High antimicrobial activity of lactoferricin-expressing Bacillus subtilis strains.

The lactoferricin expressed in Bacillus subtilis is relatively low in yield, making it hard to apply in industrial settings. We constructed a six tandem repeat of lactoferricin cDNA driven by promoter PtrnQ. After transformation, two transformants P245 and P263 possessing a stable inheritance of plasmid and high expression of lactoferricin were selected. The bactericidal activities, 1 µl of aliquot of a total 5.5 ml of solution extracted from 5 ml of cultured P245 and P263, were equivalent to the efficacy of 238.25 and 322.7 ng of Ampicillin against Escherichia coli, respectively, and 366.4 and 452.52 ng of Ampicillin against Staphylococcus epidermidis respectively. These extracts were able to kill an Ampicillin-resistant E. coli strain. The bactericidal activities of P245 and P263 equivalent to the efficacy of Tetracycline against Vibrio parahaemolyticus and V. alginolyticus were also determined. Moreover, the bactericidal activities of P245 and P263 were 168.04 and 249.94 ng of Ampicillin against Edwardsiella tarda, respectively, and 219.7 and 252.43 ng of Tetracycline against Streptococcus iniae respectively. Interestingly, the survival rate of E. tarda-infected tilapia fry fed the P263 extract displayed a significantly greater than that of the fry-fed control strain. Collectively, these B. subtilis transgenic strains are highly promising for use in animal husbandry during a disease outbreak.

Using Bacillus subtilis as a Host Cell to Express an Antimicrobial Peptide from the Marine Chordate Ciona intestinalis.

Ciona molecule against microbes-A24 (CiMAM) isolated from the marine chordate Ciona intestinalis is an antimicrobial peptide. To generate CiMAM-expressing transgenic Bacillus subtilis, we constructed a plasmid expressing recombinant CiMAM (rCiMAM) and introduced it into B. subtilis. Transgenic strains C117 and C166 were selected since they were able to highly and stably express rCiMAM. We studied the bactericidal activity of pepsin-digested extracts from rCiMAM-expressing strains against freshwater and euryhaline pathogens that commonly occur in aquaculture ponds and found no difference from that of lactoferricin-expressing strains. The bactericidal activity of 1-µL aliquot from a total 5.5 mL extracted from 5 mL of cultured C117 (1.45 × 108 CFU·mL-1) and C166 (2.17 × 108 CFU·mL-1) against halophilic bacteria was equivalent to the efficacy of 57.06 and 32.35 ng of Tetracycline against Vibrio natriegens, 47.07 and 25.2 ng against V. parahaemolyticus, and 58.17 and 36.55 ng against V. alginolyticus, respectively, indicating higher bactericidal activity of pepsin-extracts from rCiMAM-containing strains against halophilic bacteria compared to that from lactoferricin-containing strains. Since the antibacterial activity of rCiMAM-expressing B. subtilis strains shows higher competence against halophilic pathogens compared to that against freshwater and euryhaline pathogens, these strains are promising candidates to protect marine fish and shellfish from halophilic bacterial infection.

Oral administration of transgenic biosafe microorganism containing antimicrobial peptide enhances the survival of tilapia fry infected bacterial pathogen.

To develop an alternative to conventional antibiotics used in the aquaculture and livestock industries, we employed Bacillus subtilis, considered a biosafe microorganism, to express the degradable antimicrobial peptide lactoferricin. An expression plasmid pP43-6LFBII-GFP, in which reporter GFP cDNA was fused downstream of lactoferricin cDNA driven by an endogenous constitutive P43 promoter was electroporated into B. subtilis, followed by regeneration and cultivation. The putative colonies harboring plasmids were primarily screened by PCR-amplification of lactoferricin cDNA. Four transformants which were stable inheritance of plasmid containing lactoferricin cDNA included strains T1, T4, T7 and T13. Based on Western blot and Southern blot analyses, we found that transgenic strains T1 and T13 not only highly expressed exogenous recombinant lactoferricin, but also exhibited more stable inheritance of plasmids with 931 and 647 copies per cell, respectively. In the antibacterial in vitro experiment, the bactericidal activity of each microliter of cell lysate from transgenic strains T1 and T13 (5 × 108 CFU) for Escherichia coli was equivalent to 56 and 53 ng of Ampicillin dosage, respectively, while for Staphylococcus epidermidis, the equivalency T1 and T13 was 154 and 130 ng of Ampicillin dosage, respectively. Equivalencies of bacterial activity for Vibrio parahaemolyticus and Edwardsiella tarda followed suit. In the antibacterial in vivo experiment, we oral-in-tube fed tilapia fry (Oreochromis mossambicus X O. niloticus) with cell lysate from transgenic strain T1 and T13 individually. After 1-h of incubation, we immersed these treated fish fry in a water tank containing E. tarda (5 × 1011 CFU) for a 5-hr bacterial challenge. After one month cultivation, an average survival rate of 63 and 67% was observed after having fed the fish fry with transgenic strains T1 and T13, respectively. However, the average survival rate of fish fry fed with B. subtilis WT strain and transgenic strain T19 without expressing recombinant lactoferricin reached only 5 and 9%, respectively. These data indicate that the survival of fish fry infected by the intestinal pathogen tested could be significantly enhanced by feeding transgenic B. subtilis containing antibacterial peptide. Therefore, we suggest that this strategy could be applied to both aquaculture and livestock industries to (i) reduce the dependency on conventional antibiotics during seasonal outbreaks and (ii) eliminate the problem of antibiotic resistance.