Detection of -Lactamase-Mediated Resistance
David M Livermore,Specialist & Reference
Microbiology Division
Action of a -lactamase
NO
COOH
S
HN
OCOOH
S
OH
Active penicillin
Inactive penicilloateH2O
-Lactamase families
Staph penicillinase; TEM & SHV; chromosomal -lactamases of Proteus, Klebsiella & Bacteroides
Zinc types
Chromosomal AmpC -lactamases of most enterobacteria
OXA-class plasmid -lactamases
AB
C
D
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 -lactamases
• 1963 Ampicillin; 1st broad spectrum penicillin
• 1965 TEM -lactamases in E. coli
• 1969 TEM -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
-Lactamase stable cephs
CONHC
NOR
O
COOH
H2N
Oxyimino-aminothiazolylstability to classical TEM/SHV
OCH3
R
-methoxy, stability to TEM, SHV, ESBLs & Bacteroides enzymes
S NS
AmpC -lactamases
Basal in:• E. coli & shigellaeInducible in:• Enterobacter spp.• C. freundii• M. morganii• Serratia spp.• P. aeruginosa2nd, 3rd gen cephs:• Labile, but weak
inducers, select derepressed mutants
[ -lactam]
Am
t -
lact
am
ase
Derepressed
Inducible
AmpC -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 -lactamases
TEM-11965
TEM-21970
TEM-31987
Activity vs.3rd gen cephs
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
Pip + 4 mg/L taz 0.5 1 2 1
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 -lactamase of K. ascorbata
– PER- PER-1 in Turkey; PER-2 in Argentina
– ESBL OXA-2 & -10 mutants- mostly P. aeruginosa Turkey
CTX-M -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-M2Vmax
(%)Km
(M)MIC
R-
(mg/L)
MICR+
(mg/L)Cephaloridine 100 - - -
Piperacillin - - 1 1024
Cefotaxime 12.5 70 0.06 16
Ceftazidime 0.02 203 0.13 2
Imipenem 0 - 0.03 0.03
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--lactamases
• IMP, 16 types; VIM, 12 types; SPM-1
– 15% variation in families; 70% between them
• Hydrolyse -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--lactamases
MEM IMP AZT CTX CTZ CFM
R947 IMP-8TEM-1
1 2 0.03 >256 >256 32
Y580 IMP-8TEM-1
0.5 2 0.02 16 128 32
T524 IMP-8TEM-1
1 4 0.03 32 >256 32
N947C. freundii
VIM-2 0.5 1 0.06 32 64 16
Yan et al., JAC 2002, 50, 503
-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 -lactamase types. Have GNB got ?:
ESBL,
AmpC
Metallo types, VIM, IMP etc…
• Spotting unusual patterns; knowing what to refer
Some useful knowledge
AmpC
Hi-level
TEM ESBL
CTX-M K1
Ceftazidime 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 g) % Detected (n =100)
Ceftazidime +/- clav 88
Cefotaxime +/- clav 66
Both 93
M’Zali et al. 2000, JAC, 45, 881
0
10
20
30
40
50
60
-3 -1 1 3 5 7 9 11 13 15 17 19 21 23 25
Control AmpC K1 ESBL CTX-M
Zone differences (mm), Klebsiella & E. coli
c’pod/clav 10+1 g minus c’pod 10 g
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 -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--lactamase
Imipenem
Imipenem+
EDTA
Etest for metallo--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 -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, 20032 Baxter & Lambert JAC 1997, 39, 838
Activity of pip/tazo vs. ESBL +ve klebsiellae; 1994 & 1997/8
0
10
20
30
40
50
0.5 1 2 4 8 16 32 64 128 256 512 1024
MIC (mg/L)
% o
f E
SBL
pro
duce
rs
1994 1997/8
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