Mycobacteriaceae – Part I General Characteristics & Safety Created by: Michele L. Jurgensmeier MT(ASCP) Edited by: Kathy Talmon MT(ASCP)SM Voice over lecturer: Carol Larson MSEd, MT(ASCP) Clinical Laboratory Science Program

General Characteristics Found in water, soil and animals Aerobic, non spore formers Slow growing Gram stain = beaded gram-positive rod High lipid content = difficult to pick up aniline dyes Mycobacteriaceae in most laboratories are treated as a distinct separate group of bacteria based on their distinguishing characteristics and safety issues. Mycobacteriaceae can be found in water, soil and animals. They are aerobic, slow growing, non spore forming bacteria. Mycobacterial cells resist staining with commonly used basic aniline dyes at room temperature due to the high lipid content in their cell wall. Therefore, when using the gram stain procedure the organisms will stain irregularly and appear as beaded gram positive rods.

General Characteristics “Acid fast” Resist decolorization by acid alcohol Distinguishes Mycobacteria from other genera Since mycobacterial cells resist staining with basic aniline dyes at room temperature, staining techniques to detect these organisms have been developed. Mycobacterial cells are able to take up basic dye (carbolfuchsin and auramine O) with increased staining time or application of heat. Carbolfuchsin or Auramine O dyes bind to the mycolic acid, a unique fatty acid found in the mycobacterial cell wall. After the primary stain, the slide is decolorized with acid-alcohol, acids or acid-acetone solutions. Organisms are said to be acid-fast because they are able to retain the basic stain even after attempts to decolorize with acid-alcohol, acids, or acid-acetone solutions. Acid fastness distinguishes mycobacteria from other non-mycobacterial organisms. Photo courtesy of CDC public image library Content provider Dr. George P. Kubica

Growth Requirements Mycobacteria are slow growers High lipid content Hydrophobic cells clump, nutrients slow into cell Slow replication rate Range = 2 days to 8 weeks Optimal temp = 35-37°C Some species prefer 30°C Prefer CO2 for growth Mycobacteria grow more slowly than most other bacterial pathogens. Mycobacteria are said to be hydrophobic, because the high lipid content of their cell wall causes them to float. The high lipid content causes the organisms to clump, so nutrients are not easily allowed into the cell, slowing their growth. The most rapidly growing species can grow in as little as 2 to 3 days. However, most mycobacteria associated with clinical disease require 2 to 6 weeks incubation. The optimal temperature for most species is 35-37 degrees Celsius, although some species prefer 30 degrees Celsius. Mycobacteria grow best at atmosphere of 5-10% CO2.

Safety Is critical! WHY ? Serious nature of mycobacterial disease, particularly tuberculosis Health care workers are at risk for contracting tuberculosis Minimal chance of infections when proper precautions used. Safety is critical when working with patients suspected of having mycobacterial disease. Special safety precautions must be used for both personnel caring for patients suspected of having mycobacterial disease and laboratory personnel handling specimens from those patients suspected of having mycobacterial disease. Why are special safety precautions needed? The serious nature of mycobacterial disease, particularly tuberculosis is why safety is critical. Patients with active tuberculosis can be heavily infected, increasing the chance of human to human transfer. Health care workers are at risk of contracting tuberculosis and more importantly acquiring one of the more multi-drug resistant strains that have developed in the past several years. If special safety precautions are used the chance of infection is minimal.

Safety –Administrative Controls Patient screening and risk assessment Prompt isolation and appropriate therapy The hospital can protect hospital personnel including laboratory personnel that may enter patients rooms from possible infections by screening symptomatic patients for disease. Also, risk assessments can be taken to evaluate the potential for possible mycobacterial infections. If a patient is suspected of having mycobacterial disease, an isolation room can be used to limit exposure to other patients or healthcare workers. Use of respirator masks when entering patient rooms is recommended.

Safety – Monitor Personnel Purified protein derivative (PPD) skin test Quantiferon-TB® Gold test Monitoring employees can ensure prompt treatment if a laboratory acquired infection occurs. There are currently two tests available to monitor personnel. The purified protein derivative (PPD) test and the Quantiferon-TB® Gold test. These test are helpful in screening patients for M. tuberculosis infections. False negative and positive results can be obtained with both testing methods, for this reason all tests results should be correlated with clinical history, radiography and other medical diagnostic test if needed.

Safety – Monitor Personnel Purified protein derivative (PPD) skin test In vivo Administer PPD test (Mantoux) upon employment Negative - Screen regularly Positive - Counseled regularly, report changes in health status The in vivo, PPD test is based on the fact that infection with M. tuberculosis produces a delayed-type hypersensitivity skin reaction to certain components of the bacterium. Extracts of culture filtrates, known as purified protein derivative, are used for skin testing for tuberculosis. A skin reaction is observed when T cells, which have been sensitized by prior infection are recruited to the skin site and release lymphokines. The lymphokines are responsible for the induration that occurs in positive patients. Induration is a hard raised area with clearly defined margins at or around the injection site. The induration is produced through local vasodilation edema, fibrin deposits, and other inflammatory cells at the site. The Purified protein derivative skin (PPD) test should be administered upon employment. Those testing negative should be screened regularly throughout employment. Those employees testing positive and showing signs of active disease should be further evaluated for treatment. Employees testing positive and not showing active signs of disease should be counseled regularly and should report changes in health status

Safety – Monitor Personnel Quantiferon-TB® Gold test This test is performed on blood collected from the patient Measures the amount of interferon-gamma (INF-γ) secreted by a patient's lymphocytes in whole blood in response to simulated mycobacterial antigens in vitro. Detection and quantification of interferon-gamma is the basis of test The Quantiferon®-TB Gold test is an in vitro test that measures a component of cell-mediated immune reactivity to M. tuberculosis, interferon-gamma. Patient’s blood is mixed with stimulating peptide antigens that simulate mycobacterial proteins. If patient is infected, sensitized T cells recognize the tuberculin and release interferon. The concentration of interferon is measured by ELISA. The QFT results are based on the proportion of interferon-γ in response to four antigens.

References: Mahon, C.R. & Manuselis, G., Textbook of Diagnostic Microbiology, 2nd Ed., W.B. Saunders, 2000 Forbes, B.A., Sahm, D. F., & Weissfeld, A.S., Bailey and Scott’s Diagnostic Microbiology, 11th Ed., Mosby, 2002. Koneman, Color Atlas and Textbook of Diagnostic Microbiology, 5th Ed., J.B. Lippincott Co., 1997 Murray PK, et al., Manual of Clinical Microbiology, 7th ed., ASM Publishing, Washington DC, 1999. Center for disease control and prevention/ National institute of Health, Biosafety in Microbiological and Biomedical Laboratories, 4th ed., U.S. Government Printing Office, 1999. www.ehs.cornell.edu/bio/cabinets.htm www.medicinenet.com www.cdc.gov