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1 The Krean Jurnal f Micrbilgy, Vl. 45, N. 2, June 2009, p Cpyright 2009, The Micrbilgical Sciety f Krea ü ³ Klebsiella pneumniae Escherichia cliƒ w Ÿ k k (Extended-Spectrum β-lactamase, ESBL) wz x z w w w w w w Ÿ k k wz (extendedspectrum β-lactamase, ESBL) ³ wš w ESBL x w. û w z w û y w j w l w³ j y x(duble disk synergy test) ùkü 19 ³ 1 x³ kw. x methyl-red, Vges-Prskauer, Simmn s citrate, decarbxylase-dihydrlase z yw x mw Klebsiella pneumniae (3 ³ ) Escherichia cli (16 ³ )ƒ. ³ œ ³, Escherichia cli J53 (sdium azide r ) v ³ w w x w plasmid 4 ³ w. l plasmid w PCR s g wš w Klebsiella pneumniae ESBL x SHV-12 (3 ³ ) š, Escherichia cli ESBL x SHV-12/TEM-1 (1 ³ ). Key wrds ý duble disk synergy test, Escherichia cli, extended spectrum beta-lactamase (ESBL), Klebsiella pneumniae, SHV-12, TEM Klebsiella pneumniae Escherichia cli ü ³ plasmid Ÿ k k wz (extended spectrum β-lactamase, ESBL) ³ ƒ y l z ù sww s e w š (1, 18). β-lactamase (EC ) amide, amidine,»k C-N w ƒ w j z p y amide w k (5, 6). penicillin ù cephalsprin β-lactam w³ amide w, β-lactam ring ƒ w k y y k (4, 8). j TEMx SHVx ù TEM ù SHV β-lactamase 1~4 ey ù x (pint mutatin)ƒ ù (9). Bush DNA w Ambler β- lactamase» w ƒ ƒ š (2, 5). x w š ESBL ³ ü ³ (Enterbacteriaceae) ³ cephalsprin *T whm crrespndence shuld be addressed. Tel: , Fax: hunku@pknu.ac.kr 3 w³ w ü z wš. ³ DNA w» w ³ w w plasmid ³ ü š (6, 8). y ù ƒ l 3 ü z w ³ ƒ ù yw, ù s» mw y, ü wš ³ ü k š, ü ³ ƒ ù,»k ƒ w yy m w x ƒ š. ù ESBL ³ w ESBL x y l» yy l w ƒ. w w û rv,, w yy l ESBL ü ³ w, j y x, w x, x mw plasmid ESBL ³ w ESBL x w š (12). z l yw w û y û w w j w l ESBL ³ wš x q w» w. 163

2 164 Hun-Ku Lee Kr. J. Micrbil ³ w l ³ w ³ 2 w z x ƒƒ 0.1 ml w 3 cephalsprin w³ ceftazidime (4 µg/ml, Yung-Jin Pharm., Krea) ƒ w ü ³ k MacCnkey agar (Difc Labratry, USA) š w 37 C 17 w ³. 2z Brain Heart Infusin agar (BHIA, Difc) w. ³ yw w ¾ w. ³ Ewing (7). w³ ü q w³ j(bbl, Bectn Dickinsn, Micrbilgy System, USA) w j y w š q» NCCLS (15). w³ ampicillin (AM), amikacin (AN), cephalthin (CF), chlramphenicl (C), gentamicin (GM), kanamycin (K), nalidixic acid (NA) tetracycline (Te) 8. j y (Duble disk synergy) x w³ McFarland N. 0.5 w ³ ³ Mueller-Hintn agar (MHA, Difc) š w. sq ticarcillin/clavulanate (75/10 µg, Bectn Dickinsn Micrbilgy System, USA) disk š disk l 25 mm œ 3 w³ ceftaxime (30 µg), ceftazidime (30 µg), ceftriaxne (30 µg) disk š 37 C 18 w z xk w z (synergism) q w (10, 13). w³ BBL (Bectn Dickinsn Micrbilgy System) t. w x(transcnjugatin) w ü j y x ùkü ³ ESBL plasmid ü ³ (dnr) wš sdium azide (50 µg/ ml)ƒ ƒ Mueller-Hintn agar y w. E. cli J53 (sdium azide r ceftazidime ) v ³ s (recipient) w w x w. x³ BHI» w ³ w. ³ v ³ ³ 1:10 w BHI yww z 37 C 24 w. ³ ceftazidime (32 µg/ml) sdium azide (50 µg/ml)ƒ wì ƒ MacCnkey agar š wš 37 C 24 w ³ ³ wš y w mw E. cli y ³ x ³ wš w C w ³ y w ³ w (13). z ³ w PCR,», x plasmid w ü y w Gel Extractin kit (DyneBi Inc., Krea) w plasmid DNA w k(gentec, Krea) w» w.» z, NCBI ( BCM Search Launcher ( w Multiple Sequence Alignment, Sequence Utility g y wš w wš ESBL x w. x» Bush Jacby w š lahey clinical study ( temtable.asp). (Iselectric fcusing, IEF) d w ³ l crude β-lactamase, plyacrylamide gel» w š (12, 13). BHI 30 ml w 37 C 24 k w z 1,000 g 15 w e ³ 3 1ml g. q q»(ultrasnic hmgenizer Cle-Palmer Instrument C., USA) w 30 4z w. 5,000 g 5 w eppendrf tube ¼ z ¾ -20 C w (13). q pipette w š gel supprt film q» sƒ» w g. Plyacrylamide gel (3~4 ml) wš, gel gel supprt film e»sƒ» w g. w gelq xÿ 1 w g. gelq sample template š sample 2 µl lading w z 5 ew sample template w. Iselectrfcusing Chamber (Mine IEF Cell 111., Bi-Rad C., USA) w k jš gelq gel w 100 V 30, 200 V 30, 450 V 60» g. w gel (12, 13).» óù gelq l gel supprt film w nitrcefin (500 µg/ml in phsphate buffer, ph 7.0) 1~2 ml gel t z bandƒ ùkù Wattman N. 2 gel jš band t marker w q w (1, 12). Plasmid Plymer Chain Reactin (PCR) w ESBL x x³ BHI w 37 C 18 k w z 1ml 3,000 g w e ³ g 3z w. plasmid kit plasmid Minipreps DNA Purificatin kit (Injae Science C. Krea). ESBL ³ x y w primer ƒƒ TEM type

3 민락동 오수처리장 분리 ESBL 유형 165 속에서 100 V로 15분간 전기영동을 하여 각각의 전형적인 TEM (1 kb)과 SHV (800 bp)형의 band를 UV 형광등 아래에서 확인 하였다(13). Vl. 45, N. 2 결 과 ESBL 생성 장내세균의 분리동정 Fig. 1. Detectin f ESBL psitive strains by the duble-disk synergy test between 3rd generatin cephalsprins (ceftazidime, ceftriaxn, and ceftaxime) and ticacillin/cavulanic acid (center). The synergic effects between 3rd generatin cephalsprins and ticacillin/clavulanic acid are marked in arrw. SHV type의 보존적 염기서열을 이용하여 제노택(Gentec, Krea)에서 제작하였다. TEM형 primer는 F (5 -ATA AAA TTC TTG AAG ACG AAA-3 ) R (5 -GAC AGT TAC CAA TGC TTA ATC-3 )였고 SHV형 primer는 F (5 -TGG TTAT TGC GTT ATA TTC GCC3 ) R (5 - GGT TAG CGT TGC CAG TGC T-3 )였다(13) TEM형 확인을 위한 PCR 조건은 denaturatin 94 C 30초, annealing 50 C 90초, extensin 72 C 60초로 하여 30 cycle을 반복하였고, SHV형 확인은 denaturatin 96 C 30초, annealing 50 C 15초, extensin 72 C 120초로 하여 24 cycle을 반복하였다. 사용된 primix Taq은 TaKaRa 제품(TaKaRa Bi Inc., Japan)이었 고 최종량은 20 µl가 되게 조절 하였다. PCR 기기는 GeneAmp PCR system 2400 (Perkin Elmer, USA)를 사용하였다. 이와 같 이 하여 얻어진 PCR 생성물을 1.0% agarse gel에 TBE buffer 2009년 1월 민락오수처리장의 방류수를 채취하여 ceftazidime (4 µg/ml)이 첨가된 MacCnkey agar 배지 상에서 성장된 균집락 을 선별하여 이중 디스크 확산시험결과 중앙에 놓여진 ticarcillin/ clavulanic acid와 주변에 놓인 제 3세대 항균제 사이에 간섭현상 을 나타내 투명대를 형성한 19균주를 실험균주로 선택하였다 (Fig. 1). 그람염색 및 xidase 시험 결과 2종 모두 음성을 나타 내었고, catalase 시험은 양성을 나타내었다. 이들에 대한 생화학 검사결과 Klebsiella pneumniae (3주)와 E. cli 16주 등 총 19 균주의 장내세균이 동정되었다(Table 1). 각 균종에 대한 생화학 검사결과는 다음과 같았다(Table 1). E. cli는 MacCnkey 평판배지에서 전 균주가 고원(plateau)형 의 적색 집락을 형성하였고 KIA 배지 slant와 butt에서 산 생성 (), 0.4% 한천이 첨가된 nutrient 반고체 배지에서 일부 균주 들이 운동성이 있었고, indle, methyl-red 시험에서 양성을 나타 내었다. 그러나 전 균주는 VP 배지에서 acetin을 생성하지 못하 였고 Simmn s citrate 배지에서 citrate를 단독 탄소원으로 이용하 지 못하였다. Lysine decarbxylase 시험과 rnithine decarbxylase 시험은 양성반응을 나타내었고, arginine dihydrlase 시험에서는 균주에 따라 변화가 있었다. Glucse를 비롯한 여러 종류의 당을 발효시켰다. K. pneumniae는 MacCnkey 평판배지에서 전 균주가 중앙이 선명한 적색, 주변부가 투명한 동심원으로 집락 표면에 점성을 띄는 매끄러운 cnvex형태였다. KIA 배지 상에서 slant 부분과 butt 부분에서 산 생성(), 0.4% 한천이 첨가된 nutrient 반고 체 배지에서 운동성이 없었다. Indle, methyl-red 시험에서 음성 Table 1. Bichemical reactins f strains islated frm sewage plant drain water at Minragdng in Busan, Krea Strain N. Test KIA HS Indle Prductin Methyl-Red Vges-Prskauer Citrate, Simmns Lysine, Meller s Arginine, Meller s Ornithine, Meller s Mtility Gas frm Glucse

4 166 Hun-Ku Lee Kr. J. Micrbil Table 1. Bichemical reactins f strains islated frm sewage plant drain water at Minragdng in Busan, Krea (cntinued) Acid prductin frm: Glucse Lactse Sucrse Mannitl Dulcitl Salicin Adnitl Insitl Srbitl - Arabinse Raffinse Rhamnse - Trehalse Species E. cli E. cli E. cli E. cli E. cli E. cli E. cli E. cli E. cli E. cli Table 1. Bichemical reactins (cntinued) Strain N. Test KIA H 2 S Indle Prductin Methyl-Red Vges-Prskauer Citrate, Simmns Lysine, Meller s Arginine, Meller s - - Ornithine, Meller s Mtility Gas frm Glucse Acid prductin frm: Glucse Lactse Sucrse Mannitl Dulcitl Salicin - - Adnitl Insitl - - Srbitl Arabinse Raffinse Rhamnse - - Trehalse Species E. cli E. cli E. cli K. pneumniae E. cli E. cli K. pneumniae K. pneumniae E. cli

5 Vl. 45, N. 2 ESBL x 167 ùkü š VP x acetin w ùkü š, Simmn s citrate x ùkü. Lysine decarbxylase x š rnithine decarbxylase x. Glucse w z g. Glucse l ƒ w. w x(transcnjugatin) w ü Plasmid w ESBL y w» w 19³ (ceftazidime r sdium azide ) s ³, E. cli J53 (ceftazidime s sdium azide ) r v ³ w ceftazidime (32 µg/ml) sdium azide (50 µg/ml)ƒ ƒ MacCnkey agar ³ w. w x w» v ³ E. cli J53 ceftazidime (32 µg/ml) ƒ MacCnkey agar y w š, ³ sdium azide (50 µg/ml) y w. K. pneumniae 3³ (³ y c14, c17, c18) E. cli 1³ (³ y c12)ƒ w x w. w w yw w v ³ E. cli J53 ew y w. w³ j y x ùkü 19 ³ w 8 w³ x w, E. cli 16³ 8ƒ w³ w 3 l 8 w ³ ü z w. 5ƒ w³ ü z w ³ (5³ )ƒ ƒ (Table 2). K. pneumniae ³ ƒ 3³» m ù 5~6 w³ ü Table 2. Multidrug resistance patterns f the islates Species Antibitics Strains (Strain N.) E. cli K. pneumniae Nal AN K Te C Am Ce Ge 1 (8) Nal AN K Te Am Ce Ge 1 (4) AN K Am Ce Ge 1 (12) Nal K Am C Ge 3 (1, 2, 6) Nal K Am C Ce 2 (7,10) Nal Am C Ce 2 (3,9) Nal K Am Ce 2 (5,11) Nal Am Ce Ge 1 (15) Nal T Am Ce 1 (16) Nal Am Ce 1 (15) K Te C Am Ce Ge 1 (17) Nal AN Am Ce Ge 1 (14) AN K Am Ce Ge 1 (18) Abbrivatins: ampicillin (AM), amikacin (AN), cephalthin (CF), chlramphenicl (C), gentamicin (GM), kanamycin (K), nalidixic acid (NA), tetracycline (Te) Fig. 2. Iselectric fcusing pints f each ESBL prducing transcnjugants. E. cli C12 prduced the prducts f pi 5.4 and pi 8.3. K. pneumniae (C14, C17, C19) prduced nly ne prduct at pi 8.3. z w (Table 2). w β-lactamase w x w ³ w E. cli pi 5.4 pi 8.3 š(fig. 2), K. pneumniae pi 8.3 w (Fig. 2). Plasmid Plymer Chain Reactin (PCR) w ESBL» w l plasmid w PCR sw» w. E. cli 1,000 bp 800 bp x w TEMx(1,080 bp) SHVx(780 bp). ù K. pneumniae 800 bp w x w SHVx. elutin kit w w DNA w E. cli w TEM-1 SHV-12. K. pneumniae» SHV x w 3³ SHV-12 (Fig. 3). š Ÿ k k wz (extended-spectrum β-lactamase, ESBL) w ü ³ ü wš ƒ l (11, 14) y š š (3, 20). ù ù ù w yy plasmid ESBL ³ š ESBL x (12, 13). plasmid ESBL ³ wš w ESBL x w» w. w ƒ Ÿ w û y š l s ¾. «qp sww, x z lù

168 Hun-Ku Lee Kr. J. Micrbil Fig. 3. The prducts f transcnjugant SHV and TEM-type by PCR. C12 expressing PCR prducts at bth 1 kb (TEM type) and 0.8 kb (SHV type). C14, C17, and C18 expressed at 0.

6 168 Hun-Ku Lee Kr. J. Micrbil Fig. 3. The prducts f transcnjugant SHV and TEM-type by PCR. C12 expressing PCR prducts at bth 1 kb (TEM type) and 0.8 kb (SHV type). C14, C17, and C18 expressed at 0.8 kb f SHV nly. w š s w s» w ü w w y š. Ÿ wš w y«l w œ y w y. Plasmid ESBL ³ ü ³ (Enterbacteriaceae) w E. cli K. pneumniaeƒ ƒ t ³ ³»z ³ ü ³ w (18). ù TEMx, ƒ TEM-52x š š(17), SHV SHV-12ƒ š (11). y TEM x TEM-52 TEM-1ƒ š (12, 13). y SHVx w SHV-12ƒ x š(12, 13), w. ƒ š ù yy SHV x w» SHV-12ƒ ù SHV x ƒ š ƒ (13). SHV-12 SHV-1 35Leu Gln, 238Gly Ser, 240Glu Lys ey pi 8.2 SHV-5a (16). y ü j» w (19). jy x 19³ E. cli 16³, K. pneumniae 3³ š K. pneumniae plasmid ³ ƒ ESBL v E. cli J53 g E. cli 1 ³ K. pneumniae { û. Plasmid ESBL ³ x w š, w ESBL x x š š. ù w š (1, 10). ù x ESBL ³ ƒ y ù y ƒ w ƒ, w» w w, w, w yy š w, û yy y ƒ. x ¾ y E. cli K. pneumniae ü ³(Enterbacteriaceae) p ³ w š š x TEM-1, TEM-52, SHV- 12 w p plasmid ³ ü ƒ w» ƒ¾ ü ü wš ³ w x ƒ. yy l ü ³» e w³ ü z w k (12, 13) plasmid ESBL ƒ ƒ. š x 1.,,,, «x Extended brad-spectrum β-lactamase Escherichia cli Klebsiella pneumniae. 26, Ambuler, R.P., A.F.W. Culsn, N.M. Frere, J.M. Ghuysen, B. Jris, M. Frsman, R.C. Lebversque, G. Tiraby, and S.G. Waley A standard numbering scheme fr class A β-lactamases. Bichem. J. 276, Brinas, L., M. Zarazaga, Y. Saenz, F. Ruiz-Larrea, and C. Trres β-lactamases in ampicillin-resistant Escherichia cli islates frm fds, humans, and haelthy animals. Antimicrb. Agents Chemther. 46, Bush, K Characterizatin f β-lactamases. Antimicrb. Agents Chemther. 33, Bush, K., G.A. Jacby, and A.A. Medeirs A functinal classificatin scheme fr β-lactamases and its crrelatin with mlecular structure. Antimicrb. Agents Chemther. 39, Bush, K. and G. Jacby Nmenclature f TEM β-lactamases. Antimicrb. Agents Chemther. 39, Ewing, W.H The genus Klebsiella. In W.H. Ewing, Edwards and Ewing's Identificatin f Enterbacteriaceae.

7 Vl. 45, N. 2 ESBL x 169 Elsevier, N.Y., USA. 8. Jacby, G.A Genetics f extended-spectrum beta-lactamases. Eur. J. Clin. Micrbil. Infect. Dis. 13, Jacby, G.A. and I. Carreras Activities f β-lactam antibitics against Escherichia cli strains prducing extended-spectrum β-lactamases. Antimicrb. Agents Chemther. 34, Jeng, Y.S., J.C. Lee, H.Y. Kang, H.S. Yu, E.Y. Lee, C.H. Chi, S.H Tae, Y.C. Lee, D.T. Ch, and S.Y. Sel Epidemilgy f nalidixic acid resisitance and TEM-1-and TEM-52-mediated ampicillin resistance f Shigella snnei islates btained in Krea between 1980 and Antimicrb. Agents Chemther. 47, Kim, J., Y. Kwn, H. Pai, J.W. Kim, and D.T. Ch Survey f Klebsiella pneumniae strains prducing extended-spectrum β- lactamase: prevalence f SHV-12 and SHV-2a in Krea J. Clin. Micrbil. 36, Lee, H.K Typing f extended-spectrum β-lactamase (ESBL) prducing Enterbacteriaceae islated frm slaughterhuse in Pusan, Krea. Kr. J. Micrbil. 42, Lee, H.K., H.J. Kim, and G.D. Kim Typing f extendedspectrum β-lactamases f Escherichia cli and Klebsiella pneumniae islated frm rivers in Pusan, Krea. Kr. J. Micrbil. 43, Medeirs, A.A Nscmial utbreaks f multiresistant bacteria: extended- spectrum beta-lactamases have arrived in Nrth America. Ann. Intern. Med. 119, Natinal Cmmittee fr Clinical Labratry Standards Perfrmance standards fr antimicrbial disk susceptibility tests. NCCLS dcument M2-A6. NCCLS, Wayne, Pa, USA. 16 Nueesch-Inderbinen, M.T., F.H. Kayser, and H. Haechlier Survey and mlecular genetics f SHV β-lactamases in Enterbacteriaceae in Switzerland: tw nvel enzymes, SHV-11 and SHV-12. Antimicrb. Agents Chemther. 41, Pai, H.J., S. Lyu, J.H. Lee, J. Kim, and Y. Kwn Survey f extended-spectrum β-lactamase in clinical islates f Escherichia cli and Klebsiella pneumniae: prevalence f TEM-52 in Krea. J. Clin. Micrbil. 37, Philippn, A., R. Labia, and G. Jacbi Extended-spectrum β-lactamases (minireview). Antimicrb. Agents Chemther. 33, Silva, J., R. Gatca, C. Auguilar, Z. Becerra, U. Garza-Rams, M. Velzqez, G. Miranda, B. Leas, F. Slrzan, and G. Echniz Outbreak f infectin with extended-spectrum β-lactamase prducing Klebsiella pneumniae in a Mexican Hspital. J. Clin. Micrbil. 39, Teshager, T., L. Dmnguez, M.A. Mren, Y. Sanz, C. Trres, and S. Cardesa Islatin f an SHV-12 β-lactamase-prducing Escherichia cli strain frm a dg with recurrent urinary tract infectins. Antimicrb. Agents Chemther. 44, (Received May 21, 2009/Accepted June 23, 2009) ABSTRACT : The Types f Extended-Spectrum β-lactamase (ESBL) Prduced by Enteric Bacteria, Klebsiella pneumniae and Escherichia cli Islated frm Sewage f Wastewater Treatment Plant at Minragdng in Busan, Krea Hun-Ku Lee (Department f Micrbilgy, Cllege f Natural Sciences, Pukyng Natinal University, Busan , Republic f Krea) This study was perfrmed t investigate the type f extended-spectrum β-lactamases (ESBL) prduced by bacteria islated frm the sewage f wastewater treatment plant at Minragdng, Suyng-gu in Busan. The facility is lcated at sushi restaurants and guides its drain water t the wastewater treatment plant at Ynghdng, Nam-gu in Busan. Samples were cllected n January, A ttal f 19 strains were selected as ptential ESBL psitive strains thrugh a duble disk synergy test. On the basis f the results frm bichemical tests including indle, methyl-red, Vges-Prskauer, Simmn s citrate, decarbxylase-dihydrlase and sugar-fermentatin tests, the 19 strains were identified with 16 strains f Escherichia cli and 3 strains f Klebsiella pneumniae. Out f 19 strains, 4 transcnjugants against Escherichia cli J53, which is sdium azide resistant recipient strain, were btained. The plasmids islated frm transcnjugants were used fr PCR analysis. The type f each extended-spectrum β-lactamase (ESBL) prduced by the strains was determined n the basis f iselectric fcusing analysis and DNA sequencing. The results indicated that the types f ESBL frm Klebsiella pneumniae were SHV-12 (3 strains), and Escherichia cli was SHV-12/TEM-1 (1 strain), respectively.

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304.fm Journal of the Korean Housing Association Vol. 20, No. 3, 2009 yw s w - û - A Study on the Planning of Improved-Hanok - Focused on Jeon-Nam Province - y* ** z*** **** Kang, Man-Ho Lee, Woo-Won Jeong, Hun