Ewingella americana — primary results and discussion

© 2025 Iwata et al. Gut Microbes 17(1), 2599562. CC BY-NC 4.0; selected Results and Discussion text, with laboratory protocols omitted. Figures retain the authors’ claims and labels.

Results

Isolation and characterization of gut microbiota from amphibians and reptiles

The composition and diversity of gut microbiota exhibit remarkable variation across host species, with numerous factors including dietary patterns, environmental antigen exposure, infectious disease history, pharmacological interventions, genetic background, host age, and hygiene practices profoundly influencing microbial community structure and function. 22-24 Furthermore, phylogenetically distinct host species often harbor entirely unique and largely uncharacterized gut microbial communities that have co-evolved with their respective hosts over evolutionary timescales. The intricate host-microbiota interactions that emerge from these relationships play pivotal roles in determining health outcomes versus disease susceptibility. Of particular interest is the epidemiological observation that spontaneous tumor formation in wild amphibian populations occurs at remarkably low frequencies and is typically restricted to specific populations or species, contrasting markedly with neoplasm prevalence patterns observed in other vertebrate taxa. 25 This striking phenomenon suggests that amphibians may possess inherent resistance mechanisms against carcinogenesis, although the underlying molecular and cellular mechanisms responsible for this apparent cancer resistance remain incompletely elucidated and represent an area of significant scientific interest. 23

In the present investigation, we systematically isolated gut bacteria from phylogenetically diverse lower vertebrates, including two amphibian species ( Dryophytes japonicus and Cynops pyrrhogaster ) and one reptilian species ( Takydromus tachydromoides ). Figure 1a provides a comprehensive schematic illustration of the bacterial isolation methodology employed for amphibian and reptilian gut microbiota sampling. Through systematic cultivation and isolation protocols, a total of 45 distinct bacterial strains were successfully obtained and subsequently subjected to preliminary biocompatibility assessment through intravenous administration into BALB/c mice (200 μL via tail vein injection at 5 × 10 9 CFU/mL) to evaluate acute toxicity and general tolerability profiles (Supplementary Table S1).

Figure 1.

Isolation of gut microbiota from amphibians and reptiles . (a) Workflow for isolating gut bacteria from Dryophytes japonicus , Cynops pyrrhogaster and Takydromus tachydromoides . (b) Representative optical microscopy images of isolated gut bacterial strains.

Following rigorous biocompatibility screening procedures, nine bacterial strains demonstrating acceptable safety profiles were selected for comprehensive antitumor evaluation: Priestia aryabhattai (Bacterial No. 1), Rhodococcus qingshengii (Bacterial No. 2), and multiple isolates of Ewingella americana (Bacterial No. 3, 6, 7, 9, 10, and 11) obtained from Dryophytes japonicus ; Citrobacter portucalensis (Bacterial No. 16), Chryseobacterium gambrini (Bacterial No. 22), and Enterobacter ludwigii (Bacterial No. 24) isolated from Cynops pyrrhogaster ; and Rathayibacter oskolensis (Bacterial No. 28, 42), Microbacterium oxydans (Bacterial No. 29, 32, and 43), and Arthrobacter humicola (Bacterial No. 30) derived from Takydromus tachydromoides ( Figure 1b ). Bacterial species identification was confirmed through 16S rRNA gene sequencing and subsequent Basic Local Alignment Search Tool (BLAST) analyzes to ensure taxonomic accuracy (Supplementary Table S2‒S10).

Morphological and growth characteristics revealed distinct phenotypic properties among the isolated strains. On solid agar media, R. oskolensis and M. oxydans formed characteristic yellow-pigmented colonies, whereas the remaining bacterial strains produced white or cream-colored colonies. Growth optimization studies demonstrated that E. americana , R. oskolensis , and M. oxydans exhibited robust growth across multiple culture media formulations. However, to maximize bacterial yield and viability, LB medium was selected as the optimal growth medium for R. oskolensis and M. oxydans , while Pearl Core medium provided superior growth conditions for E. americana . All nine selected bacterial strains were subsequently propagated in liquid culture medium at room temperature (25 °C) under controlled conditions and maintained for subsequent experimental applications.

In vivo antitumor efficacy of isolated gut bacteria

To systematically evaluate the in vivo antitumor potential of the nine selected gut bacterial isolates, we employed a well-established murine Colon-26 carcinoma syngeneic tumor model that recapitulates key features of human colorectal cancer while maintaining immunocompetence ( Figure 2a ). This experimental approach enables assessment of both direct bacterial cytotoxicity and host immune-mediated antitumor responses in a physiologically relevant context. When established Colon-26 tumors reached approximately 200 mm 3 in volume, tumor-bearing immunocompetent mice received single intravenous administrations via tail vein injection of 200 μL bacterial suspensions (5 × 10⁹ CFU/mL) or phosphate-buffered saline (PBS) as negative controls. Tumor growth dynamics were monitored longitudinally for 40 days post-treatment to capture both acute and sustained therapeutic responses. We conducted pilot experiments testing a dose range of 2 × 10 7 to 1 × 10 9 CFU and found that 1 × 10⁹ CFU provided optimal therapeutic efficacy while maintaining excellent safety profiles (Supplementary Figure S1).

Figure 2.

Antitumor activity of gut microbiota in Colon26-bearing BALB/c mice. (a) Experimental timeline: tumor inoculation followed by a single intravenous (i.v.) administration of gut bacterial strains. (b) Antitumor efficacy of bacteria isolated from D. japonicus . Each strain administered via a single tail-vein injection (200 µL, 5 × 10⁹ CFU/mL); PBS served as control. Data shown as mean ± SEM ( n = 3). Statistical comparison versus PBS: ns, not significant; , p < 0.01; , p < 0.0001 (Student’s two-sided t-test). (c) Efficacy of bacteria from C. pyrrhogaster (200 µL, 5 × 10⁹ CFU/mL). Data: mean ± SEM ( n = 3). , p < 0.05; **, p < 0.01; , p < 0.0001 (Student’s two-sided t-test). (d) Efficacy of bacteria from T. tachydromoides (200 µL, 5 × 10⁹ CFU/mL). Data: mean ± SEM ( n = 3). , p < 0.01; , p < 0.0001 (Student’s two-sided t-test). (e) Body-weight monitoring following treatment. Data: mean ± SEM ( n = 3). ns, not significant; , p < 0.05 (Student’s two-sided t-test). Changes in body weight (Δweight, g) were monitored daily throughout the treatment period. All bacterial treatments maintained body weight within acceptable ranges, with no animals experiencing weight loss exceeding 20% of baseline (the predetermined humane endpoint criterion as described in Methods). (f) Kaplan–Meier survival curves up to day 40 post–tumor implantation ( n = 3). **, p < 0.0001 (log-rank [Mantel–Cox] test).

PBS administration served as an appropriate negative control and had no detectable impact on tumor growth kinetics, confirming that the injection procedure itself did not influence tumor progression. Among the bacterial strains isolated from Dryophytes japonicus , P. aryabhattai demonstrated no measurable antitumor activity compared with PBS-treated controls, indicating that not all gut bacteria possess intrinsic anticancer properties. In contrast, both R. qingshengii and E. americana achieved significant tumor growth suppression relative to control treatments ( Figure 2b and Supplementary Figure S2). Most remarkably, E. americana demonstrated exceptional therapeutic efficacy, achieving potent tumor suppression and complete tumor regression (complete response, CR) following a single bacterial administration. The therapeutic kinetics revealed that mice treated with R. qingshengii exhibited initial tumor suppression up to day 5 post-injection; however, tumor re-growth was subsequently observed, suggesting that while this strain possesses antitumor activity, its therapeutic effects are not sustained long-term.

Evaluation of bacterial strains isolated from Cynops pyrrhogaster revealed that C. portucalensis , C. gambrini , and E. ludwigii all significantly suppressed tumor growth compared with PBS controls ( Figure 2c and Supplementary Figure S3). Notably, treatment with C. portucalensis and E. ludwigii induced complete tumor regression by day 3 post-treatment, demonstrating rapid and potent antitumor efficacy. However, tumor recurrence was observed after day 8 in both treatment groups, indicating that while these strains can achieve initial tumor elimination, they may not provide long-term tumor control. Similarly, administration of bacterial strains isolated from Takydromus tachydromoides , including R. oskolensis , M. oxydans , and A. humicola , resulted in significant inhibition of tumor growth relative to control treatments ( Figure 2d and Supplementary Figure S4).

Comprehensive analysis of therapeutic outcomes revealed distinct patterns of antitumor activity among the nine gut bacterial isolates. Specifically, one strain ( P. aryabhattai ) exhibited no detectable antitumor activity, five strains ( R. qingshengii , C. gambrini , R. oskolensis , M. oxydans , and A. humicola ) demonstrated significant tumor growth suppression, and three strains ( E. americana , C. portucalensis , and E. ludwigii ) achieved both tumor growth suppression and active tumor regression. Importantly, safety monitoring throughout the treatment period revealed that none of the bacterial treatments induced significant body weight loss ( Figure 2e ), with body weight changes remaining within ±20% of baseline values, indicating the absence of major adverse impacts on host health and physiological homeostasis. Furthermore, E. americana administration significantly prolonged overall survival compared with PBS controls, with treated mice achieving 100% survival rates and complete response (CR) rates ( Figure 2f ).

Tumor rechallenge experiments demonstrated complete tumor rejection in all E. americana -cured mice (0/10 developed tumors) versus uniform tumor growth in naïve controls (10/10), providing evidence of durable antitumor immunity with immunological memory persisting beyond 60 days (Supplementary Figure S5). This distinguishes bacterial immunotherapy from conventional treatments that lack memory generation, suggesting applications as adjuvant therapy preventing recurrence or consolidation therapy after initial tumor reduction.

A particularly intriguing observation emerged from the analysis of bacterial characteristics associated with tumor regression capability. The three bacterial strains that successfully induced tumor regression ( E. americana , C. portucalensis , and E. ludwigii ) were all identified as facultative anaerobic bacteria. This finding is consistent with established principles of bacterial cancer therapy, as anaerobic bacteria possess the unique capability to selectively accumulate and colonize within solid tumors due to the characteristically hypoxic and immunosuppressive tumor microenvironment. 26 , 27 This selective tumor colonization likely enabled efficient intratumoral bacterial proliferation and, in conjunction with activated immune cell responses, contributed significantly to the observed tumor regression phenomena.

E. americana , isolated from Dryophytes japonicus , is a Gram-negative facultative anaerobic bacterium that belongs to the Enterobacteriaceae family. While this species has been occasionally associated with opportunistic infections in neonates and immunocompromised patients in clinical settings, it is generally recognized as having low pathogenic potential, with opportunistic rather than obligate virulence characteristics. 28 , 29 Additionally, E. americana typically exhibits limited antibiotic resistance profiles and remains susceptible to multiple clinically available antimicrobial agents, 28 , 29 suggesting that potential adverse effects could be effectively managed through targeted antibiotic intervention if required, thereby reducing the likelihood of severe toxicity complications. To our knowledge, this represents the first reported demonstration that a naturally occurring gut bacterium isolated from a wild host organism achieved complete tumor regression following a single intravenous administration. Given its exceptional antitumor efficacy combined with favorable safety characteristics, E. americana was selected as the primary candidate for subsequent detailed mechanistic investigations and comprehensive therapeutic evaluation.

Comparative analysis of E. americana anticancer efficacy versus conventional therapeutic agents

To rigorously assess the therapeutic potential of E. americana and position its efficacy within the context of established cancer treatments, we conducted comprehensive comparative studies evaluating its anticancer activity against conventional therapeutic agents. Specifically, we compared E. americana with the immune checkpoint inhibitor anti-PD-L1 antibody (Anti-PD-L1) and the widely used chemotherapeutic liposome-based nanomedicinal agent doxorubicin (DOX) using the same Colon-26 tumor-bearing mouse model ( Figure 3a ). The experimental design employed clinically relevant dosing regimens: E. americana was administered as a single intravenous injection via tail vein at a dose of 200 μL (5 × 10⁹ CFU/mL), while anti-PD-L1 and DOX were administered intravenously every other day for four total injections at 2.5 mg/kg, representing standard therapeutic protocols.

Figure 3.

Anticancer efficacy: Ewingella americana versus conventional therapies . (a) Experimental design showing treatment schedules for E. americana (single i.v. injection), anti-PD-L1 antibody (four i.v. injections every other day), and doxorubicin (four i.v. injections every other day). All treatment groups in panels (b-f) were evaluated simultaneously using a shared PBS control group to minimize inter-experimental variability and ensure rigorous comparison under identical conditions. (b) Representative mouse images post-treatment; N.A., not available. (c) Tumor response: single i.v. dose of E. americana (200 µL, 5 × 10⁹ CFU/mL); four doses of doxorubicin or anti–PD-L1 (200 µL, 2.5 mg/kg per dose); PBS as control. Data: mean ± SEM ( n = 5). *, p < 0.0001 (Student’s two-sided t-test). (d) Complete response rate at day 30 post-treatment. Data: mean ± SEM ( n = 5). , p < 0.0001 (Student’s two-sided t-test). (e) Kaplan–Meier survival curves up to day 60 ( n = 5). ***, p < 0.0001 (log-rank test). (f) Body-weight monitoring: single E. americana dose, four conventional-drug doses, or single PBS dose. Data: mean ± SEM ( n = 5). ns, not significant (Student’s two-sided t-test).

Comparative efficacy analysis revealed that both anti-PD-L1 and DOX achieved significant tumor growth inhibition compared with PBS-treated controls, confirming the antitumor activity of these established therapies in our experimental model. However, E. americana demonstrated markedly superior antitumor effects, substantially outperforming both conventional treatments ( Figure 3b, c ). While anti–PD-L1 and DOX effectively suppressed tumor progression and delayed tumor growth, only one complete response (CR) was observed in the anti–PD-L1–treated group, and neither therapy achieved consistent tumor eradication across the cohort. In striking contrast, E. americana completely eliminated tumor cells and achieved a 100% CR rate across all treated animals ( Figure 3d ). Furthermore, a single administration of E. americana extended mouse survival by at least 30 days compared with PBS-treated controls, demonstrating both superior efficacy and sustained therapeutic benefit ( Figure 3e ).

The observed superiority of E. americana over conventional therapies can be attributed to fundamental differences in their mechanisms of action and tumor-targeting capabilities. PD-L1 is a critical immune checkpoint protein that binds to the PD-1 receptor on T cells, thereby suppressing T cell activity and serving as a primary mechanism through which tumors evade anti-tumor immunity. 30-32 Tumor cells frequently exploit PD-L1 overexpression to escape immune surveillance, facilitating tumor progression and metastasis. Although PD-L1 inhibitors play increasingly important roles in modern cancer immunotherapy, they lack inherent tumor-targeting capability, resulting in systemic distribution and potentially suboptimal therapeutic efficacy. Furthermore, a significant proportion of cancer patients do not respond to PD-L1 antibody treatments, highlighting the urgent need for more effective therapeutic strategies. 33

DOX represents a cornerstone of chemotherapeutic intervention with multiple well-characterized mechanisms of action, including DNA intercalation and adduct formation, topoisomerase II (TopII) poisoning, free radical generation and oxidative stress induction, and cellular membrane damage through altered sphingolipid metabolism. 34 In this study, we employed a liposomal doxorubicin formulation, which represents an advanced drug delivery system designed to improve therapeutic efficacy while reducing systemic toxicity. Liposomal encapsulation enables enhanced tumor accumulation through the enhanced permeability and retention (EPR) effect, allowing for preferential drug release within the tumor microenvironment. 35 , 36 Despite these pharmacokinetic advantages, liposomal DOX still exhibits limitations in terms of tumor-specific targeting compared to naturally tumor-homing bacteria, and its therapeutic efficacy remains dependent on passive accumulation mechanisms rather than active tumor colonization.

In contrast to these conventional approaches, E. americana , as a facultative anaerobic bacterium, possesses intrinsic tumor-targeting properties that enable specific accumulation within tumor tissues while avoiding healthy organs. This selective tumor colonization allows E. americana to exert potent localized therapeutic effects directly within the tumor microenvironment, explaining why a single bacterial administration was sufficient to achieve dramatic and sustained tumor regression. Safety monitoring revealed that while a slight reduction in body weight was observed shortly after E. americana injection, consistent with mild acute inflammatory responses, no significant differences in body weight were noted among treatment groups by day 15 post-treatment, indicating that the bacterial therapy did not adversely affect overall mouse health or physiological homeostasis ( Figure 3f ).

Mechanistic investigation of E. americana antitumor activity

Given the exceptional anticancer efficacy demonstrated by E. americana in the Colon-26 tumor-bearing mouse model, we conducted comprehensive mechanistic investigations to elucidate the underlying biological processes responsible for its therapeutic activity. Our mechanistic analysis encompassed multiple complementary approaches to characterize both direct bacterial effects and indirect host-mediated responses.

First, to assess the tumor-targeting capability and colonization dynamics of E. americana , intratumoral bacterial colony assays were performed to quantify bacterial accumulation and proliferation within tumor tissues ( Figure 4a ). These investigations revealed that the bacterial load within tumors increased approximately 3000-fold between 3 and 24 hours after intravenous administration, demonstrating highly efficient tumor accumulation and rapid intratumoral proliferation. This dramatic increase in bacterial density within tumor tissues confirms the selective tumor-targeting properties of E. americana and suggests that the hypoxic tumor microenvironment provides favorable conditions for bacterial growth and therapeutic activity.

Figure 4.

Mechanisms underlying Ewingella americana antitumor effects. (a) Colony counts in Colon26 tumors following a single i.v. administration of E. americana (5 × 10⁹ CFU/mL). Data: mean ± SEM ( n = 3 experiments). (b) Optical microscopy of Colon26 spheroids incubated with or without E. americana (5 × 10⁸ CFU). (c) Virulence factors distribution of E. americana . (d) Tumor histology at day 1 post-treatment: H&E, TUNEL, and IHC for NKp46, F4/80, CD19, CD3, CXCR4, TNF- α , caspase-3. (e) Quantification of marker-positive cells (10 independent fields per tumor). Data: mean ± SEM. ns, not significant; , p < 0.001; , p < 0.0001 (Student’s two-sided t-test). (f) qPCR (6 h post-i.v.): expression of NKp46, F4/80, CD19, CD3, Ly6G, IFN- γ , TNF- α (log₁₀ fold-change vs untreated control; ACTB as internal control). Data: mean ± SEM ( n = 3). , p < 0.01; , p < 0.001; **, p < 0.0001 (Student’s two-sided t-test).

To evaluate the direct cytotoxic effects of E. americana against cancer cells, we employed three-dimensional Colon-26 tumor spheroid models that better recapitulate the structural organization and cellular interactions present in solid tumors compared to traditional monolayer cultures. Co-culture experiments involving tumor spheroids with E. americana at varying bacterial concentrations (5 × 10 8 , 5 × 10 7 , 5 × 10 6 , 5 × 10 5 , and 5 × 10 4 CFU) revealed dose-dependent and time-dependent spheroid disruption and cancer cell death. Spheroids treated with the highest bacterial concentration (5 × 10 8 CFU) were largely destroyed within 24 hours, with the majority of cancer cells eliminated through bacterial-mediated cytotoxicity ( Figure 4b, c , and Supplementary Data 1). Notably, even at relatively low bacterial concentrations, E. americana retained sufficient cytotoxic potency to achieve significant cancer cell destruction thanks to bacterial secreted cytolysins such as hemolysin and exotoxin (Supplementary Figure S6). These findings were corroborated by in vitro cytotoxicity assays using the Cell Counting Kit-8 (CCK-8) methodology applied to murine colorectal cancer cells (Colon-26), which demonstrated effective cancer cell elimination across all tested bacterial concentrations (Supplementary Figure S7).

To characterize intratumoral immune responses following bacterial administration, we conducted comprehensive histopathological and immunohistochemical analyzes of tumor tissue sections ( Figure 4d, e ). Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining protocols were employed to assess tissue architecture and immune cell infiltration patterns. IHC analysis revealed that tumor tissues from E. americana -treated mice exhibited prominent expression of multiple immunological biomarkers, including CD19 (B cell marker), CD3 (T cell marker), and CXCR4 (neutrophil marker). Quantitative analysis demonstrated significant increases in immune cell populations compared with control treatments: B cells (CD19⁺) increased by 3%, T cells (CD3⁺) by 5%, and neutrophils (CXCR4⁺) by 30%. These findings indicate robust recruitment and activation of multiple immune cell populations within the tumor microenvironment following E. americana treatment.

The kinetics of immune cell recruitment were further characterized through quantitative PCR (qPCR) analysis, which confirmed that B cells, T cells, and neutrophils were recruited into tumor tissues as early as 6 hours after E. americana administration ( Figure 4f ). This rapid immune cell mobilization suggests that bacterial treatment triggers immediate inflammatory responses that contribute to subsequent antitumor effects. Neutrophils, which represented the most substantially increased immune cell population, possess multiple effector mechanisms including neutrophil extracellular trap (NET) formation, direct phagocytosis of tumor cells, and secretion of pro-inflammatory cytokines and chemokines that can recruit additional immune effector cells. 37

Consistent with enhanced immune cell activation, tumor tissues treated with E. americana exhibited elevated expression of key inflammatory cytokines, including interferon- γ (IFN- γ ) and tumor necrosis factor- α (TNF- α ), relative to control treatments. These cytokines play crucial roles in antitumor immunity: IFN- γ enhances antigen presentation and promotes Th1-type immune responses, while TNF- α promotes T cell activation and proliferation, 38 thereby amplifying immune-mediated tumor destruction.

To confirm the induction of tumor cell death, we employed complementary apoptosis detection methodologies, including Caspase-3 immunostaining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Both techniques revealed widespread apoptosis throughout tumor tissues treated with E. americana , confirming extensive tumor cell death. Additionally, H&E staining demonstrated significant tissue destruction and architectural disruption in treated tumors compared with fresh tumor tissue obtained from PBS-treated control mice.

Collectively, these mechanistic investigations demonstrate that E. americana employs a multifaceted approach to achieve tumor elimination, combining direct bacterial-mediated cytotoxicity with robust activation of host immune responses ( Figure 5 ). The bacterium efficiently infiltrates and proliferates within tumors, where it exerts direct cytotoxic effects while simultaneously activating immune cells (particularly T cells, B cells, and neutrophils) to effectively eliminate cancer cells through complementary mechanisms.

Figure 5.

Schematic illustration of the proposed mechanism.

Biocompatibility and safety evaluation of E. americana

Given the critical importance of therapeutic safety in bacterial cancer therapy, we conducted comprehensive evaluations of the systemic effects and biocompatibility of E. americana administration. These safety assessments were designed to detect potential adverse effects on major organ systems and physiological parameters that could limit clinical translation.

Hematological and biochemical analyzes were performed 7 days after bacterial administration to assess systemic toxicity. Mouse blood samples were collected for complete blood count (CBC) determinations, including white blood cell count (WBC), platelet count (PLT), hematocrit (HCT), hemoglobin concentration (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), and red blood cell count (RBC). Additionally, plasma samples were analyzed for comprehensive biochemical parameters to evaluate liver function, kidney function, and metabolic status ( Figure 6a and Supplementary Table S11).

Figure 6.

Biocompatibility of Ewingella americana . (a) Complete blood counts at day 30 (5 × 10⁹ CFU/mL E. americana or PBS). Data: mean ± SEM ( n = 5). Definitions: WBC, white blood cells; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelets. ns, not significant (Student’s two-sided t-test). All values within normal physiological ranges for BALB/c mice (manufacturer’s data). (b) Histopathology (H&E) of major organs at day 30 post-treatment with E. americana or PBS.

Comprehensive analysis of hematological and biochemical parameters revealed no significant differences between E. americana -treated mice and PBS-treated control groups across all measured parameters. These findings indicate that bacterial treatment did not induce detectable hematological toxicity, hepatotoxicity, nephrotoxicity, or metabolic dysfunction, suggesting excellent systemic biocompatibility.

To evaluate potential organ-specific toxicity, major organs including liver, spleen, heart, lungs, and kidneys were harvested 30 days post-treatment and subjected to detailed histopathological analysis using hematoxylin and eosin (H&E) staining protocols ( Figure 6b ). Microscopic examination of tissue sections revealed no evidence of bacterial existence, tissue damage, inflammatory infiltration, necrosis, or other pathological alterations attributable to bacterial administration in any examined organ. These histological findings confirm that E. americana treatment does not cause detectable organ toxicity or structural damage to vital organs.

We have performed acute toxicity studies with blood collection at multiple early timepoints (24 hours, 48 hours, 72 hours, and 240 hours post-injection) (Supplementary Table S14–S17). These analyzes confirm that E. americana administration induces only transient, mild responses that resolve within 240 hours. Extended observation (60 days) confirmed sustained safety without chronic toxicity. As a naturally occurring, antibiotic-susceptible strain, 28 E. americana offers advantages over genetically engineered bacteria regarding regulatory pathways and safety intervention capabilities.

Blood bacterial colony assays performed at 0.08 (5 min), 3, 24, and 48 hours post-injection demonstrated rapid clearance kinetics, with bacteria becoming completely undetectable by 24 hours (Supplementary Figure S8). This rapid systemic clearance, coupled with tumor-exclusive colonization, explains the favorable safety profile. These results suggest that E. americana is effectively cleared by immune cells following tumor eradication and does not exert adverse effects on host physiology.

The excellent safety profile observed in our studies can be attributed to several factors. First, the naturally occurring, non-pathogenic characteristics of the gut-derived E. americana strain likely contribute to its biocompatibility. Second, the apparent effective clearance of bacteria by host immune cells following tumor eradication prevents bacterial persistence and potential long-term complications. These results suggest that E. americana represents a promising therapeutic candidate with an acceptable safety profile suitable for potential clinical development.

Discussion

In this comprehensive study, we have demonstrated that bacterial strains isolated from the intestinal microbiomes of amphibians and reptiles exhibit remarkable anticancer activity with significant therapeutic potential. Among the nine bacterial strains systematically evaluated, eight displayed significant antitumor effects following single intravenous administrations, highlighting the therapeutic richness of these unexplored microbial communities. Most notably, E. americana , isolated from the Japanese tree frog Dryophytes japonicus , achieved exceptional therapeutic outcomes including complete tumor remission (CR) due to its robust and sustained anticancer efficacy.

Our mechanistic investigations have elucidated that E. americana employs a sophisticated dual-action therapeutic mechanism involving both direct and indirect antitumor effects. The underlying therapeutic mechanism encompasses selective tumor colonization and proliferation by this facultative anaerobic bacterium, coupled with potent direct cytotoxic effects against cancer cells and comprehensive immune-mediated tumor suppression through coordinated activation of neutrophils, T cells, and B cells. The recruited immune cell populations release pro-inflammatory cytokines, particularly TNF- α and IFN- γ , which further amplify immune activation and induce substantial apoptosis throughout tumor tissues. Importantly, E. americana , being isolated from a natural host organism, demonstrated exceptional biocompatibility with no evidence of systemic toxicity or adverse effects on major organ systems, thereby establishing its promise in terms of both efficacy and safety.

Our subcutaneous Colon-26 model, while differing from orthotopic colorectal cancer models, provides critical advantages for proof-of-concept bacterial therapy studies. This model enables unambiguous demonstration of tumor-specific homing through bloodstream circulation, independent of anatomical proximity to the gastrointestinal tract. Importantly, our findings have direct clinical relevance to metastatic disease, where tumors are anatomically distant from gut microbiota (liver, lung, peritoneum). The syngeneic immunocompetent model preserves essential host-bacterial-tumor immune interactions, crucial for evaluating immunomodulatory mechanisms. Future orthotopic studies will provide complementary insights into bacterial-gut microbiota interactions.

E. americana 's exceptional tumor specificity likely arises from multiple synergistic mechanisms beyond hypoxia alone. Tumor cells overexpress CD47 (“don't eat me” signal), 39 , 40 creating locally immunosuppressed microenvironments permitting bacterial persistence, while intact immune surveillance in healthy organs rapidly clears bacteria. Additional tumor-specific features—necrotic regions, aberrant vasculature facilitating bacterial extravasation, 41 altered metabolic byproducts, and disrupted extracellular matrix—synergistically support selective colonization. Our colony assays and histopathological analyzes demonstrate bacterial recovery exclusively from tumors, with zero detectable colonization in lung, liver, spleen, kidney, or heart. Since physiological hypoxia exists in certain normal tissues (intestinal crypts, renal medulla), the complete absence of bacterial colonization in these sites confirms that hypoxia is necessary but insufficient, and the unique tumor microenvironment constellation creates a permissive niche for E. americana .

E. americana achieved superior outcomes (100% CR) versus anti-PD-L1 (tumor suppression only), reflecting fundamentally different immune activation mechanisms. Anti-PD-L1 passively removes inhibitory signals on pre-existing T cells (“releasing brakes”), whereas E. americana actively stimulates immunity through PAMPs (pathogen-associated molecular patterns), triggering robust innate immune activation through pattern recognition receptors including Toll-like receptors (TLRs) and NOD-like receptors (NLRs) 42 that recruits neutrophils, macrophages, and NK cells while initiating de novo inflammatory cascades. This mechanistic orthogonality is evidenced by distinct immune infiltration patterns— E. americana induced dramatic neutrophil recruitment 43 that directly contributes to tumor destruction, while checkpoint inhibitors minimally affect neutrophil populations. These differences suggest potential synergy in combination therapy and indicate bacterial therapy may benefit checkpoint inhibitor-resistant patients, particularly those with “cold” tumors lacking T cell infiltration, 44 which represent a significant proportion of patients who fail conventional immunotherapy.

Our comparator selection was strategic: anti-PD-L1 represents best-in-class immunotherapy with broad clinical adoption, enabling assessment of bacterial therapy advantages over current immunotherapy standards. Liposomal doxorubicin (FDA-approved nanoformulation) represents state-of-the-art drug delivery technology; demonstrating superiority establishes that biological tumor targeting outperforms sophisticated pharmaceutical delivery systems. These comparators facilitate mechanistic insights (immune-based vs. cytotoxic) and literature comparison. Future studies will include colorectal-specific regimens (5-FU, FOLFOX) for comprehensive benchmarking.

Pharmacokinetic studies revealed favorable kinetics: rapid blood clearance coupled with efficient tumor accumulation. This profile explains both excellent safety and potent efficacy with single-dose administration. Biodistribution analyzes confirmed tumor-exclusive localization. These characteristics compare favorably with chemotherapy agents exhibiting prolonged systemic exposure and normal tissue accumulation. Peak intratumoral bacterial burden (48 hours) provides rational timing for combination therapies.

The therapeutic mechanism—hypoxia-driven colonization and PAMP-mediated immune activation—relies on features common to most solid tumors, suggesting broad applicability. Literature supports this: tumor-targeting bacteria demonstrate efficacy across various cancer models. 15 , 17 , 20 Hypoxic, immunologically “cold” tumors (pancreatic cancer, triple-negative breast cancer) may be particularly responsive. Our spheroid experiments demonstrate direct cytotoxicity operating through universal cellular targets. Future studies in other cancer models will definitively establish broad-spectrum activity.

Our dose-finding studies identified 1 × 10⁹ CFU as both the optimal therapeutic dose and maximum tolerated dose, indicating a narrow therapeutic window that requires careful consideration for clinical translation. However, comprehensive safety evaluation at this dose revealed only transient, self-limiting inflammatory responses with complete resolution by 72 hours and no organ toxicity. Pharmacokinetic analysis demonstrated rapid bacterial clearance from blood (undetectable by 24 hours) with selective tumor accumulation, explaining the acceptable safety profile despite narrow margins. For clinical development, several strategies can optimize the therapeutic index: dose fractionation to avoid peak systemic burden, intratumoral injection for accessible tumors, combination with lower bacterial doses plus checkpoint inhibitors, and intensive monitoring with antibiotic intervention capability as a fail-safe mechanism. Species differences in immune responses and allometric scaling with appropriate safety factors will guide safe starting doses for human trials.

For clinical translation, key considerations include: GMP manufacturing protocols ensuring batch consistency; 45 patient selection focusing on advanced solid tumors with documented hypoxia; dose-escalation trials with intensive safety monitoring; and exploration of combination strategies with checkpoint inhibitors (potentially synergistic), chemotherapy (enhanced drug delivery), and radiation (tumor sensitization). Investigation of multiple dosing schedules, intratumoral injection for accessible tumors, and autologous microbiome screening represent promising avenues for personalized bacterial therapy.

Future investigations should explore alternative administration routes, particularly oral delivery, which could offer advantages in patient convenience and accessibility. However, oral administration of E. americana would require development of protective formulation strategies (enteric coating, acid-resistant capsules) to ensure bacterial survival through gastric acid and successful translocation from gut to tumor sites. Such formulation-based approaches would be especially relevant for treating primary colorectal tumors or liver metastases where anatomical proximity facilitates bacterial translocation. While technically challenging, oral delivery strategies could expand clinical applicability and enable investigation of administered bacteria-host microbiota interactions, representing a promising direction for future research beyond the intravenous approach validated in the current study.

Current cancer therapeutic approaches leveraging gut microbiota have primarily focused on microbiome modulation strategies or fecal microbiota transplantation protocols rather than direct bacterial administration. However, the intestinal tract represents a vast repository of bacterial diversity, harboring numerous species that remain insufficiently characterized despite possessing unique metabolic pathways, bioactive compound production capabilities, and immunomodulatory properties with potential therapeutic applications. The present study highlights the tremendous potential of systematically exploring and harnessing these underexplored microbial resources for innovative antitumor therapy approaches.

Our findings underscore the critical need for expanded research efforts focused on comprehensive bacterial strain characterization, detailed mechanistic investigations of bacterial-host interactions, and the systematic development of novel therapeutic strategies toward clinical application. The exploration of diverse bacterial communities from phylogenetically distinct host species may yield additional therapeutic candidates with complementary or enhanced anticancer properties. Furthermore, the development of optimized bacterial delivery systems, combination therapeutic approaches, and personalized treatment protocols based on individual patient microbiome profiles represents promising avenues for future investigation.

The successful identification of E. americana as a potent, naturally occurring anticancer agent establishes a proof-of-concept for microbiome-derived bacterial therapeutics and provides a foundation for the development of a new class of cancer treatments. These discoveries may ultimately lead to transformative advances in precision oncology and offer new hope for patients with treatment-refractory cancers. Future research directions should focus on expanding bacterial discovery programs, optimizing therapeutic protocols, investigating combination therapies, and advancing promising candidates toward clinical translation to fully realize the therapeutic potential of microbiome-derived cancer therapeutics.

This study provides novel insights into the therapeutic potential of previously uncharacterized gut microbes from lower vertebrates and establishes a foundation for the development of naturally occurring bacterial therapeutics in cancer treatment. Our findings demonstrate the vast untapped potential residing within diverse microbial ecosystems and highlight the critical importance of biodiversity conservation efforts in advancing medical science and therapeutic innovation.