자일리톨 처리농도에 따른 자일리톨 감성균주와 내성균주의 gtf 유전자 발현과 부착력 비교

임상욱 ( Sang Uk Im ),안서영 ( Seo Young An ),최연희 ( Youn Hee Choi ),송근배 ( Keun Bae Song ) 대한예방치과·구강보건학회 2012 大韓口腔保健學會誌 Vol.36 No.2

Objectives: Xylitol is an effective anticarious natural sugar substitute, by inhibiting the virulence of Streptococcus mutans (S. mutans). However, long-term xylitol consumption leads to an emergence of the xylitol-resistant (XR) strains. This study aimed to confirm the general characteristics, mRNA expression of gtf genes, and adhesive ability of the xylitol-sensitive (XS) and XR S. mutans, by xylitol-treated concentrations. Methods: S. mutans KCTC3065 was maintained in TYE medium, containing 0.4% glucose with 1% xylitol for 30 days at 37oC, 10% CO2 to form XR strain and the same procedures, without xylitol, were repeated for the formation of XS S. mutans. Both XS and XR were cultured by xylitol-treated concentrations (0%, 0.1% and 1%), then, general characteristics, such as growth and acid production, mRNA expression of gtf genes and adhesive ability were analyzed. Results: Xylitol reduced the cell growth of XS S. mutans in a dose-dependent manner, but did not reduce the XR. Xylitol inhibited acid production of XS in a dose-dependent manner. However, it did not inhibit that of XR. Xylitol reduced the gtfB and gtfD mRNA expression of the XS S. mutans, which the genes synthesized soluble and insoluble extracellular polysaccharides, but not reduced that of the XR. By a microtiter plate assay, biofilm formation was more reduced in the XR strains, which means biofilm`s adhesive ability of XR S. mutans was lower than that of the XS. Conclusions: These results indicate that a lower level of adhesive ability for XR S. mutans is related with mRNA expression level of gtf genes, which suggested that the XR strains may be less cariogenic than that of the XS.

자일리톨 처리 농도에 따른 자일리톨 감성균주와 내성균주의 독력 비교

임상욱 ( Sang Uk Im ),안상헌 ( Sang Hun Ahn ),송근배 ( Keun Bae Song ) 한국치위생과학회 2011 치위생과학회지 Vol.11 No.5

Streptococcus mutans (S. mutans) is the major causative bacteria in dental caries. Xylitol is effective anticarious natural sugar substitute by inhibiting the virulence of S. mutans. However, long-term xylitol consumption leads to the emergence of the xylitol-resistant (XR) strains which means xylitol is no more inhibited their growth. We therefore confirmed the general characteristics and the virulence factors of the xylitol-sensitive (XS) and XR S. mutans for different concentrations of xylitol. S. mutans KCTC 3065 was maintained in TYE medium containing 0.4% glucose with 1% xylitol during 30 days at 37oC, 10% CO2 to form XR strain. The strains were transferred to new medium every 24 hr and the same procedures without xylitol were repeated for the formation of XS S. mutans. Both XS and XR were cultured in different concentrations of xylitol (0%, 0.1% and 1%) then, cell growth, acid production and mRNA expression of gtf genes were analyzed. Xylitol reduced the cell growth of XS S. mutans in dose-dependent manner, but not reduced that of XR. Xylitol inhibited acid production of XS in dose-dependent manner, but not inhibited that of XR. Xylitol reduced the gtfB and gtfD mRNA expression of XS S. mutans which genes synthesized soluble and insoluble extracellular polysaccharides, but not reduced that of XR. These results indicate that the virulence of XR S. mutans is different characters of XS strains, which suggests XR strains may have different cariogenicity of XS strains. Further study is needed to explain the mechanism related to extracellular polysaccharide in the XR strains.

다양한 탄수화물 배양 조건에 따른 xylitol-resistant Streptococcus mutans와 xylitol-resistant Streptococcus sobrinus의 독력 비교

정선영 ( Sun Young Chung ),김지혜 ( Ji Hye Kim ),최연희 ( Youn Hee Choi ),송근배 ( Keun Bae Song ) 대한예방치과·구강보건학회 2011 大韓口腔保健學會誌 Vol.35 No.1

Objective. This study aimed to compare the cariogenic traits in xylitol-resistant (XR) mutans streptococci (MS) exposed to various carbohydrates. Methods. To select the XR MS, S. mutans KCTC 3065 and S. sobrinus KCTC 3308 were cultured in TYE medium containing 0.5% glucose and 1% (66 mM) xylitol at 37℃, 10% CO2 conditions for 30 days. Both the strains were divided into the following 4 groups in terms of the culture medium that was used: (1) 0.5% glucose containing basal culture medium (BCM), (2) BCM plus 0.5% sucrose (BCM-S), (3) BCM plus 0.5% fructose (BCM-F), and (4) BCM plus 0.5% maltose (BCM-M). To examine the cariogenic traits, cell growth, acid production, mRNA expressions related to glucan synthesis, and the formation of extracellular polysaccharides (ECPs) in each culture media, they were measured by ELISA, pH meter, real-time RT-PCR, and TEM, respectively in the 4 culture media. Results. The characteristics of xylitol-resistant MS were identified by confirming that the growth curve on glucose in the presence of xylitol was identical to that in its absence. Both the S. mutans and S. sobrinus expressed the final pH without any significant differences depending on carbohydrate sources. Cell growth, gene expression levels and the formation of ECPs were apparently distinct in S. mutans depending on sugar sources whereas these were not different in S. sobrinus except for BCM-S. Conclusions. The virulence of XR S. mutans responded differently when grown in various carbohydrates, yet the virulence of XR S. sobrinus, in general, showed no difference depending on sugar sources except for BCM-S. Further study is needed to explain the metabolite mechanism related to carbohydrate sources inside the XR MS cells.

Quantitative comparison of mRNA expression of glucosyltransferase(GTF) between xylitol-resistant(X^(R)) and xylitol-sensitive(X^(s)) mutans streptococci

Kim, Chong-Chul,Lee, Mi-Na,Kim, Young-Jae,Lee, Sung-Hoon 大韓小兒齒科學會 2006 大韓小兒齒科學會誌 Vol.33 No.1

Since the long-term exposure of mutans streptococci to xylitol is known to select for xylitol-resistant(X^(R)) natural mutants, the occurrence and survival of such X^(R) strains were performed in batch culture methods. The aim of the study was to compare the differentiation and quantification of mRNA expression of the gtf genes of X^(R) and X^(S) mutans streptococci. Using a real-time reverse-transcription polymerase chain reaction, the expression of each gtf was determined. In X^(R) strains, the relative levels of transcription of gtfB and gtfC were decreased while that of gtfD was increased, suggesting the presence of independent promoters. It also suggested that mutation related to production of glucosyltransferase occurred under the exposure of xylitol could explain the caries-preventive mechanisms of xylitol. 자일리톨에 장기간 노출된 mutans streptococci는 자일리톨에 내성이 발현되어 자일리톨 내성균주가 생성된다고 알려져 있다. 본 연구의 목적은 mutans streptococci에서 자일리톨 내성균주와 감성균주의 gtf 유전자 발현량을 각각의 유전자별로 정량적으로 분석하고 비교하는 것이다. 실시간 역전사 중합효소연쇄반응법을 이용하여 각각의 gtf 발현을 조사한 결과 gtfD는 증가한 반면. gtfB와 gtfC는 감소하였는데 이는 각 유전자의 독립된 조절기전이 존재함을 보여주는 것이다. 또한 자일리톨에 노출된 mutans streptococci에서의 glucosyltransferase와 연관된 유전자변형이 자일리톨의 치아우식증 예방효과의 작용기전 중 하나임을 알 수 있었다.

다양한 탄수화물 함유 배지 사용에 따른 자일리톨 감성 및 내성 Streptococcus mutans의 독력 비교

정선영 ( Sun Young Chung ),이영은 ( Young Eun Lee ),안상헌 ( Sang Hun Ahn ),김혜영 ( Hye Young Kim ),최연희 ( Youn Hee Choi ),송근배 ( Keun Bae Song ) 대한예방치과·구강보건학회 2011 大韓口腔保健學會誌 Vol.35 No.4

연구목적. Streptococcus mutans ( S. mutans)는 치아 우식의 중요한 병인 중 하나로, S. mutans가 자일리톨에 장기간 노출되었을때 자일리톨에 대한 감수성을 잃고 자일리톨에 의한 성장 억제를 보이지 않는 자일리톨 내성균주가 형성된다고 보고된 바 있다. 본 연구는 자일리톨 내성균주와 자일리톨 감성균주가 다양한 탄수화물 조건에서 배양되었을 때의 독력 인자 발현 양상을 비교하고자 하였다. 연구방법. S. mutans KCTC3065를 포도당이 포함된 TYE 배지에 1% 자일리톨을 처리하여 자일리톨 내성균주를 형성한 후, 자일리톨 감성균주와 함께 포도당 단독 및 포도당에 자당, 과당, 엿당이 각각 포함된 배지에서 배양하였다. S. mutans의 독력과 관련이 있는 성장과 산생성, 글루칸 합성 관련 유전자 발현 및 세포외 다당류 합성을 살펴보았다. 연구결과. 자일리톨 내성규주는 감성균주와 비교하여 pH의 차이 없이 최종 성장, 글루칸 합성 관련 유전자 발현 및 세포외 다당류 합성이 모두 저하된 양상을 나타내었다. 탄수화물 종류별 특성을 살펴보면, 자일리톨 감성균주와 내성균주 모두 포도당과 비교하여 포도당과 엿당이 혼합된 배지에서 최종 성장이 증가한 반면, gtfB와 gtfD의 발현은 감소하였다. 또한 포도당과자당 그리고 포도당과 과당이 혼합된 배지에서 최종 성장이 감소한 반면, gtfB와 gtfD의 발현은 증가하였다. 그러나 감성균주와 내성균주 모두 배양 조건과 관계없이 산생성에서는 차이를 보이지 않았다. 투과전자현미경 관찰 결과, 포도당과 엿당이 혼합된 배지에서 특히 자일리톨 내성균주의 세포외 다당류 합성이 저하된 것을 확인할 수 있었다. 결론. 자일리톨 내성균주는 감성균주에 비해 우식원성이 저하되어 있으며, 탄수화물 종류에 따라 자일리톨 내성균주와 감성균주의 독력인자가 다르게 발현되었다.