Clostridium

USDA (food safety)

Introduction

The genus Clostridium is comprised of gram-positive, anaerobic, spore-forming bacilli. The natural habitat of these organisms is the environment and the interstinal tracts of humans and other animals. Indeed, clostridia are ubiquitous; they are commonly found in soil, dust, sewage, marine sediments, decaying vegetation, and mud. Despite the identiciation of about 100 species of Clostridium, only a small number have been recognized as etiologic agents of medical and veterinary importance. Nonetheless, these species are associated with very serious diseases, including botulism, tetanus, anaerobic cellulitis, gas gangrene, bacteremia, pseudomembranous colitis, and clostridial gastroenteritis. As virtually all of these species have been siolated form fecal samples of apparently healthy persons, some of these isolates may be transient, rather than permanent residuents of the colonic flora (US20040115215).

Botulism is caused by a neurotoxin produced by a member of the genus Clostridium, usually Clostridium botulinum. Organisms classified in this species produce neurotoxins distinguished by their serologic properties into toxin types A, B, C, D, E, and F.

The endospore forming genus Clostridum includes several disease cuasing species. Members of this genus include the causative agents of tetanus (Clostridum tetani), gas gangrene (Clostridium perfringens), and botulism (Clostridium botulinum). The toxins produced by these organisms lead to a myriad of symptoms and signs, including spastic paralysis (tetanus), myonecrosis or necrotizing myositis (gas grangrene) and flaccid paralysis (botulism).

Particular Clostridium Species

Botulism: is a rare but serious illness caused by a toxin that attacks the body’s nerves and causes difficulty breathing, muscle paralysis, and even death. This toxin is made by Clostridium botulinum and sometimes Clostridium butyricum and Clostridium baratii bacteria. These bacteria can be spread by food and sometimes by other means. The bacteria that make botulinum toxin are found naturally in many places, but it’s rare for them to make people sick. These bacteria make spores, which act like protective coatings. Spores help the bacteria survive in the environment, even in extreme conditions. The spores usually do not cause people to become sick, even when they’re eaten. But under certain conditions, these spores can grow and make one of the most lethal toxins known.  See CDC

There are three types of botulism; 1) food borne, 2) infant and 3) wound. The botulinum toxin is a simple A-B toxin with a binding domain (B) and an enzymatic domain (A). There are 8 serotypes of botulinum toxin: A-H. The toxin binds to gangliosides receptor and is internalized. Each serotype cleaves a different part of the SNARE proteins that are responsible for the release of acetylcholine into the synapse from vesicles.

Botulism is a rare but serious illness caused by a toxin that attacks the body’s nerves. Botulism usually start with weakness of the muscles that control the eyes, face, mouth, and throat. This weakness may spread to the neck, arms, torso, and legs. Botulism also can weaken the muscles involved in breathing, which can lead to difficulty breathing and even death. See CDC

–C. botulinum produces the most poisonous biological toxin known. Botulinal toxin blocks nerve transmission to the muscles, resulting in flaccid paralysis. When the toxin reaches airway and respiratory muscles, it results in respiratory failure that can cause death. C. botulinum used to be a major problem in home canning. Its spore is not killed by boiling and survives inadequate pressure sterilization. Mortality can be up to 25%. Symptoms are those common for neurotoxins like muscle weakness and paralysis.C. botulinum spores are carried by dust and are found on vegetables taken from the soil, on fresh fruits and on agricultural products such as honey. Under conditions favorable to the organism, the spores germinate to vegetative cells which produces toxin. Botulims disease may be goruped into 4 types based on the method of introduction of toxin into the bloodstream. Food borne botulims results form ingesting improperly preserved and inadequately heated food that contains botulinal toxin. There were 355 cases of food borne botulism in the US between 1976 and 1984. The death rate due to botulinal toxin is 12% and can be higher in particular risk groups. Wound induced botulism results form C. botulinum penetrating traumatized tissue and producing toxin that is absorbed into the bloostream. Since 1950, 30 cases of wound botulism have been reported. Inhalation botulism results when the toxin is inhaled.

–C. butyricum: C. butyricum is a soil inhabitant in various parts of the world, has been cultured from the stool of healthy children and adults, and is common in soured milk and cheeses. butyricum strains have been reported to be pathogenic, expressing virulence factors (i.e. toxins such as enterotoxins or botulinum neurotoxin; enzymes such as neuraminidase; adhesion molecules; and secretion of high levels of butyric acid).  See Casir 

Clostridium difficile (C. difficile) is a gram positive anaerobic bacillus. C dificile produces two main toxins: enterotoxin A (TcdA) and cytotoxin B (TcdB) which can play a role in diarrhaea. It is the leading casue of hospital acquired diarrhaea. (Pituch, Internat J Antimicrobial Agents, 33(S1), 2009, S42-S45)

Clostridium difficile is a Gram-positive, sporogenic and anaerobic bacterium that causes a potentially fatal colitis. C. difficile enters the body as dormant spores that germinate in the colon to form vegetative cells that secrete toxins and cause the symptoms of infection. During transit through the intestine, some vegetative cells transform into spores, which are more resistant to killing by environmental insults than the vegetative cells. 

C. difficile is considered one of the most difficult infections to treat. In patients, CDI manifests in the colon and presents as abdominal cramping, colitis, and watery diarrhea. One of the biggest issues is the likelihood of reinfection. C. difficile casues about 500k cases of CDI in the US annually. Out of that population, 1 in 6 patients will be reinfected within 8 weeks of recovery. This is known as recurrent CDI. (Ohaka, “Decreasing difficulty levels with mRNA-LNP vaccines” Tides Global, Oct 23, 2024).

During the mid 1990s C difficile infection in acute care hospitals in the US was 30-40 cases per 100k. In 2001, this number rose to almost 50 and in 2005 was nearly three times the 1996 rate (31 per 100k). Of even greater concern are the increases in severe or fatal infection. In England, for example, C difficileinfection was listed as the primary cause of death for 599 patients in 199 and rote to 1998 in 2005 and 3393 in 2006. In addition, sporadic outbreaks have been reported in many hospitals. C dfficile infection predominantly affects elderly and frail hosptial and nursing home patients. However, a recent CDC advisory warns of the risk in populations not previously considered at risk. Metronidazole or oral vacomycin remain treatment of choice. (Kelly, NEJM, 359(18), 2008, 1932-1940)

The clinical spectrum of C difficile assocaited diase (CDAD) ranges form diarrhaea to severe life threatening psuedomembranous colitis. C difficile can be transmitted via personal contact or environmentally. C. difficile has more than 150 PCR ribotypes and 24 toxinotypes, has a pathogenicity locus (PaLoc) with genes encoding enterotoxin A (tcdA) and cytotoxin B (tcdB). (Kuijper, Clinical Microbiology and infection, 12(6), 2006, 2-18).

C difficile s also called “antibiotic associated colitis” due to disruption of intestinal flora by antibiotics which allows C. difficile to grow rapidly in unoccupied niches. Virulence factors include two toxins (A and B). Antibiotic associated pseudomenmbranous colitis results from the use of broad spectrum antibiotic agents such as clindamycin. These anti biotics cause diarrhea in about 10% of treated patietns and pseudomembranous colitis in about 1%. C. difficle causes antiotic assocaited diarrhea and almost all cases of pseudomembranous colitis. Psuedomembranous colitis results from the production of C dfficile toxin A and Toxin B in the olon. Toxin A probably caues most of the gastrointestinal symptoms because of its enterotoxic activity. The toxins may act synergistically and the initial pathology cased by toxin A allows toxin B to manifest its toxicity.

Apart from inflammation-induced oxidative and nitrosative stresses, oxygen (O2) is also a major stress that C. difficile faces during gut colonization. Indeed, although a healthy gut, with a diverse microbiota, is regarded as mainly anoxic, a longitudinal decreasing O2 gradient is observed along the gastrointestinal tract. From 4% in the small intestine lumen, which is the location of spore germination, the O2 tension decreases to 0.1 to 0.4% in the large intestine lumen, where vegetative cells multiply. A second increasing O2 gradient from the colon lumen toward the intestinal epithelium also occurs (from 0.1 to 0.4% to 5%). In addition, antibiotic-induced disruption of the host microbiota leads to an increased O2 level within the gut. Thus, O2 concentration fluctuations within the gastrointestinal tract present a challenge to anaerobic bacteria such as C. difficile. While strictly anaerobic, C. difficile is able to grow in nonstrict anoxic conditions (1 to 3% O2), indicating that this bacterium encodes an arsenal of proteins involved in O2 detoxification and protection against oxidative stress. Indeed, teins involved in O2 detoxification and protection against oxidative stress. Recently, the deletion of the iscS2 gene encoding a cysteine desulfurase likely involved in Fe-S cluster biogenesis was shown to cause a severe growth defect in the presence of 2% O2. See Kint

–Metabolism of C. difficile: C. difficile vegetative cells are considered fastidiously anaerobic and are more sensitive to oxygen than many enteric bacteria (5, 6). Nevertheless, vegetative cells are exposed to, and can tolerate, oxygen in the mammalian digestive tract. Oxygen content in the gut decreases progressively from the stomach to the small intestine and the colon. there is also an oxygen gradient from the central portion of the lumen, which is essentially anaerobic (pO2 < 1 mmHg), toward the epithelial surface, where oxygen concentrations approach 40 mmHg due to perfusion from underlying capillaries. Oxygen levels are elevated in the dysbiotic gut, and vegetative cells have been observed in close proximity to the relatively oxygen-rich mucus layer. Correspondingly, C. difficile vegetative cells tolerate low levels of oxygen, displaying no loss of viability after 15 minutes of air exposure. Recent studies have implicated a role for various C. difficile genes in resistance to reactive oxygen species. A three-gene operon (rbr1-perR-sor) encoding a rubrerythrin, the transcriptional regulator PerR, and a putative superoxide reductase (SOR; desulfoferrodoxin) was upregulated following growth in micro-aerobic conditions (1.5% oxygen) and was implicated in C. difficile oxidative stress response (24). sor encodes a functional superoxide reductase and protects C. difficile vegetative cells against oxidative stress. See Vedantam

–Structure:

—-S-Layer: C. difficile presents a paracrystalline protein array on the surface of the cell known as an S-layer. S-layers have been demonstrated to possess a wide range of important functions, which, combined with their inherent accessibility, makes them a promising drug target. The unusually complex S-layer of C. difficile is primarily comprised of the high- and low- molecular weight S-layer proteins, HMW SLP and LMW SLP, formed from the cleavage of the S-layer precursor protein, SlpA, but may also contain up to 28 SlpA paralogues. (Bradshaw “The structure of the S-layer of Clostridium difficile” J. Cell Commun. Signal. (2018) 12:319–331)

S-layers have been observed in hundreds of prokaryotic species, including a diverse range of bacteria and virtually all archaea. A typical S-layer consists of a single protein arranged in a two dimensional paracrystalline array, forming the outermost surface of the cell (Sara and Sleytr 2000; Smarda et al. 2002). An S-layer may allow the surface presentation of other proteins anchored deeper in the cell wall, but will, by far, form the majority of the externally presented cell surface (Desvaux et al. 2006). S-layer proteins can account for 15% of the total protein of a cell (Sara and Sleytr 2000), and their need for continuous replenishment necessitates the translation of around 500 molecules per second (Smarda et al. 2002). It can be inferred from the high metabolic cost of having an S-layer that it must fulfil significant and essential requirements of the cell. (Bradshaw “The structure of the S-layer of Clostridium difficile” J. Cell Commun. Signal. (2018) 12:319–331)

Unlike the majority of S-layers, which consist of a single protein, the mature S-layer of C. difficile is largely heterodimeric but may contain over 30 other proteins (Fagan et al., 2011b; Monot et al. 2011; Sebaihia et al. 2006). The majority of the S-layer is formed by the low and high molecular weight S-layer proteins (LMW SLP and HMW SLP – previously known as P36 and P47, respectively), which are coded for by a single gene: slpA (Calabi et al. 2001; Karjalainen et al. 2001). (Bradshaw “The structure of the S-layer of Clostridium difficile” J. Cell Commun. Signal. (2018) 12:319–331)

–Treatment of C. dificile:

—-Antibiotics:

Antibiotic –namely metronidazole and the glycopeptide vancomycin –are the current standard frist line therapy againt C. dificile infection.(Ohaka, “Decreasing difficulty levels with mRNA-LNP vaccines” Tides Global, Oct 23, 2024). Most patients with C. difficile associated disease are treated effectively with vancomysin or metronidazole. Other treatment includes tolevemer, a toxin binding polymer.

Although antiboiotics are generally effective, the disease frequently relapses, partly because antibotics kills not only C. difficile but also disrupt colonisation resistance of the gut microflora. Non-antibiotic strateis include probiotics, delibertate colonisation by non-toxigenic C. difficile strains, toxin binding agents, active immunisation, passive immunothepary with intravenous immunoglobulin, mAbs or bovine anti C diffcile whey concentrate and faecal transplantation. (Bauer, International J. of Antimicrobial Agents, 33(S1), 2009, S561-S56).

Between 15-30% of C. diffcile infected patients who initially respond to antimicrobial therapy experience rCDI. (Ohaka, “Decreasing difficulty levels with mRNA-LNP vaccines” Tides Global, Oct 23, 2024).

—-IG or IGM or Both with Secretory component:

Simon (US2008/0260822) discloses treatment for C difficile using antigen specific polyclonal dimeric secretory IgA or pentameric secretory IgM. Because of the secretory component, it has resistance ot degradation in the gastrointestinal tract and can be used at lower doses.

Simon (US 15/205359, published as US 2016/0319039) discloses treating C. difficile by adminstering a mixture of secretory IgA and secretory IgM.  In some embodiments the IgM or IgA is modified with secretory component.

—-Vaccination against toxins A and B stimulates active immunity against C difficile in animals. Passive immunization as by serum antibodies agaisnt C difficile have shown to protect hamsters against C. difficile after oral adminsitration. Passive immunization with bovine antibodies has been prosed as a treatment for other infectious diseases of the gastrointestinal tract, such as diseases cuased by rotavius, enteropathogenic and enterotoxigenic E. coli, Vibrio cholerae and Cryptosporidium parvum. It has been reported that bovine IgG from the colostrum of cows vacinated with C. difficile toxoid protects hamsters agaisnt antibiotic assocaited ceditis. Human intravenous immunoglobuilin dervied from plasma donors has facilitated treatment in some pateints, specially patietns who lack circulating antibodies to the C difficile toxins. In vitro experiments have shown that polymeric IgA is superior to monomeric IgA and IgG in preventing C. dfficile toxin damainge to intestinal eptihelial cell monolayers. (Brown US 14.476,559).

—-mRNA-LNP Vaccines:

Pro-Pro endopeptidase 1 (PPEP-1) is a highly conserved factor that modulates pathogen motility and adhersion through the cleavage of multiple factors on the C. difficle cell surface. PPEP-1 is a metalloprotease that plays a key role in bacterial mobility and gut colonization. It is secreted by all C. difficle strains, amking it an appealing target for such a heterogeneous family of pathogens. PPEP1 has another advantage which is that it is not present in other bacteria in the microbiome. This means that a vaccine should have a narrow-spectrum -C. difficile can be attacked without disrupting composition of the gut. As well as PPEP-1, C. difficile toxins TcdA and TcdB are targeted, further winnowing down the targets to toxigenic strains. Results showd that mRNA-LNP vaccines activate both CD4+ and CD8+ T cell resposnes, signifying an adaptive immune response. The vaccine could overcome deficits in host immunity to protect animals even after infection has occured. (Ohaka, “Decreasing difficulty levels with mRNA-LNP vaccines” Tides Global, Oct 23, 2024).

–Disinfection of C. difficle: 

One study examined vegetative cell and spore tolerances of three C. difficile strains, including 630Δerm, a 012 ribotype and a derivative of a past epidemic strain; R20291, a 027 ribotype and current epidemic strain; and 5325, a clinical isolate that is a 078 ribotype. All isolates were tested for tolerance to ethanol, oxygen, hydrogen peroxide, butanol, chloroform, heat and sodium hypochlorite (household bleach). Our results indicate that 630Δerm vegetative cells (630 spo0A) are more resistant to oxidative stress than those of R20291 (R20291 spo0A) and 5325 (5325 spo0A). In addition, 5325 spo0A vegetative cells exhibited greater resistance to organic solvents. In contrast, 630Δerm spores were more sensitive than R20291 or 5325 spores to butanol. Spores from all three strains exhibited high levels of resistance to ethanol, hydrogen peroxide, chloroform and heat, although R20291 spores were more resistant to temperatures in the range of 60-75°C. Finally, household bleach served as the only chemical reagent tested that consistently reduced C. difficile vegetative cells and spores of all tested strains. See McBridge

C tetani is a spore forming clostridium that can become deposited in tissue through wounds. Clostridium tetani is the causative organism for the disease process known as tetanus. Clostridia are anaerobic organisms with at least 209 species and five subspecies. Clostridium tetani is one of the 4 most well-known exotoxin producing pathogens within this category. Although widespread vaccination efforts have reduced the public health threat, tetanus is a potentially fatal condition. Thus, it is important to recognize the typical clinical presentation, immediate management, and treatment of C. tetani infection. See George 

Clostridium tetani is a motile, anaerobic, spore forming bacteria (terminal spores with drum stick appearance). Vegetative cells are rod shaped, pleomorphic, and occur in pairs or short chains Footnote 1. It is Gram positive in young cultures, but becomes Gram negative upon sporulation. It is catalase and superoxide dismutase negative. It produces a potent neurotoxin tetanospasmin (TeNT), which degrades the SNARE protein required for GABA-ergic neurotransmission.

Tetanus is caused by C. tetani, and has 4 different clinical manifestations: 1) local tetanus at the site of injury; 2) cephalic tetanus, which occurs due to head injuries or infections; 3) Generalized tetanus, which is the most common and represents 80% of the cases; 4) neonatal tetanus, which occurs in infants within 28 days of birth, due to infection of the umbilical stump. C. tetani colonizes small, non serious wounds such as a puncture wound with a splinter, and releases TeNT at the site of injury. The toxin rapidly enters the CNS through retrograde transport and blocks postsynaptic inhibition of spinal motor reflexes resulting in prolonged spasmodic contractions of the skeletal muscles. The first muscles to be affected are the neck and masseter muscles, causing rigidity of the neck and spasms of the jaw (lock jaw/trismus). See MSDS online

Because tetanus spores cannot be eliminated from the environment, and tetanus infection does not confer immunity, elimination requires ongoing active immunization with a tetanus toxoid–containing vaccine (TTCV). To protect infants from tetanus susceptibility at birth, women of reproductive age (usually 15–49 years) should be vaccinated with ≥2 doses of TTCV (TTCV2+), and immunization is recommended for undervaccinated pregnant women early in the third trimester. See MMWR

C. perfringens are ubiquitous and part of the normal flora. They produce a variety of toxins. The virulence of C. perfringens is mediated in large part by its intimidating toxin arsenal. It is a major cause of gas gangrene. It is also a major cause of foodborne illness, ranking as the second most common bacterial cause of food poisoning in the USA. In addition, this bacterium is responsible for approximately 5–15% of all cases of antibiotic-associated diarrhea, which develops in 5–40% of all patients receiving antibiotic therapy. It also causes an often-fatal human disease, enteritis necroticans. As an animal pathogen, C. perfringens is responsible for several serious diseases, including avian necrotic enteritis, which drains approximately US$2 billion/year from the global agricultural system. In addition, widespread vaccination is practiced to protect livestock from C. perfringens-induced enteritis and enterotoxemias, the latter characterized by intestinally produced toxins that are absorbed into the circulation and then affect other organs such as the brain. See McClane

C. perfringens is associated with myonecrosis and gas gangrene (a highly lethal, necro-
tising infection of skeletal muscle and subcutaneous tissue that is most commonly caused
by C. perfringens type A). The symptoms of gas gangrene are caused by the toxicoinfection
of traumatic wounds from spores or vegetative cells. The multiplication of C. perfrin-
gens cells causes severe necrosis of affected tissues. Clinical symptoms include fever, pain,
oedema, and progressive myonecrosis, leading to sepsis, toxic shock, and, finally, death. 

Clostridium perfringens is a Gram-positive anaerobic bacterium that can form spores that are crucial during transmission. C. perfringens spores are exceptionally resistant to stressful environments, such as high temperatures, the presence of oxygen, or low nutrient levels. These features facilitate its survival in different environmental niches, including soil, faeces, sewage, food, and the intestinal tract of humans and animals.

Clostridium perfringens is a ubiquitous, Gram-positive, oligosporogenous pathogen, present in the air, soil and water and in the intestinal tracts of humans and animals. Although C. perfringens is strictly anaerobic, it is considered to be an aerotolerant anaerobe. Indeed, vegetative and stationary-phase cells can survive in a growth-arrested state in the presence of oxygen and/or low concentrations of superoxide- or hydroxyl-radical-generating compounds. C. perfringens possesses specialized genes that might be involved in this adaptive process, such as those encoding superoxide dismutase (SOD), superoxide reductase and alkyl hydroperoxide reductase, but their contribution to the oxidative stress response and their control mechanisms are unknown. See Reysett

Corynebacterium: are gram-positive rods, often club-shaped, in singles, pairs (V-forms), or arranged in stacks (palisades) or irregular clusters. Some are found in the environment and many are in the normal flora (skin and mucous membranes). Corynebacterium species are frequent constituents of the normal bacterial flora of the conjunctival sac and are common as well on human skin and mucous membranes and in the intestines.

One important species, Corynebacterium diphtheri, is the pathogenic bacterium that causes diphtheria, an upper respiratory infection generally characterized by sore throat and high temperature. In severe diphtheria cases, respiratory obstruction due to the inflammation of the tonsils, oropharynx, and pharynx can lead to death as the worst-case scenario. However, in general, Corynebacterium species seem to have low pathogenicity; therefore, the isolation of Corynebacterium species from infected tissue has been considered to arise as a result of mishandling or contamination. Corynebacterium species can be found commonly on the ocular surface, in conjunction with other indigenous bacteria, such as Staphylococcus epidermidis and Propionibacterium acnes. Aoki 

–Corynebacterium diphtheriae (C. diptheriae (diphtheria)): is a non-endospore forming gram-positive club shaped bacterium.  C. diptheriae causes diptheria, a diseases which in the last 50 years has declined but has resurfaced because fewer people are getting vaccinated. In Venezuela in 2017, health care was disrupted and hundreds of children got diphteria.

The most striking symptom of diptheria is a characteristic membrane that forms on the tonsils or pharynx. It can eventually cut of the airway, leading to death. The major virulence factor is an exotoxin encoded by a bacteriophage of C. diptheriae. Strains of the bacterium that are not lysogenized by this phage do not cause serious disease. The rlease of diptheria toxin in the blood leads to complications in distant organds, espeically myocarditis and neuritis. Neuritis affects motor nerves and may result in temperory paralysis of limbs, the soft palate and even the diaphragm.  See Wiki