1. ESBL, AmpC -lactamase 표준진단법
Seong Geun Hong
Dept. of Lab. Medicine, CHA Bundang Medical Center, CHA University

2. Extended-spectrum β-lactamases (ESBL)

3. Extended-spectrum -lactamase (ESBL)
Inhibitor susceptible -lactamases
Hydrolyze penicillins, cephalosporins, and aztreonam
Encoded by mobile genes
Usually mutiple drug resistant aminoglycoside, trimethoprime, sulfonamides, chloramphenicol, fluoroquinolones

4. Need for ESBL detection
Breakpoint do not reliably detect -lactamases.
To improve the accuracy of susceptibility test reports by indicating which -lactam drugs may not be effective clinically. => change the interpretations of extended-spectrum cephalosporins and penicillins from “S” to “R” for ESBL producing strains.

5. New CLSI criteria for cephalosporins and aztreonam
PK-PD and limited clinical data
Routine ESBL testing: no necessary (?)
Result editing: no necessary (?)
No use of the new breakpoints - ESBL testing - Result editing
ESBL test - useful for epidemiological or infection control purposes

6. CLSI breakpoints change
Agent
Old (M100-S19)
Revised (M100-S20)
New (M100-S21)
MIC
Disk
Cefazolin
8, 32
18, 14
1, 4 -
23, 19
Cefotaxime
8, 64
23, 14
26, 22
Ceftizoxime
20, 14
25, 21
Ceftriaxone
21, 13
Ceftazidime
4, 16
21, 17
Aztreonam
22, 15

7. Escherichia coli, Klebsiella
CLSI ESBL Screening
Escherichia coli, Klebsiella
MIC Test
>2 µg/ml for cefotaxime, ceftriaxone, ceftazidime, or aztreonam
>8 µg/ml for cefpodoxime  
Disk Test
<22 mm for ceftazidime
<17 mm for cefpodoxime
<25 mm for ceftriaxone
<27 mm for cefotaxime or aztreonam
   
Proteus mirabilis
> 2 µg/ml for cefpodoxime, cefotaxime or ceftazidime
 Disk Test
< 22 mm for cefpodoxime, ceftazidime
< 27 mm for cefotaxime

8. ESBL Confirmatory Tests
Double Disk Test
CLSI ESBL Confirmatory Test
Commercial Tests

9. Detection of ESBLs: Two Approaches
1. Screening tests and confirmatory tests for positive screens
2. Confirmatory tests
Advantages
Disadvantages
Less work
Cheaper
Sensitivity less than 100%
Delayed confirmation

10. ESBL Confirmatory Tests
Double 디스크시험
CLSI 확인시험
Disk diffusion
Broth microdilution 방법
Broth microdilution - cefotaxime and ceftazidime - +/- 4 μg/ml clavulanate: ≥ 3 doubling dilution decrease with either drug
e.g. ceftazidime μg/ml ceftazidime + CA 1 μg/ml 장점
모든 세균 가능
객관적
판독 용이 단점
주관적
판독 어려움
E. coli, Klebsiella spp., P. mirabilis에 대한 규정만 있음.

11. ESBL Confirmatory Tests
상품화된 검사법
1. Vitek & Vitek 2 ESBL card
- CTX, CAZ, FEP with clav.
2. MicroScan microdilution panel
- CAZ, CPD
3. E-test strips
분자진단검사: PCR, DNA sequencing

12. ESBL Etest Use criteria for MIC ESBL confirmation
ceftazidime
cefotaxime
Use criteria for MIC ESBL confirmation
> 3 doubling dilutions
decrease of MIC in presence of
clavulanate
cefotaxime/clavulanate
ceftazidime/clavulanate
Picture from KS Thomson

13. AmpC β-lactamases

14. Amp C type β-lactamases
Class C, 염색체성
거의 모든 그람음성 간균, 균종 특이성
Chromosomal AmpC
Plasmid-mediated AmpC (PABL)
Enterobacter cloacae
Citrobacter freundii
Serratia marcescens
Morganella morganii
Yersinia enterocolitica
Pseudomonas aeruginosa
Chromosomal ampC가 없는 균종
1. Klebsiella spp.
2. Proteus mirabilis
3. Salmonella spp.
Chromosomal ampR이 없는 균종
1. E. coli

15. AmpC의 표현형 유도성 (inducible) 특정 항균제 (cefoxitin, clavulanate 등)에 노출
구조성 (constitutive)
ampD 소실에 의한 탈억제 (derepressed)
 특정 항균제 노출과 무관한 지속적인
AmpC 생성

16. AmpC β-lactamases
Resistant to - Cephalosporins (1st, 2nd, 3rd gen) - Cephamycins - Monobactams - Penicillins - β-lactamase inhibitor combinations
Susceptible to - Carbapenems - 4th Gen cephalosporins (cefepime)

17. AmpC 검출시험 Cefoxitin 감수성시험: 중간 또는 내성 AmpC 확인시험
1. Three dimensional extract 시험
2. Cefoxitin Hodge 시험
3. AmpC (TE) 디스크 시험
4. AmpC inhibitor: boronic acid

18. Disk diffusion results of cefoxitin, cefotetan and cefepime compared with results of AmpC PCR
No. of isolates
Species
AmpC PCR
Cefoxitin
Cefotetan
Cefepime S I R
E. coli + 14 4 2 8 13 1 - 51 11 21 72 7 73 6
K. pneumoniae
104 9 15 81
100 3 10 27 26
P. mirabilis 37 39 40
All
120 18 90
116
103 33
138
139
Sensitivity (%)
100.0
88.5
Specificity (%)
66.9
89.6
Lee W, et al. Korean J Lab Med 2009;5:

19. Amp C 확인시험 3-Dimensional Cefoxitin Hodge AmpC (TE) disk
Coudron PE, et al. J Clin Microbiol 2000;38:
Yong D, et al. J Microbiol Meth 2002;51:
Black JA, et al. J Clin Microbiol 2005;43:

20. B C A
Figure. Three phenotypic detection methods for the detection of AmpC -lactamase producers;
A, Cephamycin-Hodge test (yellow arrow: positive reaction)
B, Tris-EDTA disk test (yellow arrow: positive reaction)
C, Combination disk test with boronic acid. (difference of inhibition zone diameter  5 mm: positive)
ATCC 25922
Test Strain
FOX+BA
FOX
CTT
CTT+BA
Test train
TE disk inoculated
with test strain
Inoculate pre-moistened Tris/EDTA disk
heavily with test isolate
Invert disk to ensure inoculum contacts agar
Place inoculated disk beside substrate disk but not touching

21. Comparison of three phenotypic detection methods for plasmid-mediated AmpC β-lactamase producing isolates
Species
AmpC
PCR
Boronic acid
Hodge
TE-disk
Positive (%)
Negative (%)
E. coli
Positive
13(92.9)
1(7.1)
5(35.7)
9(64.3)
Negative
10(12.0)
73(88.0)
1(1.2)
82(98.8)
3(3.6)
80(96.4)
K. pneumoniae
104(99.0)
1(1.0)
86(81.9)
19(18.1)
88(83.8)
17(16.2)
2(6.5)
29(93.5)
7(22.6)
2(77.4)
P. mirabilis
3(100)
0(0.0)
2(66.7)
1(33.3)
40(100)
3(7.5)
37(92.5)
All
120(98.4)
2(1.6)
93(76.2)
29(23.8)
98(80.3)
24(19.7)
12(7.8)
142(92.2)
6(3.9)
148(96.1)
13(8.4)
141(91.6)
Efficiency
96.3%
88.6%
87.9%
Lee W, et al. Korean J Lab Med 2009;5:

22. Amp C 확인시험 Boronic acid 디스크시험
PABL 생성 K. pneumoniae
Picture from W. Song

23. Multiplex detection of Plasmid AmpC genes
FJ Perez-Perez and ND Hanson, J Clin Microbiol 2002;40(6):

24. Problems of ESBL tests Inconclusive off-scale results
False positive results - KPC -lactamses - hyperproduced K1 -lactamses
False negative results - AmpC hyperproducers

25. Inconclusive off-scale results
Ceftazidime/CA
Cefepime
Ceftazidime
Cefepime/CA
Cefotaxim
Cefotaxim/CA
More potent cephalosporin을 사용함
Picture from KS Thomson

26. K1 -lactamse hyperproducer
Some strains of K. oxytoca
Ceftazidime MIC  2 mg/L - higher MIC: ESBL생성을 의미
High K1 and ESBL production의 구별 - High K1: CRO MIC » CTX MIC - ESBL: CRO MIC  CTX MIC
MICs
PIP
CPD
CTX
CAZ
CRO
FOX
IPM
K. oxytoca
>128 4
0.5
0.25 1
MIC Data from KS Thomson

27. ESBL, AmpC 동시생성균주 배지에 AmpC inhibitor첨가 - cloxacillin
AmpC inhibitor인 boronic acid를 이용한 CLSI ESBL confirmatory test
Cefepime  Clavulanate

28. E. coli -ESBL 과 고농도의 AmpC 생성균주
MH agar with 200 µg/ml Cloxacillin
Picture from KS Thomson

29. Boronic acid 디스크시험 Boronic acid 첨가 ESBL + PABL 동시생성 K. pneumoniae
Song W et al. Diagn Microbiol Infect Dis 2007;57:315-8

30. Boronic acid 디스크시험 (A) (B) Boronic acid 첨가
ESBL 생성 S. marcescens (Chromosomal AmpC)의 CLSI 확인시험 (A) boronic acid 첨가하지 않음 (B) boronic acid 첨가.
Song W, et al. Int J Antimicrob Agents 2008;31:

31. Double disk tests (A) (B) (C) Chromosomal AmpC 생성균주
(A) ESBL 생성 E. cloacae; (B) ESBL 생성 S. marcescens; (C) ESBL 생성 E. cloacae.
Picture from W. Song

32. Accuracy of ESBL test with BA
Organism
without boronate
with boronate
Sens.
Spec.
Effi.
E. cloacae
56.5
97.5
82.5
95.7
100
98.4
E. aerogenes
85.7
96.7
94.6
S. marcescens
89.5
97.2
94.5
C. freundii
66.7
97.1
Total
73.1
97.8
91.0
98.1
99.5
Data from W. Song

33. ESBL Etest with cefepime
clavulanate
Picture from KS Thomson

34. Another problem: plasmid loss
Multiple colonies
One colony
Picture from KS Thomson

35. Summary
DDST, CLSI ESBL test without modification - E. coli, Klebsiella, P. mirabilis, Salmonella, Shigella, C. koseri (AmpC -lactamase를 얻지 않았다면)
ESBL detection in AmpC producing isolates - AmpC inhibitor 또는 4세대 cephalosporin 사용
Phenotypic AmpC detection tests - cephamycin-Hodge test - AmpC (Tris-EDTA) disk test - boronic acid를 이용한 combination disk test
Plasmid-mediated AmpC -lactamase의 검출 - multiplex AmpC PCR이 유용함
If done tests for ESBL or/and AmpC, modify the results.

36. 도움을 주신 분들
강의자료 제공 1. Song W : Hallym Univ. 2. KS Thomson : Creighton Univ. 공동연구자 2. Hong HL : Seoul Medical Science Institute 3. Shin HB : Soon Chun Hyang Univ. 4. Lee YS, Chung GT, Yoo JS : KCDC 관련기관 1. 질병관리본부 국립보건연구원 2. 대한임상미생물학회