2. Current status of  -lactamases-producing gram-negative bacilli isolated in Korea Kyungwon Lee, MD, PhD Department of Laboratory Medicine and Research Institute of Bacterial Resistance Yonsei University College of Medicine, Seoul, Korea

3. Antimicrobial Resistance of Bacteria n Serious problems in many countries many countries n In Korea, - relatively more - relatively more prevalent prevalent - has worsened during - has worsened during past several years past several years

4. Major clinical problems with antibiotic- resistant bacteria n Gram-negative bacilli : 59% - 3 rd -gen cepha.-resistant - 3 rd -gen cepha.-resistant E. coli 3,681 E. coli 3,681 K. pneumoniae 1,728 K. pneumoniae 1,728 E. cloacae 756 E. cloacae 756 S. marcescens 454 S. marcescens 454 C. freundii 271 C. freundii 271 - Carbapenem-resistant - Carbapenem-resistant P. aeruginosa 3,476 P. aeruginosa 3,476 A. baumannii 1,894 A. baumannii 1,894 n Gram-positive cocci: 41% - Methicillin-resistant Staphylococcus aureus 4,008 - Vancomycin-resistant Enterococcus faecium 1,870 - Penicillin-nonsusceptible Streptococcus pneumoniae 236 Antimicrobial resistance newsletter, 2003

5. Korean Nationwide Surveillance of Antimicrobial Resistance (KONSAR) n WHO, organized in 1997 n To monitor the nationwide the nationwide antimicrobial resistance antimicrobial resistance n 72 university or general hospitals hospitals n WHONET program KONSAR 2003 26 2 1

6. β-lactam resistance in gram-negative rods n Impermeability n Alteration of PBP n β-lactamase production: β-lactam hydrolysis - 1995, 170; 2000, 350 - 1995, 170; 2000, 350 - Novel β-lactamase - Novel β-lactamase n Most important groups of enzymes - Extended spectrum β-lactamase (ESBL) - Extended spectrum β-lactamase (ESBL) - AmpC β-lactamase - AmpC β-lactamase - Carbapenemase - Carbapenemase (Bush CID, 2001)

7. Classification of β-lactamases Ambler Bush Activity CA Ambler Bush Activity CA class group PEN CAR OXA CEP CTX AZT IMP Inh Enzymes class group PEN CAR OXA CEP CTX AZT IMP Inh Enzymes Serine Serine A 2a +++ + - +/- - - - ++ GPC pen-nase A 2a +++ + - +/- - - - ++ GPC pen-nase 2b +++ + + ++ - - - ++ TEM-1, 2, SHV-1 2b +++ + + ++ - - - ++ TEM-1, 2, SHV-1 2be +++ + + ++ ++ ++ - ++ TEM-3, SHV-2 2be +++ + + ++ ++ ++ - ++ TEM-3, SHV-2 2br +++ + + + - -- - TEM-30 2br +++ + + + - -- - TEM-30 2c ++ +++ + + - -- + PSE-1 2c ++ +++ + + - -- + PSE-1 2e ++ ++ - ++ ++ ++- ++ Induc. ceph-nase 2e ++ ++ - ++ ++ ++- ++ Induc. ceph-nase 2f ++ + ? + + ++ ++ + NMC-A, Sme-1 2f ++ + ? + + ++ ++ + NMC-A, Sme-1 C 1 ++ +/- 저해 +++ + 저해 - - GNR 의 AmpC C 1 ++ +/- 저해 +++ + 저해 - - GNR 의 AmpC D 2d ++ + +++ + - -- +/- OXA-1 D 2d ++ + +++ + - -- +/- OXA-1 - 4 ++ ++ ++ v v -- - B. cepacia - 4 ++ ++ ++ v v -- - B. cepacia penicillinase penicillinase Zinc Zinc B 3 ++ ++ ++ ++ ++ - ++ - S. maltophilia L1 B 3 ++ ++ ++ ++ ++ - ++ - S. maltophilia L1

8. Extended-spectrum TEM and SHV -lactamases Extended-spectrum TEM and SHV β-lactamases n Class A, Bush group 2be, plasmid-mediated n Europe, 1983 n Mutation - Ampicillin, 1-Gen cepha: TEM-1, 2, SHV-1 - Ampicillin, 1-Gen cepha: TEM-1, 2, SHV-1 Penicillin, 1-Gen cepha (R) Penicillin, 1-Gen cepha (R) - Extended-spectrum cepha: TEM, SHV derivatives - Extended-spectrum cepha: TEM, SHV derivatives 3-Gen cepha, monobactam (R) 3-Gen cepha, monobactam (R) Carbapenem, cephamycin (S) Carbapenem, cephamycin (S) Inhibited by -lactamase inhibitors Inhibited by β-lactamase inhibitors

9. Extended spectrum β-lactamases n TEM and SHV derivatives n Non-TEM and Non-SHV - CTX-M family, TOHO-1, TOHO-2 - CTX-M family, TOHO-1, TOHO-2 - OXA type - OXA type - Other ESBLs: PER-1, PER-2, VEB-1, etc - Other ESBLs: PER-1, PER-2, VEB-1, etc n Estimated No.: 1995, 36; 2000, 119 Bradford PA: CMR 14:933, 2001; Bush K: CID 32:1085, 2001

10. Extended-spectrum TEM and SHV -lactamases Extended-spectrum TEM and SHV β-lactamases Enzyme Amino acid 39 104 164 205 237 238 240 265 39 104 164 205 237 238 240 265 TEM-1 Gln Glu Arg Arg Ala Gly Glu Thr TEM-2 Lys TEM-13 Lys Met TEM-3 Lys Lys Ser TEM-4 Lys Ser Met TEM-5 Ser Thr Lys : SHV-1 Gln Asp Arg Arg Ala Gly Glu Leu SHV-2 Ser SHV-3 Leu Ser :

11. TEM-type ESBLs n Parent enzyme: TEM-1, 2 n Isoelectric point: 5.4-6.5 n TEM-type enzymes: > 90, ESBL, IRT, BS n TEM-type ESBLs - E. coli, K. pneumoniae: common - E. coli, K. pneumoniae: common - Other gram-negative bacilli - Other gram-negative bacilli Enterobacteriaceae - EAE, MMO, PMI, SAL Enterobacteriaceae - EAE, MMO, PMI, SAL Pseudomonas aeruginosa - TEM-42 Pseudomonas aeruginosa - TEM-42 Capnocytophaga ochracea - TEM-17 Capnocytophaga ochracea - TEM-17

12. SHV-type ESBLs n Parent enzyme: SHV-1; Isoelectric point: 7.0-8.2 n SHV-type enzymes: > 25, ESBL, IR, BS n SHV-type ESBLs K. pneumoniae > C. diversus, E. coli, P. aeruginosa K. pneumoniae > C. diversus, E. coli, P. aeruginosa n pI Enzymes Type 7.0 3, 14 ESBL 7.0 3, 14 ESBL 7.5-7.8 1, 11 BS 7.5-7.8 1, 11 BS 2, 2a, 4, 6, 7, 8, 13, 18-22, 24 ESBL 2, 2a, 4, 6, 7, 8, 13, 18-22, 24 ESBL 8.2 5, 9, 12 ESBL 8.2 5, 9, 12 ESBL 10 IR 10 IR

13. CTX-M-type ESBLs n Preferentially hydrolyze cefotaxime n Inhibited better by TZB than by SUB and CA n Salmonella enterica serovar Typhimurium, E. coli > other spp. of Enterobacteriaceae > other spp. of Enterobacteriaceae n Enzymes pI Country Bacterial spp. CTX-M-1 8.9 Germany, Italy E. coli CTX-M-1 8.9 Germany, Italy E. coli CTX-M-2 7.9 Argentina S. enterica CTX-M-2 7.9 Argentina S. enterica : : : : : : : : CTX-M-10 8.1 Spain E. coli CTX-M-10 8.1 Spain E. coli Toho-1, 2 7.8, 7.7 Japan E. coli Toho-1, 2 7.8, 7.7 Japan E. coli

14. CTX-M-type ESBLs n Phylogenetic study: 4 major groups 1) CTX-M-1 related: CTX-M-1, 3, 10, 11, 12, 22 1) CTX-M-1 related: CTX-M-1, 3, 10, 11, 12, 22 2) CTX-M-2 related: CTX-M-2, 4, 5, 6, 7, 20, Toho-1 2) CTX-M-2 related: CTX-M-2, 4, 5, 6, 7, 20, Toho-1 3) Toho-2 related: Toho-2, 3) Toho-2 related: Toho-2, CTX-M-9,13, 14, 15, 16, 18, 19, 21 CTX-M-9,13, 14, 15, 16, 18, 19, 21 4) CTX-M-8 4) CTX-M-8

15. OXA-type ESBLs n Molecular class D, functional group 2d n Hydrolytic activity against oxacillin & cloxacilllin n Poorly inhibited by CA n Mainly Pseudomonas aeruginosa n OXA-type ESBLs - OXA-10: OXA-11, 14, 16, 17 (pI 6.1-6.4) - OXA-10: OXA-11, 14, 16, 17 (pI 6.1-6.4) OXA-13, 19, 28 (pI 7.6-8.0) OXA-13, 19, 28 (pI 7.6-8.0) - OXA-2: OXA-15 (pI 8.7, 8.9) - OXA-2: OXA-15 (pI 8.7, 8.9) - OXA-9?: OXA-18 (pI 5.5) - OXA-9?: OXA-18 (pI 5.5)

16. Other novel, unrelated ESBLs Enzymes pI Substrate Country Bacterial spp. Enzymes pI Substrate Country Bacterial spp. PER-1 5.4 CAZ France P. aeruginosa PER-1 5.4 CAZ France P. aeruginosa PER-2 5.4 CAZ Argentina S. Typhimurium PER-2 5.4 CAZ Argentina S. Typhimurium VEB-1 5.35 CAZ, ATM Vietnam E. coli VEB-1 5.35 CAZ, ATM Vietnam E. coli CME-1 >9.0 CAZ C. meninosep. CME-1 >9.0 CAZ C. meninosep. TLA-1 9.0 CAZ, CTX, ATM Mexico E. coli TLA-1 9.0 CAZ, CTX, ATM Mexico E. coli SFO-1 7.3 CTX Japan E. cloacae SFO-1 7.3 CTX Japan E. cloacae GES-1 5.8 CAZ Guiana K. pneumoniae GES-1 5.8 CAZ Guiana K. pneumoniae Bradford PA: CMR 14, 2001 Bradford PA: CMR 14, 2001

17. Treatment for infections caused by ESBL-producers MIC 10 4 10 6 Clinical usefulness 3 gen cepha. Usu. high High None 4 gen cepha. Usu. low High Low, high dosage w/ aminoglycoside  -lactam-  -lactamase inh. High or low High Clin. data, limited Cephamycin Low Low Clin. data, limited attractive, 2nd-line Carbapenem Low Low 1st-line Paterson (CMI, 2000)

18. Prevalence of ESBLs n Varies depending on country or institution n USA: 3% (0-25%) n Netherlands (1999): <1% of E. coli and K. pneumoniae strains K. pneumoniae strains n France (1998): 40% of K. pneumoniae, CAZ (R) n Japan (2000): <0.1% of E. coli 0.3% of K. pneumoniae 0.3% of K. pneumoniae n Taiwan (2000): 8.5% of K. pneumoniae Bradford PA (2001)

19. Cefotaxime susceptibility of E. coli (Severance Hospital)

20. Cefotaxime susceptibility of K. pneumoniae (Severance Hospital)

21. Prevalence of ESBL-producing E. coli and K. pneumoniae in Korea (1999)

22. ESBL types n Germany: SHV-2, SHV-5 n France: TEM-3, SHV-3, SHV-4 n U.S.A.: TEM-10, TEM-12, TEM-26 n Japan: TOHO-1, TOHO-2 n Korea: TEM-52, SHV-12, SHV-2a n SHV-2, 5: widespread internationally China, Denmark, France, Spain, South Africa China, Denmark, France, Spain, South Africa Switzerland, Tunisia, UK Switzerland, Tunisia, UK Nordmann P (1998); Bradford PA (2001); Pai (1997)

23. Evolution of TEM-type ESBLs in Korea n Blood isolates of ESBL-producing E. coli and K. pneumoniae in 1994-1999 K. pneumoniae in 1994-1999 n TEM-type ESBLs: E. coli (12), K. pneumoniae (18) amino acid at: amino acid at: TEM No 104 182 196 238 TEM No 104 182 196 238 1 Glu (GAG) Met (ATG) Gly (GGC) Gly (GGT) 1 Glu (GAG) Met (ATG) Gly (GGC) Gly (GGT) 15 2 Lys (AAG) Ser (AGT) 15 2 Lys (AAG) Ser (AGT) 52 25 Lys (AAG) Thr (ACG) Ser (AGT) 52 25 Lys (AAG) Thr (ACG) Ser (AGT) 88 3 Lys (AAG) Thr (ACG) Asp (GAC) Ser (AGT) 88 3 Lys (AAG) Thr (ACG) Asp (GAC) Ser (AGT) Pai et al (AAC, 2001)

24. Evolution of TEM-type ESBLs in Korea n Nationwide study (1999-2000) n ESBL-producing E. coli (202) and K. pneumoniae (329) K. pneumoniae (329) n Novel TEM type ESBLs: TEM-106, 107 TEM-15 TEM-88 TEM-15 TEM-88 TEM-1 TEM-20 TEM-52 TEM-107 TEM-106 TEM-43 TEM-106 TEM-43

25. TEM-52 ESBL-producing non-typhoidal Salmonella isolates in Korea S. enterica Age serovar (Year) Present illness serovar (Year) Present illness Saintpaul 0.4 Loose stool Stanley 1 Diarrhea Agona 1 Diarrhea Enteritidis 25 Diarrhea 1 mon. after adm. Enteritidis 51 Diarrhea 1 mon. after adm.

26. CTX-M-type ESBLs in Korea n Pai et al (JCM, 2001): CTX-M-14 vs. CTX-M-9 (1 aa), Toho-2 (1 aa) vs. CTX-M-9 (1 aa), Toho-2 (1 aa) E. coli 1, K. pneumoniae 2 in 1995-96 E. coli 1, K. pneumoniae 2 in 1995-96 Shigella sonnei 1 in 2000 Shigella sonnei 1 in 2000 n Nationwide study (1999-2000) ESBL-producing E. coli and K. pneumoniae ESBL-producing E. coli and K. pneumoniae CTX-M-type: 27 (18.8%) of 144 CTX-M-type: 27 (18.8%) of 144 CTX-M-1 related (15): CTX-M-12 CTX-M-1 related (15): CTX-M-12 Toho-2 related (12): CTX-M-14 Toho-2 related (12): CTX-M-14

27. PER-1 ESBL n Reported only in European countries - First report (1991): P. aeruginosa, France - First report (1991): P. aeruginosa, France - Acinetobacter spp., P. aeruginosa, Turkey, Italy - Acinetobacter spp., P. aeruginosa, Turkey, Italy n PER-2: Salmonella typhimurium in Argentina n Yong et al (Korea, 2002) - PER-1: Acinetobacter spp., 53 of 97 - PER-1: Acinetobacter spp., 53 of 97 P. aeruginosa: none of 282 P. aeruginosa: none of 282

28. Amp C type β-lactamases n Class C, chromosomal n Ubiquitous in GNB, species-specific - Uninducible : E. coli etc, insignificant amount - Uninducible : E. coli etc, insignificant amount - Inducible : C. freundii, E. cloacae, S. marcescens - Inducible : C. freundii, E. cloacae, S. marcescens Absence of Absence of β-lactam: trace amount - AMP, CEP (r) Present of β-lactam Induction: 100-fold - FOX (R), CTX (S) Mutation: permanent hyperproduction, derepression FOX, CTX (R), 4-Gen Cepha (S)

30. Plasmid-mediated class C β-lactamases n Cephamycin - Second-line therapy for infections caused by ESBL-producers - Resistance is mainly due to chromosomal  -lactamases - Chromosomal ampC gene: plasmid n Plasmid-mediated class C n Plasmid-mediated class C  -lactamases - CMY-1 Korea 1989 - CMY-1 K. pneumoniae Korea 1989 MIR-1 U.S.A. 1990 - MIR-1 K. pneumoniae U.S.A. 1990 - Subsequently, over 20 types have been discovered - CMY, FOX, MOX, LAT, ACT, ACC; MIR-1, DHA, BIL Philippon (AAC, 2002)

32. Cefoxitin-resistant E. coli and K. pneumoniae n KONSAR survey (1997 vs. 2000) - Cefoxitin resistance - Cefoxitin resistance E. coli 12% vs. 9%, K. pneumoniae 16% vs.16% E. coli 12% vs. 9%, K. pneumoniae 16% vs.16% n Yong et al (1999): CMY-1  -lactamase FOX (I,R) CMY-1 DHA-1 FOX (I,R) CMY-1 DHA-1 E. coli (97) 33 24 (24.7%) 0 E. coli (97) 33 24 (24.7%) 0 K. pneumoniae (48) 10 5 (10.4%) 2 (4.2%) K. pneumoniae (48) 10 5 (10.4%) 2 (4.2%)

33. DHA-1, inducible AmpC  -lactamase n In 2002, 3109 clinical isolates in a Korean hospital Antagonistic zone between cefoxitin and Antagonistic zone between cefoxitin and aztreonam disks aztreonam disks - E. coli: 8 (0.4%) of 1937 - E. coli: 8 (0.4%) of 1937 - K. pneumoniae: 55 (6.1%) of 899 - K. pneumoniae: 55 (6.1%) of 899 - K. oxytoca: 8 (5.4%) of 149 - K. oxytoca: 8 (5.4%) of 149 - P. mirabilis: 1 (0.8%) of 124 - P. mirabilis: 1 (0.8%) of 124 bla DHA-1 alleles were detected in all of them bla DHA-1 alleles were detected in all of them n In 2003, DHA-1, 29 (10.5%) of 275 K. pneumoniae

34. Imipenem resistance of P. aeruginosa and Acinetobacter spp. at Severance hospital % resistance

35. Antimicrobial resistance in P. aeruginosa Large Seoul Non-Seoul Mean Antimicrobials >1000 1000 <1000 <1000 (5,414) (4,050) (7,740) (17,204) (5,414) (4,050) (7,740) (17,204) Piperacillin 38% 37% 42% 39% Pip/tazobactam 25 24 3628 Ceftazidime 18 19 1919 Aztreonam 22 18 1819 Imipenem 21 20 1920 Amikacin 26 35 3632 Gentamicin 41 44 4744 Fluoroquinolone 39 44 4242 (KONSAR 2000)

36. Antimicrobial resistance in P. aeruginosa Asia- Europe Latin North Antimicrobials Pacific America America (894) (1,702) (1,103) (3,270) (894) (1,702) (1,103) (3,270) Piperacillin 14 (39) 20 32 13 Pip/tazobactam 11 (28) 14 2410 Ceftazidime 14 (19) 15 2814 Aztreonam 19 (19) 15 3320 Imipenem 9 (20) 12 18 9 Amikacin 5 (32) 14 27 5 Gentamicin 16 (44) 28 3816 Fluoroquinolone 12 (42) 25 3316 SENTRY 1997-2000; ( ), KONSAR, 2000

37. Carbapenem resistance mechanism  -lactamase  -lactamase - Serine  -lactamase: Bush group 1, 2f, 2d - Serine  -lactamase: Bush group 1, 2f, 2d - Metallo-  -lactamase: IMP, VIM, SPM-1 - Metallo-  -lactamase: IMP, VIM, SPM-1 Outer membrane impermeability or efflux Outer membrane impermeability or efflux - OprD porin, MexAB-OprM overexpression - OprD porin, MexAB-OprM overexpression Penicillin binding protein Penicillin binding protein

38. Metallo-  -lactamase IMP type IMP type - IMP-1: Japan (1990s): P. aeruginosa, S. marcesens, - IMP-1: Japan (1990s): P. aeruginosa, S. marcesens, A. baumannii, K. pneumoniae A. baumannii, K. pneumoniae Korea (2000): Acinetobacter genomospecies 3 Korea (2000): Acinetobacter genomospecies 3 A. baumannii A. baumannii - IMP-2: Italy (2000), A. baumannii - IMP-2: Italy (2000), A. baumannii - IMP-3: Japan (2000), S. flexneri - IMP-3: Japan (2000), S. flexneri - IMP-4: Hong Kong, China (2001), - IMP-4: Hong Kong, China (2001), Acinetobacter, Citrobacter Acinetobacter, Citrobacter - IMP-5, 6, 7,….12: gram-negative rods in several countries - IMP-5, 6, 7,….12: gram-negative rods in several countries

39. Metallo-  -lactamase VIM type VIM type - VIM-1: Italy (1999), P. aeruginosa; - VIM-1: Italy (1999), P. aeruginosa; (2001), Achromobacter xylosoxydans (2001), Achromobacter xylosoxydans - VIM-2: France (2001), P. aeruginosa - VIM-2: France (2001), P. aeruginosa Greece (2000), P. aeruginosa Greece (2000), P. aeruginosa - VIM-3: Taiwan (2001), P. aeruginosa - VIM-3: Taiwan (2001), P. aeruginosa - VIM-4: Greece (2002), P. aeruginosa - VIM-4: Greece (2002), P. aeruginosa - Korea - Korea VIM-2: P. aeruginosa, P. putida, A. baumannii VIM-2: P. aeruginosa, P. putida, A. baumannii S. marcescens, Acinetobacter genomospecies 3 S. marcescens, Acinetobacter genomospecies 3 E. cloacae, Achromobacter xylosoxydans E. cloacae, Achromobacter xylosoxydans

40. P. aeruginosa P. putida Acinetobacter spp. P. aeruginosa P. putida Acinetobacter spp. Year No. bla VIM + No. bla VIM + No. bla VIM + bla IMP + Year No. bla VIM + No. bla VIM + No. bla VIM + bla IMP + 1994 1 0 NT NT 1994 1 0 NT NT 1995 20 5 NT NT 1995 20 5 NT NT 1996-7 138 15 1 1 NT 1996-7 138 15 1 1 NT 1998-9 324 23 8 8 28 13 1 1998-9 324 23 8 8 28 13 1 2000-1 325 46 6 6 42 6 28 2000-1 325 46 6 6 42 6 28 2002 454 38 15 15 195 34 39 2002 454 38 15 15 195 34 39 Total (%) 1262 127 (10) 30 30 265 53 (20) 68 (26) Total (%) 1262 127 (10) 30 30 265 53 (20) 68 (26) Lee et al. (AAC, 2002; JAC, 2002) Lee et al. (AAC, 2002; JAC, 2002) Prevalence of bla VIM-2 or bla IMP-1 -positive strains among imipenem-resistant isolates

41. Prevalence of MBL-producing gram-negative bacilli in Korean Hospitals Organism No. (%) hospital No. (%) of isolates Tested MBL+ Tested VIM-2+ IMP-1+ Total Tested MBL+ Tested VIM-2+ IMP-1+ TotalPseudo- monas spp. 21 11 (52.4) 387 44 0 44 (11.4) monas spp. 21 11 (52.4) 387 44 0 44 (11.4)Acineto- bacter spp. 24 10 (41.7) 267 27 11 38 (14.2) bacter spp. 24 10 (41.7) 267 27 11 38 (14.2) Total 28 17 (60.7) 654 71 11 82 (12.5) Lee et al (ICAAC, 2002)

42. A. baumannii YMC 98-7-R363 (3 kb; AF324464) IntI1bla VIM-2 aacA7aadA1 qacE  1 Acinetobacter genomospecies 3 YMC 99-11-U160 (5 kb; AF369871) IntI1bla VIM-2 aacA4aadA1“orfII”“orfIII” qacE  1 P. aeruginosa YMC 95-1-704 (5.6 kb; AY029772) IntI1 aacA4bla VIM-2 “orfI”aadA1“orfII”“orfIII” qacE  1 S. marcescens YMC 00-4-1591 (4 kb; AY030343) IntI1 qacbla VIM-2 “orfII”“orfIII” qacE  1 P. putida YMC 97-8-322 (3 kb; AF327064) IntI1bla VIM-2 aacA7aadA1 qacE  1 Structure of integrons found

43. Conclusions n ESBL- or plasmid-mediated AmpC- producing gram-negative bacilli - More prevalent in Korea - More prevalent in Korea - High levels of antimicrobial selective pressure - High levels of antimicrobial selective pressure - Nosocomial spread - Nosocomial spread n Carbapenem-resistant P. aeruginosa and Acinetobacter spp. - New threats in Korea - New threats in Korea