Antimicrobial Activity of an Extract of Hermetia illucens Larvae Immunized with Lactobacillus casei against Salmonella Species

Simple Summary In this investigation, we found antimicrobial activity of the extract of Lactobacillus casei–infected Hermetia illucens larvae (HIL) against Salmonella species. The result demonstrates that the extract is a powerful natural antibiotic and preservative. Abstract The expressions of antimicrobial peptides (AMPs) in the larvae of the black soldier fly, Hermetia illucens, were significantly increased by pathogen or stimulant induced innate immunity activation. We immunized H. illucens fifth instar larvae with five different Lactobacillus species, that is, Lactobacillus acidophilus, L. brevis, L. casei, L. fermentum, or L. delbrueckii, to induce the mass production of AMPs and selected optimal immune inducers. Antimicrobial activities in hemolymph and H. illucens larvae (HIL) extract were evaluated against three salmonella species (Salmonella pullorum, Salmonella typhimurium, and Salmonella enteritidis). Highest antimicrobial activity was shown by the hemolymph of HIL immunized by L. casei and its activity was closely linked with the inductions of cecropin 1 (HiCec1) and defensin 1 (HiDef1) gene expressions. Furthermore, antimicrobial activity in hemolymph was stable to heat and pH and the growth of three Salmonella species were dramatically suppressed by HIL hemolymph and extract after immunization with L. casei. The minimal inhibitory concentration (MICs) of L. casei-immunized HIL extract against Staphylococcus aureus, Escherichia coli, and Salmonella species ranged from 100~200 µg/100 µL and no cytotoxicity to CaCo-2 and L929 cells were observed in the concentration range 100~40,000 µg/100 µL. Taken together, the present investigation demonstrates that L. casei-immunized HIL extract is a powerful natural antibiotic and preservative that can prevent contamination by Salmonella species.


Introduction
Antibiotics are used to treat infectious diseases and prevent infections in humans and animals and to promote animal growth and thus have played leading roles in the intensive farming of livestock. However, abuse of in-feed antibiotics has caused many problems, such as the appearance of antibiotic-resistant bacteria, antibiotic residues in livestock products, weakening of disease resistance in livestock, and ecosystem pollution [1][2][3]. To address these problems, the European Union prohibited the use of veterinary antibiotics for growth promotion from 2006, whereas in Korea, after a phased reduction

Evaluation of the Antimicrobial Activities in HIL Hemolymph and Extract
The antimicrobial activities of HIL extracts were assessed using a radial diffusion assay (RDA) using Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, S. pullorum, S. typhimurium, and S. enteritidis. To perform RDA, autoclaved underlay gel [9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4, 1% low electroendosmosis agar, and 0.03% tryptic soy broth (TSB)] was mixed with each bacterium and hardened in a 100 mm square plate. Then, we prepared 3.5 mm-diameter of well onto underlay gel and 10 µL of hemolymph or extract of HIL was injected into the well. The underlay gel was held at 37 • C for 3 h and then covered with a sterilized overlay gel (6% TSB and 1% low electroendosmosis agar). Gel plates were incubated for 18 h at 37 • C, and antimicrobial activities were assessed by measuring the widths of clear zones.

Determination of Minimum Inhibitory Concentrations (MICs) of HIL Extracts
Firstly, we determined cfu/mL of each bacterial by serial tittering and then measured absorbance of each cfu/mL at 600 nm. Then, S. aureus and E. coli were used to determine the MICs of extracts obtained after immunization with L. casei. Determination of MICs was also used for evaluation of antimicrobial activity against Salmonella species. Bacteria, such as S. aureus, E. coli, S. pullorum, S. typhimurium, and S. enteritidis, were inoculated into liquid medium, respectively, and grown at 37 • C for 18 h in a shaking incubator (shaking speed: 200 rpm), and then further cultured to 4 × 10 6 cfu/mL over 2.5 h under the same conditions. A total of 90 µL (1 × 10 6 cfu/mL) of bacterial culture mixtures were transferred to a 96-well plate and 10 µL of serially diluted extracts were then added to wells and incubated at 37 • C for 18 h. Absorbances were measured at 600 nm to determine MICs. Melittin was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) was used as control.

Analysis of AMPs Transcription
Lactobacillus-infected HIL were held at RT for defined times and then total RNA was extracted. First-strand cDNA was synthesized using a high capacity reverse transcription kit (Applied Biosystems, Waltham, MA, USA), and the cDNA obtained was assessed for the gene expressions of cecropin 1 (HiCec1) and defensin 1 (HiDef1). Primers for HiCec1 and HiDef1 were designed using the Primer 3 program (http://simgene.com/Primer3; accessed on 13 June, 2016) and gene expressions were normalized versus the Drosophila melanogaster actin 5C (DmAct5C) gene. Primer sequences are provided in Table 1. Table 1. Primer sequences used to analyze cecropin 1 and defensin 1 gene expressions by real-time PCR.

Cytotoxicity Analysis by MTT Assay
We investigated the cytotoxicity of Lactobacillus-immunized HIL extract using CaCo-2 human intestinal cells and L929 mouse fibroblasts. Cells were seeded in a 96-well plates and allowed to attached for 24 h in a 5% CO 2 atmosphere at 37 • C. Then, culture medium was removed and then cells were treated with 180 µL of various concentrations of Lactobacillus-immunized HIL extract (0~5000 µg/100 µL) or melittin (0~32 µg/µL) for 24 h. Cells were then administered 20 µL of 5 mg/mL MTT reagent and left for 4 h in the dark. Media were then carefully discarded and 200 µL of DMSO was added to each well to dissolve the formazan. Absorbances were measured at 540 nm using an ELISA reader.

Statistical Analysis
The analysis was performed using the Student's t-test and one-way analysis of variance. Experiments were independently performed at least three times, and results are presented as means ± standard deviations (SDs). p-value of less than 0.05 was regarded as statistically different.

Optimization of Lactobacillus Species for the Mass Production of AMPs in H. illucens
We used five types of Lactobacillus to activate the innate immune system of H. illucens. RDA results showed normal HIL hemolymph did not exhibited antimicrobial activity against E. coli or S. aureus ( Figure 1). Furthermore, we did not observed death of any HIL infected by Lactobacillus species (data not shown). In contrast, although the induction of antimicrobial activity was depended on Lactobacillus species type, increased antimicrobial activities to E. coli and S. aureus were observed in the hemolymph of Lactobacillus-immunized HIL ( Figure 1). Highest antimicrobial activity against E. coli and S. aureus were observed for the hemolymph of L. casei-infected larvae, and notably, this was greater than that of Enterococcus faecalis (KACC 11859) or Serratia marcescens (KACC 11961) challenged larvae ( Figure 1) [23]. Therefore, L. casei was chosen as an activator of the innate immune system of H. illucens.
Insects 2020, 11, x FOR PEER REVIEW 4 of 12 MTT reagent and left for 4 h in the dark. Media were then carefully discarded and 200 µL of DMSO was added to each well to dissolve the formazan. Absorbances were measured at 540 nm using an ELISA reader.

Statistical Analysis
The analysis was performed using the Student′s t-test and one-way analysis of variance. Experiments were independently performed at least three times, and results are presented as means ± standard deviations (SDs). p-value of less than 0.05 was regarded as statistically different.

Optimization of Lactobacillus Species for the Mass Production of AMPs in H. illucens
We used five types of Lactobacillus to activate the innate immune system of H. illucens. RDA results showed normal HIL hemolymph did not exhibited antimicrobial activity against E. coli or S. aureus ( Figure 1). Furthermore, we did not observed death of any HIL infected by Lactobacillus species (data not shown). In contrast, although the induction of antimicrobial activity was depended on Lactobacillus species type, increased antimicrobial activities to E. coli and S. aureus were observed in the hemolymph of Lactobacillus-immunized HIL ( Figure 1). Highest antimicrobial activity against E. coli and S. aureus were observed for the hemolymph of L. casei-infected larvae, and notably, this was greater than that of Enterococcus faecalis (KACC 11859) or Serratia marcescens (KACC 11961) challenged larvae ( Figure 1) [23]. Therefore, L. casei was chosen as an activator of the innate immune system of H. illucens.

Determination of Optimal Conditions for the Mass Production of AMPs
The results obtained showed that antimicrobial activity in hemolymph was increased by L. casei infection in a concentration-dependent manner ( Figure 2A). We also evaluated changes in antimicrobial activity over the time after L. casei infection. As shown in Figure 2B, highest activity was observed at 24 h after infection. Furthermore, the gene expressions of HiCec1 and HiDef1 (major AMPs produced by H. illucens) peaked at 24 h ( Figure 2C). This result showed that the antimicrobial activity of L. casei-infected HIL hemolymph was closely linked with the induced gene expressions of HiCec1 and HiDef1.

Determination of Optimal Conditions for the Mass Production of AMPs
The results obtained showed that antimicrobial activity in hemolymph was increased by L. casei infection in a concentration-dependent manner ( Figure 2A). We also evaluated changes in antimicrobial activity over the time after L. casei infection. As shown in Figure 2B, highest activity was observed at 24 h after infection. Furthermore, the gene expressions of HiCec1 and HiDef1 (major AMPs produced by H. illucens) peaked at 24 h ( Figure 2C). This result showed that the antimicrobial activity of L. casei-infected HIL hemolymph was closely linked with the induced gene expressions of HiCec1 and HiDef1.

Analysis of the Antimicrobial Activities of HIL Hemolymph against Salmonella Species
We investigated the antimicrobial activities of L. casei-immunized HIL hemolymph against three Salmonella species using RDA. Salmonella species are pathogens parasitic on gut and major causes of food poisoning. In fact, 75% of food poisoning cases are due to Salmonella species, and reductions in livestock productivity are closely associated with Salmonella contamination [24,25]. We found that the antimicrobial activities of HIL hemolymph against S. enteritidis, S. typhimurium, and S. pullorum were dramatically enhanced by L. casei-immunization and that their activities were greater than that of 1 µg of melittin ( Figure 3A). Furthermore, the antimicrobial activity of L. casei-immunized HIL hemolymph was maintained after heating (60, 70, 80, or 90 • C) for 24 h and over the pH range 2~10 (Figure 3B,C). We also found that antimicrobial activity of hemolymph was lowest at pH 7.0 ( Figure 3C). These results suggest that AMPs produced by L. casei-immunized HIL effectively suppress the growth of Salmonella species and is stable to heat and a wide pH range.

Analysis of the Antimicrobial Activities of HIL Hemolymph against Salmonella Species
We investigated the antimicrobial activities of L. casei-immunized HIL hemolymph against three Salmonella species using RDA. Salmonella species are pathogens parasitic on gut and major causes of food poisoning. In fact, 75% of food poisoning cases are due to Salmonella species, and reductions in livestock productivity are closely associated with Salmonella contamination [24,25]. We found that the antimicrobial activities of HIL hemolymph against S. enteritidis, S. typhimurium, and S. pullorum were dramatically enhanced by L. casei-immunization and that their activities were greater than that of 1 µg of melittin ( Figure 3A). Furthermore, the antimicrobial activity of L. casei-immunized HIL hemolymph was maintained after heating (60, 70, 80, or 90 °C) for 24 h and over the pH range 2~10 ( Figure 3B,C). We also found that antimicrobial activity of hemolymph was lowest at pH 7.0 ( Figure  3C). These results suggest that AMPs produced by L. casei-immunized HIL effectively suppress the growth of Salmonella species and is stable to heat and a wide pH range.

Determination of the MICs of L. casei-Immunized HIL Extract
We investigated whether L. casei-immunized HIL extract has the potential to be used as a natural antibiotic or feed additive with antimicrobial activity. Antimicrobial activities were evaluated by determining MICs against E. coli and S. aureus. The MIC range of melittin to E. coli and S. aureus was 7~10 µg/100 µL and the L. casei-immunized HIL extract had an MIC range of 100~200 µg/100 µL ( Figure 4A,B). The result reveals that both bacteria, Gram-negative E. coli and Gram-positive S. aureus, show same sensitivity to antimicrobial substances in the extract. Although MICs of the extract were greater than that of melittin, the manufacturing cost of L. casei-immunized HIL extract is much lower than that of melittin, and thus, the extract is more cost-effective.

Determination of the MICs of L. casei-Immunized HIL Extract
We investigated whether L. casei-immunized HIL extract has the potential to be used as a natural antibiotic or feed additive with antimicrobial activity. Antimicrobial activities were evaluated by determining MICs against E. coli and S. aureus. The MIC range of melittin to E. coli and S. aureus was 7~10 µg/100 µL and the L. casei-immunized HIL extract had an MIC range of 100~200 µg/100 µL ( Figure 4A,B). The result reveals that both bacteria, Gram-negative E. coli and Gram-positive S. aureus, show same sensitivity to antimicrobial substances in the extract. Although MICs of the extract were greater than that of melittin, the manufacturing cost of L. casei-immunized HIL extract is much lower than that of melittin, and thus, the extract is more cost-effective.

Analysis of the Antimicrobial Activities of L. casei-Immunized HIL Extract against Salmonella Species
We assessed the antimicrobial activities of L. casei-immunized HIL extract against S. enteritidis, S. typhimurium, and S. pullorum by determining MICs. The results obtained showed that th MICs of melittin to Salmonella species were 10 µg/100 µL and at a concentration of >200 µg/100 µL the extract completely blocked the growth of all Salmonella species (MICs range 100~200 µg/100 µL) ( Figure  5A,B).

Analysis of the Antimicrobial Activities of L. casei-Immunized HIL Extract against Salmonella Species
We assessed the antimicrobial activities of L. casei-immunized HIL extract against S. enteritidis, S. typhimurium, and S. pullorum by determining MICs. The results obtained showed that th MICs of melittin to Salmonella species were 10 µg/100 µL and at a concentration of >200 µg/100 µL the extract completely blocked the growth of all Salmonella species (MICs range 100~200 µg/100 µL) ( Figure 5A,B).

Analysis of the Cytotoxicities of L. casei-Immunized HIL Extracts to Mammalian Cells
The cytotoxic effects of antimicrobial in human and animals are key obstacles to clinical, livestock, and feed applications. Therefore, we examined the cytotoxic activities of melittin and of L. casei-immunized HIL extract in CaCo-2 and L929 cells (human intestinal and cell mouse fibroblast cell lines, respectively). No cytotoxic effect was observed on against CaCo-2 and L929 cells at extract concentrations from 100 to 4000 µg/100 µL or from 100 to 2000 µg/100 µL, respectively ( Figure 6A).

Analysis of the Cytotoxicities of L. casei-Immunized HIL Extracts to Mammalian Cells
The cytotoxic effects of antimicrobial in human and animals are key obstacles to clinical, livestock, and feed applications. Therefore, we examined the cytotoxic activities of melittin and of L. casei-immunized HIL extract in CaCo-2 and L929 cells (human intestinal and cell mouse fibroblast cell lines, respectively). No cytotoxic effect was observed on against CaCo-2 and L929 cells at extract concentrations from 100 to 4000 µg/100 µL or from 100 to 2000 µg/100 µL, respectively ( Figure 6A). In contrast, although cell survival was not reduced by melittin at 1 to 4 µg/100 µL, it was found to have potent cytotoxic effects at > 8 µg/100 µL ( Figure 6B).

Analysis of the Cytotoxicities of L. casei-Immunized HIL Extracts to Mammalian Cells
The cytotoxic effects of antimicrobial in human and animals are key obstacles to clinical, livestock, and feed applications. Therefore, we examined the cytotoxic activities of melittin and of L. casei-immunized HIL extract in CaCo-2 and L929 cells (human intestinal and cell mouse fibroblast cell lines, respectively). No cytotoxic effect was observed on against CaCo-2 and L929 cells at extract concentrations from 100 to 4000 µg/100 µL or from 100 to 2000 µg/100 µL, respectively ( Figure 6A). In contrast, although cell survival was not reduced by melittin at 1 to 4 µg/100 µL, it was found to have potent cytotoxic effects at > 8 µg/100 µL ( Figure 6B).

Discussion
Synthetic antibiotics and antimicrobials have contributed to public health and promoted the growth of livestock. However, overuse and abuse of antibiotics and antimicrobial drugs are now known to be important causes of drug-resistant bacteria, which threaten public and livestock health. Natural antibiotics, especially AMPs, are considered as promising novel candidates for overcoming drug-resistance [26]. Several investigations have demonstrated insects manufacture AMPs and that

Discussion
Synthetic antibiotics and antimicrobials have contributed to public health and promoted the growth of livestock. However, overuse and abuse of antibiotics and antimicrobial drugs are now known to be important causes of drug-resistant bacteria, which threaten public and livestock health. Natural antibiotics, especially AMPs, are considered as promising novel candidates for overcoming drug-resistance [26]. Several investigations have demonstrated insects manufacture AMPs and that these acts as potent natural antibiotics [7,11,12,27]. One investigation showed that a HIL extract prevented growth of plant pathogens and suggested its use as a natural antimicrobial [28]. Furthermore, Lactobacillus species activated innate immune response in insects and contributed to reduction of susceptibility in pathogen infected-insects [29][30][31][32]. In this investigation, we also found HIL extract immunized with L. casei exhibited antimicrobial activity against three Salmonella species ( Figure 4B), which demonstrates that this extract offers a potential means of preventing and treating plant pathogen infections and protecting against Salmonella contamination.
In general, insect AMPs are induced when the innate immune system is activated and are present at much lower levels under normal conditions. In this investigation, we used five Lactobacillus species to activate the innate immunity system. As shown Figure 1, highest antimicrobial activities against Gram-positive S. aureus and Gram-negative E. coli were observed in the hemolymph of L. casei-infected H. illucens, and these activities were higher than those observed for the hemolymphs of HIL challenged by E. faecalis or S. marcescens (Figure 1). E. faecalis and S. marcescens are well known human and animal pathogens [33,34]. Although these bacteria also effectively activated the innate immune system of H. illucens, they pose risks to human and animal health. In contrast, L. casei is a beneficial bacterium in human and its use induced significant increases in HiCec1 and HiDef1 levels and superior antimicrobial activity in the hemolymph and the extract of HIL (Figures 2 and 4). Furthermore, we found that the antimicrobial activity of hemolymph was stable for 24 h over the temperature range (60-90 • C) and the pH range (pH 2-11) ( Figure 3B,C). In addition, the extract of HIL immunized with L. casei did not adversely affect the survival of L929 or CaCo-2 cells at concentrations up to 2000 µg/100 µL or 4000 µg/100 µL, respectively ( Figure 6A). These results demonstrate that L. casei-immunized HIL offer a safe antibiotic candidate for the treatment of bacterial infections in human and livestock and for food preservation.
Melittin is major component of bee venom and has been reported to be a powerful antimicrobial against human and animal pathogens and against methicillin-resistant S. aureus [17,35,36]. However, the production cost of melittin and purified bee venom is very high and their applications in the medical and food industries are limited by cytotoxic effects on normal cells and allergic responses ( Figure 6B) [37]. In the present study, cytotoxic concentration of melittin to Caco-2 and L929 were dramatically lower than MIC to E. coli and S. aureus ( Figure 6B). In contrast, L. casei-immunized HIL extract could be produced cheaply and this extract proved to be relatively non-toxic to normal animal cells ( Figure 6A). In fact, the MICs of L. casei-immunized HIL extract to E. coli and S. aureus were 20to 50-fold lower than those to normal animal cells (Figures 4A and 6A). Consequently, the present investigation shows that L. casei-immunized HIL extract provides a potential cost-effective means of producing a natural antibiotic with applications in medicine and livestock. However, the allergic effects of this extract have yet to be investigated.
To prevent Salmonella contamination and growth, synthetic preservatives, such as ethylparaben, methylparaben, benzoice acid, sorbic acid, and propanoic acid, are added to foodstuffs, including meat products [38]. Although synthetic preservatives are effective and economical, many have reported harmful effects [39], and thus, there is a considerable demand for a safe and economical natural preservative. We report the hemolymph and extract of L. casei-immunized HIL exhibit powerful antimicrobial activities against three Salmonella species (S. enteritidis, S. typhimurium and S. pullorum) as determined by RDA and MIC assays (Figures 3 and 5). Furthermore, L. casei-immunized HIL extract was non-toxic to normal animal cells at concentrations up to 4000 µg/100 µL ( Figure 6A). Our results demonstrate that L. casei-immunized HIL extract should be considered a potential natural preservative for the prevention of food poisoning caused by Salmonella species and a functional feed additive that promotes livestock growth.

Conclusions
Summarizing, we show that Lactobacillus can be used as an AMP inducer in HIL and that the extract of L. casei-immunized HIL prevents the growth of Salmonella species without inducing cytotoxicity in normal animal cells. Furthermore, the antimicrobial activity of L. casei-immunized HIL extract remained stable over the temperature (60-90 • C) for 24 h and the pH range (pH 2-11), respectively. In conclusion, the present investigation reveals that L. casei-immunized HIL extract is a potent natural antibiotic and preservative with potential applications the medical, food, and livestock industries.