The Korean Journal of Microbiology, Vol. 44, No. 2, June 2008, p. 130-134 Copyrightc2008, The Microbiological Society of Korea 자동배양기를이용한미생물검출 성혜란 김일회 김지연 이종길 정연복 한상배 송석길 * 충북대학교약학대학약동력학국가지정연구실, 충북 BIT 연구중심대학육성사업단 미생물자동배양기를이용한감염성물질의검출은시험적오차의경감은물론분리율향상과시간단축을가능하게하였다. BacT/ALERT 3D 자동배양기는미생물성장시발생되는이산화탄소를비색법으로검출하는장비로서임상시료를이용한미생물배양과검출에이용되어왔다. 자동배양기의효율성을검증하고의약품의무균시험에적용가능한지를평가하기위하여 6 종의세균을이용하여증식및검출특성을분석하였다. 3 종의호기성세균 Pseudomonas aeruginosa, Micrococcus luteus, Bacillus subtilis 와 Staphylococcus aureus 는 1 CFU 가 31.44 시간내에검출되었고, 혐기성균인 Clostridium sporogenes 는 15.96 시간에균의증식이감지되었다. 저성장혐기성세균인 Propionibacterium acnes 는 10 4 CFU 의균수에서검출에 129.36 시간이소요되었다. 이같은결과는전통적직접배양법보다검출감도에있어서 10~10 5 배높고, 검출시간을 2~10 시간단축한것으로서자동배양법이미생물증식과검출에효율적임을말해준다. 따라서본시험에서이용한자동배양법은임상시료에서의감염원진단뿐아니라생물의약품의무균시험에이용가능하다고판단된다. Key words automated culture system, BacT/ALERT, sterility 혈액이나감염조직으로부터감염원을검출하기위하여약 30 년전부터 BACTEC 460 (Becton Dickinson, USA) 을비롯하여자동배양시스템이개발되어왔다. 이러한자동배양기의기본원리는미생물증식에의해생성되는 CO 2 로인해생기는 ph의변화, 산화환원전위변화등을감지하고분석하여미생물의증식여부를판단하는것이다 (1, 8). 가장먼저도입된 BACTEC 460 (Becton Dickinson, USA) 은방사성탄소의분석분석방식으로방사능배지의취급이나폐기에주의가필요하므로이후비방사성분석방식의 BACTEC 660/730/860 (Becton Dickinson, USA), BioArgos (Diagnostics Pasteur, France) 등이개발되었지만, 이들은자동배양은가능할지라도연속적인검출이불가능하여위양성결과가나타나기쉬운단점이있었다 (2,8,16). 이러한단점을보완하여연속모니터링이가능한색변화검출방식의 BacT/ALERT (biomerieux, Inc., USA), 형광검출방식의 Vital (biomerieux vitek, France), BACTEC 9240 (Becton Dickinson, USA), 압력측정방식의 ESP (Difco, USA), o.a.s.i.s (Oxoid Automated Septicaemia Investigation System Unipath, United Kingdom) 등의여러배양기들이개발되었다 (3,6,7,9,13,17). BacT/ALERT 3D 자동배양기 (biomerieux, Inc., USA) 는연속모니터링이가능한최초의자동배양기인 BacT/ALERT의최근모델로미생물의증식에따라 CO 2 양이증가하면배양액의 ph가변화하고배양용기바닥에부착된 LED 발광센서의색깔이청색 ( 음성 ) 에서황색 ( 양성 ) 으로변한다. 이러한변화는매10분마다자 *To whom correspondence should be addressed. Tel: 82-43-261-2817, Fax: 82-43-268-2732 E-mail: songs@chungbuk.ac.kr 동으로탐지자에의해감지되어전극신호로변환되고이는컴퓨터에서자동으로분석함으로써효과적으로미생물을검출하는시스템이다 (5, 6, 13, 16). 최근생물의약품의미생물학적안전성을확보하고자미국 FDA에서는선진화된자동배양시스템을도입하여생물의약품에적합한대체무균시험법으로공인하였으며, 종합병원의임상병리실등에서도전통적인맹계대 (blind subculture) 혈액배양법을자동화된배양시스템으로대체함으로써노력과비용을절감하고신속하고효과적인진단을하고있다 (3,4,10, 11, 12). 혈액제제및생물의약품에서오염, 전파가능한미생물오염원중무균시험법의표준균주를포함한 3종의호기성세균 (Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus luteus), 1종의통성혐기성균 (Staphylococcus aureus) 및 2종의혐기성세균 (Clostridium sporogenes, Propionibacterium acnes) 을대상으로 BacT/ALERT 3D 자동배양기의검출감도와유용성을평가하고자본연구를수행하였다. 균주및배양용시료의제조 재료및방법 American Type Culture Collection (ATCC) 또는 Korean Collection for Type Cultures (KCTC) 에서구입한 Pseudomonas aeruginosa (KCTC 2513), Bacillus subtilis (KCTC 1021), Micrococcus luteus (KCTC 1071), Staphylococcus aureus (KCTC 1927), Clostridium sporogenes (ATCC 11437), Propionibacterium acnes (ATCC 33179) 6종의미생물균주를이용하였다. 먼저자동배양기를이용한시험에사용하기위한표준배양균액를준비하기위해각각의미생 130
Vol. 44, No. 2 자동배양기를이용한무균시험 131 물마다적합한배지에접종하여포화될때까지충분히배양하였다. P. aeruginosa, M. luteus, S. aureus 3종의미생물은 thioglycollate 배지에 (Sigma, USA 또는 Merck, USA) 접종하였고, B. subtilis는 tryptic soy broth (BD, USA) 배지에, 혐기성세균인 Propionibacterium acnes와 C. sporogenes는각각 potato dextrose 배지와 reinforced clostridial medium (Merck, USA) 배지에접종하여배양하였다. 배양한균액에 20% 의 glycerol을첨가하고이를 0.2 ml씩분주하여 70 o C에보관하였다. BacT/ALERT 3D 자동배양 준비된배양용시료는자동배양용호기성표준배지 (SA media; 1.7% of pancreatic digest of casein, 0.3% of papaic digest of soybean meal, 0.035% of sodium polyanetholesulfonate, 0.001% of pyridoxine HCl) 또는혐기성표준배지 (SN media; 1.36% of pancreatic digest of casein, 0.24% of papaic digest of soybean meal, 0.035% of sodium polyanetholesulfonate, 0.00005% of menadione, 0.0005% of hemin, 0.376% of yeast extract, 0.0008% of pyridoxine HCl, 0.08% of pyruvic acid sodium salt) 에접종하여 BacT/ALERT 3D 자동배양장비에서 5일간배양하였다. 호기성표준배지 (SA) 와혐기성표준배지 (SN) 모두배지의성분은일반적으로다양한미생물의배양에널리이용되는 tryptic soy broth가주성분이며혈액샘플의배양이용이하도록하는 sodium polyanetholesulfonate 항응고제가첨가되어있다. 혐기성표준배지 (SN) 는난배양성미생물들의성장을돕는 Hemin, Vitamin K, Pyridoxine 등이추가되어있다. 매시험시마다초저온냉동고에보관된표준배양균액을꺼내각각의배지에 duplicate로접종하고적합한배양온도및조건에서 18시간동안배양하였다. 이를 10배계열희석한후일회용주사기를이용하여 5ml씩호기성표준배지 (SA) 또는혐기성표준배지 (SN) 에접종하고 BacT/ALERT 3D 자동배양기에서 5일간배양하였다. 생균수의측정 BacT/ALERT 3D 자동배양에접종된검체의균수를확인하기위하여가장보편적으로사용되는평판계수법을이용하여생균수를측정하였다. 각배양검체 250 µl를평판고체배지에골고루도말하고항온기에서 2일이상배양한후생성된집락의수를헤아려생균수를측정하였다. 평판고체배지에서형성된집락의수는검체내에포함되어있는집락형성이가능한살아있는세균의숫자 (CFU; colony forming unit) 를의미한다. 자동배양기를이용한세균검출시험에서실제로각배양병에접종된검체의양은 5ml이므로헤아린집락수에 20배를곱하고, 접종한균액의희석배율을곱해실제검체에존재하는초기세균수를구하였다. 직접배양법에의한세대시간측정 자동배양기를이용한검출방법과직접배양법에의한미생물의검출을비교할수있는객관적지표를설정하기위하여직접배 양시각미생물의세대시간 (generation time) 을구함으로써검출감도와검출한계를측정하고자하였다. 배양하는동안혐기적조건에서수시로배양액을채취하는것이불가능한혐기성균을제외하고 P. aeruginosa, M. luteus, S. aureus 3종의미생물은 thioglycollate (FTM) 배지, B. subtilis는 tryptic soy broth (TSB) 배지에접종하여 37 o C에서배양하면서매 30분마다흡광도를측정하였다. 측정한흡광도를바탕으로증식이활발히이루어지는지수기 (exponential phase) 의초기균수 (A) 와말기균수 (B) 를구하고이두시점사이의시간 (T) 값들을 Tg=T log2(1/logb 1/logA) 공식에대입, 계산하여각미생물의증식세대시간 (Tg) 을구하였다 (Table 2). 그람염색및현미경관찰 BacT/ALERT 3D 자동배양기에의한미생물의검출결과가신뢰성있는결과인지확인하기위하여양성으로판정된각각의배양병으로부터균을일부채취하여그람염색법에의해염색하고현미경 (Leica RXA2, Germany) 으로균의모양을관찰함으로써검출된균을확인하였다. 결과및고찰 임상시료나생물의약품에서의미생물검출은전통적으로직접배양법에의존해왔다. 그러나직접배양법은시험자의조작에따른개인편차가크고긴시간이요구되므로무균시험이나미생물검출법이다양한생물학적원리를응용하여개선되어왔다. 최근에는분자생물학적기법이적용된핵산분석법 (NAT; nucleic acid technology), 중합효소연쇄반응 (PCR; polymerase chain reaction) 등을이용한검출법이적용되어왔다 (7). 그러나이들기법은단일대상미생물유전자의특정염기서열을검출하는기법이므로다양한미생물을단일시험으로검출하기어려운단점을지닌다. 따라서검출대상의다양성때문에최근에직접배양법에의한미생물검출이주목받고있다. 하지만직접배양법역시배양이까다로운미생물의검출이어렵고노력과시간이많이소요된다는단점이있다 (1, 3, 8). 따라서이러한단점을보완하고미지의다양한오염원을자동으로배양가능하며미생물의성장을연속적으로검출할수있는자동배양기가이용되어오고있다. 자동배양기는이미초기스크리닝단계로임상시료분석, 백신의무균시험, 식품분야및혈액제제의미생물검출및무균시험법으로이용되고있다 (6, 14, 15). 그러므로 3종의호기성세균 (Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus luteus), 1종의통성혐기성균 (Staphylococcus aureus), 2종의혐기성세균 (Clostridium sporogenes, Propionibacterium acnes) 을대상으로직접배양법에따른미생물검출법과호기성표준배지 (SA) 및혐기성표준배지 (SN) 를사용한 BacT/ALERT 3D 자동배양기를이용한미생물검출법의효율성및검출감도를비교평가하고자하였다. 각미생물의표준배양균액을재배양하여호기성또는통성혐기성균은약 0.01 CFU부터약 1,000 CFU까지계열희석하였고혐기성균은약 0.01 CFU부터약 1,000,000 CFU까지계열희석
132 Hyeran Sung et al. Kor. J. Microbiol 하였다. 이를자동배양용호기성표준배지 (SA) 및혐기성표준배지 (SN) 에각각반복배수로접종하고 BacT/ALERT 3D 자동배양에서 5일간배양하여검출감도를확인하였다. 직접배양시배양조건이까다로운혐기성세균인 Propionibacterium acnes와 C. sporogenes은 5일이상배양, 관찰하였다. 자동배양기를이용한시험결과, 통성혐기성균인 S. aureus와호기성균인 B. subtilis는호기성표준배지 (SA) 와혐기성표준배지 (SN) 에서모두약 1 CFU 까지검출가능하였다. S. aureus는호기성표준배지 (SA) 에서 18 Table 1. Growth detection of 6 microorganisms in both aerobic (SA bottle) and anaerobic (SN bottle) culture conditions Growth condition SA SN B. subtillis Aerobic C. sporogenes Anaerobic M. luteus Aerobic P. acnes Anaerobic P. aeruginosa Aerobic S. aureus Facultative * ; detected, ; not detected 시간, 혐기성표준배지 (SN) 에서는 21.84시간후에검출되었으며, B. subtilis는호기성표준배지 (SA) 에서 12.32시간, 혐기성배지 (SN) 에서는 18.36시간후에검출되었다. 그러나 P. aeruginosa와 M. luteus는혐기성표준배지 (SN) 에서는전혀검출되지않았고, 호기성표준배지 (SA) 에서각각약 1CFU까지 18.48시간, 31.44 시간후에검출되었다. 혐기성세균인 Propionibacterium acnes와 C. sporogenes은모두호기성표준배지 (SA) 에서는전혀검출되지않았고혐기성표준배지 (SN) 에서 C. sporogenes는약 1CFU까지 15.96시간후에검출되었다. 직접배양시배양조건이매우까다로운 Propionibacterium acnes는 129.36시간후에약 10,000 CFU 이상에서검출되었다 (Table 1 and Fig. 1). 직접배양법의미생물검출감도와비교하기위하여자동배양기를이용한미생물검출법의검출감도를다음의방법에따라환산하여직접배양법과비교하였다. 각미생물의최초접종균수와세대시간 (Tg), 그리고약 1CFU로접종한미생물이 BacT/ALERT 3D 자동배양기의호기성표준배지 (SA bottle) 에서양성으로판정되는데까지걸린시간 (Tb) 을이용한 Pb=2 (Tb/Tg) 공식에근거하여양성판정시점의실제균수 (Pb) 를구하였다 (Table 3). 이때, 각미생물의세대시간 (Tg) 은 Table 2를참고하였다. 이결과, BacT/ALERT 3D 자동배양기를이용한미생물을검출법으로 B. subtilis는 2.98 10 3 개의균을 12.12시간에검출할수있었고, S. aureus는 18시간내에 1.05 10 6 개의균을, P. aeruginosa는 18.48시간내에 1.52 10 6 개의균을검출하였다. 또한, M. luteus는 7.71 10 7 개의균을 31.44시간내에검출가능하였다. 전통적으로미생물검출에이용되어온직접배양법은육안으로배양액의탁도를관찰하여미생물의검출여부를판정하는것이일반적이다. 보통배양액의탁도를육안으로보아미생물의증식 Table 2. Generation time and viable cell numbers per OD 600 Detection time (hr) 100 CFU 10 CFU 1 CFU Bacillus subtilis 10.08 11.04 12.12 Clostridium sporogenes 12.24 14.88 15.96 Micrococcus luteus 25.32 29.04 31.44 Pseudomonas aeruginosa 15.48 18.00 18.48 Staphylococcus aureus 15.60 17.28 18.00 Fig. 1. Detection time of 5 microorganisms in BacT/ALERT 3D automated culture system at 37 o C under aerobic (SA) or anaerobic (SN) bottles. The indicated number of cells from subculture of B. subtilis, M. luteus, P. aeruginosa, and S. aureus were inoculated in standard aerobic (SA) bottles and cultured until flagged as positive or for 5 days. The anaerobic bacteria C. sporogenes was inoculated in standard anaerobic (SN) bottles and cultured until flagged as positive or for 7 days. 1 CFU, inoculated about 1 cell; 10 CFU, inoculated about 10 cells 100 CFU, inoculated about 100 cells. Generation time (min) Viable cell numbers (cells/od 600 ml) B. subtilis 63 1 10 9 M. luteus 72 1 10 9 P. aeruginosa 54 1 10 10 S. aureus 54 1 10 10 Table 3. Detection sensitivity of 4 bacteria cultured by conventional culture method and BacT/ALERT 3D automated culture system Conventional culture method BacT/ALERT 3D system Tc (hr) Pc Tb (hr) Pb B. subtilis 21.5 5 10 8 12.12 2.98 10 3 M. luteus 22.8 5 10 8 31.44 7.71 10 7 P. aeruginosa 20.9 5 10 9 18.48 1.52 10 6 S. aureus 22.8 5 10 9 18.00 1.05 10 6 * Tc; Time to reach at 0.5 OD 600, Pc; Probable number of cells at time, Tc, Tb; time to automatic detection, Pb; Probable number of cells at time Tb
Vol. 44, No. 2 자동배양기를이용한무균시험 133 Fig. 2. Growth curves of 4 microorganisms under aerobic conditions. s was inoculated about less than 0.1 of optical density at 600 nm and cultured with rapid shaking. At every 30 min, the 1 ml samples of culture were taken and measured the optical density by spectrophotometer at 600 nm wavelength. And the results were plotted the optical density (OD 600 ) vs. time (hr). B. subtilis and M. luteus was cultured in tryptic soy broth and thioglycollate media at 30, respectively. P. aeruginosa and S. aureus were cultured in thioglycollate media at 37 o C. 이확인되는시점의흡광도가대략 OD 600 =0.5 정도이므로이시점을양성으로판정되는시점으로결정하였다. 이를근거로직접배양법에의한미생물검출법의검출감도를다음과같이구하였다. 흡광도측정이불가능한혐기성미생물을제외한 4종의미생물을각미생물의최적배양조건에서 OD 600 =0.1 이하 ( 약 1 CFU 접종시와같은조건 ) 로접종한후배양하면서매 30분마다 600 nm 파장에서흡광도값을측정하여각미생물의성장곡선을나타내었다 (Fig. 2). 성장곡선을토대로각미생물의세대시간 (Tg) 및 OD 600 =1당세균수, 그리고 OD 600 =0.5가되기까지걸리는시간즉, 직접배양법에의해양성으로판정되는데걸리는시간 (Tc) 을구하였다. 또한직접배양법에의해양성으로판정되는시점 (OD 600 =0.5) 의실제균수 (Pc) 는각미생물의 OD 600 =1 당세균수 (cells/od 600 =1) 를이용해서구하였다 (Table 2 and 3). 직접배양법으로배양한결과 S. aureus와 M. luteus는배양 22.8 시간후에양성으로판별되었으며이시점의실제균수는각각약 5 10 9, 약 5 10 8 정도였다. P. aeruginosa 역시같은조건에서약 5 10 9 정도증식하였을때양성으로판별되었으며, 양성으로판정되기까지는 20.9시간이걸렸다. B. subtilis는배양후 21.5시간이지나서양성으로판별되었고, 이때의실제균수는약 5 10 8 정도였다 (Table 3). 미생물의특성상직접배양법에의한미생물의검출감도측정이불가능한혐기성미생물을제외한 4종의미생물에대하여호기성표준배지 (SA) 를사용하는 BacT/ALERT 3D 자동배양기에의해양성으로판정될때까지걸리는시간및검출감도를직접배양법과비교한결과, 자동배양기를이용한검출법이직접배양법보다약 10~10만배더검출감도가높았으며검출시간도약 2~12시간정도단축하였다 (Table 3). 그러므로자동배양기에의한검출법이적어도호기성미생물의검출에있어서는직접배양법에비해훨씬더감도가높은효율적인방법이라고판단된다. 또한, 혐기성균에대해서도검출시간이직접배양시와거의유사하거나단축되는결과를관찰하였다. 호기성세균이자동배양기에서검출되는시점에서의 OD 600 값은육안으로관찰이용이하지않은 0.1 이하였으므로자동배양기의검출능은감지시스템의우수성에서기인한다고판단된다. 또한, 세포배양액에 spiking하여일정양의미생물에대한검출감도를비교하여세포배양액이미치는영향을검토한결과, 세포배양액에항생물질이포함되어있지않는한미생물검출에어떠한영향도미치지않음을확인할수있었다. 그러므로혈액제제뿐만아니라세포배양으로제조되는각종생물의약품에대한미생물검출법으로도적용가능함을확인하였다. 감사의말 We appreciate the technical support of Moo-Jin Kim for operation of BacT/ALERT 3D (biomerieux Korea Inc). 이논문은 2006년충북대학교학술연구지원사업의연구비지원에의하여수행되었음 (This work was supported by the research grant of the Chungbuk National University in 2006.). 참고문헌 1. Alfa, M., W. Sanche, S. Roman, Y. Fiola, P. Lenton, and G. Harding. 1995. Continuous quality improvement for introduction of automated blood culture instrument. J. Clin. Microbiol. 33, 1185-1191. 2. Courcol, T.J., M. Duhamel, A. Decoster, V.M. LeMaire, M.L. Rastorgoueff, D. Ochin, and G.R. Martin. 1992. BioArgos: a fully automated blood culture system. J. Clin. Microbiol. 30, 1995-1998. 3. Gimenez, M., C. Part, X. Valles, and L. Matas. 2002. Evaluation of the VITAL (biomeriuex) automatic blood culture system using blind subcultures. Clin. Microbiol. Infect. 8, 222-228. 4. Hardy, D.J., B.B. Hulbert, and P.C. Migneault. 1992. Time to detection of positive BacT/Alert blood cultures and lack of need for routine subculture of 5- to 7 day negative cultures. J. Clin. Microbiol. 30, 2743-2745. 5. Kocoglu, M.E., A. Bayram, and I. Balci. 2005. Evaluation of negative results of BacT/Alert 3D automated blood culture system. J. Microbiol. 43, 257-259. 6. Mirrett, S., K.E. Hanson, and L.B. Reller. 2007. Controlled clinical comparison of VersaTREKand BacT/ALERT blood culture systems. J. Clin. Microbiol. 45, 299-302. 7. Qian, Q., Y.W. Tang, C.P. Kolbert, C.A. Torgerson, J.G. Hughes, E.A. Vetter, W.S. Harmsen, S.O. Montgomery, F.R. Cockerill, 3rd., and D.H. Persing. 2001. Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rrna gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results. J. Clin. Microbiol. 39, 3578-3582. 8. Reimer, L.G., M.L. Wilson, and M.P. Weinstein. 1997. Update on detection of Bacteremia and Fungemia. Clin. Microbiol. Rev. 10, 444-465. 9. Rohner, P., B. Pepey, and R. Auckenthaler. 1995. Comparison of BacT/Alert with signal blood culture system. J. Clin. Microbiol.
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