Clostridium novyi 

1. Overview

Clostridium novyi is a Gram-positive, anaerobic, spore-forming bacillus of the family Clostridiaceae and genus Clostridium, and is the causative agent of severe infectious diseases in animals and humans, including gas gangrene, myositis, necrotic hepatitis, and sepsis. As an obligate anaerobe, it is motile and capable of forming endospores, which persist in the environment for extended periods.

C. novyi is classified into three main types (A, B, and C) based on the toxins produced. Type A primarily causes gas gangrene and soft tissue infections in humans, Type B causes infectious necrotic hepatitis (black disease) in sheep and cattle, and Type C is less common. Both Type A and Type B C. novyi produce a lethal alpha-toxin (Tcnα) , which is encoded by a bacteriophage and is the primary determinant of bacterial pathogenicity.

In recent years, the most notable application of C. novyi has been the breakthrough progress of its attenuated strain C. novyi-NT (Nontoxic) in oncolytic bacterial therapy. This strain is attenuated by deleting the phage episode containing the major systemic toxin gene and is capable of selectively germinating and proliferating within the hypoxic and necrotic regions of solid tumors, lysing tumor cells through the secretion of phospholipase C, proteases, and lipases. A Phase Ib clinical trial published in 2025 demonstrated that C. novyi-NT combined with pembrolizumab achieved a 25% confirmed objective response rate with a median response duration of 10.93 months in treatment-refractory solid tumors.

2. Background Information

2.1 What is Clostridium novyi?

C. novyi (formerly known as Clostridium oedematiens) is a Gram-positive, anaerobic, motile, spore-forming bacillus of the family Clostridiaceae. The bacterium is widely distributed in soil and the intestinal tracts of animals, forming part of the normal gut microbiota in pigs. Under anaerobic conditions (such as deep wounds or necrotic tissue), spores germinate into vegetative cells and produce toxins, causing severe infection.

C. novyi strains differ in biochemical characteristics and pathogenicity, and are divided into types A, B, and C based on fermentation capacity and toxin production. All strains are highly sensitive to penicillin G, rifampicin, and other antibiotics.

Schematic representation of Clostridium novyi life cycle. (PMID: 33854487)

2.2 Genome & Classification

The genome size of C. novyi is approximately 2.55 Mb (as in C. novyi-NT). C. novyi, C. botulinum (group III), and C. haemolyticum share a close genetic relationship and are collectively referred to as C. novyi sensu lato. The pathogenic traits of these species (such as botulinum neurotoxin and novyi alpha toxin) are mainly linked to a large plasmidome consisting of plasmids and circular prophages.

A large-scale genomic study published in 2014 sequenced 24 C. novyi sensu lato strains, identifying 61 plasmids, of which 28 were completed. Genomic comparisons revealed four separate lineages, and plasmids were categorized into 13 different plasmid groups. The study found that plasmids and toxin genes had moved across lineage boundaries, a dynamic process primarily driven by phages. The alpha-toxin gene is present in all C. novyi strains but is located on different plasmids (PG9 and PG10).

2.3 What Are C. novyi Virulence Factors？

Alpha-toxin (Tcnα) is the major lethal toxin of C. novyi types A and B and belongs to the large clostridial toxin (LCT) family. Tcnα consists of four functional domains: a glucosyltransferase domain (GTD) , an autocatalytic cysteine protease domain (APD) , a delivery and receptor-binding domain (DRBD) , and the combined repetitive oligopeptides (CROPs) domain.

2.4 Mechanism of Infection and Pathogenesis

2.4.1 Spore Germination and Toxin Production

Spores of C. novyi enter the body through contaminated soil, feed, and water via ingestion, or through broken skin and mucous membranes. After ingestion, spores are transported to the liver via the lymphatic system and germinate when hepatic tissue is damaged (e.g., by migrating immature liver flukes).

2.4.2 Molecular Mechanism of Action of Tcnα

The mechanism of action of Tcnα involves the following key steps:

a. Cell surface attachment: Tcnα first binds to cell-surface sulfated glycosaminoglycans (sGAG) for cell-surface attachment.

b. Receptor-mediated endocytosis: Tcnα utilizes low-density lipoprotein receptor (LDLR) family members for rapid entry. A 2022 study published in Communications Biology demonstrated that LRP1 and Megalin can also facilitate Tcnα cellular entry. LDLR, LRP1, and Megalin recognize Tcnα via their ligand-binding domains (LDL receptor type A repeats) . Notably, LDLR and LRP1 have contrasting expression levels in different cells, and thus the dominant entry receptor for Tcnα is cell-type dependent.

c. Endosomal acidification and translocation: The low pH of endosomes induces structural changes in the toxin, and the GTD is delivered across the endosomal membrane and released into the cytoplasm.

d. Rho/Ras GTPase glycosylation: The GTD glycosylates small GTPases of the Rho and Ras families in the cytoplasm, leading to cytoskeleton disruption and cell death.

2.4.3 Immune Evasion and Immune Response

C. novyi evades host immune recognition through its extremely rapid disease progression. Toxin production is rapid, causing severe tissue damage and systemic intoxication before the immune system can mount an effective response. Furthermore, Tcnα directly impairs phagocyte function by disrupting Rho/Ras GTPases, weakening the host's ability to clear the bacteria.

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2.4.4 Unique Mechanism of Oncolytic Bacterial Therapy

The oncolytic mechanism of C. novyi-NT is based on the unique physiological features of the tumor microenvironment. Solid tumors develop hypoxic and necrotic regions as a consequence of rapid cell proliferation and insufficient blood supply; these regions are inhospitable for normal cells but provide an ideal niche for obligate anaerobes.

The mechanisms of action include:

a. Tumor-selective germination: C. novyi-NT spores selectively germinate within hypoxic and necrotic tumor regions. Spores in normal tissues cannot germinate due to higher oxygen concentrations, eliminating the possibility of germination in normal tissues.

b. Bacterial proliferation and tumor lysis: Germinated bacteria proliferate within the tumor, lysing tumor cells through the secretion of phospholipase C, proteases, and lipases.

c. Immunogenic cell death: Bacterially mediated tumor destruction promotes immunogenic cell death, releasing tumor-associated antigens and damage-associated molecular patterns (DAMPs) that recruit innate immune cells and facilitate cytotoxic T-cell priming, contributing to systemic antitumor immunity.

d. Synergistic enhancement: C. novyi-NT can synergize with chemotherapy, radiotherapy, and immune checkpoint inhibitors, overcoming hypoxia-associated treatment resistance.

2.5 Transmission Routes and Process

Transmission of C. novyi occurs primarily through the following routes:

l Ingestion: Spores are ingested through contaminated soil, feed, and water; this is the primary route of animal infection. The source of infection is fecal contamination of pastures by carrier animals, leading to ingestion of spores by herd mates.

l Wound infection: Spores enter through broken skin and mucous membranes, germinating in deep soft tissues to cause gas gangrene and myositis.

l Injection site infection: Among people who inject drugs, subcutaneous or intramuscular injection of contaminated spores can cause severe soft tissue infection and sepsis.

l Farm-to-farm spread: Via the movement of infected animals, contaminated soil, and carriage during flooding.

Process: Spores enter the body → germinate under anaerobic conditions → vegetative cells produce Tcnα → toxin spreads via blood and lymph → binds target cell receptors → endocytosis and translocation → glycosylation of Rho/Ras GTPases → cytoskeleton disruption and tissue necrosis → systemic intoxication and death.

2.6 Symptoms and Treatment

2.6.1 Clinical Symptoms

Animal infections:

l Infectious necrotic hepatitis (black disease) : Primarily affects sheep and cattle, caused by type B; characterized by acute hepatic necrosis and systemic intoxication, with high mortality

l Gas gangrene and malignant oedema: Present with severe swelling, crepitation, and necrosis at the infection site

l Sudden death: Sudden death cases have been observed in pigs

Human infections:

l Gas gangrene: Deep soft tissue infection presenting with severe pain, swelling, skin discoloration, and systemic intoxication

l Myositis and necrotizing fasciitis

l Sepsis: Can rapidly progress to septic shock and multi-organ failure

2.6.2 Treatment

Antimicrobial therapy: C. novyi is highly sensitive to penicillin G, the first-line antibiotic. Alternatives include rifampicin, clindamycin, and metronidazole. In severe infections, maximum doses of antimicrobials should be used in combination with antitoxin serum to neutralize circulating toxin.

Surgical intervention: For gas gangrene, thorough debridement to remove necrotic tissue and foreign bodies is critical; amputation may be necessary.

Antibiotic management in C. novyi-NT therapy: In oncolytic bacterial therapy, antibiotics (such as amoxicillin/clavulanate and metronidazole) are administered 2 weeks after C. novyi-NT administration to control bacterial proliferation and limit adverse effects.

Supportive care: Includes intravenous fluids, anti-inflammatory therapy, and organ function support.

2.7 Prevention

l Animal vaccination: Vaccines containing C. novyi toxoid are the core measure for preventing black disease and gas gangrene in animals. Commercial multivalent vaccines (such as Ultrabac® 7/Somubac® and Covexin 10) contain inactivated cultures and toxoids of C. novyi type B for active immunization of cattle and sheep. The standard vaccination protocol is a two-dose primary series separated by 4–6 weeks, followed by annual boosters.

l Liver fluke control: Because the occurrence of black disease is closely associated with liver damage caused by migrating liver flukes, controlling liver fluke infection is a key preventive measure.

l Pasture management: Avoid grazing on marshy ground and heavily irrigated pastures, especially during dry summers and droughts, to reduce spore exposure risk.

l Wound management: Thoroughly clean and debride contaminated wounds to reduce the anaerobic environment.

l Risk education for people who inject drugs: Avoid using contaminated injection equipment to reduce the risk of injection site infection.

2.8 Targets for Intervention

2.8.1 Pathogen Targets

2.8.2 Host Targets

2.9 Vaccine Types and Development Progress

2.9.1 Animal Vaccines (Licensed)

Multivalent clostridial vaccines are the primary means of preventing C. novyi infection in animals. Typical products include:

l Ultrabac® 7/Somubac® (Pfizer) : For use in healthy cattle and calves 3 months of age or older, preventing blackleg caused by C. chauvoei, malignant oedema caused by C. septicum, black disease caused by C. novyi, and diseases caused by C. perfringens types C and D.

l Covexin 10 (MSD) : For active immunization of sheep and cattle against C. perfringens types A, B, C, D, C. novyi type B, C. sordellii, C. tetani, etc. Protection against C. novyi type B lasts at least 12 months.

2.9.2 Alpha-Toxin Epitope Vaccine (2025 New Development)

A 2025 study published in Microorganisms screened immunogenic epitopes of C. novyi alpha-toxin using in silico methods and optimized epitope production using recombinant E. coli BL21 pLysS (DE3) strains.

l Epitope selection: Ep1 from the N-terminal region (amino acids 1–583) and Ep2 from the C-terminal region (amino acids 1599–2178) were selected.

l Bioprocess optimization: In fed-batch cultures, dry mass yield reached 1.03 g/L, and in a scale-up system without mechanical agitation, 1.20 g/L. Ep1 yield was 0.56 mmol/mL and Ep2 was 0.61 mmol/mL, with no acetic acid production.

l Immunoreactivity validation: Both Ep1 and Ep2 reacted with serum from animals infected with wild-type active alpha-toxin.

l Significance: This system provides cost savings and technological advances for the veterinary vaccine industry, addressing the challenge of obtaining alpha-toxin from C. novyi as a fastidious organism.

2.9.3 Multi-Epitope Vaccine Design (2025 New Development)

Another 2025 study used in silico vaccine design to target highly epitopic regions of C. perfringens type D epsilon toxin and C. novyi type B alpha toxin, achieving optimal immunogenicity through fusion gene design.

2.10 Drugs

2.10.1 Pathogen-Targeted Drugs

In vitro susceptibility data: C. novyi type A has a MIC90 of 0.06 μg/mL for clindamycin and is highly sensitive to penicillin G, rifampicin, and reverin. However, in vivo animal studies showed that reverin and rifampicin failed to protect infected guinea pigs, suggesting a discrepancy between in vitro susceptibility and in vivo efficacy.

2.10.2 Antitoxin Therapy

Antitoxin serum (containing neutralizing antibodies against C. novyi alpha-toxin) can be used to neutralize circulating toxin and should be administered in combination with antimicrobial therapy.

2.10.3 C. novyi-NT (Oncolytic Bacterial Therapy)

C. novyi-NT is an attenuated strain of C. novyi obtained by deleting the phage episode containing the major systemic toxin gene, and is a representative agent in oncolytic bacterial therapy.

Clinical research progress:

l Phase I clinical trial: Intratumoral injection of C. novyi-NT monotherapy in patients with refractory solid tumors showed evidence of tumor colonization and partial tumor regression.

l Phase Ib clinical trial (NCT03435952) : The Phase Ib study published in Clinical Cancer Research in 2025 (16 patients with advanced solid tumors, median age 62.5 years) evaluated the safety and efficacy of intratumoral injection of C. novyi-NT combined with pembrolizumab. Results showed a 25% confirmed objective response rate (4/16) with a median response duration of 10.93 months, demonstrating a promising therapeutic strategy in treatment-refractory solid tumors.

l Combination strategies: A 2025 review examined the potential strategy of irreversible electroporation (IRE) ablation combined with C. novyi-NT for colorectal liver metastases.

l Microsphere delivery system: A 2025 study developed polylactic acid-based multifunctional porous microspheres (MPMs) loaded with C. novyi-NT spores for image-guided local tumor therapy.

2.11 Recent Research

2.11.1 Diagnostic Technology Advances

PCR detection: A 2025 study identified C. novyi type B isolates from Iranian slaughterhouse samples by PCR amplification of the alpha-toxin gene, confirming PCR as an effective tool for C. novyi diagnosis.

Multiplex PCR detection system: In December 2025, Russian scientists developed a PCR-based test system capable of simultaneously detecting and differentiating 9 Clostridium species (including C. chauvoei, C. novyi, and C. septicum), providing an efficient tool for veterinary diagnostics.

Fluorescent antibody and immunohistochemistry: In cases of bacillary hemoglobinuria in cattle, C. novyi was confirmed by fluorescent antibody testing of liver impression smears (11/12 positive) and immunohistochemistry of liver sections (7/7 positive), demonstrating the feasibility of histological diagnosis.

2.11.2 Molecular Epidemiology and Genomics

The 2014 large-scale genomic study, through sequencing 24 C. novyi sensu lato strains, revealed the dynamic process of plasmid and toxin gene transfer across lineages, primarily driven by phages. This finding explains the "mixed" pathogenic traits among C. novyi, C. botulinum, and C. haemolyticum—strains of different lineages have acquired different pathogenic capabilities through plasmid exchange.

2.11.3 New Discoveries in Pathogenesis

A 2022 study published in Communications Biology overturned the previous view that Tcnα uses only LDLR as its entry receptor, demonstrating that LRP1 and Megalin can also facilitate Tcnα cellular entry, with the dominant entry receptor differing among cell types. This finding provides a theoretical basis for the design of cell-type-specific toxin inhibition strategies.

2.11.4 Expanded Applications of Oncolytic Bacterial Therapy

HPV-positive cervical cancer model: In the HPV-positive TC-1 cervical cancer model, C. novyi-NT spore treatment activated mitochondrial apoptosis, characterized by cytochrome C release and caspase-9/-3 activation, alongside suppression of anti-apoptotic Bcl-2 signaling.

Immunotherapy synergy: C. novyi-NT-mediated tumor destruction promotes immunogenic cell death, releasing tumor-associated antigens and DAMPs, recruiting innate immune cells, and facilitating cytotoxic T-cell priming, producing synergistic antitumor effects when combined with immune checkpoint inhibitors.

2.10.5 Epidemiological Updates

A 2025 case report documented a rare case of dog splenitis caused by C. novyi type B, suggesting potential pathogenicity in companion animals. Additionally, C. novyi has caused outbreaks among people who inject drugs, characterized by large soft tissue infections around injection sites with severe septic/toxic presentation and high lethality.

3. Related Products & Services

l Recombinant Antigens & Receptors

l Antibodies

l Detection Kits

l Recombinant Protein Expression Services

l Antibody Development Services

4. Resources

5. References

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2. Junior, C. A. O., Silva, R. O. S., Lobato, F. C. F., et al. (2020). Gas gangrene in mammals: a review. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, 32(2), 175–183.

3. Skarin, H., Segerman, B. (2014). Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens. PloS one, 9(9), e107777.

4. Bettegowda, C., Huang, X., Lin, J., Cheong, I., Kohli, M., Szabo, S. A., Zhang, X., Diaz, L. A., Jr, Velculescu, V. E., Parmigiani, G., et al. (2006). The genome and transcriptomes of the anti-tumor agent Clostridium novyi-NT. Nature biotechnology, 24(12), 1573–1580.

5. Busch, C., Schömig, K., Hofmann, F., et al. (2000). Characterization of the catalytic domain of Clostridium novyi alpha-toxin. Infection and immunity, 68(11), 6378–6383.

6. Ziegler, M. O., Jank, T., Aktories, K., et al. (2008). Conformational changes and reaction of clostridial glycosylating toxins. Journal of molecular biology, 377(5), 1346–1356.

7. Zhou, Y., Li, D., Luo, J., et al. (2021). Sulfated glycosaminoglycans and low-density lipoprotein receptor mediate the cellular entry of Clostridium novyi alpha-toxin. Cell research, 31(8), 935–938.

8. Zhou, Y., Li, D., Li, D., et al. (2022). LDLR, LRP1, and Megalin redundantly participate in the uptake of Clostridium novyi alpha-toxin. Communications biology, 5(1), 906.

9. Dailey, K. M., Jacobson, R. I., Johnson, P. R., et al. (2021). Methods and Techniques to Facilitate the Development of Clostridium novyi NT as an Effective, Therapeutic Oncolytic Bacteria. Frontiers in microbiology, 12, 624618.

10. Staedtke, V., Roberts, N. J., Bai, R. Y., et al. (2016). Clostridium novyi-NT in cancer therapy. Genes & diseases, 3(2), 144–152.

11. Roberts, N. J., Zhang, L., Janku, F., et al. (2014). Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses. Science translational medicine, 6(249), 249ra111.

12. Janku, F., Zhang, H. H., Pezeshki, A., et al. (2021). Intratumoral Injection of Clostridium novyi-NT Spores in Patients with Treatment-refractory Advanced Solid Tumors. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(1), 96–106.

13. Nelson, B. E., Janku, F., Fu, S., et al. (2025). Phase Ib Study of Pembrolizumab in Combination with Intratumoral Injection of Clostridium novyi-NT in Patients with Advanced Solid Tumors. Clinical cancer research : an official journal of the American Association for Cancer Research, 31(18), 3864–3875.

14. Félix, M. K. C., Deusdará, T. T., Brito, H. S., et al. (2025). Optimization of the Production of Vaccine Epitopes from Clostridium novyi Alpha-Toxin Using Strains of Recombinant Escherichia coli. Microorganisms, 13(7), 1481.

15. Sharafabad, B. E., Abdoli, A., Abdolmohammadi Khiav, L., et al. (2023). Therapeutic Potential of Clostridium novyi-NT in Cancer: Current Knowledge and Future Perspectives. Current cancer drug targets, 23(9), 682–696.