Mycobacterium tuberculosis

Mycobacterium tuberculosis (TB) is a Gram-positive, non-spore forming bacterium with a thick, lipid-rich cell wall.  M. tuberculosis is endemic worldwide and a leading cause of disability and death; an estimated one-third of the world’s population is latently infected with TB.  Isolates which are multi-drug resistant (MDR-TB), being resistant to at least isoniazid and rifampicin, and extensively drug-resistant (XDR-TB) are complicating the treatment regimes for this disease.  A global movement, the Stop TB Partnership, strives to create a tuberculosis-free world by 2050. 

Potential Health Hazards

Many initial infections with M. tuberculosis go unnoticed; only 5-10% of people infected with M. tuberculosis become sick or infectious during their lifetime.  The host’s immune response results in the formation of granulomas, lung nodules which contain dormant bacilli.  Tuberculosis may persist for a lifetime as a latent infection or become active when host immune defenses are suppressed, such as with HIV infection.  Exogenous re-infection of exposed individuals has also been documented as a cause of active infection. 
  
Progression to pulmonary tuberculosis, resulting in fibrosis and/or cavitation of the lungs, can occur following an active infection.  Extrapulmonary involvement (miliary, meningeal) is the result of bacilli entering the bloodstream and spreading throughout the body.  Initial infections in infants and children more frequently lead to serious outcomes.  

Modes of Transmission

M. tuberculosis is often transmitted through aerosolization of the bacterium.  The main infection route of the bacterium is through the lungs; however, invasion of mucous membranes or breaks in the skin are also possible.  Direct person to person transmission of M. tuberculosis occurs through exposure to airborne bacilli in the sputum of infected persons.  Medical personnel are at risk of infection during autopsies, intubation, bronchoscopies or through dermal inoculation.

Human infection can result from inhalation of as few as 10 colony forming units (CFU). 

Laboratory Acquired Infections

Laboratory acquired infections with M. tuberculosis are the fourth most commonly reported.  Infection can result from accidental inoculation, exposure to aerosols and infectious droplets, ingestion, and direct contact of mucous membranes with infectious materials.  Infected non-human primates are also a known source of human infection.
Infectious materials include sputum, gastric lavage fluids, cereobrospinal fluid, urine, lesions, and a variety of tissues.  The litter of infected rodents can be contaminated and generate infectious aerosols.  Additionally, infectious bacteria may survive in heat-fixed smears or be aerosolized during the preparation of frozen sections and during the handling of liquid cultures.

Host Range

Humans, primates and rodents can serve as a host for M. tuberculosis.

Survival

While M. tuberculosis is a non-spore forming bacterium, this organism can survive for weeks in dust, on carpet or clothes, or in animal carcasses and for months in sputum.

Laboratory Practices

The appropriate biosafety level (BSL) practices and facilities will depend on the biological material that the laboratory is manipulating.  

Biosafety level 2 practices and facilities, at a minimum, must be used for activities involving primary cultures of sputum and preparing smears from clinical material.  As a result of the low infectious dose (10 CFU), specimens from suspected or known cases of tuberculosis must be considered as potentially infectious.  

  • Biohazard signs and labels must be displayed in areas and on equipment where bacteria are used and stored.  This includes, but is not limited to, laboratory entrance doors, biological safety cabinets, incubators, refrigerators, and freezers.
  • Use a biological safety cabinet (BSC, a.k.a., tissue culture hood) for manipulations that can generate aerosols, such as pipetting, harvesting, infecting cells, filling tubes/containers, and opening sealed centrifuge canisters.  
  • Use a slide-warming tray rather than a flame to heat-fix slides.
  • Use aerosol containment devices when centrifuging.  These include sealed canisters that fit in the centrifuge bucket, covers for the centrifuge bucket, heat sealed tubes, or sealed centrifuge rotors.  Rotors should be removed and opened inside a BSC.  Centrifuge tubes should be filled and opened in a BSC.
  • Vacuum lines must be protected with liquid disinfectant traps and/or micron filters.
  • Policies for handling and disposing of sharps (needles, pipettes, broken glassware, etc.) must be implemented.  Work practice controls and engineering devices should be implemented to reduce sharps injuries.
  • Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant.
  • All cultures, stocks and other potentially infectious materials should be decontaminated prior to disposal with an effective method, such as autoclaving or treatment with a bleach solution.
  • Potentially infectious materials must be placed in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility.
  • Personal protective equipment (PPE) requirements are outlined below.

Biosafety level 3 practices and facilities must be used when preparing or manipulating cultures and with experimental animal studies involving M. tuberculosis, or any subspecies of the M. tuberculosis complex.

  • Directional airflow is provided, drawing air into the laboratory from “clean” areas and toward “contaminated” areas.
  • A BSL3 specific biosafety manual and biosafety precautions are incorporated into standard operating procedures.
  • Respiratory protection is required.
  • Policies and procedures are developed such that only persons who have been advised of the potential biohazards, who meet any specific entry requirements, and who comply with all entry and exit procedures may enter the lab.
  • BSL3 policies and standard operating procedures are outlined in manuals specific to the BSL3 laboratory.
  • To ensure that no cross contamination occurs, work with only one strain of Mycobacterium species at a time.

Personal Protective Equipment

Personal protective equipment (PPE) for the BSL2 laboratory includes, but is not limited to:

  • Disposable gloves (nitrile, latex, etc.).
  • Lab coat when working in the area.  Remove when leaving the laboratory.
  • Face and eye protection must be used when potentially infectious material is handled outside of the biosafety cabinet or containment device.
  • Protective laboratory clothing must be laundered by the University and not taken home for cleaning.

PPE requirements for the BSL3 laboratory are outlined in the BSL3 specific manuals.

Precautions When Using Animals

Animal use requests are made to the Institutional Animal Care and Use Committee (IACUC) by submitting an Animal Care and Use Form (ACURF).
When animals are infected with M. tuberculosis, the animal biosafety level (ABSL) will be assigned as ABSL-3.  

Recombinant M. tuberculosis Research

All protocols involving recombinant M. tuberculosis must be approved by the Institutional Biosafety Committee (IBC); complete an online rDNA Registration Document”.

Medical Surveillance

Annual or semi-annual skin tests with purified protein derivative (PPD) are used to monitor previously skin-test-negative personnel. All personnel working in labs that culture M. tuberculosis must have an annual PPD test done initially and at least annually. The PPD test is performed by University Employee Health Clinic; call 356-3631.

Employee Exposure

Eye Exposure – Rinse eyes in an eyewash for at least 15 minutes.
Skin Exposure – Wash skin with soap and water.
Accidental Needlestick Injury – Scrub contaminated skin with soap and water.
Report Incidents and Seek Treatment – Report actual or suspected exposure incidents to your supervisor immediately and seek treatment at the Worker’s Health Clinic.  It is located on the first floor of Boyd Tower – General Hospital.  The clinic’s phone number is 353-8653.  If the incident occurs after 4:30pm, during the weekend, or on a holiday, proceed to UIHC’s Emergency Treatment Center (ETC).  The phone number is 356-2233.

  • Incubation Period – The incubation period from infection to primary lesion or tuberculin reaction varies from 4 to 12 weeks.  The greatest risk of progressive pulmonary or extrapulmonary tuberculosis is 1 to 2 years after infection.
  • Symptoms – Progression to pulmonary tuberculosis manifests as fatigue, fever, cough, weight loss, chest pain, and hemoptysis (coughing up blood). 
  • Immunizations – Mycobacterium bovis – Bacille Calmette-Guèrin (BCG) has been licensed as an attenuated live vaccine.  However, this vaccine is not used in the United States.
  • Prophylaxis – Preventative treatment involves the anti-bacterial drug, isoniazid (INH); however, there is risk of hepatitis in patients over 35 years old. 

Spill and Disposal Procedures

For spills outside a biological safety cabinet, leave the area while holding your breath.  Once outside the area, wash hands and face with soap and water.  Do not allow anyone inside the area or room where the spill occurred.  Allow 30 minutes for the aerosols to settle.  Enter the room wearing required protective clothing, carefully cover the spill with paper towels, and apply disinfectant starting at the perimeter and working towards the center.  Allow the disinfectant to remain on the spill for at least 30 minutes before initiating spill clean up.  After initial clean up, disinfect the area a second time.

For spills inside a biological safety cabinet, cover the spill with paper towels or wipes.  Carefully pour disinfectant over the spill area.  Let the disinfectant soak for 30 minutes before cleaning up the spill.  After initial clean up, disinfect the area a second time.
Contaminated materials must be disposed of as biohazardous waste.
Decontaminate adjacent surfaces with a bleach solution.

For spills in the BSL3 laboratory, follow the specific procedures outlined in the BSL3 biosafety manual.

Disinfectants

Disinfectants should be allowed a minimum of 20-30 minutes contact time.  Use one of the following:

  • Sodium hypochlorite (20% dilution of fresh bleach).
  • Phenol (5%).
  • Glutaraldehyde.
  • Formaldehyde.

Decontamination

Autoclave cultures and other laboratory waste for 60 minutes at 121° C or 250° F (15 lbs per square inch of steam pressure).
Infected animal carcasses must be autoclaved before disposing with the biohazardous waste.

Disinfect work surfaces using an effective germicide (see above).  This may be followed by an alcohol wipe to lessen the corrosive nature of the germicide.

Transport Requirements 

Both importation and domestic transport of M. tuberculosis may require CDC/USDA/APHIS permits.  Department of Commerce permits may be required to export this organism to another country. 

Materials must be appropriately contained and labeled for transport within the University.  Shipping infectious substances, diagnostic specimens, and/or shipping with dry ice off-campus require training and certification.  See EHS’s fact sheet “Transporting and Shipping Infectious Substances”.

If you have questions

Contact Haley Sinn, Biosafety Officer (335-9553), at EHS.

Information and References