ABSTRACT
The ubiquitous mold Aspergillus fumigatus (A. fumigatus) is one of the main fungal pathogens causing invasive infections in immunocompromised humans. Conventional antifungal agents exhibit limited efficacy and often cause severe side effects. Nanoparticle-based antifungal delivery provides a promising alternative, which can increase local drug concentration; while, mitigating toxicity, thereby enhancing treatment efficacy. Previous research underscores the potential of poly(glycidol)-based nanogels (NG) with negative surface charge as carriers for delivering antifungals to A. fumigatus hyphae. In this study, NG is tailored with 2-carboxyethyl acrylate (CEA) or with phosphoric acid 2-hydroxyethyl acrylate (PHA). It is discovered that quenching with PHA clearly improves the adhesion of NG to hyphal surface and the internalization of NG into the hyphae under protein-rich conditions, surpassing the outcomes of non-quenched and CEA-quenched NG. This enhancement cannot be solely attributed to an increase in negative surface charge but appears to be contingent on the functional group of the quencher. Further, it is demonstrated that itraconazole-loaded, PHA-functionalized nanogels (NGxPHA-ITZ) show lower MIC in vitro and superior therapeutic effect in vivo against A. fumigatus compared to pure itraconazole. This confirms NGxPHA as a promising antifungal delivery system.
ABSTRACT
Lysocin E (1 a) and WAP-8294A2 (2 a) are peptidic natural products with 37- and 40-membered macrocycles, respectively. Compounds 1 a and 2 a have potent antibacterial activities against Gram-positive bacteria and share a unique mode of action. The electron-rich indole ring of d-Trp-10 of 1 a and 2 a interacts with the electron-deficient benzoquinone ring of menaquinone, which is a co-enzyme in the bacterial respiratory chain. Formation of the electron-donor-acceptor complex causes membrane disruption, leading to cell death. Despite the promising activities of 1 a and 2 a, the susceptibility of Trp-10 to oxidative degradation potentially deters the development of these compounds as antibacterial drugs. To address this issue, we replaced the indole ring with more oxidation-resistant aromatics having a similar shape and electron-rich character. Specifically, analogues with benzofuran (1 b/2 b), benzothiophene (1 c/2 c), and 1-naphthalene (1 d/2 d) rings were designed, and chemically prepared by full solid-phase total syntheses. Antibacterial assays of the six analogues revealed similar activities of 1 d/2 d and markedly reduced activities of 1 b/2 b and 1 c/2 c compared with 1 a/2 a. Equipotent 1 d and 2 d both showed high resistance to oxidation by peroxyl radicals. Hence, the present study demonstrates a new molecular editing strategy for conferring oxidation stability on natural products with pharmacologically useful functions.
Subject(s)
Anti-Bacterial Agents , Biological Products , Anti-Bacterial Agents/chemistry , Vitamin K 2 , Microbial Sensitivity TestsABSTRACT
Tryptophan (Trp) plays a unique role in peptides and proteins as its indole ring possesses an electron-rich character and an N1-H hydrogen-bond donor. Because of its non-rotationally symmetric structure, synthetic alterations of the orientation of the indole ring would modulate the intrinsic structures and functions of peptides and proteins. Here we developed synthetic routes to the five Trp isomers in which the C3-substitution of the indole ring was changed to the C2/4/5/6/7-substitutions, and applied the five monomers to Fmoc-based solid-phase peptide synthesis. Specifically, the five monomers were prepared via Negishi cross-coupling reactions of C2/4/5/6/7-iodoindoles. To demonstrate the applicability of the monomers to the solid-phase synthesis, the five Trp isomers of macrocyclic antibiotic lysocin E were selected as target molecules and synthesized through peptide elongation, on-resin macrocyclization, and global deprotection. The Trp isomers displayed markedly weaker antibacterial activity than the parent natural product, revealing the biological importance of the precise three-dimensional shape of the original Trp residue of lysocin E. The present methods for the preparation and application of these five Trp isomers provide a new strategy for analyzing and modifying the specific functions of numerous Trp-containing peptides and proteins beyond this study.
Subject(s)
Solid-Phase Synthesis Techniques , Tryptophan , Tryptophan/chemistry , Peptides/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , IndolesABSTRACT
We found the activity of arylsulfatase in the midgut contents of the silkworm, Bombyx mori. We identified a 60-kDa protein that comigrates with the activity on a column chromatography following ammonium sulfate precipitation. Based on its partial amino acid sequence, we searched for its coding gene using Basic Local Alignment Search Tool (BLAST) and identified KWMTBOMO05106. Transcriptional data suggest a specific expression of the gene in middle silk glands. The majority (80%) of arylsulfatase activity was found in the silk glands, concurring the specific transcription in the silk gland. Observing the feeding behaviour of the silkworm, we found that silkworms smear a mucus secretes from the spinneret on the food pellet as they feed on. Arylsulfatase activity was also detected in the food pellet bitten by the silkworm as well as in the gut content. Furthermore, arylsulfatase activity was not detected either in the food pellet and in the gut content when silkworms had obstructed the spinneret. These results suggest that arylsulfatase is secreted from the silk glands and may contribute to digestive function.
Subject(s)
Bombyx , Animals , Bombyx/chemistry , Silk/genetics , Silk/metabolism , Arylsulfatases/metabolism , Insect Proteins/genetics , Insect Proteins/metabolismABSTRACT
Staphylococcus aureus RN4220 has been extensively used by staphylococcal researchers as an intermediate strain for genetic manipulation due to its ability to accept foreign DNA. Despite its wide use in laboratories, its complete genome is not available. In this study, we used a hybrid genome assembly approach using minION long reads and Illumina short reads to sequence the complete genome of S. aureus RN4220. The comparative analysis of the annotated complete genome showed the presence of 39 genes fragmented in the previous assembly, many of which were located near the repeat regions. Using RNA-Seq reads, we showed that a higher number of reads could be mapped to the complete genome than the draft genome and the gene expression profile obtained using the complete genome also differs from that obtained from the draft genome. Furthermore, by comparative transcriptomic analysis, we showed the correlation between expression levels of staphyloxanthin biosynthetic genes and the production of yellow pigment. This study highlighted the importance of long reads in completing microbial genomes, especially those possessing repetitive elements.
ABSTRACT
Male crickets emit acoustic signals (i.e., songs) by chirping using their forewings. Although the mechanisms and adaptive functions of these songs are well studied, knowledge about how songs develop within a generation is relatively scarce. Our previous work demonstrated a stable peak frequency at 5.7 kHz in the calling songs recorded from mature adult male crickets (Gryllus bimaculatus). In the present study, we monitored changes in the frequency component over time from the sexual maturity stage (early adult stage). We recorded 300 calling songs from a pool of 122 adults. The peak frequency distribution was lower and unstable (i.e., greater coefficient of variance) in the early adult stage. The mean peak frequency was 4.9 kHz on day 3, but gradually converged to 5.8 kHz over the 2-week adult stage. Immature adult males (emitting immature songs) produced an appropriately tuned song with a peak frequency of 5.8 kHz in an environment of 80% helium and 20% oxygen. These results suggest that the frequency component of the calling song is acquired during the early to mid-adult stage, and may be related to sexual maturation in males. Findings from the helium substitution experiment revealed that physical resistance from surrounding gas molecules negatively affect the stability of male singing, and that muscle development and forewing hardening may contribute to the maturation of singing, suggesting that females may adaptively select sexually mature males based on song traits.
Subject(s)
Gryllidae , Male , Adult , Female , Humans , Animals , Helium , PhenotypeABSTRACT
In the development and manufacture of fermented foods, it is crucial to control and manage the bacterial species used in the products. We previously reported a complete genome sequence analysis of the Enterococcus faecalis EF-2001 strain that was used for supplements. By comparing this sequence to the publicly available complete genome sequence of E. faecalis strains, we were able to identify specific sequences of the EF-2001 strain. We designed primer sets to amplify these specific regions and performed a polymerase chain reaction (PCR). We confirmed that the DNA fragments were specifically amplified in the genome of the EF-2001 strain, but not those of other lactic acid bacteria (LABs) or strains of the same genus. Furthermore, these primers amplified DNA fragments even in genomic DNA extracted from heat-treated bacteria at 121°C and foods containing the EF-2001 strain. These results suggest that this method allows for simple and highly accurate identification of specific fermentation strains, such as LABs at the strain level, which will be useful for controlling the quality of fermented foods.
ABSTRACT
Tuberculosis remains a public health crisis and a health security threat. There is an urgent need to develop new antituberculosis drugs with novel modes of action to cure drug-resistant tuberculosis and shorten the chemotherapy period by sterilizing tissues infected with dormant bacteria. Lysocin E is an antibiotic that showed antibacterial activity against Staphylococcus aureus by binding to its menaquinone (commonly known as vitamin K2). Unlike S. aureus, menaquinone is essential in both growing and dormant Mycobacterium tuberculosis. This study aims to evaluate the antituberculosis activities of lysocin E and decipher its mode of action. We show that lysocin E has high in vitro activity against both drug-susceptible and drug-resistant Mycobacterium tuberculosis var. tuberculosis and dormant mycobacteria. Lysocin E is likely bound to menaquinone, causing M. tuberculosis membrane disruption, inhibition of oxygen consumption, and ATP synthesis. Thus, we have concluded that the high antituberculosis activity of lysocin E is attributable to its synergistic effects of membrane disruption and respiratory inhibition. The efficacy of lysocin E against intracellular M. tuberculosis in macrophages was lower than its potent activity against M. tuberculosis in culture medium, probably due to its low ability to penetrate cells, but its efficacy in mice was still superior to that of streptomycin. Our findings indicate that lysocin E is a promising lead compound for the development of a new tuberculosis drug that cures drug-resistant and latent tuberculosis in a shorter period.
Subject(s)
Antitubercular Agents , Mycobacterium tuberculosis , Peptides, Cyclic , Adenosine Triphosphate/metabolism , Animals , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Mice , Mycobacterium tuberculosis/drug effects , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Staphylococcus aureus/metabolism , Streptomycin/pharmacology , Tuberculosis , Vitamin K 2/metabolismABSTRACT
We performed in vivo RNA-sequencing analysis of Staphylococcus aureus in infected mouse liver using the 2-step cell-crush method. We compared the transcriptome of S. aureus at 6, 24, and 48 h post-infection (h.p.i) in mice and in culture medium. Genes related to anaerobic respiration were highly upregulated at 24 and 48 h.p.i. The gene expression patterns of virulence factors differed depending on the type of toxin. For example, hemolysins, but not leukotoxins and serine proteases, were highly upregulated at 6 h.p.i. Gene expression of metal transporters, such as iron transporters, gradually increased at 24 and 48 h.p.i. We also analyzed the transcriptome of mouse liver infected with S. aureus. Hypoxia response genes were upregulated at 24 and 48 h.p.i., and immune response genes were upregulated from 6 h.p.i. These findings suggest that gene expression of S. aureus in the host changes in response to changes in the host environment, such as the oxygenation status or immune system attacks during infection.
Subject(s)
Staphylococcal Infections , Staphylococcus aureus , Animals , Liver , Mice , Staphylococcal Infections/genetics , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Transcriptome , Virulence Factors/geneticsABSTRACT
In this study, we investigated immunoreactivity of peanut (Arachis hypogaea) oil using the silkworm (Bombyx mori) model. The peanut oil induced melanin formation when injected to the silkworm hemocoel. We then purified the active substance and identified the triacylglycerols (TAGs) as the responsible molecule for the melanin-forming effect of peanut oil. Also, the peanut TAGs induced the muscle contraction of the silkworm (i.e., cleavage of the insect cytokine BmPP) and the TNF-α production by cultured mouse macrophage cells. The muscle contraction activity of the peanut TAGs was reduced by saponification reaction, indicating that the TAG (not the degraded fatty acids) moiety is responsible for the activity. The muscle contraction effects of other TAGs of olive, lard, and beef oil were comparable with that of peanut TAGs. Nevertheless, for the melanin formation, the effect of peanut TAGs was outstanding. The fatty acid composition of peanut TAGs was distinct from that of olive TAGs. These results suggest that TAGs are immunoreactive and induces cytokines both in insect and mammalian immune systems. Also, the differential effects of peanut and olive TAGs for the melanin formation may suggest that TAGs with different fatty acid compositions are distinguished by the immune system.
Subject(s)
Arachis , Melanins , Animals , Arachis/metabolism , Cattle , Fatty Acids/metabolism , Immunity, Innate , Insecta/metabolism , Mammals/metabolism , Melanins/metabolism , Mice , Peanut Oil/metabolism , Triglycerides/metabolismABSTRACT
A total of 1253 compounds approved as therapeutic drugs in Japan (Pharmaceuticals and Medical Devices Agency (PMDA)-approved compounds) were screened for their therapeutic effects against Staphylococcus aureus infection using the silkworm infection model. In the first stage of screening with an index of prolonged survival, 80 compounds were identified as hits. Of these, 64 compounds were clinically used as antimicrobial agents, and the remaining 16 compounds were not. The 16 compounds were examined for their dose-dependent therapeutic effects on the silkworm model as a second screening step, and we obtained five compounds as a result. One of the compounds (capecitabine) had no documented in vitro minimum inhibitory concentration (MIC) value against S. aureus. The MIC value of capecitabine against S. aureus strains ranged from 125 to 250 µg/ml, and capecitabine was therapeutically effective at a dose of 200 mg/kg in a murine model of S. aureus infection. These results suggest that silkworm-based drug repositioning studies are of potential value. Furthermore, the therapeutic effects of capecitabine demonstrated in this study provide an important scientific rationale for clinical observational studies examining the association between staphylococcal infection events and capecitabine administration in cancer chemotherapy patients.
ABSTRACT
Mucormycosis, a rare but highly fatal infection, is caused by fungi of the order Mucorales. Due to their ubiquitous nature, reduced susceptibility to antifungals, acid tolerance, and ability to infect immunocompromised patients through rapid dissemination, these fungi have been frequently reported to infect the COVID-19 patients. In order to develop strategies to overcome mucormycosis, it is essential to understand and identify novel Mucorales present in the environment. In this study, we report the identification of four novel pathogenic Mucorales using the silkworm (Bombyx mori) model. The strains' phylogeny was analyzed using the genome sequence of the large subunit ribosomal ribonucleic acid (LSU rRNA) and the internal transcribed spacer (ITS) region, where strains 1-3, 5-3, and S286-1101 claded with Mucor orantomantidis, and strain 827-14 claded with Backusella lamprospora. All the strains had a cold-sensitive phenotype with their inability to grow prominently at 4 °C. Mucor sp. 1-3 and 5-3 were characterized by their filamentous and yeast-like growth under aerobic and anaerobic conditions, respectively. The yeast colonies of Mucor sp. 5-3 had multipolar budding cells often observed with cleaved cell surfaces under a scanning electron microscope. We further found that these strains were able to kill immunocompromised mice suggesting their pathogenicity to mammals. Our study established an invertebrate model-based screening system to identify novel pathogenic Mucorales from the natural environment and provided a clue towards the rapid increase in COVID-19 related mucormycosis.
ABSTRACT
Lysocin E is a lipopeptide with antibiotic activity against methicillin-resistant Staphylococcus aureus. For unclear reasons, the antibacterial activity of lysocin E in a mouse systemic infection model is higher than expected from in vitro results, and the in vitro activity is enhanced by addition of bovine serum. Here, we confirm that serum from various species, including humans, increases lysocin E antimicrobial activity, and identify apolipoprotein A-I (ApoA-I) as an enhancing factor. ApoA-I increases the antibacterial activity of lysocin E when added in vitro, and the antibiotic displays reduced activity in ApoA-I gene knockout mice. Binding of ApoA-I to lysocin E is enhanced by lipid II, a cell-wall synthesis precursor found in the bacterial membrane. Thus, the antimicrobial activity of lysocin E is potentiated through interactions with host serum proteins and microbial components.
Subject(s)
Anti-Bacterial Agents/pharmacology , Apolipoprotein A-I/blood , Methicillin-Resistant Staphylococcus aureus/drug effects , Peptides, Cyclic/pharmacology , Staphylococcal Infections/drug therapy , Animals , Disease Models, Animal , Female , Lipopeptides/pharmacology , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Microbial Sensitivity Tests , Staphylococcal Infections/blood , Staphylococcal Infections/microbiologyABSTRACT
The discovery of novel therapeutic antimicrobials has become an urgent issue in response to the global crisis of the spread of multi-drug-resistant bacteria. In this report, we propose an efficient screening method for antimicrobial agents with therapeutic potential from soil bacteria. With this method, colonies of the soil bacteria were formed first on agar plates containing only an extract of soil, followed by an overlay of soft agar containing the pathogens, an antibiotic target. Then, we selected the colonies that formed the inhibitory zones on soft agar and evaluated the therapeutic efficacy of their culture supernatants using a silkworm bacterial infection model. Using Staphylococcus aureus as an indicator strain to obtain bacteria that produce therapeutically effective antimicrobials, we succeeded in reducing the screening size by 20-fold compared to the conventional method. An analysis of 86 antibiotics producers identified in this study indicated that the majority belonged to Streptomyces sp. and Lysobacter sp., well-known producers of secondary metabolites. Besides, the presence of eight genera and 37 species among the identified species indicated the diversity of antibiotic producers. Based on the finding of our study, we propose this method as an efficient way to discover novel antimicrobial agents that are therapeutically effective.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/metabolism , Soil Microbiology , Animals , Anti-Bacterial Agents/isolation & purification , Bacteria/isolation & purification , Bacterial Infections/drug therapy , Bacterial Infections/microbiology , Bombyx/microbiology , Drug Discovery/methods , Drug Resistance, Multiple, Bacterial , Secondary MetabolismABSTRACT
Various stresses (high temperature, starvation, or sublethal Cryptococcal infection) increased the susceptibility of silkworms to bacterial infection by up to 100-fold, confirming the stress-induced immunosuppression reported in a range of species. When the silkworm was injected with a steroidal drug, betamethasone (1 mg/larva), the susceptibility of the silkworm to bacterial infection increased about 100-fold. This indicates that the immune function of the silkworm can be suppressed by a known compound that shows immunosuppressive effects in humans. We further tested the immunosuppressive effect of the culture supernatants (acetone extracts) of soil bacteria, and 24 out of 193 isolates showed the immunosuppressive activity. These results suggest that it is possible to search for immunosuppressive agents targeting innate immunity by using a silkworm bacterial infection model as a screening system, and that there may be candidate compounds for immunosuppressive agents among the substances produced by soil bacteria.
Subject(s)
Bombyx , Disease Models, Animal , Immunosuppression Therapy , Staphylococcal Infections/immunology , Animals , Anti-Inflammatory Agents/pharmacology , Betamethasone/pharmacology , Cryptococcosis/complications , Cryptococcosis/immunology , Disease Susceptibility , Heat-Shock Response/immunology , Immunosuppressive Agents/pharmacology , Starvation/complications , Starvation/immunologyABSTRACT
In this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.
Subject(s)
Cytokines/genetics , Fish Diseases/microbiology , Goldfish/immunology , Pseudomonas Infections/genetics , Animals , Eye/immunology , Eye/microbiology , Fish Diseases/genetics , Fish Proteins/genetics , Gene Expression Regulation , Goldfish/microbiology , Hot Temperature , Pseudomonas Infections/immunology , Pseudomonas Infections/veterinary , Pseudomonas aeruginosa/immunologyABSTRACT
Nocardia is a ubiquitous environmental microbe that causes nocardiosis against immunosuppressed and immunocompromised hosts. The assay system for the quantitative evaluation of virulence of Nocardia sp. or therapeutic effectiveness of antimicrobials for treatment of nocardiosis is not established so far. In this study, we established an infection model of Nocardia sp. using silkworm as an alternative animal model. We found that all tested Nocardia sp. such as Nocardia asiatica, Nocardia elegans, Nocardia exalbida, Nocardia farcinica, and Nocardia nova killed silkworm and their killing ability were different by species. N. farcinica showed higher pathogenicity among tested strain, similar to the mouse model as previously reported. In addition, we found that antimicrobials such as amikacin and minocycline showed therapeutic effectiveness in silkworms infected with N. farcinica, and we could determine effective doses 50 (ED50) values. These results suggest that silkworm is a useful alternative animal to evaluate the pathogenicity of Nocardia pathogen and the therapeutic effects of antimicrobials against Nocardia sp. in a quantitative manner.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bombyx/drug effects , Nocardia Infections/drug therapy , Nocardia/drug effects , Animals , Bombyx/microbiology , Dose-Response Relationship, Drug , Humans , Immunocompromised Host , Japan/epidemiology , Mice , Models, Animal , Nocardia/pathogenicity , Treatment Outcome , Virulence/drug effectsABSTRACT
Bombyx mori, the silkworm, has biological functions in common with mammals, including humans. Since the molecular design of silkworm's innate immune system is analogous to that of mammals, understanding the silkworm's innate immunity is expected to contribute to the control of infection in humans. It is also possible to use silkworms to explore foodstuffs that activate innate immunity. Lactic acid bacteria have long been used in the production of fermented foods, and in recent years, their use as supplements has been attracting attention. Using silkworms, which are laboratory animals, functional lactic acid bacteria can be explored and isolated at low cost. Fermented foods produced by this method are expected to contribute to the maintenance of human health. In addition to the immune system, humans and silkworms share a common mechanism for maintaining blood glucose homeostasis, and it is possible to construct a pathological model of diabetes and search for therapeutic substances using silkworms. Taken together, we propose that the silkworm is useful for assessing the functions of lactic acid bacterial for health purposes.
Subject(s)
Bombyx/immunology , Fermented Foods/adverse effects , Lactobacillales/immunology , Animals , Bacterial Physiological Phenomena , Bombyx/microbiology , Drug Discovery/methods , Fermented Foods/microbiology , Glycemic Control , Humans , Immunity, Innate , Infection ControlABSTRACT
In this study, we designed a method for conducting a human study by the following process. (1) The host computer stores the subject information. (2) The sample preparer prepares a food sample. (3) The subject (healthy human volunteer) sends the information of an intake of the food sample to the host computer, which creates an event entry for the event. (4) The medical professional (typically a physician) collects and stores the subject's blood sample in a container with the subject's identification (e.g., ID number). (5) The sample analyst analyzes the blood biochemical profiles. (6)The host computer stores the blood biochemical data, and by matching the blood biochemical data with the subject IDs, a final analysis report will automatically be created. In this study, we also run a test case, based on this design, where we obtained a blood biochemical dataset from healthy volunteers. This scheme can reduce the cost of human trials for functional foods and will help acquiring the scientific basis of functional foods.
Subject(s)
Clinical Trials as Topic , Functional Food , Research Design , HumansABSTRACT
Enterococcus faecalis is a common human gut commensal bacterium. While some E. faecalis strains are probiotic, others are known to cause opportunistic infections, and clear distinction between these strains is difficult using traditional taxonomic approaches. In this study, we completed the genome sequencing of EF-2001, a probiotic strain, using our in-house hybrid assembly approach. Comparative analysis showed that EF-2001 was devoid of cytolysins, major factors associated with pathogenesis, and was phylogenetically distant from pathogenic E. faecalis V583. Genomic analysis of strains with a publicly available complete genome sequence predicted that drug-resistance genes- dfrE, efrA, efrB, emeA, and lsaA were present in all strains, and EF-2001 lacked additional drug-resistance genes. Core- and pan-genome analyses revealed a higher degree of genomic fluidity. We found 49 genes specific to EF-2001, further characterization of which may provide insights into its diverse biological activities. Our comparative genomic analysis approach could help predict the pathogenic or probiotic potential of E. faecalis leading to an early distinction based on genome sequences.