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

Extended-spectrum β-lactamases (ESBL)

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

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.

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

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

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

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

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

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 8 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.

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

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

AmpC β-lactamases

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

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

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

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

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;5:448-54.

Amp C 확인시험 3-Dimensional Cefoxitin Hodge AmpC (TE) disk Coudron PE, et al. J Clin Microbiol 2000;38:1791-6. Yong D, et al. J Microbiol Meth 2002;51:407-10. Black JA, et al. J Clin Microbiol 2005;43:3110-3.

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

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;5:448-54.

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

Multiplex detection of Plasmid AmpC genes FJ Perez-Perez and ND Hanson, J Clin Microbiol 2002;40(6):2153-2162

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

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

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

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

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

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

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;31:467-71.

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

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

ESBL Etest with cefepime clavulanate Picture from KS Thomson

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

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.

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