Enterococcus faecalis biofilm의 Chlorhexidine에 대한 저항성의 비교연구

Abstract

[한글]Enterococcus faecalis는 근관치료의 실패에 있어서 중요한 원인인자로 생각되고 있다. 세균과 관련하여, 또 다른 중요한 문제는 근관 내에서 세균은 biofilm을 형성하며 존재하여 약제에 대한 저항성을 가진다는 사실이다. 이 실험의 목적은 실험실상에 재현된 E. faecalis biofilm의 Chlorhexidine(CHX)에 대한 저항성을 planktonic 상태의 세균의 저항성과 비교하고, 나아가 CHX의 농도 및 처리시간을 달리하여 그 영향을 비교 분석하는 것이다.이 실험은 Luppens가 제시한 방법으로 E. faecalis biofilm 을 각각 1-day, 7-day동안 형성하고, 추가로 planktonic cell group을 형성하여, 각각 group간에 CHX 처리시간을 달리하여 CHX에 대한 저항성을 비교하는 방법으로 이루어졌다.7-day group에서의 세균 감소량이 planktonic이나 1-day group에 비하여 유의차있게 나타났으므로, 세균의 약제에 대한 저항성에 대한 실험에 있어서 7-day 동안 배양한 biofilm의 사용이 추천된다. 또한 E. faecalis biofilm이 형성되어 있는 근관내에서는 단시간의 CHX 근관내 세척제 사용보다는 장시간 효과가 발현될 수 있는 근관내 약제로써의 CHX 사용이 추천된다.

[영문]I. Objectives: Enterococcus faecalis has been suggested to be an important etiological agent in endodontic failure. The purpose of this study was to evaluate the antibacterial efficacy of chlorhexidine on the biofilm, of E. faecalis depending on the time of application and on the concentration of chlorhexidine , antimicrobacterial efficacy of biofilm with planktonic cell was compared.

II. Materials & Methods: Experimental specimens were divided to three groups. Group I was planktonic cell group ( as control ) and Groups II & III were the Biofilm groups. Biofilms were grown in a simple apparatus that consisted of a vessel containing 1/10 Blood Heart Infusion (BHI), a micro pump, a culture container (perfusion culture container; Minucells and Minutissue. Bad Abbach, Germany), and a vessel with waste, all connected by silicon tubing. Twenty coupons (diameter 13mm), each held by a coupon carrier (Minusheet; Minucells and Minutissue) were placed in the culture container and used as surfaces for biofilm formation. All coupons and spports were cautoclaved before use. In group II, biofilms were allowed to develop for 24 hours at 30 degrees with a constant nutrient flow, and in group III, the biofilms were obtained by same method for 7days. In group II & III, Each specimens were removed by container and washed by 1/10 BHI for remove unattatched cells. And then, the specimens were soaked with 3ml test solution(0.2% CHX, 2% CHX, DW(control)) for determined time (4min, 8min, 15min). These coupons were Detoxified with solution (containg 3% Tween 80 + 0.3% L- a Lecithin) for 1min and vortexed for 1min to re-suspend the organisms. Ten-fold serial dilution were generated in DW by 1ml of each suspensions. Each dilution was seeded onto BHI agar plates , and the colony-forming units copunted after 24h of incubation.

III. Results: The difference in gradients of bacterial killing between group I (planktonic cell group) and group III (7-day biofilm group) was significant (p<0.0001).IV. Conclusion: E. faecalis 7-day biofilm groups have more resistant to CHX comparing with 1-day biofilm groups or planktonic cell groups