대한임상검사학회지 : 38 권제 3 호, 173-178, 2006 Multiplex Polymerase Chain Reaction 을이용한 Extended-Spectrum β-lactamase 생성 Klebsiella pneumoniae 균주의검출 진주보건대학임상병리과 양병선 Detection of Extended-Spectrum β-lactamase Producing Klebsiella pneumoniae by Multiplex Polymerase Chain Reaction Byoung-Seon Yang Department of Clinical Pathology, Jinju Health College, Jinju 660-757, Korea The production of extended-spectrum β-lactamases (ESBL S ) is the main mechanism of bacterial resistance to third-generation cephalosporins and monobactams, whose prevalence varies depending on the different geographical areas. In the last years it has increased notably to the point of being considered a health problem of great importance. The characterization of the ESBLs producing Klebsiella penumoniae strains present in clinical isolates is time-consuming. I describe here the development of a new system, which consists of a multiplex PCR. I found 51 K. pneumoniae strains to be presumptive strains ESBLs producers by clinical and laboratory standards institute (CLSI) guidelines. The double disc synergy test showed 47 positive K. pneumoniae, which were K. pneumoniae isolates. All ESBLs producing K. pneumoniae strains were resistant to antibiotic amikacin, gentamicin and ciprofloxacin. By multiplex PCR analysis, bla TEM gene in 17 strains 44 bla SHV genes and bla CTX genes in 33 strains were identified. In this study, the multiplex polymerase chain reaction (PCR) assay was a good method to detect and differentiate ESBLs producing K. penumoniae strains in clinical isolates. Key words : Klebsiella penumoniae, Extended-spectrum β-lactamases, Multiplex polymerase chain reaction 1)II. 서 론 광범위내성 β-lactamases 세균은 extended spectrum cephalosporin(cefotaxime, ceftazidime 등 ), aztreonam 및 oxyimino-β-lactams 뿐만아니라, 이전세대의 penicillin 과 cephalosporins 항생제에내성을나타내는세균이다. Klebsiella pneumoniae는 2% 에서 5% 정도가원내감염의 교신저자 : 양병선, ( 우 )660-757 경남진주시상봉서동 1142 진주보건대학임상병리과 Tel : 055-740-1851, 016-835-7191 E-mail : ybseon@jhc.ac.kr 원인균으로잘알려져있으며, 특히하부호흡계와비뇨기계에감염을일으키는것으로잘알려져있다 (Gazouli 등, 1997; Kil 등, 1997; Pnaiara 등, 2000; Silva 등, 2001). K. pneumoniae가 cephalosporins계에다제내성을나타내는것은 1980년대초에발견되었으며 (Bingen 등, 1993), 1986년에그추세가점점더증가하였다. ESBL 생성균주의역학적조사는이미실시해오고있으며, 광범위내성 K. pneumoniae 분리균주의감염과집락화에관여하는몇몇위험인자들의연구도시행되어져왔다 (Macrae 등, 2001). 173
PCR을기초로하는다른 typing 방법으로 randomly amplified polymorphic DNA(RAPD) 분석은보다더빠르고, 수행이수월한방법이다. 그러나, typing에있어비교성이나, 재현성, 분석력그리고이러한방법의효율성의전반적인조사는아직이루어지지않고있다. Pulsed-field gel electrophoresis(pfge) 분석에따른유전자의분석방법으로다른균주로부터의 K. pneumoniae의균주를비교동정할수있다. 그러나이방법은기술적으로어려움이있으며, 시간이많이소모되고, 특수한장비를필요로한다. 그러므로, 이러한방법들이널리이용되고있기는하지만, ESBL 생성 K. pneumoniae의 typing에있어, 제일좋은분석력이나재현성을나타내는방법을확립하여야만한다 (Monica 등, 2004). Multiplex PCR 방법을이용한 ESBL 생성 K. pneumoniae의 typing 방법은 ESBL 생성 K. pneumoniae 임상분리균주의검출과분석의기술적인어려움이나, 시간적인소모그리고특수한장비가있어야 되는문제점을보완하고, 분석력이나재현성이좋은방법으로잘알려져있다. 본연구에서는 ESBL 생성 K. pneumoniae 균주를대학병원으로부터분리하여 double disc synergy test(ddst) 를실시하고 TEM, SHV, CTX 유전자를대상으로하여 multiplex PCR을실시하여표현형과유전자형을비교분석하였다. II. 재료및방법 1. 재료 3개의대학병원 ( 충북, 충남그리고경상대학병원 ) 에서 VITEK기기 (Biomerieux, Marcy, France) 로 ESBL 생성 K. pneumoniae로동정된 51개의균주를대상으로하였다 (Table 1). Table 1. List of ESBL producing K. pneumoniae isolates used in this study Strains Source AST AST Strains Source ATM CIP CRO CAZ CF AZT CIP CRO CAZ CF CB1 CBUH R I S R R CN14 " R R S R R CB2 " R S S R R CN15 " R R I R S CB3 " R R S R S CN16 " R I I R R CB4 " R R R R R CN17 " R R I R R CB5 " R R S R R CN18 " R R S R S CB6 " R R S R R CN19 " R R S R S CB7 " R S I R R CN20 " R S S R S CB8 " R I R R R CN21 " R R I R S CB9 " R R S R R KS1 KSUH R R I R S CB10 " R R S R S KS2 " R R R R S CB11 " R I I R R KS3 " R R R R S CB12 " R R R R R KS4 " R S R R S CB13 " R R I R R KS5 " R S R R S CN1 CNUH R R I R R KS6 " R R R R S CN2 " R R I R R KS7 " R R R R S CN3 " R R I R R KS8 " R R R R R CN4 " R R I R R KS9 " R R R R R CN5 " R R S R R KS10 " R R R R S CN6 " R R S R S KS11 " R R R R S CN7 " R R S R S KS12 " R R R R S CN8 " R R I R S KS13 " R R R R S CN9 " R I S R R KS14 " R R R R S CN10 " R S S R R KS15 " R R R R S CN11 " R S I R R KS16 " R R R R S CN12 " R S S R R KS17 '' R R R R S CN13 " R R S R R CBUH, Chungbuk University Hospital; CNUH, Chungnam University Hospital; KSUH, Kyoungsang University Hospital; AST, antibiotic susceptibility test; ATM, aztreonam; CIP, ciprofloxacin; CRO, ceftriaxone; CAZ, ceftazidime; CF, cephalothin; R, resistant; I, intermediate; S, sensitive. 174
2. 균주로부터의 DNA 분리 51개의 K. pneumoniae 균주를 QIAamp DNA Mini kit(cat. No. 51304) 를이용하여 DNA를추출하였다. 순수분리된 51개균주를 LB media(bd, Detroit, USA) 를이용하여 37 shaking incubator에서하루동안진탕배양하였다. 배양액을 13,000 rpm에서 3분간원심후상등액은버리고침전물을이용하여 QIAamp DNA Mini Kit가제시한방법으로 DNA를추출하였다. 3. Double disc synergy 시험세균부유액을 Mueller-Hinton 한천 (BD, Detroit, USA) 에고르게접종한후, 배지의중앙에는 ticarcillin-clavulanic acid(tim) 또는 cefotaxime-clavulanic acid(ctx/ CLA) 를, 주위에는 30 μg의 cefotaxime(ctx), ceftaxidime(caz) 및 aztreonam(atm) 디스크를놓되, 중앙과주변디스크의가장자리간격은 2 cm가되도록하였다. 접종배지는 37 항온기에 18시간배양후결과를판독하였다. CTX, CAZ 및 ATM 디스크에의한억제대가 TIM 또는 CTX/CLA 디스크쪽으로커지거나없던억제대가새로생기면양성으로판정하였다. 4. Multiplex PCR을위한 primer의합성 Table 2. Primer sets used in charactering β-lactamases Gene TEM SHV CTX Primer sequence 5'-ATAAAATTCTTGAAGACGAA-3' 5'-GACAGTTACCAATGCTTAAT-3' 5'-TGGTTATGCGTTATATTCGC-3' 5'-GGTTAGCGTTGCCAGTGCT-3' 5'-TTTGCGATGTGCAGTACCAG-3' 5'-GATATCGTTGGTGGTGCCAT-3' PCR product (bp) 1080 870 543 70 volt, 100 ma로 1시간동안전기영동을실시한다음, agarose gel을 ethidium bromide(0.5 μg/ml) 수용액에서 20분간염색하고증류수에 10분간탈색하여 UV transilluminator로관찰하고, polaroid camera로사진촬영하였다. III. 결 1. Double disk synergy 시험 과 Double disk synergy 확인시험에서는충북대병원의경우 CB5 균주, 충남대병원의경우 CN5 균주, 경상대병원의경우 KS11, KS14 균주가음성으로나타나총 47 균주가양성으로판명되었다 (Fig. 1). β-lactamase의특성을분석하기위한 primer를다음과같이제작하였다 (Table 2). 5. Multiplex PCR을이용한 ESBL 유전자의증폭 Multiplex PCR을위한 PCR 혼합액 dntp( 각기 2.5 mm), 10 PCR buffer 2 ul, primer 3쌍 (TEM, SHV, CTX) 10 pmol 각각 1 μl씩넣었고, genomic DNA(25 μg) 1 μl, Taq DNA polymerase(2 unit) 1 μl, 최종반응액의총량은증류수로 20 μl 되게하였다. DNA thermal cycler (Perkin Elmer, Wellesley, USA) 에서 94 에서 1분간변성, 58 에서 1분간접합, 72 에서 1분간확장의순서로 35회를시행하고 72 에서 10분간최종확장하였다. 6. PCR 산물의확인 PCR 증폭산물을 2% agarose gel에 1 TAE buffer에서 2. ESBL 유전형 Multiplex PCR에서는충북대병원의경우 CB5 균주, 경상대병원의경우 KS7, KS11 균주가음성으로나타나총 48균주가양성으로나타났고, 33% 가 TEM으로, 86% 가 SHV 그리고 67% 가 CTX로분류할수있었다 (Table 3. Fig. 2-4). IV. 고찰 최근에 β-lactam 항균제에내성인세균에의한감염의증가가중요한문제로대두되고있다. 세균이 β-lactam 항균제에대한내성을획득하는기전은 β-lactamase 생성, penicillin-binding protein의변성, β-lactam 항균제의세포막투과성저하, 세포밖으로의항균제유출등다양하며, 장내세균은흔히 β-lactamase 생성에의하여 β-lactam 175
Strains (A) (B) Fig. 1. Detection of ESBL production of CB1 strain in double disc synergy tests. (A) discs. left, CTX; centre, TIM; right, CAZ (B) discs. left, CTX; centre, CTX/CLA; right, CAZ Table 3. Genotyping of ESBL producing K. pneumoniae from university hospital isolates TEM/SHV/CTX type DDST (CAZ+TIM) Strains TEM/SHV/CTX type DDST (CAZ+TIM) CB1 TEM, SHV, CTX + CN14 SHV, CTX + CB2 TEM, SHV, CTX + CN15 SHV, CTX + CB3 SHV, CTX + CN16 SHV + CB4 SHV, CTX + CN17 SHV, CTX + CB5 - - CN18 TEM + CB6 SHV + CN19 SHV, CTX + CB7 SHV, CTX + CN20 SHV, CTX + CB8 SHV, CTX + CN21 SHV, CTX + CB9 SHV + KS1 TEM, SHV, CTX + CB10 SHV, CTX + KS2 TEM, SHV, CTX + CB11 TEM, SHV, CTX + KS3 SHV + CB12 SHV + KS4 TEM, SHV, CTX + CB13 TEM + KS5 TEM, SHV, CTX + CN1 TEM, SHV, CTX + KS6 TEM, SHV, CTX + CN2 TEM, SHV, CTX + KS7 - + CN3 SHV, CTX + KS8 TEM, SHV, CTX + CN4 SHV, CTX + KS9 SHV + CN5 SHV - KS10 SHV + CN6 TEM, CTX + KS11 - - CN7 SHV, CTX + KS12 TEM, SHV, CTX + CN8 SHV, CTX + KS13 TEM, SHV, CTX + CN9 SHV, CTX + KS14 SHV - CN10 SHV + KS15 TEM, SHV, CTX + CN11 SHV, CTX + KS16 TEM, SHV, CTX + CN12 SHV, CTX + KS17 SHV + CN13 SHV, CTX + DDST, double disc synergy test; CAZ, ceftazidime; TIM, ticarcillin-clavulanic acid 176
Fig. 2. Electrophoresis of the amplified products of TEM, SHV, CTX genes by a mutiplex PCR in a 2% agarose gel. Lane M, 100-bp DNA ladder; Lanes 1 to 13, Chungbuk University Hospital isolates. The amplified products and their sizes are indicated on the left. M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 TEM(1080bp) SHV(87bp) CTX(543bp) Fig. 3. Electrophoresis of the amplified products of TEM, SHV, CTX genes by a mutiplex PCR in a 2% agarose gel. Lane M, 100-bp DNA ladder; Lanes 1 to 21, Chungnam University Hospital isolates. The amplified products and their sizes are indicated on the left. M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 TEM(1080bp) SHV(87bp) CTX(543bp) Fig. 4. Electrophoresis of the amplified products of TEM, SHV, CTX genes by a mutiplex PCR in a 2% agarose gel. Lane M, 100-bp DNA ladder; Lanes 1 to 17, Gyoungsang University Hospital isolates. The amplified products and their sizes are indicated on the left. 177
항균제에대한내성을획득한다. 호흡기감염, 요로감염증, 패혈증, 수막염등다양한감염증의발생원인이며, 화학요법제에저항성이강하기때문에기회감염증과균교대증의원인이되는 K. pneumoniae는현재지난 20년동안가장현저하게증가한원내감염원인균으로대두되고있으며, 3세대 cephem계다제내성을나타내는 ESBL로증가되고있는추세이다 (Galani 등, 2002). 지금까지의 ESBL생성 K. pneumoniae의검출에는표현형특성을이용하여검출하는방법이널리알려져있다. 그러나, 표현형적방법은검출에시간이많이소요된다는것과, 접종량이정확하지않으면정확한결과를얻기가힘들다. 또한, ESBL 매개화학요법제내성유전자의정확한동정은, 병원에서의원내감염의감시와역학에대단히중요한역할을할수있다. Chia 등 (2005) 은 K. pneumoniae의 ESBL 생성유전자를검출하는데 multiplex PCR 방법을이용하면유전자들을검출하는데있어서가장정확하고재현성이있으며또한빠른시간에검사가가능한방법이며, 지역적인분포도뿐만아니라다른균주에서의 ESBL 생성유전자의검출에적용하는것도아주용이하게접근할수있는방법이라고주장하였다. 본실험결과표현형적방법으로분리된 ESBL 생성 K. pneumoniae 51 균주중 CN5, KS14 균주의경우 DDST 결과가음성으로나타났으나 SHV 유전자를가지고있었고, KS7 균주의경우 DDST 결과는양성으로나타났으나 TEM, SHV, CTX 유전자검출에서음성으로나타나 ESBL 유전자자형에대한보다많은연구가필요하다. 그리고, 대부분의 ESBL 생성 K. pneumoniae 균주에서는 SHV 유전자를포함하고있는것으로나왔으며, 이는 ESBL 유전자의검출과이유전자의 monitoring에중요한역할을할것이라생각되어진다. 따라서, 분자생물학적기법인 multiplex PCR을이용한 ESBL 내성유전자의검출은표현형적방법에서의문제점의해결이나, 원내감염의역학에아주유용한방법이라사료된다. 참고문헌 1. Bingen EH, Desjardins P, Arlet G, Bourgeois F, Mariani-Kurkdjian P, Lambert-Zechovsky NY, Denamur E, Philippon A, Elion J. Molecular epidemiology of plasmid spread among extended broad-spectrum beta-lactamase producing Klebsiella pneumoniae isolates in a pediatric hospital. J Clin Microbiol 31:179-184, 1993. 2. Chia JH, Chu C, Su LH, Chiu CH, Kuo AJ, Sun CF, Wu TL. Development of a Multiplex PCR and SHV Melting-Curve Mutation Detection System for Detection of Some SHV and CTX-M β-lactamases of Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae in Taiwan. J Clin Microbiol 43:4486-4491, 2005. 3. Galani I, Russell EG, Tymms M, Roper EJ, Grayson ML, Turnidge J. Transferable plasmid mediating resistance to multiple antimicrobial agents in Klebsiella pneumoniae isolates in Greece. Clin Microbiol Infect 8:579-588, 2002. 4. Gazouli M, Kaufmann ME, Tzelepi E, Dimopoulou H, Paniara O, Tzouvelekis LS. Study of an outbreak of cefoxitin-resistance Klebsiella pneumoniae in a general hospital. J Clin Microbiol 35:508-510, 1997. 5. Kil KS, Darouiche RO, Hull RA, Mansouri MD, Musher DM. Identification of a Klebsiella pneumoniae strain associated with nosocomial urinary tract infection. J Clin Microbiol 35:2370-2374, 1997. 6. Macrae MB, Shannon KP, Rayner DM, Kaiser AM, Hoffman PN, French GL. A simultaneous outbreak on a neonatal unit of two strains of multiply antibioticresistant Klebsiella pneumoniae controllable only by ward closure. J Hosp Infect 49:183-192, 2001. 7. Monica C, Sonia MT, Alejandro P, Angeles BN, David D, Francisca V, Rosa M, German V. Risk Factors for Colonization and Infection in a hospital Outbreak Caused by a Strain of Klebsiella pneumoniae with Reduced Susceptibility to Expanded -Spectrum Cephalosporins. J Clin Microbiol 42:42-49, 2004. 8. Pnaiara E, Platouka H, Dimopoulou E, Tzelepi B, Miriagou, Tzouvelekis LS. Diversity of beta-lactam resistance levels among related Klebsiella pneumoniae strains isolated in an intensive care unit. J Chemother 12:204-207, 2000. 9. Silva J, Gatica R, Aguilar C, Becerra Z, Garza-Ramos U, Velazquez M, Miranda G, Leanos B, Solorzano F, Echaniz G. Outbreak of infection with extendedspectrum beta-lactamase producing Klebsiella pneumoniae in a Mexican hospital. J Clin Microbiol 39:3193-3196, 2001. 178