Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Antibacterial Activity and Probiotic Potential of Lactobacillus plantarum HKN01: A New Insight into the Morphological Changes of Antibacterial Compound-Treated Escherichia coli by Electron Microscopy

  • Sharafi, Hakimeh (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Maleki, Hadi (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Ahmadian, Gholamreza (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Zahiri, Hossein Shahbani (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Sajedinejad, Neda (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Houshmand, Behzad (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Vali, Hojatollah (Facility for Electron Microscopy Research, McGill University) ;
  • Noghabi, Kambiz Akbari (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB))
  • Received : 2012.08.02
  • Accepted : 2012.09.24
  • Published : 2013.02.28
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Abstract

Among several bacteria examined, an antibacterial-producing Lactobacillus strain with probiotic characteristics was selected and identified based on 16S rRNA gene sequencing. Subsequent purification and mode of action of the antibacterial compounds on target cells including E. coli were investigated. Maximum production of the antibacterial compound was recorded at 18 h incubation at $30^{\circ}C$. Interestingly, antibacterial activity remained unchanged after heating at $121^{\circ}C$ for 45 min, 24 h storage in temperature range of $70^{\circ}C$ to room temperature, and 15 min exposure to UV light, and it was stable in the pH of range 2-10. The active compounds were inactivated by proteolytic enzymes, indicating their proteinaceous nature, and, therefore, referred to as bacteriocin-like inhibitory substances. Isolation and partial purification of the effective agent was done by performing ammonium sulfate precipitation and gel filtration chromatography. The molecular mass of the GFC-purified active compound (~3 kDa) was determined by Tris-Tricine SDS-PAGE. To predict the mechanisms of action, transmission electron microscopy (TEM) analysis of ultrathin sections of E. coli before and after antibacterial treatment was carried out. TEM analysis of antibacterial compounds-treated E. coli demonstrated that the completely altered bacteria appear much darker compared with the less altered bacteria, suggesting a change in the cytoplasmic composition. There were also some membrane-bound convoluted structures visible within the completely altered bacteria, which could be attributed to the response of the E. coli to the treatment with the antibacterial compound. According to the in vivo experiments oral administration of L. plantarum HKN01 resulted in recovery of infected BALB/c mice with Salmonella enterica ser. Typhimurium.

Keywords

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