Gowrishankar Shanmugaraj, Poornima Balan, Pandian Shunmugiah Karutha
Department of Biotechnology, Alagappa University, Science Campus, Karaikudi 630004, Tamil Nadu, India.
Department of Biotechnology, Alagappa University, Science Campus, Karaikudi 630004, Tamil Nadu, India.
Res Microbiol. 2014 May;165(4):278-89. doi: 10.1016/j.resmic.2014.03.004. Epub 2014 Apr 1.
Since Streptococcus mutans is the principal etiologic agent causing dental caries, by encompassing an array of unique virulence traits, emerging treatment strategies that specifically target the virulence of this pathogen may be promising as alternative approaches compared to conventional antibiotic therapy. In this perspective, we investigated chloroform extract of cell-free culture supernatant from mangrove rhizosphere bacterium Bacillus amyloliquefaciens (MMS-50) in terms of anticariogenic properties of S. mutans, without suppressing its viability. Crude chloroform extract of MMS-50 was subjected to column and high performance liquid chromatographic techniques to obtain the active fraction (AF), and MMS-50 AF was used for all further assays. GC-MS and FT-IR were carried out to identify the major components present in MMS-50 AF. Comparative gene expression analysis of some biofilm-forming and virulence genes (vicR, comDE, gtfC, and gbpB) was done by real-time PCR. Cyclo(L-leucyl-L-prolyl) was found to be the chief compound in MMS-50 AF responsible for bioactivity. The minimum and maximum inhibitory concentrations of MMS-50 AF against S. mutans were found to be 100 and 250 μg/mL, respectively. Anti-virulence assays performed using below-sub-MIC levels of MMS-50 AF (30 μg/mL) resulted in significant reduction in adherence (68%), acid production, acid tolerance, glucan synthesis (32%), biofilm formation (53.5%) and cell surface hydrophobicity, all devoid of affecting its viability. The micrographs of CLSM and SEM further confirmed the antibiofilm and anti-virulence efficacies of MMS-50 AF. Expression data showed significant reduction in expression of all studied virulence genes. Thus, the current study unveils the anticariogenic potential of cyclo(L-leucyl-L-prolyl) from B. amyloliquefaciens, as well as its suitability as a novel and alternative anticariogenic agent against dental caries.
由于变形链球菌是导致龋齿的主要病原体，通过包含一系列独特的毒力特性，与传统抗生素疗法相比，专门针对这种病原体毒力的新兴治疗策略可能是有前景的替代方法。从这个角度来看，我们研究了红树林根际细菌解淀粉芽孢杆菌（MMS - 50）无细胞培养上清液的氯仿提取物对变形链球菌的防龋特性，同时不抑制其活力。对MMS - 50的粗氯仿提取物进行柱色谱和高效液相色谱技术以获得活性组分（AF），并将MMS - 50 AF用于所有进一步的测定。进行气相色谱 - 质谱联用（GC - MS）和傅里叶变换红外光谱（FT - IR）以鉴定MMS - 50 AF中存在的主要成分。通过实时聚合酶链反应（PCR）对一些生物膜形成和毒力基因（vicR、comDE、gtfC和gbpB）进行比较基因表达分析。发现环（L - 亮氨酰 - L - 脯氨酰）是MMS - 50 AF中负责生物活性的主要化合物。发现MMS - 50 AF对变形链球菌的最小和最大抑制浓度分别为100和250μg/mL。使用低于最低抑菌浓度水平的MMS - 50 AF（30μg/mL）进行的抗毒力测定导致黏附（68%）、产酸、耐酸性、葡聚糖合成（32%）、生物膜形成（53.5%）和细胞表面疏水性显著降低，且所有这些均未影响其活力。共聚焦激光扫描显微镜（CLSM）和扫描电子显微镜（SEM）的显微照片进一步证实了MMS - 50 AF的抗生物膜和抗毒力功效。表达数据显示所有研究的毒力基因的表达均显著降低。因此，当前研究揭示了解淀粉芽孢杆菌中环（L - 亮氨酰 - L - 脯氨酰）的防龋潜力，以及其作为一种新型替代防龋剂对抗龋齿的适用性。
