Published online Feb 7, 2015. doi: 10.3748/wjg.v21.i5.1510
Peer-review started: July 13, 2014
First decision: August 15, 2014
Revised: October 10, 2014
Accepted: October 21, 2014
Article in press: October 21, 2014
Published online: February 7, 2015
AIM: To investigate the antibacterial effects of a crude extract of maggots against Escherichia coli (E. coli) and the underlying mechanisms, and to separate and purify the crude extract of maggots to assess the antibacterial effects of the active ingredients in the crude extract.
METHODS: Different concentrations of the crude extract of maggots were incubated with E. coli (O157:H7) and cultured. The optical density (OD) was measured at different time points to plot the OD-T curve. The effects of different concentrations of the crude extract on bacterial membrane permeability were determined by fluorescence probe technique. The effects of different concentrations of the crude extract on plasmid DNA replication were determined by agarose gel electrophoresis. DEAE-Sepharose ion exchange chromatography and Sephacryls-200HR gel filtration chromatography were used to separate and purify the crude extract of maggots. The molecular weight of proteins in the purified crude extract was determined by SDS-PAGD electrophoresis, and its antibacterial effects were determined by turbidimetric method.
RESULTS: The antibacterial effects of the crude extract of maggots at concentrations > 0.5 mg/mL were significant. The antibacterial effects of the crude extract at concentrations of 1.0, 1.5 and 2.0 mg/mL did not differ significantly. Fluorescence probe analysis showed that the rate of membrane permeability change was 1223.1% in bacteria incubated with 2 mg/mL of the crude extract, and 1300.0% in those incubated with 80 mg/mL of the crude extract. Plasmid DNA was undetectable in E. coli incubated with 2 and 80 mg/mL of the crude extract. A low molecular weight protein band (about 15 kDa) was detected in the crude extract of maggots and eluent, but not in eluant, from DEAE-Sepharose ion exchange chromatography. The antibacterial effects of the crude extract of maggots and eluent were superior to those of eluant, with the antibacterial effects of eluents being better than those of the crude extract of maggots. Of 24 tubes of filtrates, the antibacterial effects of filtrates in tubes 4, 5 and 11 were significantly higher than those of the control. The molecular weight of the protein in filtrates in tubes 4, 5 and 11 was about 15 kDa.
CONCLUSION: The crude extract of maggots exhibits obvious, dose-dependent antibacterial effects. The crude extract exerts antibacterial effects by changing the bacterial membrane permeability and inhibiting plasmid DNA replication. The protein that has antibacterial effects in the crude extract of maggots has a molecular weight of about 15 kDa.
Core tip: Previous studies have found the antibacterial activity of the maggots, but studies on the underlying mechanisms are still lacking. In this study, we separated and purified the crude extract of maggots using DEAE-Sepharose ion exchange chromatography and Sephacryls-200HR gel filtration chromatography. Agarose gel electrophoresis and fluorescence probe technique were used to investigate the antibacterial mechanism of the crude extract of maggots. We found that the protein of maggots weighing around 15 KD had the antibacterial effect and the crude extracts played the antibacterial role by destroying the cell membrane of Escherichia coli and disturbing the DNA replication of plasmids.