Joseph Baby, Dhas Berlina, Hena Vimalin, Raj Justin
Interdisciplinary Research Centre, Department of Biotechnology, Malankara Catholic College, Mariagiri, Kaliakkavilai, Tamil Nadu, India.
Asian Pac J Trop Biomed. 2013 Dec;3(12):942-6. doi: 10.1016/S2221-1691(13)60183-5.
To isolate and identify Bacillus subtilis (B. subtilis) from soil and to characterize and partially purify the bacteriocin. To evaluate the antimicrobial activity against four diabetic foot ulcer bacterial pathogens.
Genotypic identification was done based on Bergey's manual of systemic bacteriology. Antimicrobial susceptibility test was done by Kirby-Bauer disc diffusion method. Colonies were identified by colony morphology and biochemical characterization and also compared with MTCC 121 strain. Further identification was done by 16S rRNA sequencing. Inhibitory activities of partially purified bacteriocin on all the DFU isolates were done by agar well diffusion method. The strain was identified to produce bacteriocin by stab overlay assay. Bacteriocin was extracted by organic solvent extraction using chloroform, further purified by HPLC and physical, and chemical characterization was performed.
The four isolates showed high level of resistance to amoxyclav and sensitivity to ciprofloxacin. HPLC purification revealed that the extracts are bacteriocin. The phylogenetic tree analysis results showed that the isolate was 99% related to B. subtilis BSF01. The results reveled activity to all the four isolates and high level of activity was seen in case of Klebsiella sp.
Partially purified bacteriocin was found to have antimicrobial activity against the four diabetic foot ulcer bacterial pathogens, which can thus be applied as a better drug molecule on further studies. The strain B. subtilis are found to be safe for use and these antimicrobial peptides can be used as an antimicrobial in humans to treat DFU bacterial pathogens.
从土壤中分离和鉴定枯草芽孢杆菌，并对其细菌素进行表征和部分纯化。评估其对四种糖尿病足溃疡病原菌的抗菌活性。
根据《伯杰氏系统细菌学手册》进行基因型鉴定。采用 Kirby-Bauer 纸片扩散法进行药敏试验。通过菌落形态和生化特性鉴定菌落，并与 MTCC 121 菌株进行比较。通过 16S rRNA 测序进行进一步鉴定。采用琼脂孔扩散法检测部分纯化细菌素对所有糖尿病足溃疡分离株的抑制活性。通过穿刺覆盖试验鉴定该菌株是否产生细菌素。使用氯仿通过有机溶剂萃取法提取细菌素，进一步通过高效液相色谱法纯化，并进行物理和化学表征。
这四种分离株对阿莫西林克拉维酸表现出高度耐药性，对环丙沙星敏感。高效液相色谱法纯化显示提取物为细菌素。系统发育树分析结果表明，该分离株与枯草芽孢杆菌 BSF01 的亲缘关系为 99%。结果显示对所有四种分离株均有活性，在克雷伯菌属中观察到较高水平的活性。
发现部分纯化的细菌素对四种糖尿病足溃疡病原菌具有抗菌活性，因此在进一步研究中可作为一种更好的药物分子应用。发现枯草芽孢杆菌菌株使用安全，这些抗菌肽可作为人类治疗糖尿病足溃疡病原菌的抗菌剂。
