Definition/Introduction

Bacterial species have different coping mechanisms with selective harsh environmental conditions. One of the most common coping mechanisms for bacteria is forming spores to protect themselves against ecological degrading agents. Bacterial spores are the most dormant form of bacteria since they exhibit minimal metabolism and respiration, as well as reduced enzyme production.

Typically, Gram-positive bacteria are best known for producing intracellular spores called endospores as a survival mechanism. Endospores are highly retractile and thick-walled structures formed inside the bacterial cells. It is most common for Bacillus species as well as Clostridium species to create endospores.[1] B. cereus is a member of the Bacillus species and is well-known for its ability to cause foodborne illness as a result of its spores surviving various temperatures. Similarly, C. perfringens spores are acid-soluble proteins that show high resistance to chemicals and heat.[2]

Endospores can resist inactivation from ethanol treatment.[3] They also can survive high temperatures for up to 150°C, making specific Gram-positive species heat resistant. Further, bacterial spores can show typical viability signs at temperatures near the absolute zero. Endospores are resistant to the chemical agents, e.g., triphenylmethane dyes, and can even protect the bacterial cells against ultraviolet radiation, extreme pH gradients, drought, and nutrition depletion.

Endospores germinate back into vegetative cells (an active bacterial cell that undergoes metabolism) when surrounding environmental conditions favor bacterial growth and reproduction. Several stimulants revert bacterial cells to their active vegetative cells, such as optimal close-to-body temperature and diffusion of nutrients and water through bacterial cell walls through alteration of their surface tension.

The process of spore formation is a multistep process. It starts from replication of the bacterial DNA, followed by the formation of the forespore, which is, by definition, pinching of the cellular plasma membrane between the replicated chromosome. Then, a cortex forms between the inner and outer membrane by extending the second cellular membrane to enclose the forespore with calcium and dipicolinic acid. Finally, the external spore coat surrounds the endospore before its release.[4]

Microscopic examination to delineate the morphology of endospores involves differential staining processes such as malachite green and fluorescence staining techniques. Staining dormant bacterial samples with malachite green as the primary stain and safranine as the counteract stain results in the appearance of green oval endospores enclosed inside pink vegetative bacterial cells.[5] There are different locations of the endospores inside the bacterial cell. For instance, central endospores are located in the middle of the bacterial cell, while the terminal endospore appears at the end. There is also a subterminal type of endospores that appears between the middle and the end of the cell.[6]

Despite their sturdy and resistant nature to environmental threats, endospores can get affected by certain eradication factors. During the 17th century, John Tyndall, a famous European physicist, discovered Tyndallization. The latter is the process of heating liquids and objects at a temperature of 80 to 100°C for 30 minutes; then, the sample is incubated. The procedure is repeated for three consecutive days. The principle behind successive heating for three days is that heating endospores for the first-time results in reverting them into vegetative cells killed through repetitive heat in the second and third days.[7]

Issues of Concern

Anthrax Bioweapon (Bioterrorism)

B. anthracis is a gram-positive spore-forming bacteria that are commonly found in the soil of endemic areas. It is one of the most common agents used in biological warfare. Many factors make B. anthracis a good bioweapon. Its endospores can be placed into food, water, powder, and sprays, spreading the anthrax infection without anyone's knowledge, as endospores are microscopic, cannot be tasted or felt. In 2001, anthrax spores were used as a bioweapon in the U.S. They were distributed into letters delivered by the United States Postal system spreading anthrax infection among 22 mail handlers and customers. Anthrax bioterrorism attacks can take several other forms. B. anthracis endospores can be released into food and water, sprayed from the air or high buildings, or even carried on clothes or shoes. B. anthracis endospores can cast a high risk of misuse and pose severe threats to public safety and health.[8]

Clostridium difficile Colitis

C. difficile infections (CDI) are associated with high morbidity, mortality, and healthcare costs. The cost of CDI is estimated at $5.4 billion in the United States - $4.7 billion (86.7%) from healthcare settings and $725 million (13.3%) incurred in the community[9]. The morbidity includes an increased need for colectomies, discharges to nursing homes, and readmissions.[10] The morbidity and mortality rates of pseudomembranous colitis are 10% to 20% in untreated elderly individuals. In patients with toxic megacolon, the mortality can be as high as 35% despite surgical intervention.[11]

Clinical Significance

Anthrax:

Endospores of B. anthracis cause anthrax. It has four types according to its mode of infection and the affected system:

1. Cutaneous anthrax
2. Gastrointestinal anthrax
3. Inhalational anthrax
4. Injection anthrax

Cutaneous anthrax develops as a result of wound contamination with B. anthracis endospores while handling contaminated animal products. It is characterized by a painless skin ulcer with a black center and edematous blisters that may be itchy. Gastrointestinal anthrax develops as a result of the ingestion of water or food contaminated with B. anthracis endospores. The patients can present with fever, nausea, bloody vomiting, bloody diarrhea, painful swellings of the neck lymph nodes, and flushing of the face and the eyes. Inhalational anthrax results from the inhalation of bacterial endospores while handling contaminated animal materials, such as wools or feces. It characteristically presents with fever, shortness of breath, cough, chest discomfort, body ache, sweating, and nausea.[12]. Injection anthrax develops from using syringes contaminated with bacterial endospores. It clinically presents with the development of deep abscesses under the skin.

Appropriate specimen collection is crucial to confirm the diagnosis. For inhalational anthrax, gram stain and PCR of pleural, bronchial, and CSF (if meningitis) is usually required. Skin swabs for gram stain, PCR, and biopsy of the skin lesion are performed in cutaneous anthrax. Similarly, gram stain, culture, and PCR of ascitic fluid, oral lesion, or rectal swab can be collected in gastrointestinal anthrax. 

Systemic or disseminated anthrax is treated with a combination of intravenous antibiotics (ciprofloxacin, meropenem, and linezolid ) and antitoxin therapy.

Tetanus

It is a bacterial infection caused by contamination of open wounds with C. tetani endospores. The most common lesions owing to tetanus infection are contaminated puncture wounds, infected foot ulcers, surgical wounds, and animal bites. Within the incubation period of tetanus (7 to 10 days), the bacterial endospores produce a neurotoxin called tetanospasmin that impaired motor nerves causing the characteristic clinical picture of tetany. The most common symptoms of tetanus are trismus (stiffness of the jaw muscles), painful body spasms, especially in the neck and abdominal muscles triggered by a noise or physical touch, in addition to difficulties in swallowing and fever. Tetanic contractions strengthen through the course of infection and may cause fractures and pulmonary embolism, which eventually leads to death. The diagnosis of tetanus can be through toxin assays in the blood. Tetanus has no treatment. However, prevention of tetanus is through vaccination with the DTap vaccine given as a series of injections during childhood (with the first delivered at two months of age while the last vaccination given at the age of 4). Also, for further prevention from the infection, booster doses against the toxin are given once every ten years.[13]

Food Poisoning

Endospores causing food poisoning include:

• Bacillus cereus
• Clostridium perfringes

B. cereus endospores are among the leading organisms causing food poisoning. B. cereus food poisoning is divided into emetic and diarrheal subtypes and is caused mainly by the ingestion of raw and contaminated food with B. cereus endospores. Emetic food poisoning results from B. cereus endospores that produce cereulide toxin, which nausea and vomiting. Diarrheal food poisoning results from the ingestion of meat products, milk, or vegetable contaminated with enterotoxin secreting endospores. Diarrheal syndrome characteristically presents with bloody or mucoid diarrhea and abdominal pain. 

C. perfringens is naturally present in the intestinal microbiota. Ingestion of food contaminated with human or animal feces containing C. perfringens endospores causes food poisoning. The symptoms usually appear 6 to 24 hours following the ingestion of contaminated food and are characterized by abdominal cramps and watery diarrhea. The diagnosis is through stool analysis and culturing bacteria on differential microbiological media. Diagnosis of both bacterial strains can be confirmed by isolating bacteria from feces or vomitus and culturing it on a differential media plate. Patients usually recover without the administration of antibiotics. Supportive treatment with fluid administration is the key due to excessive diarrhea and vomiting.[14]

Clostridial Myonecrosis

Clostridial myonecrosis (gas gangrene) is a bacterial infection caused by clostridial endospores (especially C. perfringens) and most commonly affects the upper and lower extremities. There are two types of gas gangrene: Traumatic and Non-traumatic syndromes. The traumatic syndrome is the most common and includes contamination of open wounds with Clostridium endospores. In contrast, the non-traumatic syndrome develops diminished perfusion from vascular diseases (such as atherosclerosis) or diabetes mellitus, leading to clostridial spores and endotoxin-mediated gas gangrene. The common symptoms of gas gangrene are blisters with a foul smell, painful edema around the wound, air under the skin, fever, and jaundice (yellow skin and eyes) at later stages. The diagnosis involves skin culture to test for C. perfringens. Surgical evaluation is necessary to limit the spread of gangrene. The treatment rests on prompt administration of antibiotics and surgical removal of necrotic tissues. Late-stage may require limb amputation.[15] 

Clostridium difficile Colitis:

C. difficile is part of the healthy intestinal microbiota. However, overgrowth of the bacteria due to prolonged intake of antibiotics (such as fluoroquinolones, clindamycin, and penicillin) disrupts the balance of colon microbiota, causing pseudomembranous colitis. This syndrome is commonly considered a nosocomial infection. C. difficile colitis contributes to about 15% to 30% of antibiotic-associated diarrhea. Common symptoms of this condition include fever, watery diarrhea, nausea, mucous in stool, and abdominal cramps. About 8% of C. difficile infections develop a fulminant infection.[16]

Botulism

C. botulinum endospores cause four significant types of botulism syndrome according to the mode of infection. Foodborne botulism is caused by contamination of food with bacterial endotoxin and spores. Wound botulism is caused by the toxin produced by the colonization of C. botulinum inside open wounds. Intestinal colonization of C. botulinum in infants and adults (though it is rare) causing infant botulism. Inhalation botulism is caused by inhaling C. botulinum toxins.[17][18] Besides, there is another rare form of botulism called iatrogenic botulism that results from the injection of C. botulinum toxins, such as rarely failed botox injection procedures during cosmetic surgeries. 

The clinical picture of all types of botulism consists of the symptoms starting from symmetrical cranial nerve paralysis, followed by descending symmetrical flaccid paralysis of voluntary muscles. Those symptoms progress to respiratory muscle paralysis leading to respiratory failure and death. The exact dose of the lethality of botulism toxins is not accurately defined. However, the commonly estimated lethal dose of pure type A botulinum crystalline is about 0.1 micrograms for a 70-kg man.[19]

The mortality rates attributed to botulism have improved since 1910 due to the development of intensive care techniques, such as mechanical ventilation.[20] Patients with suspected botulism should be promptly referred to intensive care units with closer monitoring in case of progressive respiratory failure. Paralysis caused by botulinum toxin is prolonged and could last for several weeks or months. Rehabilitation of paralyzed patients is crucial.

The treatment of botulism is antitoxin therapy. Antitoxin therapy is usually given during the first 24 hours after the exhibition of symptoms. Botulinum toxin can be absorbed through mucus membranes of the eye, mouth, and nose even though they are not transmitted through direct skin contact. Therefore proper isolation and PPE (personal protective equipment) are essential.

Nursing, Allied Health, and Interprofessional Team Interventions

The diseases associated with bacterial spores can affect different organ systems and present with a myriad of symptoms. An interprofessional team approach by physicians, nurses, pharmacists, physiotherapists, and laboratory personnel is vital for the best patient outcomes. [Level 5]