J Bacteriol Virol.  2013 Jun;43(2):120-130. 10.4167/jbv.2013.43.2.120.

The Synergistic Effects of Antimicrobial Peptides on the Growth Inhibition of Salmonella Typhimurium through Imd Pathway in Drosophila Intestine

  • 1Department of Microbiology, Cancer Research Institute, College of Medicine, Chungnam National University, Daejeon, Korea. jekpark@cnu.ac.kr


Some Bacillus species present in fermented foods are regarded as probiotics because of their ability to modulate the prevention of some intestinal infections and the modulation of the inflammatory immune response. We isolated bacteriocin-like substances producing Bacillus subtilis and B. lentus from Cheonggukjang, a traditional Korean fermented soybean paste having an inhibitory effect against Salmonella Typhimurium using a well diffusion inhibition assay and a broth co-culturing method. B. subtilis or B. letus was fed to Drosophila melanogaster alone as well as in combination with Salmonella Typhimurium and survival was monitored daily. The survival rates by oral feeding B. subtilis, B. lentus and Salmonella Typhimurium separately resulted in 85, 90 and 75%, respectively. In contrast, survival rates of co-feeding of B. lentus with Salmonella Typhimurium were increased from 75 to 90% during 7 days post-feeding as compared to Salmonella Typhimurium alone. However, B. subtilis in co-feeding with Salmonella Typhimurium significantly reduced D. melanogaster survival rate (85 to 70%). We found that the immune response to B. lentus and Salmonella Typhimurium is characterized synergistic activation of antimicrobial peptide gene expression by Imd pathway. In conclusion, the in vitro and natural-route infection of the D. melanogaster digestive system can result in the use of the probiotic B. lentus for effective treatment of Salmonella Typhimurium infection. We therefore propose the strain B. lentus as a suitable candidate probiotics for use in the prevention and treatment of the intestinal infections caused by Salmonella Typhimurium.


Antimicrobial peptide; B. lentus; Salmonella Typhimurium; Drosophila melanogaster

MeSH Terms

Bacillus subtilis
Digestive System
Drosophila melanogaster
Gene Expression
Salmonella typhimurium
Sprains and Strains
Survival Rate


  • Figure 1. Detection of antimicrobial activity of B. subtilis (BS) or B. lentus (BL) with Salmonella Typhimurium (ST) by agar well diffusion assay. The indicator organism (Salmonella Typhimurium) in the 0.7% soft agar was spread on the surface of solid agar medium. Pasteur pipette was used to create 5 mm wells in the overlaid base agar plate. The well was filled with 30 μl of the cell free supernatant of Bacillus species. The plates were incubated and examined for the clear zones around the wells. Bacillus species isolated from a traditional fermented soybean paste displayed a wide range of antimicrobial activity against Salmonella Typhimurium. S. typhi: Salmonella typhimurium, 1800: Bacillus subtilis, 1900: Bacillus lentus-1, 2720: Bacillus lentus-2.

  • Figure 2. The effect of co-cultured B. subtilis (BS) or B. lentus (BL) with Salmonella Typhimurium (ST) to inhibit the growth of ST in broth. BS or BL were mixed at a ratio of approximately 1:1 and co-cultured in broth at 24 h interval for 7 days. They were plated on MacConkey agar plate to determine viable cell count of ST. BL resulted in the complete killing of ST within 7 days co-cultured in broth, but BS did not observed the bactericidal effect.

  • Figure 3. Survival analysis of D. melanogaster that were oral feeding with B. subtilis (BS), B. lentus (BL), Salmonella Typhimurium (ST), BS/ST and BL/ST. Adult female flies were fed 5% sucrose in nutrient broth containing BS, BL, ST, BS/ST and BL/ST for 24 h, then transferred to sterile food. Survival rate (%) were determined over indicated time.

  • Figure 4. Effect of oral feeding with B. lentus (BL) or B. subtilis (BS) on eradication of Salmonella Typhimurium (ST) in D. melanogaster. Adult female flies were fed 5% sucrose in nutrient broth containing ST, BL/ST and BS/ST for 24 h, and then transferred to sterile food. Guts were dissected and crushed at various times after ingestion using micropestle, and then homogenate was serially diluted in LB medium. The number of colony forming units (CFU) was determined through growth overnight at 37°C on MacConkey agar.

  • Figure 5. Synergistic induction of antimicrobial peptides (AMPs) genes of D. melanogaster that were oral feeding with B. subtilis (BS), B. lentus (BL), Salmonella Typhimurium (ST), BS/ST and BL/ST. Total RNA was isolated from fly intestine treated with different combinations of BS, BL and ST for 6, 24 and 48 h as indicated. The RNA samples were analyzed by RT-PCR. rp49 was used as the experimental expression standard.


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