Evaluation of the Antimicrobial Activity of an Extract of Lactobacillus casei-Infected Hermetia illucens Larvae Produced Using an Automatic Injection System

Simple Summary In this investigation, an automatic mass-injection system was developed to produce an extract of Lactobacillus casei–infected Hermetia illucens larvae (HIL) at low cost. The extract produced was found to be a novel natural antibiotic candidate with a wide range of applications, especially in the food, animal feed, and medicinal industries. Abstract In the present study, we developed an automatic mass-injection system (AMIS) to produce an extract of infected H. illucens larvae (iHIL-E) and then evaluated antimicrobial peptide (AMP) expressions and assessed the antimicrobial activity of iHIL-E against various pathogens and Lactobacillus species. AMP gene expressions were assessed by real-time quantitative polymerase chain reaction (PCR) and the antimicrobial activities of iHIL-E were estimated using a radial diffusion assay and by determining minimal inhibitory concentrations. Results showed that the antimicrobial activity of HIL extract was effectively enhanced by L. casei infection and that the gene expressions of cecropin 3 and defensin 3 (antimicrobial peptides) were up-regulated. iHIL-E also prevented the growths of Enterococcus faecalis, Streptococcus mutans, and Candida vaginitis (MICs 200, 500, and 1000 µg/100 µL, respectively) and demonstrated high protease resistance. Moreover, the growths of methicillin-resistant Staphylococcus aureus, antibiotic-resistant Pseudomonas aeruginosa and AMP-resistant bacteria, Serratia marcescens, and Pseudomons tolaasii were significantly suppressed by iHIL-E. In addition, although iHIL completely cleared Salmonella species at concentrations of >200 µg/100 µL, Lactobacillus species were unaffected by iHIL at concentrations of <1000 µg/100 µL. The present investigation shows that the devised automatic mass injection system is effective for the mass production of the extract of infected HIL and that this extract is a novel, natural, protease-resistant, antibiotic candidate with broad-spectrum antibiotic activity.


Introduction
Antibiotics play important roles in human and livestock health as they effectively treat and prevent infectious diseases caused by pathogens. Antibiotics have also been used to enhance animal growth and have enabled the development of intensive farming and the large-scale commercialization of

Radial Diffusion Assay
To evaluate iHIL-E protease resistance and evaluate the suitability of the AMIS for the mass production of L. casei-infected HIL, the antimicrobial activity of iHIL-E was assessed using a radial diffusion assay (RDA). First, bacteria were mixed with autoclaved liquid underlay gel (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4, 1% low electroendosmosis agar and 0.03% tryptic soy broth (TSB)) and the mixture was poured into a 100 mm square plate and allowed to harden. A 3.5 mm-diameter well was prepared on the underlay gel, 10 µL of iHIL-E incubated with or without 50 nM protease was loaded into the well, kept for 3 h at 37 • C, and then covered with sterilized overlay gel (6% TSB and 1% low electroendosmosis agar). The gel plate was then incubated for 18 h at 37 • C and then the diameter of the clear zone was measured and used to assess relative antimicrobial activity. Melittin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and purified bee venom (Chungjin Biotech Co., Ltd., Ansan, Korea) were used as controls.

Analysis of AMP Transcriptions
HIL were infected with L. casei and held at RT for defined times prior to total RNA extraction from hemolymph. To extract total RNA, HIL were transferred to sterilized microtubes and 1 mL of TRIzol (Thermo Fisher Scientific; Waltham, MA, USA) was added to each tube. HIL were then homogenized and total RNA was extracted according to manufacturer's instruction. cDNA was synthesized using a high capacity reverse transcription kit (Applied Biosystems, Waltham, MA, USA), and the cDNA was used as a template to assess the gene expressions of cecropin 3 (HiCec3) and defensin 3 (HiDef3) in iHIL-E. We used the Primer 3 program (http://simgene.com/Primer3) to design primers for HiCec3 and HiDef3, and the Drosophila melanogaster actin 5C (DmAct5C) gene was used as an internal control to normalize gene expressions. Primer sequences are provided in Table 1.

Determination of the Minimum Inhibitory Concentrations (MICs) of HIL Extract versus Pathogens and Lactobacillus Species
Pathogens and Lactobacillus species were inoculated into liquid medium and cultured at 200 rpm in a shaking incubator at 37 • C to 4 × 10 6 cfu/mL. Bacterial cultures were then diluted to 1 × 10 6 cfu/mL placed in the wells of a 96-well plate and 90 µL/well of diluent was added. The bacterial cultures were then treated with 10 µL of serially diluted extracts and incubated for 18 h at 37 • C. Absorbances were measured at 600 nm to determine MICs. Absorbance in each well was normalized by the absorbance of diluted extract solution of the same concentration.

Statistical Analysis
Data from three independent experiments were analyzed using the Student's t-test and one-way analysis of variance, and values are presented as means± standard deviations (SDs). Statistical significance was accepted for p values < 0.05. Table 1. Primer sequences used for real-time polymerase chain reaction (PCR) of the HiCec 3 and HiDef 3 genes.

Analysis of Antimicrobial Activities and AMP Gene Expressions in iHIL-E
In a previous study, we reported that L. casei injection activated the immune system in HIL and induced AMP expression [23]. Therefore, we developed an AMIS using fine needles to inject HIL with L. casei in quantity ( Figure 1A,D). The system was able to inject L. casei to 142500 HIL/h, but the manual method could inject it to 720 HIL/h ( Figure 1C). The antimicrobial activities of iHIL-E using RDA and MIC were assessed. RDA showed that the antimicrobial activities of all HIL extracts were significantly enhanced by L. casei infection ( Figure 1B). In contrast, when L. casei were injected by manual method into HIL, induced antimicrobial activities were observed in 86.1% of HIL extracts ( Figure S1). We also evaluated whether the gene expressions of AMPs were induced by L. casei infection and found HiCec3 and HiDef3 gene expressions were dramatically enhanced by injection ( Figure 2). Furthermore, iHIL-E prevented the growths of E. faecalis, S. mutans, and C. vaginitis with MICs of 200, 500, and 1000 µg/100 µL, respectively ( Figure 3). Animals 2020, 10, x 6 of 13

Analysis of Protease Resistance of iHIL-E
The antimicrobial activities of AMPs can be substantially decreased by proteases [24]. Therefore, we assessed changes in the antimicrobial activity of iHIL-E caused by protease treatment using the RDA. The results showed that antimicrobial activities of melittin and purified bee venom were almost lost after treatment with 50 nM trypsin or 50 nM chymotrypsin for 60 min ( Figure 4A). Furthermore, when 20 µg melittin or 20 µg purified bee venom was treated with trypsin plus chymotrypsin for 60 min or 24 h, their antimicrobial activities were completely lost ( Figure 4A,B). In contrast, iHIL-E (5000 µg) was unaffected by trypsin or chymotrypsin after 60 min ( Figure 4A). Moreover, iHIL activity was almost maintained after treatment with trypsin plus chymotrypsin for 1 h or 24 h ( Figure 4A,B). RDA. The results showed that antimicrobial activities of melittin and purified bee venom were almost lost after treatment with 50 nM trypsin or 50 nM chymotrypsin for 60 min ( Figure 4A). Furthermore, when 20 µg melittin or 20 µg purified bee venom was treated with trypsin plus chymotrypsin for 60 min or 24 h, their antimicrobial activities were completely lost ( Figure 4A,B). In contrast, iHIL-E (5000 µg) was unaffected by trypsin or chymotrypsin after 60 min ( Figure 4A). Moreover, iHIL activity was almost maintained after treatment with trypsin plus chymotrypsin for 1 h or 24 h ( Figure 4A,B).

Analysis of Antimicrobial Activities of iHIL-E against Antibiotic-and AMP-Resistant Bacteria
The antimicrobial activities of iHIL-E against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA) was evaluated. The results showed that the growth of MRSA and MDRPA were dramatically inhibited by iHIL-E (MICs ~300 µg/100 µL; Figure 5A,B).

Analysis of Antimicrobial Activities of iHIL-E against Antibiotic-and AMP-Resistant Bacteria
The antimicrobial activities of iHIL-E against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA) was evaluated. The results showed that the growth of MRSA and MDRPA were dramatically inhibited by iHIL-E (MICs~300 µg/100 µL; Figure 5A,B).
Some authors have reported the appearance of AMP-resistant bacteria [25][26][27], and thus, we evaluated the antimicrobial activities of iHIL-E against the AMP-resistant bacteria, S. marcescens and P. tolaasii. We found that the growths of both bacteria were effectively suppressed by iHIL-E (MICs~300 µg/100 µL) but not by melittin ( Figure 5C). Some authors have reported the appearance of AMP-resistant bacteria [25][26][27], and thus, we evaluated the antimicrobial activities of iHIL-E against the AMP-resistant bacteria, S. marcescens and P. tolaasii. We found that the growths of both bacteria were effectively suppressed by iHIL-E (MICs ~300 µg/100 µL) but not by melittin ( Figure 5C).

Confirmation of the L. casei Infection by AMIS
To confirm whether AMIS effectively infected HIL with L. casei, we checked the anti-microbial activity of iHIL-E after AMIS injection using E. coli (Gram-negative) and S. aureus and Salmonella species (S. pullorum, S. tylpimurium and S. enteritidis) (all Gram-positive) by determining MICs. We found that the growths of E. coli and S. aureus were completely suppressed by iHIL-E (MICs ~200 µg/100 µL; Figure 6A), and that it also inhibited the growth of S. pullorum, S. tylpimurium, and S.

Confirmation of the L. casei Infection by AMIS
To confirm whether AMIS effectively infected HIL with L. casei, we checked the anti-microbial activity of iHIL-E after AMIS injection using E. coli (Gram-negative) and S. aureus and Salmonella species (S. pullorum, S. tylpimurium and S. enteritidis) (all Gram-positive) by determining MICs. We found that the growths of E. coli and S. aureus were completely suppressed by iHIL-E (MICs~200 µg/100 µL; Figure 6A), and that it also inhibited the growth of S. pullorum, S. tylpimurium, and S. enteritidis (MICs 200 µg/100 µL; Figure 6B). These results confirmed that AMIS induced antimicrobial activity in HIL by successfully introducing L. casei infection.

Analysis of the Antimicrobial Activity of iHIL-E against Lactobacillus Species
The antimicrobial activity of iHIL against the probiotics L. brevis, L. casei, and L. fermentum, was assessed to investigate its antimicrobial selectivity. Probiotics were completely destroyed by melittin at 1 µg/100 µL ( Figure 7A). In contrast, the MIC of iHIL-E for Lactobacillus species was ~2000 µg/100 µL ( Figure 7B). However, the viability of Lactobacillus species was significantly diminished by iHIL-E at 1000 µg/100 µL ( Figure 7B). This result suggests that iHIL-E could be used to prevent contamination of fermented foods without seriously depleting probiotic levels.

Analysis of the Antimicrobial Activity of iHIL-E against Lactobacillus Species
The antimicrobial activity of iHIL against the probiotics L. brevis, L. casei, and L. fermentum, was assessed to investigate its antimicrobial selectivity. Probiotics were completely destroyed by melittin at 1 µg/100 µL ( Figure 7A). In contrast, the MIC of iHIL-E for Lactobacillus species was~2000 µg/100 µL ( Figure 7B). However, the viability of Lactobacillus species was significantly diminished by iHIL-E at 1000 µg/100 µL ( Figure 7B). This result suggests that iHIL-E could be used to prevent contamination of fermented foods without seriously depleting probiotic levels.

Analysis of the Antimicrobial Activity of iHIL-E against Lactobacillus Species
The antimicrobial activity of iHIL against the probiotics L. brevis, L. casei, and L. fermentum, was assessed to investigate its antimicrobial selectivity. Probiotics were completely destroyed by melittin at 1 µg/100 µL ( Figure 7A). In contrast, the MIC of iHIL-E for Lactobacillus species was ~2000 µg/100 µL ( Figure 7B). However, the viability of Lactobacillus species was significantly diminished by iHIL-E at 1000 µg/100 µL ( Figure 7B). This result suggests that iHIL-E could be used to prevent contamination of fermented foods without seriously depleting probiotic levels.

Discussion
AMPs are well-known potent natural antibiotics and may in time replace synthetic antibiotics [28]. Furthermore, reports show that AMPs can be produced in abundance by insects [7,11,12,29] and that AMP production in insects is enhanced by bacterial infection [30][31][32]. In a previous study, we showed that the antimicrobial activities of and AMP expressions in hemolymph and in a HIL extract were significantly enhanced by bacterial infection [22,33]. These results suggest that insects could be used to produce AMPs in quantity and that iHIL-E has potential use as a commercial natural antibiotic.
Although natural antibiotics are attractive options in terms of overcoming antibiotics resistance and preventing environmental pollution, the industry requires that they can be produced cheaply and are biologically stable. Therefore, we developed an automatic mass injection system (AMIS) to reduce the cost of producing iHIL-E. Using this AMIS, 1000 HIL were perfectly injected at once and the iHIL-E extracts produced effectively suppressed the growths of pathogens like E. coli, S. aureus, and S. tryphimurium ( Figure 1). As mentioned above, when L. casei was injected by manual method, antimicrobial activities were induced in 86.1% of HIL ( Figure S1). The injection of L. casei into HIL by manual method is time-consuming and the induction rate of antimicrobial activity is also lower than that of AMIS. Furthermore, a lot of labor is necessary for mass production, and the amount of production per day is limited when the injection is performed by hand. In contrast, AMIS can inject L. casei into 2500 HIL at once without HIL death and failure of AMP induction. As shown in Figure 1C, to inject L. casei into HIL by the manual method, at least 19.79 persons are required to inject the same number of larvae as AMIS, if all persons can inject perfectly. Consequently, present investigation shows that AMIS is a novel and highly economical injection system for mass production of iHIL-E that can significantly reduce production cost by reducing production time and labor.
In this investigation, the result showed that the gene expressions of the antimicrobial peptides HiCec3 and HiDec3 were induced by L. casei infection in HIL (Figure 2). Moreover, iHIL-E exhibited strong antimicrobial activities against E. faecalis, S. mutans, C. albicans, E. coli, S. aureus, and Salmonella species (Figures 4 and 6). Furthermore, iHIL-E completely blocked the growth of MRSA and MDRPA and of the antimicrobial peptide-resistant bacteria S. marcescens and P. tolaasii ( Figure 5). These results indicate that the antimicrobial activity of iHIL-E was caused by the induction of AMP and that iHIL-E has a wide antimicrobial spectrum that extends from bacteria to fungi and can kill drug-and antimicrobial peptide-resistant bacteria.
The biological stabilities of AMPs are closely associated with their protease resistance. Many insect-originated AMPs involving melittin are easily degraded by proteases. Therefore, AMPs stable in the presence of proteases are effective natural antibiotic candidates because the shelf lives of foods are closely associated with the long-term stabilities of AMPs. In the present study, we found that the antimicrobial activity of iHIL-E was not significantly affected by trypsin and/or chymotrypsin treatment for 24 h, although both completely abrogated the antimicrobial activities of melittin and purified bee venom (Figure 3). In addition, our findings show that the antimicrobial activity of iHIL-E is caused by the inductions of AMPs (Figures 1 and 2). Thus, our results show antimicrobial activity in iHIL-E is protease-stable and indicate that the extract has potential use as a protease-stable natural antibiotic. Moreover, our results demonstrate that AMPs in iHIL-E is resistant to protease. Several investigations showed that insect cecropins might be degraded by proteases secreted by pathogen, implying insect AMPs are sensitive to protease [34]. Evidences that endogenous protease inhibitors are present in insect hemolymph has been reported by several studies [35][36][37][38]. Therefore, although the results do not fully describe why the AMPs contained into iHIL-E are resistant to protease, it assumes that AMP itself is not resistant to protease, but AMPs in iHIL-E should be protected by an endogenous inhibitor in hemolymph from protease.
We also found that iHIL-E exhibited little toxicity against Lactobacillus species, such as L. brevis, L. casei, and L. fermentum, but potent activity against other bacteria and C. albicans (Figures 4, 6 and 7). In addition, it inactivates E. faecalis, E. coli, and S. aureus, and Salmonella species at 200 µg/100 µL and MRSA, MDRPA, S. marcescens, and P. tolaasii at 300 µg/100 µL ( Figures 4A, 5 and 6). A narrow antimicrobial spectrum is a major disadvantage of natural preservatives, but iHIL-E completely inhibited the growths of various types of microorganisms, including bacterial and fungi, showed no antimicrobial activity against Lactobacillus species at concentrations <1000 µg/100 µL, and cleared antibiotic-resistant bacteria ( Figure 5). These results demonstrate that iHIL-E may be used as a powerful potent natural preservative with little toxicity against probiotics.

Conclusions
In summary, this investigation showed that the iHIL-E prepared using the AMIS system exhibited potent wide spectrum, antimicrobial activities against bacteria and fungi, and was stable in the presence of proteases. Furthermore, the devised AMIS system enabled the production of iHIL-E in quantity.
Taken together, this investigation shows that the AMIS system can substantially reduce the cost of producing iHIL-E and that this extract is a novel natural antibiotic with a wide range of applications, especially in the food, animal feed, and medicinal industries.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2615/10/11/2121/s1, Figure S1: RDA results of the antimicrobial activities of iHIL-E against E. coli. L. casei were injected into HIL by manual method and held at room temperature for 24 h.