Bacteriocin from Bacillus subtilis as a novel drug against diabetic foot ulcer bacterial pathogens

Baby Joseph; Berlina Dhas; Vimalin Hena; Justin Raj

摘要

Objective: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. Methods: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. Results: 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. Conclusions: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.

机译：目的：从土壤中分离鉴定枯草芽孢杆菌（B.subtilis），并鉴定和部分纯化细菌素。为了评估对四种糖尿病足溃疡细菌病原体的抗菌活性。方法：根据Bergey的全身细菌学手册进行基因型鉴定。抗菌药敏试验采用Kirby-Bauer纸片扩散法进行。通过菌落形态和生化特征鉴定菌落，并与MTCC 121菌株进行比较。通过16S rRNA测序进一步鉴定。部分纯化的细菌素对所有DFU分离物的抑制活性均通过琼脂井扩散法进行。通过刺穿覆盖测定法鉴定该菌株产生细菌素。细菌素通过使用氯仿的有机溶剂萃取来萃取，通过HPLC进一步纯化并进行物理和化学表征。结果：四个分离株对阿莫克拉韦耐药，对环丙沙星敏感。 HPLC纯化表明提取物是细菌素。系统进化树分析结果表明，分离株与枯草芽孢杆菌BSF01有99％的相关性。结果显示出对所有四个分离物的活性，并且在克雷伯菌属的情况下观察到高水平的活性。结论：发现部分纯化的细菌素对四种糖尿病足溃疡细菌病原体具有抗菌活性，因此可以作为更好的药物分子用于进一步的研究。发现枯草芽孢杆菌菌株是安全使用的，并且这些抗微生物肽可以用作人类的抗微生物剂以治疗DFU细菌病原体。

Bacteriocin from Bacillus subtilis as a novel drug against diabetic foot ulcer bacterial pathogens

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