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

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

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 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

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

β-lactam resistance in gram-negative rods n Impermeability n Alteration of PBP n β-lactamase production: β-lactam hydrolysis , 170; 2000, , 170; 2000, 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)

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 /- 저해 저해 - - GNR 의 AmpC C /- 저해 저해 - - GNR 의 AmpC D 2d /- OXA-1 D 2d /- OXA v v -- - B. cepacia v v -- - B. cepacia penicillinase penicillinase Zinc Zinc B S. maltophilia L1 B S. maltophilia L1

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

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

Extended-spectrum TEM and SHV -lactamases Extended-spectrum TEM and SHV β-lactamases Enzyme Amino acid 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 :

TEM-type ESBLs n Parent enzyme: TEM-1, 2 n Isoelectric point: 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

SHV-type ESBLs n Parent enzyme: SHV-1; Isoelectric point: 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 , 11 BS , 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

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 Germany, Italy E. coli CTX-M Germany, Italy E. coli CTX-M Argentina S. enterica CTX-M Argentina S. enterica : : : : : : : : CTX-M Spain E. coli CTX-M Spain E. coli Toho-1, 2 7.8, 7.7 Japan E. coli Toho-1, 2 7.8, 7.7 Japan E. coli

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

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 ) - OXA-10: OXA-11, 14, 16, 17 (pI ) OXA-13, 19, 28 (pI ) OXA-13, 19, 28 (pI ) - 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)

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

Treatment for infections caused by ESBL-producers MIC 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)

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)

Cefotaxime susceptibility of E. coli (Severance Hospital)

Cefotaxime susceptibility of K. pneumoniae (Severance Hospital)

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

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)

Evolution of TEM-type ESBLs in Korea n Blood isolates of ESBL-producing E. coli and K. pneumoniae in K. pneumoniae in n TEM-type ESBLs: E. coli (12), K. pneumoniae (18) amino acid at: amino acid at: TEM No TEM No 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) Lys (AAG) Thr (ACG) Ser (AGT) 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)

Evolution of TEM-type ESBLs in Korea n Nationwide study ( ) 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

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.

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 E. coli 1, K. pneumoniae 2 in Shigella sonnei 1 in 2000 Shigella sonnei 1 in 2000 n Nationwide study ( ) 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

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

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)

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 CMY-1 K. pneumoniae Korea 1989 MIR-1 U.S.A MIR-1 K. pneumoniae U.S.A Subsequently, over 20 types have been discovered - CMY, FOX, MOX, LAT, ACT, ACC; MIR-1, DHA, BIL Philippon (AAC, 2002)

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) (24.7%) 0 E. coli (97) (24.7%) 0 K. pneumoniae (48) 10 5 (10.4%) 2 (4.2%) K. pneumoniae (48) 10 5 (10.4%) 2 (4.2%)

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 E. coli: 8 (0.4%) of K. pneumoniae: 55 (6.1%) of K. pneumoniae: 55 (6.1%) of K. oxytoca: 8 (5.4%) of K. oxytoca: 8 (5.4%) of P. mirabilis: 1 (0.8%) of 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

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

Antimicrobial resistance in P. aeruginosa Large Seoul Non-Seoul Mean Antimicrobials > <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 Ceftazidime Aztreonam Imipenem Amikacin Gentamicin Fluoroquinolone (KONSAR 2000)

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) Pip/tazobactam 11 (28) Ceftazidime 14 (19) Aztreonam 19 (19) Imipenem 9 (20) Amikacin 5 (32) Gentamicin 16 (44) Fluoroquinolone 12 (42) SENTRY ; ( ), KONSAR, 2000

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

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

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

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 NT NT NT NT NT NT NT NT NT NT Total (%) (10) (20) 68 (26) Total (%) (10) (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

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 (52.4) (11.4) monas spp (52.4) (11.4)Acineto- bacter spp (41.7) (14.2) bacter spp (41.7) (14.2) Total (60.7) (12.5) Lee et al (ICAAC, 2002)

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

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