Clostridioides difficile (formally Clostridium)

Characteristics
Morphology	A member of the Peptostreptococcaceae family it is a Gram-positive, motile, anaerobic, and spore-forming (forms subterminal spores) bacteria. Vegetative cells are rod shaped, pleomorphic, and occur in pairs or short chains and lacks both catalase and superoxide dismutase. It produces 2 types of toxins: enterotoxin A and cytotoxin B, which disrupts cytoskeleton signal transductions in the host.
Growth Conditions	Defined amino acid growth medium under anaerobic conditions. Can be found in air, soil, and water in nature along with sewage and contaminated meat. First isolated from human fecal matter

Characteristics

Morphology

A member of the Peptostreptococcaceae family it is a Gram-positive, motile, anaerobic, and spore-forming (forms subterminal spores) bacteria. Vegetative cells are rod shaped, pleomorphic, and occur in pairs or short chains and lacks both catalase and superoxide dismutase. It produces 2 types of toxins: enterotoxin A and cytotoxin B, which disrupts cytoskeleton signal transductions in the host.

Growth
Conditions

Defined amino acid growth medium under anaerobic conditions. Can be found in air, soil, and water in nature along with sewage and contaminated meat. First isolated from human fecal matter

Health Hazards
Host Range	Humans, pigs, calves, and other mammals
Modes of Transmission	Fecal-oral contact; evidence for transmission via fomites and hands exists (often nosocomial in nature)
Signs and Symptoms	Most infections present during or shortly after a course of antimicrobial therapy Mild or moderate diarrhea, may be associated with the passage of mucus or occult blood in stool, fever, vomiting, loss of appetite, cramping, abdominal discomfort, nausea Pseudomembranous colitis (often fatal), peripheral leukocytosis (fewer than half of patients)
Infectious Dose	Unknown, but will vary depending on the transmission source
Incubation Period	5-10 days with a range of 1 day to weeks following antibiotic treatment for AAD (antibiotic associated diarrhea)

Medical Precautions/Treatment
Prophylaxis	Antibacterial prophylaxis is not recommended for C. difficile since antibiotics can have an adverse effect on the normal gut flora
Vaccines	None publically available
Treatment	Antibiotic treatment is available, but outside of that supportive treatment with rebalancing of fluid levels and electrolytes is recommended
Surveillance	Monitor for symptoms. Detection of toxins in stool specimens is the Gold Standard test for diagnosis of C. difficile infection. Can be diagnosed by Polymerase Chain reaction (PCR) and pulse field gel electrophoresis (PFGE)
GWU Requirements	Report all incidents to the Office of Risk Management as well as the Office of Research Safety (ORS) IMMEDIATELY FOLLOWING THE OCCURRENCE.

Containment
BSL-2	Risk Group 2 classification is applied to C. difficile. Containment Level 2 facilities, equipment, and operational practices for work involving infectious or potentially infectious material. All procedures that may produce aerosols, or involve high concentrations or large volumes should be conducted in a biological safety cabinet (BSC).
ABSL-2	For work with C. difficile in animals.

Spill Procedures
Small Spills (<1 liter)	If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for 15 minutes before continuing with the spill cleanup. C. difficile can become aerosolized and contaminate surrounding surfaces, caution should be taken during a spill outside of a BSC. Leave the room immediately and allow the aerosols to dissipate for 15 minutes. Notify others working in the lab. Don appropriate PPE. Cover area of the spill with paper towels or any absorbent material and apply an EPA registered disinfectant effective against Gram-positive bacteria (bleach, glutaraldehyde, cavicide), working from the perimeter towards the center. Allow 30 minutes of contact time before disposal and cleanup of spill materials.
Large Spills	Alert lab personnel in the laboratory to the spill and keep people out of the area to prevent spread of the contamination. Check if you have been contaminated or if any of your PPE has been breached. If so follow exposure procedures. Remove any contaminated clothing and place it the biohazard waste. Wash your hands and post a sign on the door. Notify your supervisor of the incident and call ORS (4-8258) for assistance. If the situation involves an imminently life-threatening injury or has catastrophic potential, call 911.

Spill Procedures

Small Spills (<1 liter)

If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for 15 minutes before continuing with the spill cleanup.

C. difficile can become aerosolized and contaminate surrounding surfaces, caution should be taken during a spill outside of a BSC. Leave the room immediately and allow the aerosols to dissipate for 15 minutes. Notify others working in the lab. Don appropriate PPE. Cover area of the spill with paper towels or any absorbent material and apply an EPA registered disinfectant effective against Gram-positive bacteria (bleach, glutaraldehyde, cavicide), working from the perimeter towards the center. Allow 30 minutes of contact time before disposal and cleanup of spill materials.

Large Spills

Alert lab personnel in the laboratory to the spill and keep people out of the area to prevent spread of the contamination. Check if you have been contaminated or if any of your PPE has been breached. If so follow exposure procedures. Remove any contaminated clothing and place it the biohazard waste. Wash your hands and post a sign on the door. Notify your supervisor of the incident and call ORS (4-8258) for assistance. If the situation involves an imminently life-threatening injury or has catastrophic potential, call 911.

Exposure Procedures
Mucus Membrane	Flush eyes, mouth or nose for 15 minutes at eyewash station.
Other Exposure	For an area not protected by skin, wash with soap and water for 15 minutes (open wounds, sores, etc.), and a minimum of 20 seconds of soap and water for areas with intact skin.
Reporting	Report ALL injuries to the PI immediately and reported to the Office of Risk Management at [email protected] IMMEDIATELY FOLLOWING THE OCCURRENCE. Exposures that involve a bloodborne-pathogen or recombinant DNA also need to be reported to the Office of Research Safety at [email protected]. If the injury requires immediate medical attention, call GWPD at 202-994-6111 or call 911.
Medical Monitoring	Seek immediate medical evaluation, treatment, and post exposure follow-up at the Employee Health Office at GWU Hospital (900 23rd St., NW, Suite G-1090, Phone: 202-715-4275). Students should go to the Students Health Office at Marvin Center. After hours treatment can be received at the GWU hospital emergency room.

Stability
Disinfection	Spores are fairly resistant; moderate susceptibility to 1% sodium hypochlorite; susceptible to high level disinfectants (>2% glutaraldehyde) with prolonged contact time (around 30 minutes)
Inactivation	Spores of the genus Clostridioides are generally heat resistant and can withstand temperature of 116°C for 3 hours, vegetative cells can be rapidly killed by temperatures of only 55-65°C. Most spores can be inactivated by moist heat at 121°C for 15-30 minutes
Survival Outside Host	C. difficile is able to survive in a multitude of environments, both in nature and man-made, for extended periods of time. Spores are even more resistant and can persist in harsher conditions

Personal Protective Equipment (PPE)
Minimum PPE Requirements	At minimum, personnel are required to don gloves, closed toed shoes, lab coat, and appropriate face and eye protection prior to working with C. difficile. Additional PPE may be required depending on lab specific SOPs.
Additional Precautions	Additional protection may be worn over laboratory clothing when infectious materials are directly handled, such as solid-front gowns with tight fitting wrists, gloves, and respiratory protection. Wash hands with soap and water after removing gloves.

References
CDC (https://www.cdc.gov/cdiff/index.html) BMBL 6th Edition (https://www.cdc.gov/labs/BMBL.html) Aerosolization of Clostridium difficile (https://www.sciencedirect.com/science/article/pii/S0195670111003392?via…) Canadian PHAC (https://www.canada.ca/en/public-health/services/laboratory-biosafety-bi…) EPA (https://www.epa.gov/sites/default/files/2021-02/documents/02-22-2021_li…)