!!
• Risk is mutation, not inducibility per se
• Best to identify & predict risk from species
• Biggest risk Enterobacter & C freundii
• Avoid cephalosporins against them
Identify means identify TO SPECIES LEVEL all
Enterobacteriaceae (‘coliforms’) ex serious infections
AmpC & ESBLs- what to refer
to ARMRL
• E. coli & Klebsiella suspected of AmpC
– ? Have plasmid types ? Recent travel
• ESBL +ves from community
– ? CTX-M types
• Enterobacters suspected of having ESBLs
Double disc antagonism for inducible AmpC
Cefoxitin
Ceftazidime
Cheapskate’s insurance vs.
lawyers for AmpC derepression
• Test cefoxitin
• Enterobacter & C. freundii with inducible
AmpC are clearly R
• M. morganii & Serratia aren’t R; but carry
lower derepression risk
• Species without inducible AmpC are S
ARMRL recommendations for
carbapenem R isolates
• Enterobacteriaceae & Acinetobacter
– Send in to ARMRL
– Except Proteeae weakly R to imipenem only
• P. aeruginosa
– Screen with EDTA synergy test
– Send to ARMRL if +ve
S. maltophilia…. Please DON’T send
Etest for metallo-b-lactamase
Imipenem
Imipenem
+
EDTA
Etest for metallo-b-lactamase
Imipenem
Imipenem
+
EDTA
Carbapenem R isolates at
ARMRL
• Screened with imipenem/EDTA Etest
• Spectrophotometry with imipenem
• PCR for carbapenemase genes
• DNA sequencing
Weaknesses of strategy
• False positives with Etest MBL tests
– 9/23 MBL Etest+ve P. aeruginosa hydrolysis -ve &
negative for blaIMP & blaVIM
• Class D b-lactamases v. weak activity
– Difficult to detect hydrolysis
• Sometimes you wouldn’t guess to look!
Why false +ves with Etest MBL?
• EDTA may permeabilise the outer membrane
• Zinc suppresses OprD in P. aeruginosa,
inducing imipenem resistance1 So?? lack of
zinc may induce OprD. Sensitising the bug??
• Zinc inactivates imipenem!2
1Carmen-Conjeho
et al., ECCMID, 2003
2 Baxter & Lambert JAC 1997, 39, 838
Activity of pip/tazo vs. ESBL
+ve klebsiellae; 1994 & 1997/8
50
% of ESBL producers
1994
1997/8
40
30
20
10
0
0.5
1
2
4
8
16
32
64
128
256
512 1024
MIC (mg/L)
The message
• Beta-lactamases are getting more complex
• Full I/D needs complex molecular methods
Much can be inferred from simple tests.
Needs I/D
Testing wide panels of antibiotics; synergy tests
Knowledge of what’s unusual