Antibiotic Candidates Research Articles

Antimicrobial Activity and Action Mechanisms of Arg-Rich Short Analog Peptides Designed from the C-Terminal Loop Region of American Oyster Defensin (AOD).

American oyster defensin (AOD) was previously purified from acidified gill extract of the American oyster, Crassostrea virginica. AOD is composed of 38 amino acids with three disulfide bonds and exhibits strong antimicrobial activity against Gram-positive bacteria as well as significant activity against Gram-negative bacteria. Here, to develop promising peptides into antibiotic candidates, we designed five arginine-rich analogs (A0, A1, A2, A3, and A4), predicted their loop and extended strand/random structures—including nine amino acids and a disulfide bond derived from the C-terminus of AOD—and described their antimicrobial and cytotoxic effects, as well as their modes of action. In our experimental results, the A3 and A4 analogs exhibited potent antimicrobial activity against all test organisms—including four Gram-positive bacteria, six Gram-negative bacteria, and Candida albicans—without cell toxicity. A sequence of experiments, including a membrane permeabilization assay, DNA binding study, and DNA polymerization inhibition test, indicated that the two analogs (A3 and A4) possibly did not act directly on the bacterial membrane but instead interacted with intracellular components such as DNA or DNA amplification reactions. AOD analogs also showed strong bacterial inhibition activity in the plasma environment. In addition, analog-treated microbial cells clearly exhibited membrane disruption, damage, and leakage of cytoplasmic contents. Collectively, our results suggest that two analogs, A3 and A4, have potent antimicrobial activity via DNA interaction and have the potential for development into novel antimicrobial agents.

Presence of Biosynthetic Gene Clusters (NRPS/PKS) in Actinomycetes of Mangrove Sediment in Semarang and Karimunjawa, Indonesia

Actinomycetes are a group of bacteria that are widely distributed in soil, litter, water, and other natural sources. These Gram positive bacteria can produce hundreds of bioactive compounds, especially antibiotics. This research isolated culturable actinomycetes from mangrove sediments in the Semarang and Karimunjawa Island areas. The isolates that produce potential antibacterial compounds were identified by qualitative screening using the Biosynthetic Gene Cluster (NRPS/PKS) prediction approach. This research was conducted from June to November 2020. A total of 19 actinomycetes from Semarang and 17 actinomycetes from Karimunjawa were found to have at least one type of Biosynthetic Gene Cluster (NRPS, Type I or Type II PKS), but only three isolates had antibacterial activity against S. aureus, E. coli, and L. monocytogenes. Molecular identification found that the bacteria were similar to Brachybacterium paraconglomeratum (99.92%), Streptomyces pluripotens (100%), and Micromonospora chersina (99.08%). Results of the study concluded that the three bacterial isolates that had bacterial activity have similar genes with known antibiotic-producing genes and can potentially provide new antibiotic candidates.

Brevibacillin 2V Exerts Its Bactericidal Activity via Binding to Lipid II and Permeabilizing Cellular Membranes.

Lipo-tridecapeptides, a class of bacterial non-ribosomally produced peptides, show strong antimicrobial activity against Gram-positive pathogens, including antibiotic-resistant Staphylococcus aureus and Enterococcus spp. However, many of these lipo-tridecapeptides have shown high hemolytic activity and cytotoxicity, which has limited their potential to be developed into antibiotics. Recently, we reported a novel antimicrobial lipo-tridecapeptide, brevibacillin 2V, which showed no hemolytic activity against human red blood cells at a high concentration of 128 mg/L, opposite to other brevibacillins and lipo-tridecapeptides. In addition, brevibacillin 2V showed much lower cytotoxicity than the other members of the brevibacillin family. In this study, we set out to elucidate the antimicrobial mode of action of brevibacillin 2V. The results show that brevibacillin 2V acts as bactericidal antimicrobial agent against S. aureus (MRSA). Further studies show that brevibacillin 2V exerts its bactericidal activity by binding to the bacterial cell wall synthesis precursor Lipid II and permeabilizing the bacterial membrane. Combined solid-state NMR, circular dichroism, and isothermal titration calorimetry assays indicate that brevibacillin 2V binds to the GlcNAc-MurNAc moiety and/or the pentapeptide of Lipid II. This study provides an insight into the antimicrobial mode of action of brevibacillin 2V. As brevibacillin 2V is a novel and promising antibiotic candidate with low hemolytic activity and cytotoxicity, the here-elucidated mode of action will help further studies to develop it as an alternative antimicrobial agent.

Practical Gram-Scale Synthesis of Iboxamycin, a Potent Antibiotic Candidate.

A gram-scale synthesis of iboxamycin, an antibiotic candidate bearing a fused bicyclic amino acid residue, is presented. A pivotal transformation in the route involves an intramolecular hydrosilylation-oxidation sequence to set the ring-fusion stereocenters of the bicyclic scaffold. Other notable features of the synthesis include a high-yielding, highly diastereoselective alkylation of a pseudoephenamine amide, a convergent sp3-sp2 Negishi coupling, and a one-pot transacetalization-reduction reaction to form the target compound's oxepane ring. Implementation of this synthetic strategy has provided ample quantities of iboxamycin to allow for its in vivo profiling in murine models of infection.

Comparative in vitro susceptibility of a novel fluoroquinolone antibiotic candidate WFQ-228, levofloxacin, and moxifloxacin against Mycobacterium tuberculosis

ObjectiveWFQ-228 is a novel developed fluoroquinolone (FQ) displaying potent antimicrobial activity against various clinical isolates of pathogens, including FQ-resistant isolates. The aim was to comparatively analyze in vitro susceptibilities of WFQ-228, levofloxacin (LFX), and moxifloxacin (MFX) against Mycobacterium tuberculosis (MTB) isolates, especially with gyrA mutations. MethodsWe selected a panel of 75 MTB isolates, consisting of 25 FQ-susceptible and 50 FQ-resistant isolates determined by conventional drug susceptibility testing. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of FQs to MTB isolates were assessed. ResultsMFX exhibited the most potent activity against FQ-susceptible MTB, demonstrating a MIC50 of 0.031 mg/L, which was lower than that of LFX and WFQ-228. Against FQ-resistant MTB isolates, the MIC50 of WFQ-228 was higher than that of MFX but lower than that of LFX. For WFQ-228, there was a significant overlap existing in the MIC distributions between the probable susceptible (PS) and probable resistant (PR) groups. Six out of 50 PR isolates were classified as susceptible based on a proposed critical concentration (CC) of 0.5 mg/L, yielding a poor sensitivity of 88.0%. These discordant isolates had GyrA substitution in Ala90Val, Ser91Pro, and Asp94Tyr. Additionally, MFX exhibited bactericidal activity against MTB isolates without gyrA mutations, which was significantly higher than that of isolates with gyrA mutations. ConclusionWFQ-228 is more efficacious than LFX in isolates with specific mutations conferring low-level FQ resistance. The bactericidal effect is noted more frequently in FQ-susceptible isolates than FQ-resistant isolates for MFX.

Antibiotic Drug screening and Image Characterization Toolbox (A.D.I.C.T.): a robust imaging workflow to monitor antibiotic stress response in bacterial cells in vivo

The search for novel drugs that efficiently eliminate prokaryotic pathogens is one of the most urgent health topics of our time. Robust evaluation methods for monitoring the antibiotic stress response in prokaryotes are therefore necessary for developing respective screening strategies. Besides advantages of common in vitrotechniques, there is a growing demand for in vivoinformation based on imaging techniques that allow to screen antibiotic candidates in a dynamic manner. Gathering information from imaging data in a reproducible manner, robust data processing and analysis workflows demand advanced (semi-)automation and data management to increase reproducibility. Here we demonstrate a versatile and robust semi-automated image acquisition, processing and analysis workflow to investigate bacterial cell morphology in a quantitative manner. The presented workflow, A.D.I.C.T, covers aspects of experimental setup deployment, data acquisition and handling, image processing (e.g. ROI management, data transformation into binary images, background subtraction, filtering, projections) as well as statistical evaluation of the cellular stress response (e.g. shape measurement distributions, cell shape modeling, probability density evaluation of fluorescence imaging micrographs) towards antibiotic-induced stress, obtained from time-course experiments. The imaging workflow is based on regular brightfield images combined with live-cell imaging data gathered from bacteria, in our case from recombinant Shewanellacells, which are processed as binary images. The model organism expresses target proteins relevant for membrane-biogenesis that are functionally fused to respective fluorescent proteins. Data processing and analysis are based on customized scripts using ImageJ2/FIJI, Celltool and R packages that can be easily reproduced and adapted by users. Summing up, our approach aims at supporting life-scientists to establish their own imaging-pipeline in order to exploit their data as versatile as possible and in a reproducible manner.

Amphipathic Peptide Antibiotics with Potent Activity against Multidrug-Resistant Pathogens.

The emergence and prevalence of multidrug-resistant (MDR) bacteria have posed a serious threat to public health. Of particular concern are methicillin-resistant Staphylococcus aureus (MRSA) and blaNDM, mcr-1 and tet(X)-positive Gram-negative pathogens. The fact that few new antibiotics have been approved in recent years exacerbates this global crisis, thus, new alternatives are urgently needed. Antimicrobial peptides (AMPs) originated from host defense peptides with a wide range of sources and multiple functions, are less prone to achieve resistance. All these characteristics laid the foundation for AMPs to become potential antibiotic candidates. In this study, we revealed that peptide WW307 displayed potent antibacterial and bactericidal activity against MDR bacteria, including MRSA and Gram-negative bacteria carrying blaNDM-5, mcr-1 or tet(X4). In addition, WW307 exhibited great biofilm inhibition and eradication activity. Safety and stability experiments showed that WW307 had a strong resistance against various physiological conditions and displayed relatively low toxicity. Mechanistic experiments showed that WW307 resulted in membrane damage by selectively targeting bacterial membrane-specific components, including lipopolysaccharide (LPS), phosphatidylglycerol (PG), and cardiolipin (CL). Moreover, WW307 dissipated membrane potential and triggered the production of reactive oxygen species (ROS). Collectively, these results demonstrated that WW307 represents a promising candidate for combating MDR pathogens.

Antibiotic candidates from IBM

Researchers say they have come up with two new antibiotic peptide candidates using machine learning, molecular dynamics simulations, and experiments ( Nat. Biomed. Eng. 2021, DOI: 10.1038/s41551-021-00689-x ). Payel Das of IBM Research and colleagues trained a machine-learning method called a deep generative model on a database of all known peptides to create 90,000 peptide sequences that the computer predicted were plausible antimicrobials. The team then mapped the sequences in virtual space according to their properties. Das says the map let the researchers more efficiently tackle the next step: using machine learning to screen for peptides with predicted antimicrobial activity and low human toxicity. A second screening of 163 peptides used molecular dynamics simulations; Das describes the second step as a way to add the human expertise built into those techniques. Her group made the resulting 20 peptides and tested them in lab experiments and in mice. Two candidates emerged

Missense Mutations in the CrrB Protein Mediate Odilorhabdin Derivative Resistance in Klebsiella pneumoniae.

NOSO-502 is a preclinical antibiotic candidate of the Odilorhabdin class. This compound exhibits activity against Enterobacteriaceae pathogens, including carbapenemase-producing bacteria and most of the Colistin (CST)-resistant strains. Among a collection of CST-resistant Klebsiella pneumoniae strains harboring mutations on genes pmrAB, mgrB, phoPQ, and crrB, only those bearing mutations in gene crrB were found to be resistant to NOSO-502.CrrB is a histidine kinase which acts with the response regulator CrrA to modulate the PmrAB system, which finally induces the restructuring of the lipopolysaccharide present on the outer membrane and thus leading to CST resistance. Moreover, crrB mutations also enhance the transcription of neighboring genes such as H239_3063, an ABC transporter transmembrane region; H239_3064, a putative efflux pump also known as KexD; and H239_3065, a N-acetyltransferase.To elucidate the mechanism of resistance to NOSO-502 induced by CrrB missense mutations in K. pneumoniae, mutants of NCTC 13442 and ATCC BAA-2146 strains resistant to NOSO-502 and CST with single amino acid substitutions in CrrB (S8N, F33Y, Y34N, W140R, N141I, P151A, P151L, P151S, P151T, F303Y) were selected. Full susceptibility to NOSO-502 was restored in crrA or crrB deleted K. pneumoniae NCTC 13442 CrrB(P151L) mutants, confirming the role of CrrAB in controlling this resistance pathway. Deletion of kexD (but no other neighboring genes) in the same mutant also restored NOSO-502-susceptibility. Upregulation of the kexD gene expression was observed for all CrrB mutants. Finally, plasmid expression of kexD in a K. pneumoniae strain missing the locus crrABC and kexD significantly increased resistance to NOSO-502.

An Exceptional Broad-Spectrum Nanobiocide for Multimodal and Synergistic Inactivation of Drug-Resistant Bacteria

An Exceptional Broad-Spectrum Nanobiocide for Multimodal and Synergistic Inactivation of Drug-Resistant Bacteria

D-Leucine Modified CM11 Peptide has Admissible Antibacterial Activity with Low Cytotoxic Properties Against Vero Cell Line

Discovering novel antimicrobials is highly crucial to combat drug-resistant bacteria. Antimicrobial peptides (AMPs) are one of the compounds that are being studied as suitable candidates for antibiotics. Therefore, the aim of the present study was to investigate the antibacterial and cytotoxic properties of the d-leucine modified CM11 peptide. To assess the antibacterial activity, minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and bactericidal killing assay were utilized against multidrug-resistant (MDR)clinical isolates and standard strains of methicillin-resistant Staphylococcus aureus (MRSA),Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica serovartyphi. In addition, the scanning electron microscope (SEM) was used to evaluate the effect of the peptide on the bacterial cell structure and morphology. Also, MTT assay was used to determine the cytotoxicity potential of the d-leucine modified CM11 peptide on the Vero cell line. d-leucine modified CM11 exhibited good bactericidal properties and the MIC and MBC values were ranged from 4 to 32 and 8–64 µg ml−1, respectively. The effect of the d-leucine modified CM11 peptide on the morphology and bacterial structures was confirmed by the SEM. The IC50 value of the d-leucine modified CM11 peptide against Vero cells was 160 µl.ml−1.Also, there was no significant difference between the toxicity of the d-leucine modified CM11 peptide with the original CM11 peptide (p > 0.05).Antibacterial and non-toxicity of the d-leucine modified CM11 peptide, which is reported for the first time in this study, speculate the use of this peptide as safe antibacterial agent.

Hydrous manganese dioxide modified poly(sodium acrylate) hydrogel composite as a novel adsorbent for enhanced removal of tetracycline and lead from water

In this study, hydrous manganese dioxide (HMO) modified poly(sodium acrylate) (PSA) hydrogel was produced for the first time to remove tetracycline(TC) and lead(Pb(II)) from water. The as-prepared composite was characterized using various techniques, such as SEM-EDS, FTIR, XRD, BET, and XPS, to elucidate the successful loading of HMO and analyze subsequent sorption mechanisms. Different influencing parameters such as adsorbent dose, initial concentration of adsorbates, reaction time, solution pH, and temperature were also investigated. The adsorption kinetic studies of both TC and Pb(II) removal indicated that equilibrium was achieved within 12 h, with respective removal rates of 91.9 and 99.5%, and the corresponding adsorption data were fitted to the second-order kinetics model. According to the adsorption isotherm studies, the sorption data of TC best fitted to the Langmuir isotherm model while the adsorption data of Pb(II) were explained by the Freundlich isotherm model. The maximum adsorption capacities of both TC and Pb(II) were found to be 475.8 and 288.7 mg/g, respectively, demonstrating excellent performances of the adsorbent. The uptake capacity of PSA-HMO was significantly influenced by the level of solution pH, in which optimum adsorption amount was realized at pH 4.0 in the TC and Pb(II) systems, respectively. Thermodynamic studies showed the process of TC and Pb(II) adsorptions were endothermic and spontaneous. Overall this study elucidated that PSA-HMO composite can be a promising candidate for antibiotics and heavy metal removal in water treatment applications.

ПРОГРАММНЫЙ КОМПЛЕКС ДЛЯ ПРОГНОЗИРОВАНИЯ И ИССЛЕДОВАНИЯ ХАРАКТЕРИСТИК ПРОТИВОГРИБКОВЫХ АНТИБИОТИКОВ

In this work we present a software system that enables antifungal antibiotic drug candidate toxicity and likelihood of drug binding prediction. The system is composed of a number of machine learning models and deterministic algorithms. Its implementation utilizes modern software development practices including a client-server architecture with a thin web-client. Testing showed the models’ accuracy and viability for predicting antifungal antibiotics’ properties.

Advances on chemically modified antimicrobial peptides for generating peptide antibiotics.

Antimicrobial peptides (AMPs) are pinpointed as promising molecules against antibiotic-resistant bacterial infections. Nevertheless, there is a discrepancy between the AMP sequences generated and the tangible outcomes in clinical trials. AMPs' limitations include enzymatic degradation, chemical/physical instability and toxicity toward healthy human cells. These factors compromise AMPs' bioavailability, resulting in limited therapeutic potential. To overcome such obstacles, peptidomimetic approaches, including glycosylation, PEGylation, lipidation, cyclization, grafting, D-amino acid insertion, stapling and dendrimers are promising strategies to fine-tune AMPs. Here we focused on chemical modifications applied for AMP optimization and how they have helped these peptide-based antibiotic candidates' design and translational potential.

Synthesis, biological activities and docking studies of pleuromutilin derivatives with piperazinyl urea linkage

Antibiotics resistance is becoming increasingly common, involving almost all antibiotics on the market. Diseases caused by drug resistant bacteria, such as MRSA, have high mortality and negatively affect public health. The development of new drugs would be an effective means of solving this problem. Modifications based on bioactive natural products could greatly shorten drug development time and improve success rate. Pleuromutilin, a natural product from the basidiomycete bacterial species, is a promising antibiotic candidate. In this study, a series of novel pleuromutilin derivatives possessing piperazinyl urea linkage were efficiently synthesised, and their antibacterial activities and bactericidal properties were evaluated via MIC, MBC and Time-kill kinetics assays. The results showed that all compounds exhibited potent activities against tested strains, especially MRSA strains with MIC values as low as 0.125 μg/mL; 8 times lower than that of marketed antibiotic Tiamulin. Docking studies indicate substituted piperazinyl urea derivatives could provide hydrogen bonds and initiate π-π stacking between molecules and surrounding residues.

Development of an in vivo-mimic silkworm infection model with Mycobacterium avium complex.

In vivo-mimic silkworm infection models with Mycobacterium avium and Mycobacterium intracellulare were newly established to evaluate the therapeutic effects of anti-M. avium complex (MAC) antibiotics. Silkworms raised at 37°C died within 72 hours of an injection of M. avium or M.intracellulare (2.5 × 107 colony-forming unit (CFU)/larva·g) into the hemolymph. Clinical anti-mycobacterial (tuberculosis) antibiotics were evaluated under these conditions. Clarithromycin, kanamycin, streptomycin, amikacin, and ciprofloxacin exerted therapeutic effects in a dose-dependent manner, which was consistent with those in the mouse model. Furthermore, three effective actinomycete culture broths were selected in the screening program of our microbial broth library using the silkworm model, and four active metabolites, ohmyungsamycins A and B (1 and 2), chartreusin (3), and griseoviridin (4), were identified. Among these compounds, 1 showed the lowest 50% effective dose (ED50) value (8.5 µg/larva·g), while 3 had the best ED50/minimum inhibitory concentration (MIC) ratio (7.4). These results indicate that silkworm models are a useful tool for identifying anti-MAC antibiotics candidates with veritable therapeutic effects.

The Antibacterial Drug Candidate SBC3 is a Potent Inhibitor of Bacterial Thioredoxin Reductase.

Antibiotic resistance is a growing problem for public health and associated with increasing economic costs and mortality rates. Silver and silver-related compounds have been used for centuries due to their antimicrobial properties. In this work, we show that 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene silver(I) acetate/NHC*-Ag-OAc (SBC3) is a reversible, high affinity inhibitor of E. coli thioredoxin reductase (TrxR; Ki =10.8±1.2 nM). Minimal inhibition concentration (MIC) tests with different E. coli and P. aeruginosa strains demonstrated that SBC3 can efficiently inhibit bacterial cell growth, especially in combination with established antibiotics like gentamicin. Our results show that SBC3 is a promising antibiotic drug candidate targeting bacterial TrxR.

Navigating changes in Clostridioides difficile prevention and treatment.

Clostridioides difficile (C. difficile, previously known as Clostridium difficile) infections are a major health care concern. The Centers for Disease Control and Prevention (CDC) estimates that C. difficile causes almost half a million illnesses in the United States yearly, and approximately 1 in 5 patients with a C. difficile infection (CDI) will experience 1 or more recurrent infections. The incidence of infection has risen dramatically in recent years, and infection severity has increased due to the emergence of hypervirulent strains. There have been noteworthy advances in the development of CDI prevention and treatment, including a growth in the understanding of the role a patient's gut microbiome plays. The 2017 Infectious Diseases Society of America (IDSA) guidelines made a significant change in treatment recommendations for first time CDI episodes by recommending the use of oral vancomycin or fidaxomicin in place of metronidazole as a first-line treatment. The guidelines also included detailed recommendations on the use of fecal microbiota transplant (FMT) in those patients who experience 3 or more recurrent CDI episodes. A number of novel therapies for the treatment of CDI are in various stages of development. Treatments currently in phase 3 trials include the antibiotic ridinilazole, the microbiome products SER-109 and RBX2660, and a vaccine. All of these agents have shown promise in phase 1 and 2 trials. Additionally, several other antibiotic and microbiome candidates are currently in phase 1 or phase 2 trials. A qualitative review and evaluation of the literature on the cost-effectiveness of treatments for CDI in the U.S. setting was conducted, and the summary provided herein. Due to the higher cost of newer agents, cost-effectiveness evaluations will continue to be critical in clinical decision making for CDI. This paper reviews the updated CDI guidelines for prevention and treatment, the role of the microbiome in new and recurrent infections, pipeline medications, and comparative effectiveness research (CER) data on these treatments. DISCLOSURES: Durham and Le have nothing to disclose. Cassano reports consulting fees from Baxter Healthcare. Peer reviewers Drs. Ami Gopalan and Mark Rubin and Ms. Kathleen Jarvis have nothing to disclose. Planners Dr. Christine L. Cooper and Ms. Susan Yarbrough have nothing to disclose.

박테리오파지를 이용한 병원성 세균의 억제

Excessive use of antibiotics in various industries including agriculture, fisheries, and pharmaceutical industry has led to the emergence of superbacteria which is antibiotic resistant pathogenic bacteria. For that reason, alternative antibiotics have been researched and developed to replace the drugs. Bacteriophage, which is human-friendly and has host-specific characteristics, is one of the alternative antibiotics candidates and can effectively inhibit the growth of pathogenic bacteria such as Staphylococcus aureus, Salmonella, Bacillus cereus, Escherichia coli, and Listeria monocytogenes. In this paper, status of antibiotic resistant pathogenic bacteria and treatment of bacteriophages for the control of the pathogenic bacteria are shortly reviewed.

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

Simple SummaryIn 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.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.