Clostridium novyi
Encyclopedia
Clostridium novyi a Gram-positive
, endospore
- forming, obligate
anaerobic
bacteria
of the class clostridia
. It is ubiquitous, being found in the soil and faeces. It is pathogenic, causing a wide variety of diseases in man and animals. It comes in three types, labelled A, B, and a non-pathogenic type C distinguished by the range of toxins they produce. Some authors include Clostridium haemolyticum as Clostridium novyi type D. C novyi is closely related to Clostridium botulinum
types C and D as Yoshimasa Sasaki et al. have demonstrated by 16S rDNA sequence analysis. The PAGE analysis reported in ref 1 seems to indicate that the differences between these closely related types is a matter of gene
expression rather than major genetic differences. For example type C can be induced to produce the lethal alpha-toxin.
Growth in culture proceeds through 3 stages: Initial growth wherein no toxin is produced; vigorous growth wherein toxin is produced; and spore formation wherein endospores are formed and toxin production decreases. It is suggested that type C may be type B that forms spores more readily so does not go through the toxin-production stage.
Isolating and identifying C novyi is difficult due to its extreme anaerobic nature. Commercial kits may not be adequate.
It is also fastidious and difficult to culture, requiring the presence of thiols.
The alpha-toxin of Clostridium botulinum types C and D, is similar to the C novyi beta-toxin.
The A and B toxins of Clostridium difficile show homology
with the alpha-toxin of C novyi as does the lethal toxin of clostridium sordellii.
Alpha toxin
The alpha-toxin is characterised as lethal and necrotizing.
The type A alpha-toxin is oedematising
. It acts by causing morphological changes to all cell types especially endothelial cells by inhibition of signal transduction pathways, resulting in the breakdown of cytoskeletal structures. The cells of the microvascular system become spherical and the attachments to neighbouring cells are reduced to thin strings. This results in leakage from the capillaries, leading to oedema. The threshold concentration for this action to occur is 5 ng/ml (5 parts per billion) with 50% of cells rounded at 50 ng/ml.
Spontaneous infection is mostly associated with predisposing factors of hematologic or colorectal malignancies and with diabetes mellitus, although Gram-negative organisms, including Escherichia coli, may lead to a gas gangrene-like syndrome in diabetic patients. This presents with cellulitis and crepitus, and may be mistaken for gas gangrene.
Spontaneous, nontraumatic, or intrinsic infections from a bowel source have been increasingly reported recently.
C novyi has been implicated in mortality among injecting illegal drug users.
A review of the literature reveals that the organism Clostridium novyi type A is capable of inducing most of the symptoms of type 2 diabetes and its complications. There is also anecdotal evidence that claim that antibiotic treatment for this organism produces remission of these symptoms.
Testing is problematical with figures presented by McLauchlin and Brazier [cited above] suggesting a false negative rate of about 40% under ideal conditions. Only positive results may be regarded as reliable. In the absence of a positive test, C. novyi type A may be inferred from characterisation by clinical observation, table 2.
Chronic infection leading to leaky capillaries may also cause retinal haemorrhages and oedema in the lower extremities leading to necrosis and gangrene. Leaky nephrons may compromise the ability of kidneys to concentrate urine leading to frequent urination and dehydration.
Much of the core within a tumor core is necrotic, however some live tumor cells reside there often in a quiescent state. These cells are often quite resistant to standard treatments such as radiotherapy (which relies heavily on DNA damage from radiation-induced oxygen-based free radical species) and chemotherapy
which has poor access to the poorly perfused tumor core and a weak effect on non-dividing quiescent cells. As a result, cells in the hypoxic tumor core often survive treatment and become a source for subsequent cancer recurrence and spread.
Clostridium novyi-NT is a genetically modified form of Clostridium novyi that lacks a major toxin. Because C. novyi-NT is a strict anaerobe; it grows selectively in hypoxic tumor cores; elsewhere, it tends to exist as inactive spores. C. novyi-NT activates and effectively infects and lyses tumor cells in hypoxic tumor cores.
Early work on use of strict anaerobes in tumors goes back several decades. Strongly lytic, infective bacteria tended to be the most effective (however, most earlier research was abandoned due to the risk of toxicity from release of toxins).
One major limitation on the use of C. novyi-NT or other strict anaerobes in cancer treatment is that it tends to affect only the hypoxic tumor core, leaving the active cancer cells in the well-perfused tumor rim alive and intact.
It is thus not surprising that this has led to attempts to combine C. novyi-NT with traditional chemotherapy and/or with radiotherapy (both of which tend to be preferentially effective within the well-perfused tumor rim).
A variety of other clever approaches are under continuing investigation, these include :
In summary, C. novyi-NT is a promising new component to the treatment of solid tumors - effectively targeting the hypoxic tumor cores that were a source of ongoing treatment resistance and recurrence. It is likely that additional modalities will be needed to treat the well-perfused tumor rims.
Gram-positive
Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain and appearing red or pink...
, endospore
Endospore
An endospore is a dormant, tough, and temporarily non-reproductive structure produced by certain bacteria from the Firmicute phylum. The name "endospore" is suggestive of a spore or seed-like form , but it is not a true spore . It is a stripped-down, dormant form to which the bacterium can reduce...
- forming, obligate
Obligate anaerobe
Obligate anaerobes are microorganisms that live and grow in the absence of molecular oxygen; some of these are killed by oxygen. -Metabolism:...
anaerobic
Anaerobic organism
An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. It could possibly react negatively and may even die if oxygen is present...
bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
of the class clostridia
Clostridia
The Clostridia are a class of Firmicutes, including Clostridium and other similar genera. They are distinguished from the Bacilli by lacking aerobic respiration. They are obligate anaerobes and oxygen is toxic to them. Species of the genus Clostridium are all Gram-positive and have the ability to...
. It is ubiquitous, being found in the soil and faeces. It is pathogenic, causing a wide variety of diseases in man and animals. It comes in three types, labelled A, B, and a non-pathogenic type C distinguished by the range of toxins they produce. Some authors include Clostridium haemolyticum as Clostridium novyi type D. C novyi is closely related to Clostridium botulinum
Clostridium botulinum
Clostridium botulinum is a Gram-positive, rod-shaped bacterium that produces several toxins. The best known are its neurotoxins, subdivided in types A-G, that cause the flaccid muscular paralysis seen in botulism. It is also the main paralytic agent in botox. C. botulinum is an anaerobic...
types C and D as Yoshimasa Sasaki et al. have demonstrated by 16S rDNA sequence analysis. The PAGE analysis reported in ref 1 seems to indicate that the differences between these closely related types is a matter of gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
expression rather than major genetic differences. For example type C can be induced to produce the lethal alpha-toxin.
Growth in culture proceeds through 3 stages: Initial growth wherein no toxin is produced; vigorous growth wherein toxin is produced; and spore formation wherein endospores are formed and toxin production decreases. It is suggested that type C may be type B that forms spores more readily so does not go through the toxin-production stage.
Isolating and identifying C novyi is difficult due to its extreme anaerobic nature. Commercial kits may not be adequate.
It is also fastidious and difficult to culture, requiring the presence of thiols.
Toxins
- The toxins are designated by Greek letters. The toxins normally produced by the various types are shown in table 1
Table 1 C novyi type Toxins A alpha, gamma, delta, epsilon B alpha, beta, zeta C gamma
The alpha-toxin of Clostridium botulinum types C and D, is similar to the C novyi beta-toxin.
The A and B toxins of Clostridium difficile show homology
Homology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
with the alpha-toxin of C novyi as does the lethal toxin of clostridium sordellii.
Alpha toxinAlpha toxinAlpha Toxin or alpha-toxin refers to several different protein toxins produced by bacteria. Alpha toxin may be:*Staphylococcus aureus alpha toxin, a membrane-disrupting toxin that creates pores causing hemolysis and tissue damage....
The alpha-toxin is characterised as lethal and necrotizing.The type A alpha-toxin is oedematising
Edema
Edema or oedema ; both words from the Greek , oídēma "swelling"), formerly known as dropsy or hydropsy, is an abnormal accumulation of fluid beneath the skin or in one or more cavities of the body that produces swelling...
. It acts by causing morphological changes to all cell types especially endothelial cells by inhibition of signal transduction pathways, resulting in the breakdown of cytoskeletal structures. The cells of the microvascular system become spherical and the attachments to neighbouring cells are reduced to thin strings. This results in leakage from the capillaries, leading to oedema. The threshold concentration for this action to occur is 5 ng/ml (5 parts per billion) with 50% of cells rounded at 50 ng/ml.
- The duodenumDuodenumThe duodenum is the first section of the small intestine in most higher vertebrates, including mammals, reptiles, and birds. In fish, the divisions of the small intestine are not as clear and the terms anterior intestine or proximal intestine may be used instead of duodenum...
is particularly sensitive to the toxin. Injection into dogs resulted in extreme oedema of the submucosal tissues of the duodenum while leaving the stomach uninjured. Injection into the eye resulted in lesions similar to flame haemorrhages found in diabetic retinopathyDiabetic retinopathyDiabetic retinopathy is retinopathy caused by complications of diabetes mellitus, which can eventually lead to blindness....
.
- The toxin is a large 250-kDaAtomic mass unitThe unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
proteinProteinProteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
the active part of which is the NH2-terminal 551 amino acidAmino acidAmino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
fragment. Alpha-toxins are glycosyltransferases, modifying and thereby inactivating different members of the Rho and Ras subfamily of small GTP-binding proteins. C novyi type A alpha-toxin is unique in using UDP-N-acetylglucosamineUDP-N-acetylglucosamineUridine diphosphate N-acetylglucosamine or UDP-GlcNAc is a nucleotide sugar and a coenzyme in metabolism. It is used by glycosyltransferases to transfer N-acetylglucosamine residues to substrates. D-Glucosamine is made naturally in the form of glucosamine-6-phosphate, and is the biochemical...
rather than UDP-glucose as a substrate.
Epsilon-Toxin
The epsilon-toxin is characterised as lecithino-vitelin and thought to be responsible for the pearly layer found in cultures.Human diseases
The type and severity of the disease caused depends on penetration of the tissues. The epithelium of the alimentary tract, in general, provides an effective barrier to penetration. However, spores may escape from the gut and lodge in any part of the body and result in spontaneous infection should local anaerobic conditions occur.Tissue penetration
Wound infection by C novyi and many other clostridium species cause gas gangreneGas gangrene
Gas gangrene is a bacterial infection that produces gas tissues in gangrene. It is a deadly form of gangrene usually caused by Clostridium perfringens bacteria. It is a medical emergency....
Spontaneous infection is mostly associated with predisposing factors of hematologic or colorectal malignancies and with diabetes mellitus, although Gram-negative organisms, including Escherichia coli, may lead to a gas gangrene-like syndrome in diabetic patients. This presents with cellulitis and crepitus, and may be mistaken for gas gangrene.
Spontaneous, nontraumatic, or intrinsic infections from a bowel source have been increasingly reported recently.
C novyi has been implicated in mortality among injecting illegal drug users.
A review of the literature reveals that the organism Clostridium novyi type A is capable of inducing most of the symptoms of type 2 diabetes and its complications. There is also anecdotal evidence that claim that antibiotic treatment for this organism produces remission of these symptoms.
Epithelial infections
Symptoms are often non-specific including, colitis, oedematous duodenitis, and fever with somnolence.Testing is problematical with figures presented by McLauchlin and Brazier [cited above] suggesting a false negative rate of about 40% under ideal conditions. Only positive results may be regarded as reliable. In the absence of a positive test, C. novyi type A may be inferred from characterisation by clinical observation, table 2.
| Table 2 | |
|---|---|
| Observation | Comment |
| Oedema | Especially if extreme with rapid onset. In view of the sensitivity of the duodenum to the alpha-toxin, oedematous duodenum is always suspect. |
| Anaerobic | Infection occurs at an anaerobic site such as the gut or salivary gland. It may also occur at a site temporarily made anaerobic by occlusion and maintained in this state by oedema. |
| Gram positive | If penicillin causes remission of oedema then a Gram positive organism is the causative agent. |
Chronic infection leading to leaky capillaries may also cause retinal haemorrhages and oedema in the lower extremities leading to necrosis and gangrene. Leaky nephrons may compromise the ability of kidneys to concentrate urine leading to frequent urination and dehydration.
Clostridium novyi-NT - Potential Therapeutic Uses in Cancers
In general, solid tumors are characterized by hypoxic areas in the tumor core. This is due to irregular and insufficient tumor vessel growth and heavy metabolic demands of the surrounding tumor cells.Much of the core within a tumor core is necrotic, however some live tumor cells reside there often in a quiescent state. These cells are often quite resistant to standard treatments such as radiotherapy (which relies heavily on DNA damage from radiation-induced oxygen-based free radical species) and chemotherapy
Chemotherapy
Chemotherapy is the treatment of cancer with an antineoplastic drug or with a combination of such drugs into a standardized treatment regimen....
which has poor access to the poorly perfused tumor core and a weak effect on non-dividing quiescent cells. As a result, cells in the hypoxic tumor core often survive treatment and become a source for subsequent cancer recurrence and spread.
Clostridium novyi-NT is a genetically modified form of Clostridium novyi that lacks a major toxin. Because C. novyi-NT is a strict anaerobe; it grows selectively in hypoxic tumor cores; elsewhere, it tends to exist as inactive spores. C. novyi-NT activates and effectively infects and lyses tumor cells in hypoxic tumor cores.
Early work on use of strict anaerobes in tumors goes back several decades. Strongly lytic, infective bacteria tended to be the most effective (however, most earlier research was abandoned due to the risk of toxicity from release of toxins).
One major limitation on the use of C. novyi-NT or other strict anaerobes in cancer treatment is that it tends to affect only the hypoxic tumor core, leaving the active cancer cells in the well-perfused tumor rim alive and intact.
It is thus not surprising that this has led to attempts to combine C. novyi-NT with traditional chemotherapy and/or with radiotherapy (both of which tend to be preferentially effective within the well-perfused tumor rim).
A variety of other clever approaches are under continuing investigation, these include :
- "RAIT" (radioactive immunotherapy) Radioactive monoclonal antibodiesMonoclonal antibodiesMonoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
have been used as a means of targeting radiotherapy to tumors using common tumor antigens such as CEACarcinoembryonic antigenCarcinoembryonic antigen is a glycoprotein involved in cell adhesion. It is normally produced during fetal development, but the production of CEA stops before birth. Therefore, it is not usually present in the blood of healthy adults, although levels are raised in heavy smokers...
). This approach could be used to target antigenic epitopes on C. novyi-NT itself, using the tumor-localized vegetative forms to deliver radiation to specifically tumor cells thus sparing more healthy tissue. This could be viewed as a form of molecular brachytherapyBrachytherapyBrachytherapy , also known as internal radiotherapy, sealed source radiotherapy, curietherapy or endocurietherapy, is a form of radiotherapy where a radiation source is placed inside or next to the area requiring treatment...
). - Prodrug converting enzymes can be produced by further genetic modification of C. novyi-NT causing the activation of chemotherapeutic prodrugs at the tumor site.
- Anti-cancer drugs may be packaged in liposomes and then specifically released at the tumor site by tumor-localized C. novyi-NT bacteria, improving the effectiveness and safety of the therapy. This approach exposes the tumors to a six times greater concentration of chemotherapy compared to the liposomal drug alone, without increasing the levels of chemotherapy in healthy tissue. Drug release from the liposomes is mediated by an enzyme secreted by C. novyi-NT called liposomase.
- Other chemotherapy delivery technologies using minicells may be used to more specifically deliver chemotheraputic agents to the site of the remaining tumor rim. This could be achieved for example through the conjugation of bispecific antibodies targeted to epitopes on C. novyi-NT. Minicells are a very promising technology in themselves that use bispecific antibodies to dramatically increase the delivery specificity of chemotheraputic drugs by several orders of magnitude, potentially allowing effective chemotheraputic dosages that are hundreds of times the current tolerated systemic levels. Nevertheless, minicells are limited by perfusion access to tumor cores so combination with C. novyi-NT may provide an excellent complement.
- Various genetic modifications to C. novyi-NT seek to further stimulate local inflammation and immune response to boost the immunogenicity of the tumor rim. Many of these approaches secrete immunomodulators/cytokines; others try to use siRNASírnaSírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
or other approaches to further shut down tumor cells. - The hypoxic core can be made temporarily wider by use of drugs like dolastatin, or by temporarily reducing oxygenation. This then allows C. novyi-NT to lyse more of the tumor.
- Most approaches have used single administrations of C. novyi-NT, but it may be useful to give repeated injections to promote an immune response in the area of the active bacteria (the former tumor core and adjacent rim) to create a "bystander effect" on the nearby tumor cells (e.g., boosting the bystander immune response to the tumor cells). It may also help slow relapses by colonizing metastases early after they became large enough to have a significantly sized hypoxic core.
In summary, C. novyi-NT is a promising new component to the treatment of solid tumors - effectively targeting the hypoxic tumor cores that were a source of ongoing treatment resistance and recurrence. It is likely that additional modalities will be needed to treat the well-perfused tumor rims.