mycobacterium/AFB:
Up until 1985 cases of tuberculosis in the USA had been steadily decreasing, however, with the rise of immunocompromised patients, cases of tuberculosis and non-tuberculosis Mycobacterial infections have been on the rise.
Mycobacterium Species:
Phylum: Actinobacteria
Family: Mycobacteriaceae
Genus: Mycobacterium
Species: According to the American Society for Microbiology, there are >170 species of recognized Mycobacterium (Forbes, Betty; DOI: 10.1128/JCM.01287-16; Mycobacterial Taxonomy; Journal of Clinical Microbiology; American Society for Microbiology; https://jcm.asm.org/content/55/2/380.full; 12/07/2-16)
FACTS:
Mycobacteria are sometimes referred to as “acid-fast bacilli (AFB),” a term referencing their response to a laboratory staining technique. This simply means that when microscopic slides of these bacteria are rinsed with an acidic-alcohol solution (HCl acid + denatured ethanol/methanol), they retain a red dye. They can be described as Gram-positive, nonmotile and acid-fast rods (1-3 µm x 0.2-0.4 µm). They are very thin, long rods with occasional beaded or swollen cells having non-acid-fast ovoid bodies at one end and they may exhibit cording.
The cell wall of Mycobacterium is full of mycolic acids, such as N-glycolylmuramic acid, and a high content of lipids and waxes, existing as fatty acids in long chains. This renders the cell wall hydrophobic, or "water-hating", making it difficult for nutrients to enter the cell. Because of this, they retain the carbolfuchsin stain and are not easily decolorized by the acid-alcohol decolorizer.
There are at least 71 known species of Mycobacterium. Of those, the most significant is MTB Complex, consisting of Mycobacterium tuberculosis, M. bovis, M. africanum. Any or all of these can cause tuberculosis. M. tuberculosis causes the majority of cases of human tuberculosis. M. bovis affects the gastrointestinal tract, oropharynx, and infection occurs via ingestion of contaminated milk or dairy products or inhalation of aerosolized particles. Scientists think there may be a link between this type of mycobacterium and some cases of Crohn's disease (ulcerative colitis), but this is still under research. This is one of the reasons why pasteurization of milk and dairy products is so important prior to human consumption, as well as consuming properly cooked meats. M. africanum occurs via inhalation of aerosolized particles and is found in East/West Africa. These are slow growers that take 6-8 weeks to grow and are nonpigmented.
NTMs, or non-TB Mycobacterium (also referred to as "MOTT", or Mycobacterium Other Than Tuberculosis), also exist, some causing respiratory symptoms similar to tuberculosis, others causing cutaneous (skin surface) and subcutaneous (underneath the skin) symptoms, and still others causing no symptoms at all and simply existing as contaminants and are naturally found in reservoirs of soil and water. Some infect only animals and others infect both animals and humans. Some of them are pigmented.
Pathogenesis of Mycobacterium tuberculosis:
-Low-grade fevers and chills
-Night sweats
-Fatigue
-Anorexia (loss of appetite)
-Weight loss
-Cough (chronic), sometimes bloody
-Myalgias (muscle pains)
-TB may affect any organ of the body, not just the respiratory tract
Testing:
-Chest X-rays
-Skin Test (PPD)
-Quantiferon (serum) (set of 3 blood tubes)
-AFB smear and culture
-Auramine-Rhodamine Fluorescent Stain
-Acid-Fast Stain (Kenyoun or Ziehl-Neelsen)
-Culture in Broth (MB7H9, BacT or Bactec Bottles containing BHI broth)
-Culture on Media:
-MB7H11 (plate or slant)
-Lowenstein-Jensen Medium and Lowenstein-Jensen Gruft
-Chocolate Agar (for recovery of M. haemophilum spp)
-PCR
-DNA Probe
-Biopsy of Tissue/Nodules
-Mass Spectrophotometry
Media:
1) Middlebrook 7H9 broth or 7H11 broth (liquid mediums)
3) Middlebrook 7H10 and 7H11 agar (solid mediums)
7) Other aids: MB7H9 broth, TSB broth, sterile water or sterile saline, BHI broth, or thio (for small amounts of specimen, sterile body fluids, tissue or bone)
8) BD MGIT broth in incubator, with chemiluminescent technology embedded in silicone in the tube to detect bacterial and mycobacterial growth
9) Chocolate agar for recovery of species such as Mycobacterium haemophilum
Incubation of solid media plates (MB7H11 agar) occurs in the dark, plates are shrink-seal-wrapped, and LJ/LJG slants are incubated with the screwcaps loose for 1 week, in a slanted position, then may be kept slanted placed upright for the remaining 7 weeks provided there is room in your laboratory's incubator(s). Slants are incubated from 35-37 degrees Celsius in 5-10% CO2 in high humidity and examined weekly for up to 8 weeks for rate of growth, colony morphology, pigmentation, and amount of growth. Updates should be given at 3-4 weeks, and final reports given at 6-8 weeks. Some infectious disease doctors prefer for the laboratory to hold onto the slants for up to a couple of months in case they wish to later order sensitivities or additional sensitivities.
Liquid media is broth-based. BacT or Bactec or Bactec BX MGIT bottles are incubated for up to 8 weeks. Should they become positive, slides are made for acid-fast stains (AFS) with carbolfuchshin and a Gram stain and test media is inoculated (my current lab inoculates a CHOC plate and then if AFB is seen on the AFS and GS, it is subbed to 2 LJ slants, MB7H11 agar, MB7H9 broth, and BD MGIT broth). MB 7H9 broth (5 mL) may be incubated with several colonies from growth on solid media and incubated at 35 degrees Celsius in 5-10% CO2 for 5-7 days with daily Vortex mixing, then inoculated to test media and slides. Additionally, this broth may be used to homogenize tissue, or to inoculate swabs in if they are received for AFB culture and smear. Positives are typically inoculated to a chocolate plate, 2 LJ slants (some laboratories utilize one 1 regular LJ and 1 LJ Gruft slant), a MB7H11 agar plate, MB 7H9 broth, and are saved for identification (GenProbe or Vitek Mass Spec. or send out to a reference lab) and sensitivities upon request.
Other Supplies:
1) 50 mL conical tubes (Collect 5 CC's/mL's of sample) (keep refrigerated)
2) Alcohol pads
3) Syringes and needles for making antibiotic solution and for inoculating media
4) Sterile pipettes for thick samples and for inoculating plates
5) Various size racks
6) Slides, coverslips
7) Loops
8) Pencils and marking pens
Stains:
1) Auramine-rhodamine (fluorochrome)
2) Kinyoun (cold method) or Ziehl-Neesen (warm method) acid-fast stain
3) Modified Acid-Fast Stain
Testing:
1) GenProbe DNA probe testing
2) PCR
3) Mass Spectrophotometry
Growth:
1) Some are fast-growers and will grow in <7 days (M. tuberculosis, M. bovis, M. africanum)
2) Most are slow-growers and require >7 days and up to 8 weeks to grow (NMTB's/MOTT's/Atypical)
SLOW GROWERS: (>7 days)
Carotenoids:
Carotenoids are a group of pigments that range from yellow to orange to rust/red in varying amounts (beta carotene), just like carrots, yellow and orange veggies, and Autumn leaves. They are organized into 3 groups based on their pigment production:
I. Photochromogens: (potential pathogens)
-Slow-growing NMTBs
-Colonies become pigmented when they are exposed to light
-They take >7 days to grow
-Produce pigment after just one hour of exposure to light
-Slow-growing NMTBs
-Colonies become pigmented whether or not they are grown in the dark or in the light and will produce a deep yellow-to-orange pigmentation of the colonies BOTH in the dark AND in the light
-Take >7 days to grow
-Slow-growing NTM's
-Colonies do not produce any pigment
-Take >7 days to grow
RAPID GROWERS:
(<7 days), Lower virulence, Stain irregularly, More susceptible to conventional antibacterial antibiotics, Rarely cause disseminated infections, Typically introduced by trauma or iatrogenic infections
I. Potential Pathogens:
-M. agri
-M. aichiense
-M. austroafricanum
-M. aurum
-M. chitae
-M. chubuense
-M. diernhoferi
-M. duvalii
-M. fallax
-M. flavescens (eye)
-M. gadium
-M. gilvum
-M. komossense
-M. moriokaense
-M. neoaurum
-M. parafortuitum
-M. obuense
-M. phlei
-M. pulveris
-M. rhodesiae
-M. senegalense
-M. sphagni
-M. thermoresistable
-M. tokaiense
-M. vaccae
-M. triviale (atypical)
Noncultivatable NTM:
M. leprae-Causative agent of leprosy or Hansen's Disease, a chronic disease of the skin, mucous membranes and nerve tissue
Mycobacterium Species:
Phylum: Actinobacteria
Family: Mycobacteriaceae
Genus: Mycobacterium
Species: According to the American Society for Microbiology, there are >170 species of recognized Mycobacterium (Forbes, Betty; DOI: 10.1128/JCM.01287-16; Mycobacterial Taxonomy; Journal of Clinical Microbiology; American Society for Microbiology; https://jcm.asm.org/content/55/2/380.full; 12/07/2-16)
FACTS:
- Aerobic, Acid-Fast Bacilli (Rods) (straight, slightly curved, and/or exhibiting "beading" or "cording")
- 1.0-10 micrometers in length
- 0.2-0.6 micrometers in width
- Nonmotile (except M. marinum)
- Outer membrane
- Encapsulated
- Non-spore forming
- Cell wall (thick, waxy, hydrophobic, lipid-rich, rich in mycolic acids, peptidoglycan, polysaccharide arabinogalactan)
- Hardy
- Can survive bleach, detergents, acids, alkalis, oxidative burst, the immune system (complement), many antibiotics (this is why discarded biohazard material must be autoclaved prior to removal from an AFB laboratory)
- Virulent
- Mycosides can destroy the cell wall (phenolic alcohols)
- Utilize ammonia, amino acids, glycerol, mineral salts
- Ideal temperature for growth ranges from 25->50 degrees Celsius
- Growth is enhanced by 5-10% CO2
- Growth is enhanced by a pH medium of pH 6.5-6.8
- Photochromogens (Group 1)
- Scotochromogens (Group 2)
- Non-chromogens (Groups 3-4)
- Pathogenic stains
- Opportunistic pathogenic strains
- Nonpathogenic strains
Mycobacteria are sometimes referred to as “acid-fast bacilli (AFB),” a term referencing their response to a laboratory staining technique. This simply means that when microscopic slides of these bacteria are rinsed with an acidic-alcohol solution (HCl acid + denatured ethanol/methanol), they retain a red dye. They can be described as Gram-positive, nonmotile and acid-fast rods (1-3 µm x 0.2-0.4 µm). They are very thin, long rods with occasional beaded or swollen cells having non-acid-fast ovoid bodies at one end and they may exhibit cording.
The cell wall of Mycobacterium is full of mycolic acids, such as N-glycolylmuramic acid, and a high content of lipids and waxes, existing as fatty acids in long chains. This renders the cell wall hydrophobic, or "water-hating", making it difficult for nutrients to enter the cell. Because of this, they retain the carbolfuchsin stain and are not easily decolorized by the acid-alcohol decolorizer.
There are at least 71 known species of Mycobacterium. Of those, the most significant is MTB Complex, consisting of Mycobacterium tuberculosis, M. bovis, M. africanum. Any or all of these can cause tuberculosis. M. tuberculosis causes the majority of cases of human tuberculosis. M. bovis affects the gastrointestinal tract, oropharynx, and infection occurs via ingestion of contaminated milk or dairy products or inhalation of aerosolized particles. Scientists think there may be a link between this type of mycobacterium and some cases of Crohn's disease (ulcerative colitis), but this is still under research. This is one of the reasons why pasteurization of milk and dairy products is so important prior to human consumption, as well as consuming properly cooked meats. M. africanum occurs via inhalation of aerosolized particles and is found in East/West Africa. These are slow growers that take 6-8 weeks to grow and are nonpigmented.
NTMs, or non-TB Mycobacterium (also referred to as "MOTT", or Mycobacterium Other Than Tuberculosis), also exist, some causing respiratory symptoms similar to tuberculosis, others causing cutaneous (skin surface) and subcutaneous (underneath the skin) symptoms, and still others causing no symptoms at all and simply existing as contaminants and are naturally found in reservoirs of soil and water. Some infect only animals and others infect both animals and humans. Some of them are pigmented.
Pathogenesis of Mycobacterium tuberculosis:
- It is estimated that approximately 1/3rd of the world's population, or around 1.7 billion people, are infected with Mycobacterium tuberculosis complex.
- About 8 million new cases are estimated annually across the globe
- There are about 2.9 million deaths annually worldwide due to the disease
- In the United States alone, there are approximately >20,000 cases a year, and there are currently about 10 million people infected
- Persons with HIV/AIDS or who are otherwise immunocompromised are at higher risk of infection
- Infection may occur via inhalation of aerosols containing a single viable organism
- Of those who are infected, about 15-20% develop disease and symptoms of disease, typically years later
-Low-grade fevers and chills
-Night sweats
-Fatigue
-Anorexia (loss of appetite)
-Weight loss
-Cough (chronic), sometimes bloody
-Myalgias (muscle pains)
-TB may affect any organ of the body, not just the respiratory tract
Testing:
-Chest X-rays
-Skin Test (PPD)
-Quantiferon (serum) (set of 3 blood tubes)
-AFB smear and culture
-Auramine-Rhodamine Fluorescent Stain
-Acid-Fast Stain (Kenyoun or Ziehl-Neelsen)
-Culture in Broth (MB7H9, BacT or Bactec Bottles containing BHI broth)
-Culture on Media:
-MB7H11 (plate or slant)
-Lowenstein-Jensen Medium and Lowenstein-Jensen Gruft
-Chocolate Agar (for recovery of M. haemophilum spp)
-PCR
-DNA Probe
-Biopsy of Tissue/Nodules
-Mass Spectrophotometry
Media:
1) Middlebrook 7H9 broth or 7H11 broth (liquid mediums)
- Liquid growth medium for Mycobacterium recovery
- Includes nutrients such as
- Glycerol
- Oleic acid
- Albumin
- Dextrose
- Ammonium sulfate
- L-glutamic acid
- Sodium citrate
- Biotin
- Pyridoxine
- Disodium phosphate
- Monopotassium phosphate
- Ferric ammonium citrate
- Magnesium sulfate
- Calcium chloride
- Zinc sulfate
- Copper sulfate
3) Middlebrook 7H10 and 7H11 agar (solid mediums)
- MB7H11 is identical to MB7H10 except that it has a pancreatic digest of casein to aid in the growth of fastidious strains of M. tuberculosis
- Contains ammonium sulfate
- Agar
- Malachite green
- Biotin
- Pyridoxine hydrochloride
- Ferric ammonium citrate
- L-glutamic acid
- Copper sulfate
- Zinc sulfate
- Magnsium sulfate
- Sodium citrate
- Disodium phosphate
- Monopotassium phosphate
- Read within 5-7 days and once a week for up to 8 weeks
- Also known as an "LJ"
- Contains malachite green
- Glycerol
- Asparagine
- Potato starch
- Coagulated eggs
- Mineral salts
- Sodium citrate
- Magnesium sulfate
- Potassium dihydrogen phosphate
- Penicillin
- Nalidixic acid
- Must be stored in the refrigerator
- Must be used within a month
- Once inoculated with sample, it is incubated for up to 8 weeks (for slow-growers) and slanted
- Selective and Differential
- Read 5-7 days after inoculation with sample and at least once a week for up to 8 weeks
- Looks identical to the regular LJ slant, but is meant to aid in isolating Mycobacterium from contaminated specimens
- Additionally, contains ribonucleic acid
7) Other aids: MB7H9 broth, TSB broth, sterile water or sterile saline, BHI broth, or thio (for small amounts of specimen, sterile body fluids, tissue or bone)
8) BD MGIT broth in incubator, with chemiluminescent technology embedded in silicone in the tube to detect bacterial and mycobacterial growth
9) Chocolate agar for recovery of species such as Mycobacterium haemophilum
Incubation of solid media plates (MB7H11 agar) occurs in the dark, plates are shrink-seal-wrapped, and LJ/LJG slants are incubated with the screwcaps loose for 1 week, in a slanted position, then may be kept slanted placed upright for the remaining 7 weeks provided there is room in your laboratory's incubator(s). Slants are incubated from 35-37 degrees Celsius in 5-10% CO2 in high humidity and examined weekly for up to 8 weeks for rate of growth, colony morphology, pigmentation, and amount of growth. Updates should be given at 3-4 weeks, and final reports given at 6-8 weeks. Some infectious disease doctors prefer for the laboratory to hold onto the slants for up to a couple of months in case they wish to later order sensitivities or additional sensitivities.
Liquid media is broth-based. BacT or Bactec or Bactec BX MGIT bottles are incubated for up to 8 weeks. Should they become positive, slides are made for acid-fast stains (AFS) with carbolfuchshin and a Gram stain and test media is inoculated (my current lab inoculates a CHOC plate and then if AFB is seen on the AFS and GS, it is subbed to 2 LJ slants, MB7H11 agar, MB7H9 broth, and BD MGIT broth). MB 7H9 broth (5 mL) may be incubated with several colonies from growth on solid media and incubated at 35 degrees Celsius in 5-10% CO2 for 5-7 days with daily Vortex mixing, then inoculated to test media and slides. Additionally, this broth may be used to homogenize tissue, or to inoculate swabs in if they are received for AFB culture and smear. Positives are typically inoculated to a chocolate plate, 2 LJ slants (some laboratories utilize one 1 regular LJ and 1 LJ Gruft slant), a MB7H11 agar plate, MB 7H9 broth, and are saved for identification (GenProbe or Vitek Mass Spec. or send out to a reference lab) and sensitivities upon request.
Other Supplies:
1) 50 mL conical tubes (Collect 5 CC's/mL's of sample) (keep refrigerated)
2) Alcohol pads
3) Syringes and needles for making antibiotic solution and for inoculating media
4) Sterile pipettes for thick samples and for inoculating plates
5) Various size racks
6) Slides, coverslips
7) Loops
8) Pencils and marking pens
Stains:
1) Auramine-rhodamine (fluorochrome)
2) Kinyoun (cold method) or Ziehl-Neesen (warm method) acid-fast stain
3) Modified Acid-Fast Stain
Testing:
1) GenProbe DNA probe testing
2) PCR
3) Mass Spectrophotometry
Growth:
1) Some are fast-growers and will grow in <7 days (M. tuberculosis, M. bovis, M. africanum)
2) Most are slow-growers and require >7 days and up to 8 weeks to grow (NMTB's/MOTT's/Atypical)
SLOW GROWERS: (>7 days)
Carotenoids:
Carotenoids are a group of pigments that range from yellow to orange to rust/red in varying amounts (beta carotene), just like carrots, yellow and orange veggies, and Autumn leaves. They are organized into 3 groups based on their pigment production:
I. Photochromogens: (potential pathogens)
-Slow-growing NMTBs
-Colonies become pigmented when they are exposed to light
-They take >7 days to grow
-Produce pigment after just one hour of exposure to light
- M. kansasii-Found in tap water and may cause chronic pulmonary disease, cutaneous disease or cervical lymphadenitis in immunocompromised patients; Produces orange pigment due to beta carotene
- M. asiaticum-Causes pulmonary disease (rare)
- M. marinum-Has been found in both fresh and salt water and is associated with cutaneous disease, especially in those who have fish aquariums or who work closely with marine life
- M. intermedium-Causes pulmonary disease
-Slow-growing NMTBs
-Colonies become pigmented whether or not they are grown in the dark or in the light and will produce a deep yellow-to-orange pigmentation of the colonies BOTH in the dark AND in the light
-Take >7 days to grow
- M. szulgai-Found in water and soil; Associated with pulmonary disease, cervical adenitis, and bursitis, but not common
- -Scotochromogen at 35 degrees C; Nonphotochromogen at 25-30 degrees C
- M. scrofulaceum-Found in raw milk, unpasteurized dairy, soil, and water; Causes cervical adenitis in children
- M. interjectum-Causes chronic lymphadenitis
- M. gordonae-Non-pathogen found in soil and water; a contaminant; Produces an orange pigment in both dark and light; This organism is known for its ability to hydrolyze tween
- M. cookeii-Non-pathogen found in water
- M. hiberniae-Non-pathogen found in water
-Slow-growing NTM's
-Colonies do not produce any pigment
-Take >7 days to grow
- M. avium complex (MAC or MACO)-Associated with HIV/AIDS patients; This species is the most resistant
- M. avium, M. intracellulaire, M. paratuberculosis, M. lepraemurium (MAIC)
- On agar, these organisms form small, cream-colored, round colonies that tend to slightly "yellow" the LJ slant
- On agar, these organisms form small, cream-colored, round colonies that tend to slightly "yellow" the LJ slant
- M. paratuberculosis infects cattle and has been associated in some cases of Crohn's Disease in humans (still being studied/researched)
- M. celatum-NEW; Causes disseminated systemic disease in HIV/AIDS patients; Rare
- M. gastri-Non-pathogenic
- M. genavense-Carried by pet birds and some dogs; Rare cause of disease in HIV/AIDS patients
- M. haemophilum-Causes disseminated systemic disease; Cutaneous, Lymphatic, Skin, Bursa; This species only grows on CHOC agar
- M. malmoense-Causes chronic pulmonary disease or cervical lymphadenitis; Carried by armadillos
- M. shimoidei-Causes TB-like pulmonary disease and disseminated systemic disease (rare)
- M. simiae-Causes TB-like pulmonary disease in HIV/AIDS patients
- M. ulcerans-Found in the tropics; Causes cutaneous/subcutaneous disease and Buruli Ulcer disease in W. Africa
- M. tuberculosis-causative agent of tuberculosis; Results in walled-off tubercles in the lungs; May or may not cause symptoms; Grows as dry, crumbly, cream-colored colonies that look like breadcrumbs on LJ agar; M. tuberculosis is both nitrate + and niacin +, urease -
- M. xenopi-Associated with water and may contaminate hospital hot water systems; Causes Pneumonia, pulmonary disease, bone, lymph node, or sinus disease; Grows best at 42 degrees Celsius (perform a temp. challenge)
- M. terrae complex-Non-pathogenic
- -M. terrae, M. triviale, M. nonchromogenicum
RAPID GROWERS:
(<7 days), Lower virulence, Stain irregularly, More susceptible to conventional antibacterial antibiotics, Rarely cause disseminated infections, Typically introduced by trauma or iatrogenic infections
I. Potential Pathogens:
- M. fortuitum-Common; Post-operative infections, skin/soft tissue infections, nose infections, pulmonary disease; This organism does not fluoresce on auramine-rhodamine stain so it could potentially be missed and and so an AFS should be performed ("fortium forfeits fluorescence")
- M. chelonae-Post-operative infections, skin/soft tissue infections, post-shaving infections/rashes, keratitis
- M. abscessus-Disseminated systemic infections, central nervous system, eye, middle ear, skin/soft tissue infections, pulmonary infections; multi-drug resistant; rapid grower; common soil and water contaminant; Fails to reduce nitrate, take up iron, is tolerant to saline media, will grow on egg agar and Sauton agar medium; this organism is common and grows as a flat, dry, flaky, matte organism on CHOC and MB7H11 agars
- M. smegmatis-Skin/soft tissue infections; resembles M. tuberculosis on LJ slant, since it grows as cream-colored, dry, crumbly colonies resembling breadcrumbs
- M. peregrinum-Skin/soft tissue infections (rare)
- M. mucogenicum (uncommon)-Post-traumatic wound infections; catheter-related sepsis; Looks like M. abscessus on CHOC and MB7H11 agars
- M. immunogenicum-associated with pneumonitis
-M. agri
-M. aichiense
-M. austroafricanum
-M. aurum
-M. chitae
-M. chubuense
-M. diernhoferi
-M. duvalii
-M. fallax
-M. flavescens (eye)
-M. gadium
-M. gilvum
-M. komossense
-M. moriokaense
-M. neoaurum
-M. parafortuitum
-M. obuense
-M. phlei
-M. pulveris
-M. rhodesiae
-M. senegalense
-M. sphagni
-M. thermoresistable
-M. tokaiense
-M. vaccae
-M. triviale (atypical)
Noncultivatable NTM:
M. leprae-Causative agent of leprosy or Hansen's Disease, a chronic disease of the skin, mucous membranes and nerve tissue
Lowenstein-Jensen medium:
Lowenstein-Jensen agar (LJ Slant) is a medium containing the following ingredients for the recovery of Mycobacterium spp:
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Common supplies in the afb laboratory:
Mycobacterium spp and the diseases they cause:
MOTT: Mycobacterium Other Than Tuberculosis:
According to the CDC, symptoms of MOTT depend upon the site, since virtually any organ can be affected, many tend to be rapid growers, and since some are ubiquitous, most healthy persons are not affected by them, but those who are immunosuppressed, immunocompromised, or have underlying health conditions are at higher risk for infection. Those with lung infections tend to have symptoms including cough, shortness of breath, sometimes bloody sputum, or even a rash. Fever, weight loss, loss of appetite, decreased energy, and night sweats are also common symptoms. Other types of infections may include skin or soft tissue, device-associated infections resulting in bloodstream infections, artificial implant infection, pacemaker infection, exit wound site infection, lymph nodes, blood, etc...Below are listed many of the most common MOTTS seen in the healthcare setting. MOTT infections overall are rare, however, they are on the rise and many of these organisms are growing more resistant to treatment.
Mycobacterium avium complex (MAC or MACO) and avium-intracellulare (MAIC):
This ubiquitous organism is associated with fatal infections in HIV/AIDS patients with CD4 cell counts of less than 100/uL, or immunocompromised patients, especially senior citizens or cystic fibrosis patients, causing infection through inhalation or ingestion. In fact, a significant number of HIV/AIDS patients are affected by this organism, and it can produce disseminated illness with symptom of fever, night sweats, weight loss and loss of appetite. It can also infect patients who do not have HIV/AIDS, producing a chronic, persistent cough in those who are infected and who inhale the organism. Its point of entry is the lungs or the gastrointestinal tract. Patients who have ingested the organism and become infected develop diarrhea, fevers, chills, fatigue, abdominal pain, malabsorption, loss of appetite, and dissemination to the bone marrow.
Studies have shown that it can grow on shower heads or thrive in warm indoor swimming pool environments and become inhaled by HIV/AIDS patients, resulting in opportunistic infection.
Treatment consists of prophylaxis of clarithromycin and/or azithromycin or rifampicin, rifabutin, ciprofloxacin, amikacin, ethambutol, streptomycin.
Colonies are slow-growers, thin, opaque, transparent, smooth and homogenous and are typically nonpigmented but may turn yellowish with age. They produce heat-stable catalase.
Mycobacterium abscessus:
This species is a quick-grower and a water contaminant. It is also found in soil and dust. Patients who are immunocompromised are at higher risk for chronic pulmonary lung disease, disseminated cutaneous infections, or post-traumatic wound infections caused by this organism. It also causes chronic middle ear infections. Some infections have been known to cause infection years to decades later after an initial wound-causing injury. On LJ agar, colonies grow as either rough or smooth, gray or white to off-white, and are non-photochromogenic. On starch-based agar, it grows as yellow colonies or streaks. It can be distinguished from other species of Mycobacterium by its failure to take up iron or reduce nitrate.
According to the CDC, this organism can cause a variety of infections or it may contaminate medications, medical devices or medical products or even showerheads. In the healthcare setting, the most common infections caused by this organism involve skin and soft tissue. Signs of infection include warmth, redness and tenderness. Boils, pus-filled vesicles, fever, chills, aches and pains, and general illness are also experienced with the skin or soft tissue infection associated with this organism.
Mycobacterium asiaticum:
This rarely causes infection. Colonies are non-pigmented to buff but yellow when exposed to light. It produces catalase.
Mycobacterium bovis:
This is a slow-grower that causes tuberculosis in cattle (very very rarely in the USA), but can also cause tuberculosis in humans. Infected milk or inhalation of aerosols can transmit the infection to humans. Infections are rare, since pasteurization pretty much kills the microbe during the treatment process of milk. Other animals can also be infected by the microbe, including white-tailed deer, pigs, llamas, domestic cats, racoons, opossums, and coyotes. Treatment for infections consists of isoniazid and rifampin for 6 months. This microbe is inherently resistant to pyrazinamide.
Colonies are small, granular, rounded, white with irregular margins after about 21 days of incubation at 37 degrees Celsius and colonies are non-pigmented. They are niacin negative, do not reduce nitrite to nitrate, do not grow in the presence of THC, and grow on Middlebrook 7H10 medium as colonies similar to M. tuberculosis, but smaller. A small part of this microbe is used in making vaccinations.
Mycobacterium chelonae:
This species of Mycobacterium is found in sewage and tap water and is able to cause opportunistic infection in humans. Infections are typically abscesses that form at the site of injection from contaminated equipment used to perform biomesotherapy. This alternative, homeopathic practice consists of injecting saline subcutaneously at acupuncture or trigger points for pain management.
Mycobacterium cosmeticum:
Dr. Robert Cooksey, a microbiologist with the CDC, discovered the bacterium, which can live and thrive in places like salons, healthcare settings, and other types of clinics, and out of all the Mycobacterium spp discovered so far, it is the newest discovered. It is one of the rapid-growers, and has been linked to outbreaks. It is hardy, thermotolerant, an extremophile, able to tolerate extremes of environment and harsh conditions, even in biofilms. It is linked to abscesses, many of which require months-to-years of antibiotic therapy to treat, and sometimes, even drainage and surgery, resulting in disfigurement. Ironically, infection usually occurs during plastic surgery or cosmetic surgery or other procedures.
Mycobacterium flavescens:
This organism is usually a contaminant if isolated in a human clinical specimen. Soft, yellow-orange, butyrous colonies are produced, which hydrolyze Tween 80, reduce nitrate, and tolerate 5% NaCl.
Mycobacterium fortuitum:
This fast-grower (3-4 days) can cause infections, and it one of the non-tuberculosis Mycobacterium spp. The various types of infections this strain causes include skin (wound or abscesses), bone, joint, and eye infections. It is rarely a cause of pulmonary infections, and is occasionally the cause of nosocomial infection (hospital-acquired). It has also been linked to cases of endocarditis and meningitis. It is found worldwide in tap water, rivers, lakes, sewage, dust and dirt. If wounds or surgical equipment are inadvertently exposed to contaminated water before, during or after a surgical procedure, the wounds or surgical sites may become infected. Additionally, implanted devices, such as catheters, pacemakers, intravenous lines, or endoscopes or injection equipment that is contaminated can all cause infection.
Colonies are non-pigmented or cream-colored "donut"-shaped colonies on LJ, fast-growers, grow on MacConkey agar without crystal violet, produce arylsulfatase, and the colonies are rough or smooth, creamy white or buff.
Colony characteristics
Colonies are smooth and round, cream-to-buff colored, waxy, butyrous, multilobate ("brain-like") or rosette-clusters that look like "donuts" on Lowenstein-Jensen media, and some colonies are even able to absorb the malachite green dye in the LJ media
Physiology
This species grows quickly within 2-4 days on LJ agar. It does not grow at 45 degrees Celsius, but it does grow on MacConkey agar.
Differential characteristics
M. fortuitum is able to utilize L-glutamate but unable to utilize acetamide as a nitrogen and carbon source. It may or may not fluoresce with the Auramine stain.
Treatment:
Treatment may include the macrolides and quinolones, doxycycline and minocycline, and sulfonamides. Isolates of this mycobacterium are susceptible to the carbopenams, such as Imipenem. Imipenem is a broad spectrum antibiotic. Patients with severe infections may be given an IV treatment combined with oral antibiotics for up to several months. The guideline recommends “for serious skin, bone, and soft tissue M fortuitum disease, a minimum of 4 months of therapy with at least two agents with in vitro activity against the clinical isolate is necessary to provide a high likelihood of cure. Surgery is generally indicated with extensive disease, abscess formation, or where drug therapy is difficult.”
Mycobacterium gastri
is a soil organism rarely found in gastric lavage or sputum specimens as a contaminant. Colonies are slow-growers, rough and buff or non-pigmented.
Mycobacterium genavense:
This organism has been linked to disseminated infection in AIDS patients. It is a slow-grower, is fastidious, and has been recovered in BACTEC cultures. It is positive for production of catalase, pyrazinamidase, and urease.
Mycobacterium gordonae:
Scotochromogenic colonies (yellow or orange) on LJ agar, and ubiquitous organism that is rarely ever implicated in disease. It is commonly isolated from tap water and soil and is a common contaminant. Infections are rarely caused meningitis in patients with ventriculoatrial shunts, hepatoperitoneal disease, synovitis, endocarditis, wound infections and pulmonary disease. Colonies are smooth and yellow-orange. It hydrolyzes Tween 80 and produces heat-stable catalase.
Mycobacterium haemophilum:
This organism causes rare infections in patients who are immunocompromised. Cases have occurred in patients with Hodgkin's lymphoma and AIDS. Painful swellings, ulcers progressing to abscesses, draining fistulas, subcutaneous nodules, and submandibular lymphadenitis are manifestations of infections with this mycobacterium. It requires hemin or hemoglobin for growth, so it can grow on chocolate agar, Mueller-Hinton agar, or Lowenstein-Jensen medium containing 2% ferric ammonium citrate. Colonies are rough to smooth and are not pigmented. It grows on CHOC agar.
Mycobacterium kansasii:
This organism can cause a chronic human pulmonary disease that is similar to tuberculosis. It affects the upper lobe of the lungs. Rarely, it causes skin infections and lesions. The major reservoir is considered to be tap water.
Colonies are slow-growing and if grown in the dark, are non-pigmented or buff. In the light, colonies appear yellow-orange. Most strains are catalase positive. They hydrolyze Tween 80 in 3 days, reduce nitrite to nitrate, and produce pyrazinamidase.
Mycobacterium malmoense:
This organism is associated with pulmonary disease and cervical adenitis and many strains are drug-resistant. Color of colonies is non-pigmented or buff and they are slow-growers.
Mycobacterium marinum:
This free-living organism causes opportunistic infections in humans. Occasionally, it is associated with cutaneous and deeper infections that cause aquarium granuloma, associated with home fish tanks or those who work in aquariums or work with fish. Accidental infection has occurred when cleaning the tank or changing the water without wearing gloves, where the organism has entered cuts or open wounds, or was causing by fish fins or bites. Lesions produced by the microbe may be single or multiple. Superficial nodules, papules or plaques may form at the site of infection. Untreated, this infection may spread to the deeper tissues and even bone. Lesions may be very painful or painless. The incubation period is about 2-4 weeks, and infections are commonly misdiagnosed at first. The organism is isolated on LJ slants after 7-21 days after incubation at 30-33 degrees Celsius. At first, colonies are cream colored, but they may turn yellow if exposed to light, because they are photochromogens. Treatment consists of either clarithromycin, minocycline or ethambutol and sometimes, surgery. Patients should be on the lookout for future lesions, especially if the infection has disseminated.
Colonies are slow-growing, smooth to rough, wrinkled on egg medium, and non-pigmented to buff in color when grown in the dark. In the light, they are deep yellow. Some strains produce niacin and they hydrolyze Tween 80 and produce both urease and pyrazinamidase.
Mycobacterium paratuberculosis:
This is the causative agent of Johne's disease, an intestinal infection resulting in chronic diarrhea in goats, sheep, cows and ruminants.
Mycobacterium phlei:
This is an environmental microbe that is occasionally associated with human infection. Colonies are rough or coarsely wrinkled with a deep yellow or orange pigment. Sometimes strains will produce smooth and butyrous colonies that are heaped with dense centers. They take up iron.
Mycobacterium scrofulaceum:
This strain is associated with cervical lymphadenitis in children, resulting in one ore more enlarged lymph nodes in the neck, with little to no pain. Surgical incision and drainage, as well as antituberculosis drugs, are the treatment of choice. It is a slow-grower, producing smooth colonies with dense centers ranging from light yellow to bright orange. They produce urease and are catalase-producers.
Mycobacterium simiae:
This organism has been isolated from tap water and has rarely been associated with pulmonary disease in patients with existing lung damage. Smooth colonies are non-pigmented to buff in the dark and yellow when exposed to light. It accumulates niacin and produces heat-stable catalase.
Mycobacterium smegmatis:
Most of the time, this is a nonpathogen. In fact, this is one of the commonly utilized QC (quality control) strains for testing the quality of the agar, reagents, and for the QC on the Vitek Mass Spec. Rarely, has it been associated with disease. Rarely, it causes pulmonary, skin, soft-tissue or bone infections. Colonies are rough, creamy in texture, wrinkled, coarsely folded, or smooth, glistening, and butyrous with dense centers, non-pigmented or creamy white, buff or pink.
Mycobacterium szulgai:
It causes pulmonary disease but can also cause bursitis or lymphadenitis. Smooth and rough colonies are produced, which are non-pigmented to buff in the dark, and are yellow to orange in the light. It is a slow hydrolyzer of Tween 80, reduces nitrate, and will not grow in the presence of 5% NaCl.
Mycobacterium leprae:
This microbe is found in warm, tropical countries and is the causative agent of leprosy (Hansen's Disease). This slow grower takes several weeks to mature, and will stain with carbol fuschin due to its waxy coating. It is acid-fast.
Mycobacterium terrae-triviale complex:
There have only been a few reported cases of illness associated with this microbe, including septic arthritis, synovitis, osteomyelitis, tenosynovitis, and respiratory infection. Colonies are rough, dry, and heaped. They hydrolyze Tween 80, reduce nitrate, and produce catalase.
Mycobacterium thermoresistibile:
This rarely causes disease in humans. Colonies are smooth or rough yellow.
Mycobacterium tuberculosis:
This species is the causative agent of tuberculosis, or TB. TB is on the rise, particularly in HIV/AIDS patients who are immunocompromised. Mycolic acid present in the cell wall renders this bacterium waxy and acid-fast. It is a highly obligate aerobic microorganism that needs lots of oxygen to thrive, and it is a very slow grower. It is able to survive weak disinfectants and dry conditions. Its resistance and virulence is growing and on the rise. It is a primary pathogen of the respiratory system, affecting the lungs. Tuberculosis is diagnosed by a combination of acid-fast and auramine stains, the tuberculin skin test, quantiferon levels, and chest X-rays. Patients typically present with fever, sweats, fatigue, heavy cough, and production of bloody sputum during the acute phase. Within the lungs, caseous granulomas may form, along with Langhans giant cells, due to the macrophage's inability to digest the organism. Patients may be asymptomatic. Organisms may be identified by their red color standing out against a blue background in Ziehl-Neelsen stain.
Colonies are thin, flat, spreading, friable, crumbly, rough in appearance, resemble breadcrumbs, buff in color (see images below). Optimum growth is 35-37 degrees Celsius. Biochemically, M. tuberculosis is positive for niacin reduction, and it reduces nitrate to nitrite (so positive for nitrite), produces catalase, which is destroyed after heating. It is inhibited by NAP. It is urease negative.
Mycobacterium ulcerans:
This organism is a very rare cause of mycobacteriosis, causing a painless nodule under the skin after a prior trauma, followed by development of a shallow ulcer, fever, and systemic symptoms. Colonies are smooth and rough, non-pigmented or buff, and produce heat-stable catalase.
Mycobacterium vaccae:
This organism is a rapid grower producing smooth, moist and shiny colonies that are buff to orange in color.
Mycobacterium xenopi:
This organism has been recovered from hot and cold tap water, storage tanks, hospital potable water systems, and birds. It is linked to slowly progressive pulmonary infections. Colonies are small with dense centers and edges that appear filamentous. On cornmeal agar, young colonies appear with stick-looking projections and look like a "bird's nest". colonies are non-pigmented but may appear bright yellow in the absence of light when exposed to the light. They accumulate niacin and reduce nitrate, produce heat-stable catalase, arylsulfatase, and pyrazinamidase. This organism grows best at 42 degrees C.
According to the CDC, symptoms of MOTT depend upon the site, since virtually any organ can be affected, many tend to be rapid growers, and since some are ubiquitous, most healthy persons are not affected by them, but those who are immunosuppressed, immunocompromised, or have underlying health conditions are at higher risk for infection. Those with lung infections tend to have symptoms including cough, shortness of breath, sometimes bloody sputum, or even a rash. Fever, weight loss, loss of appetite, decreased energy, and night sweats are also common symptoms. Other types of infections may include skin or soft tissue, device-associated infections resulting in bloodstream infections, artificial implant infection, pacemaker infection, exit wound site infection, lymph nodes, blood, etc...Below are listed many of the most common MOTTS seen in the healthcare setting. MOTT infections overall are rare, however, they are on the rise and many of these organisms are growing more resistant to treatment.
Mycobacterium avium complex (MAC or MACO) and avium-intracellulare (MAIC):
This ubiquitous organism is associated with fatal infections in HIV/AIDS patients with CD4 cell counts of less than 100/uL, or immunocompromised patients, especially senior citizens or cystic fibrosis patients, causing infection through inhalation or ingestion. In fact, a significant number of HIV/AIDS patients are affected by this organism, and it can produce disseminated illness with symptom of fever, night sweats, weight loss and loss of appetite. It can also infect patients who do not have HIV/AIDS, producing a chronic, persistent cough in those who are infected and who inhale the organism. Its point of entry is the lungs or the gastrointestinal tract. Patients who have ingested the organism and become infected develop diarrhea, fevers, chills, fatigue, abdominal pain, malabsorption, loss of appetite, and dissemination to the bone marrow.
Studies have shown that it can grow on shower heads or thrive in warm indoor swimming pool environments and become inhaled by HIV/AIDS patients, resulting in opportunistic infection.
Treatment consists of prophylaxis of clarithromycin and/or azithromycin or rifampicin, rifabutin, ciprofloxacin, amikacin, ethambutol, streptomycin.
Colonies are slow-growers, thin, opaque, transparent, smooth and homogenous and are typically nonpigmented but may turn yellowish with age. They produce heat-stable catalase.
Mycobacterium abscessus:
This species is a quick-grower and a water contaminant. It is also found in soil and dust. Patients who are immunocompromised are at higher risk for chronic pulmonary lung disease, disseminated cutaneous infections, or post-traumatic wound infections caused by this organism. It also causes chronic middle ear infections. Some infections have been known to cause infection years to decades later after an initial wound-causing injury. On LJ agar, colonies grow as either rough or smooth, gray or white to off-white, and are non-photochromogenic. On starch-based agar, it grows as yellow colonies or streaks. It can be distinguished from other species of Mycobacterium by its failure to take up iron or reduce nitrate.
According to the CDC, this organism can cause a variety of infections or it may contaminate medications, medical devices or medical products or even showerheads. In the healthcare setting, the most common infections caused by this organism involve skin and soft tissue. Signs of infection include warmth, redness and tenderness. Boils, pus-filled vesicles, fever, chills, aches and pains, and general illness are also experienced with the skin or soft tissue infection associated with this organism.
Mycobacterium asiaticum:
This rarely causes infection. Colonies are non-pigmented to buff but yellow when exposed to light. It produces catalase.
Mycobacterium bovis:
This is a slow-grower that causes tuberculosis in cattle (very very rarely in the USA), but can also cause tuberculosis in humans. Infected milk or inhalation of aerosols can transmit the infection to humans. Infections are rare, since pasteurization pretty much kills the microbe during the treatment process of milk. Other animals can also be infected by the microbe, including white-tailed deer, pigs, llamas, domestic cats, racoons, opossums, and coyotes. Treatment for infections consists of isoniazid and rifampin for 6 months. This microbe is inherently resistant to pyrazinamide.
Colonies are small, granular, rounded, white with irregular margins after about 21 days of incubation at 37 degrees Celsius and colonies are non-pigmented. They are niacin negative, do not reduce nitrite to nitrate, do not grow in the presence of THC, and grow on Middlebrook 7H10 medium as colonies similar to M. tuberculosis, but smaller. A small part of this microbe is used in making vaccinations.
Mycobacterium chelonae:
This species of Mycobacterium is found in sewage and tap water and is able to cause opportunistic infection in humans. Infections are typically abscesses that form at the site of injection from contaminated equipment used to perform biomesotherapy. This alternative, homeopathic practice consists of injecting saline subcutaneously at acupuncture or trigger points for pain management.
Mycobacterium cosmeticum:
Dr. Robert Cooksey, a microbiologist with the CDC, discovered the bacterium, which can live and thrive in places like salons, healthcare settings, and other types of clinics, and out of all the Mycobacterium spp discovered so far, it is the newest discovered. It is one of the rapid-growers, and has been linked to outbreaks. It is hardy, thermotolerant, an extremophile, able to tolerate extremes of environment and harsh conditions, even in biofilms. It is linked to abscesses, many of which require months-to-years of antibiotic therapy to treat, and sometimes, even drainage and surgery, resulting in disfigurement. Ironically, infection usually occurs during plastic surgery or cosmetic surgery or other procedures.
Mycobacterium flavescens:
This organism is usually a contaminant if isolated in a human clinical specimen. Soft, yellow-orange, butyrous colonies are produced, which hydrolyze Tween 80, reduce nitrate, and tolerate 5% NaCl.
Mycobacterium fortuitum:
This fast-grower (3-4 days) can cause infections, and it one of the non-tuberculosis Mycobacterium spp. The various types of infections this strain causes include skin (wound or abscesses), bone, joint, and eye infections. It is rarely a cause of pulmonary infections, and is occasionally the cause of nosocomial infection (hospital-acquired). It has also been linked to cases of endocarditis and meningitis. It is found worldwide in tap water, rivers, lakes, sewage, dust and dirt. If wounds or surgical equipment are inadvertently exposed to contaminated water before, during or after a surgical procedure, the wounds or surgical sites may become infected. Additionally, implanted devices, such as catheters, pacemakers, intravenous lines, or endoscopes or injection equipment that is contaminated can all cause infection.
Colonies are non-pigmented or cream-colored "donut"-shaped colonies on LJ, fast-growers, grow on MacConkey agar without crystal violet, produce arylsulfatase, and the colonies are rough or smooth, creamy white or buff.
Colony characteristics
Colonies are smooth and round, cream-to-buff colored, waxy, butyrous, multilobate ("brain-like") or rosette-clusters that look like "donuts" on Lowenstein-Jensen media, and some colonies are even able to absorb the malachite green dye in the LJ media
Physiology
This species grows quickly within 2-4 days on LJ agar. It does not grow at 45 degrees Celsius, but it does grow on MacConkey agar.
Differential characteristics
M. fortuitum is able to utilize L-glutamate but unable to utilize acetamide as a nitrogen and carbon source. It may or may not fluoresce with the Auramine stain.
Treatment:
Treatment may include the macrolides and quinolones, doxycycline and minocycline, and sulfonamides. Isolates of this mycobacterium are susceptible to the carbopenams, such as Imipenem. Imipenem is a broad spectrum antibiotic. Patients with severe infections may be given an IV treatment combined with oral antibiotics for up to several months. The guideline recommends “for serious skin, bone, and soft tissue M fortuitum disease, a minimum of 4 months of therapy with at least two agents with in vitro activity against the clinical isolate is necessary to provide a high likelihood of cure. Surgery is generally indicated with extensive disease, abscess formation, or where drug therapy is difficult.”
Mycobacterium gastri
is a soil organism rarely found in gastric lavage or sputum specimens as a contaminant. Colonies are slow-growers, rough and buff or non-pigmented.
Mycobacterium genavense:
This organism has been linked to disseminated infection in AIDS patients. It is a slow-grower, is fastidious, and has been recovered in BACTEC cultures. It is positive for production of catalase, pyrazinamidase, and urease.
Mycobacterium gordonae:
Scotochromogenic colonies (yellow or orange) on LJ agar, and ubiquitous organism that is rarely ever implicated in disease. It is commonly isolated from tap water and soil and is a common contaminant. Infections are rarely caused meningitis in patients with ventriculoatrial shunts, hepatoperitoneal disease, synovitis, endocarditis, wound infections and pulmonary disease. Colonies are smooth and yellow-orange. It hydrolyzes Tween 80 and produces heat-stable catalase.
Mycobacterium haemophilum:
This organism causes rare infections in patients who are immunocompromised. Cases have occurred in patients with Hodgkin's lymphoma and AIDS. Painful swellings, ulcers progressing to abscesses, draining fistulas, subcutaneous nodules, and submandibular lymphadenitis are manifestations of infections with this mycobacterium. It requires hemin or hemoglobin for growth, so it can grow on chocolate agar, Mueller-Hinton agar, or Lowenstein-Jensen medium containing 2% ferric ammonium citrate. Colonies are rough to smooth and are not pigmented. It grows on CHOC agar.
Mycobacterium kansasii:
This organism can cause a chronic human pulmonary disease that is similar to tuberculosis. It affects the upper lobe of the lungs. Rarely, it causes skin infections and lesions. The major reservoir is considered to be tap water.
Colonies are slow-growing and if grown in the dark, are non-pigmented or buff. In the light, colonies appear yellow-orange. Most strains are catalase positive. They hydrolyze Tween 80 in 3 days, reduce nitrite to nitrate, and produce pyrazinamidase.
Mycobacterium malmoense:
This organism is associated with pulmonary disease and cervical adenitis and many strains are drug-resistant. Color of colonies is non-pigmented or buff and they are slow-growers.
Mycobacterium marinum:
This free-living organism causes opportunistic infections in humans. Occasionally, it is associated with cutaneous and deeper infections that cause aquarium granuloma, associated with home fish tanks or those who work in aquariums or work with fish. Accidental infection has occurred when cleaning the tank or changing the water without wearing gloves, where the organism has entered cuts or open wounds, or was causing by fish fins or bites. Lesions produced by the microbe may be single or multiple. Superficial nodules, papules or plaques may form at the site of infection. Untreated, this infection may spread to the deeper tissues and even bone. Lesions may be very painful or painless. The incubation period is about 2-4 weeks, and infections are commonly misdiagnosed at first. The organism is isolated on LJ slants after 7-21 days after incubation at 30-33 degrees Celsius. At first, colonies are cream colored, but they may turn yellow if exposed to light, because they are photochromogens. Treatment consists of either clarithromycin, minocycline or ethambutol and sometimes, surgery. Patients should be on the lookout for future lesions, especially if the infection has disseminated.
Colonies are slow-growing, smooth to rough, wrinkled on egg medium, and non-pigmented to buff in color when grown in the dark. In the light, they are deep yellow. Some strains produce niacin and they hydrolyze Tween 80 and produce both urease and pyrazinamidase.
Mycobacterium paratuberculosis:
This is the causative agent of Johne's disease, an intestinal infection resulting in chronic diarrhea in goats, sheep, cows and ruminants.
Mycobacterium phlei:
This is an environmental microbe that is occasionally associated with human infection. Colonies are rough or coarsely wrinkled with a deep yellow or orange pigment. Sometimes strains will produce smooth and butyrous colonies that are heaped with dense centers. They take up iron.
Mycobacterium scrofulaceum:
This strain is associated with cervical lymphadenitis in children, resulting in one ore more enlarged lymph nodes in the neck, with little to no pain. Surgical incision and drainage, as well as antituberculosis drugs, are the treatment of choice. It is a slow-grower, producing smooth colonies with dense centers ranging from light yellow to bright orange. They produce urease and are catalase-producers.
Mycobacterium simiae:
This organism has been isolated from tap water and has rarely been associated with pulmonary disease in patients with existing lung damage. Smooth colonies are non-pigmented to buff in the dark and yellow when exposed to light. It accumulates niacin and produces heat-stable catalase.
Mycobacterium smegmatis:
Most of the time, this is a nonpathogen. In fact, this is one of the commonly utilized QC (quality control) strains for testing the quality of the agar, reagents, and for the QC on the Vitek Mass Spec. Rarely, has it been associated with disease. Rarely, it causes pulmonary, skin, soft-tissue or bone infections. Colonies are rough, creamy in texture, wrinkled, coarsely folded, or smooth, glistening, and butyrous with dense centers, non-pigmented or creamy white, buff or pink.
Mycobacterium szulgai:
It causes pulmonary disease but can also cause bursitis or lymphadenitis. Smooth and rough colonies are produced, which are non-pigmented to buff in the dark, and are yellow to orange in the light. It is a slow hydrolyzer of Tween 80, reduces nitrate, and will not grow in the presence of 5% NaCl.
Mycobacterium leprae:
This microbe is found in warm, tropical countries and is the causative agent of leprosy (Hansen's Disease). This slow grower takes several weeks to mature, and will stain with carbol fuschin due to its waxy coating. It is acid-fast.
Mycobacterium terrae-triviale complex:
There have only been a few reported cases of illness associated with this microbe, including septic arthritis, synovitis, osteomyelitis, tenosynovitis, and respiratory infection. Colonies are rough, dry, and heaped. They hydrolyze Tween 80, reduce nitrate, and produce catalase.
Mycobacterium thermoresistibile:
This rarely causes disease in humans. Colonies are smooth or rough yellow.
Mycobacterium tuberculosis:
This species is the causative agent of tuberculosis, or TB. TB is on the rise, particularly in HIV/AIDS patients who are immunocompromised. Mycolic acid present in the cell wall renders this bacterium waxy and acid-fast. It is a highly obligate aerobic microorganism that needs lots of oxygen to thrive, and it is a very slow grower. It is able to survive weak disinfectants and dry conditions. Its resistance and virulence is growing and on the rise. It is a primary pathogen of the respiratory system, affecting the lungs. Tuberculosis is diagnosed by a combination of acid-fast and auramine stains, the tuberculin skin test, quantiferon levels, and chest X-rays. Patients typically present with fever, sweats, fatigue, heavy cough, and production of bloody sputum during the acute phase. Within the lungs, caseous granulomas may form, along with Langhans giant cells, due to the macrophage's inability to digest the organism. Patients may be asymptomatic. Organisms may be identified by their red color standing out against a blue background in Ziehl-Neelsen stain.
Colonies are thin, flat, spreading, friable, crumbly, rough in appearance, resemble breadcrumbs, buff in color (see images below). Optimum growth is 35-37 degrees Celsius. Biochemically, M. tuberculosis is positive for niacin reduction, and it reduces nitrate to nitrite (so positive for nitrite), produces catalase, which is destroyed after heating. It is inhibited by NAP. It is urease negative.
Mycobacterium ulcerans:
This organism is a very rare cause of mycobacteriosis, causing a painless nodule under the skin after a prior trauma, followed by development of a shallow ulcer, fever, and systemic symptoms. Colonies are smooth and rough, non-pigmented or buff, and produce heat-stable catalase.
Mycobacterium vaccae:
This organism is a rapid grower producing smooth, moist and shiny colonies that are buff to orange in color.
Mycobacterium xenopi:
This organism has been recovered from hot and cold tap water, storage tanks, hospital potable water systems, and birds. It is linked to slowly progressive pulmonary infections. Colonies are small with dense centers and edges that appear filamentous. On cornmeal agar, young colonies appear with stick-looking projections and look like a "bird's nest". colonies are non-pigmented but may appear bright yellow in the absence of light when exposed to the light. They accumulate niacin and reduce nitrate, produce heat-stable catalase, arylsulfatase, and pyrazinamidase. This organism grows best at 42 degrees C.
photo gallery of Mycobacterium spp:
MYCOBACTERIUM TUBERCULOSIS (TB): Microscopic and Macroscopic Morphology
MYCOBACTERIUM FORTUITUM:
MYCOBACTERIUM AVIUM-INTRACELLULARE (MAICO) and MYCOBACTERIUM AVIUM COMPLEX (MAC/MACO):
MYCOBACTERIUM ABSCESSUS:
MYCOBACTERIUM MARINUM:
MYCOBACTERIUM AVIUM-INTRACELLULARE COMPLEX:
MYCOBACTERIUM BOVIS:
MYCOBACTERIUM CHELONAE:
In California and Oregon, from 2000-2004, there were several outbreaks of this organism in nail salons across the state. Infections left victims with painful, unsightly, cutaneous sores that left scarring and were difficult to treat. It can cause abscesses and cellulitis. This is a rapid-grower that is ubiquitous in soil and water, even chlorinated water, and swimming pools. Most infections are associated with post-surgical infections due to contaminated equipment with water that has contaminated surgical devices or equipment or wound dressings. The infections in the images below were due to improperly cleaned, sanitized and disinfected foot spas in salons. Natural biofilms forming on the skin, hair, oils, lotions, creams, and other residues provides a suitable breeding environment for Mycobacterium to grow and thrive. Cuts or open wounds due to to trauma or even shaving of the legs puts persons most at risk for infection. M. fortuitum, a close relative to M. chelonae, has also caused such infections and breakouts of them.
http://www.oregon.gov/OHLA/COS/pages/features/bacterial_skin_infections.aspx
http://www.oregon.gov/OHLA/COS/pages/features/bacterial_skin_infections.aspx
MYCOBACTERIUM COSMETICUM:
By Photo Credit: James GathanyContent Providers(s): CDC/ CDC Connects - This media comes from the Centers for Disease Control and Prevention's Public Health Image Library (PHIL), with identification number #7888.Note: Not all PHIL images are public domain; be sure to check copyright status and credit authors and content providers.English | Slovenščina | +/−Transferred from en.wikipedia to Commons by Optigan13., Public Domain, https://commons.wikimedia.org/w/index.php?curid=4405721
MYCOBACTERIUM KANSASII:
MYCOBACTERIUM GORDONAE:
MYCOBACTERIUM LEPRAE:
MYCOBACTERIUM SMEGMATIS:
MYCOBACTERIUM IMMUNOGENICUM:
MYCOBACTERIUM SPP:
https://images.search.yahoo.com/images/view;_ylt=AwrTcYF2NRhVTBwACyyJzbkF;_ylu=X3oDMTI0OWN2NDZwBHNlYwNzcgRzbGsDaW1nBG9pZAM1NjkxMjBmNWUzZWFmNGJiYjU2MzE3ZGJlNGUxY2E0YwRncG9zAzE1NARpdANiaW5n?.origin=&back=https%3A%2F%2Fimages.search.yahoo.com%2Fyhs%2Fsearch%3Fp%3DMycobacterium%2Bgordonae%26fr2%3Dpiv-web%26hsimp%3Dyhs-001%26hspart%3Dmozilla%26nost%3D1%26tab%3Dorganic%26ri%3D154&w=640&h=337&imgurl=www.telmeds.org%2Fwp-content%2Fuploads%2F2009%2F10%2Fmycobacterias2.jpg&rurl=http%3A%2F%2Fwww.telmeds.org%2Fatlas%2Fbacteriologia%2Fmycobacterias-nocardia-y-actinomyces%2Fcultivos-de-mycobacterium%2Fcultivo-de-mycobacterias-atipicas%2F&size=97.4KB&name=Mycobacterias+At%C3%ADpicas&p=Mycobacterium+gordonae&oid=569120f5e3eaf4bbb56317dbe4e1ca4c&fr2=piv-web&fr=&tt=Mycobacterias+At%C3%ADpicas&b=121&ni=21&no=154&ts=&tab=organic&sigr=14c2umrt2&sigb=146po3ot5&sigi=11t2h5941&sigt=10nek3dav&sign=10nek3dav&.crumb=kZjTZuzNe/G&fr2=piv-web&hsimp=yhs-001&hspart=mozilla
Processing afb: preparation
- Get all supplies out and ready ahead of time and organized, preferably on a cart
- Mycobacterium is processed in a BLS II/III laboratory setting in a negative pressure room utilizing extreme caution, performing all procedures under the biological hood, utilizing aseptic technique, and wearing appropriate PPE, to include: disposable gown (never comes out of the room), N95 Respirator Mask or PAPR, Gloves, Goggles or Face Shield, Bleach, Cavicide Wipes, Tuberculocide, Incinerator for Metal Loops
- Always place specimens on a bleach wipe under the biological hood with the fan and light running and on
- Soak specimens and any disposable loops or pipettes in bleach or other tuberculocide prior to disposing of them in the autoclavable red biohazard bag (underneath the biological hood)
- Soak for a minimum of 30 min.
- Autoclave all biohazardous waste prior to disposal
- Place a moistened bleach or cavicide wipe underneath your work in the biological hood for extra protection (or paper towels soaked in tuberculocide)
processing afb: STEP 1: the digestion-decontamination procedure and reagents:
I. nalc/NAC (n-acetyl-l-cysteine)-2.9%: digestion/liquefaction/DECONTAMINATION
- NALC powder is added to NaOH (sodium-hydroxide) base and allowed to sit for about 30 min. or so to completely dissolve and create a solution
- Digestant
- Contains the enzymes mucolysin and sputolysin
- Breaks down mucus
- Liquefies the specimen
- Helps release Mycobacterium and other bacteria from the cells and mucus (clearing the debris)
- Encourages the Mycobacterium to grow by removal of the normal commensal flora
- Once made into a solution, it should sit for 15-30 minutes prior to using it, for complete dissolution
- Once equal parts NALC are added to the specimen, it should sit 15 minutes to break down mucus and saliva
- Once the solution is made, it is good for just 24 hours. The date/time made should be documented on the conical tube it is made in, along with the expiration date.
II. naoh (sodium hydroxide)-4.0% (50 mL or 5 g): decontamination
- Sodium citrate binds heavy metal ions that might otherwise inactivate NALC powder
- NALC powder is added to this base and must sit for 30 minutes so that it can dissolve and activate the necessary enzymes for digestion-decontamination
- Decontaminant
- Mucolytic
- Alkali/basic
- Emulsifier (stabilizes the solution by increasing its kinetic stability)
- Surfactant with a hydrophobic side and a hydrophilic side
- Water-soluble
- Removes decontaminating bacteria from cells
- Only 50-70% of the Mycobacterium survive this process
- Toxic on its own, but the addition of the NALC powder decreases the concentration of NaOH required and it shortens the time, and it helps in the recovery of acid-fast bacteria
- After 30 minutes, 5-10 mL of specimen/sputum is put into a 50 mL conical tube
- An equal amount of the digestant-decontaminant solution is added to the specimen (5-10 mL) and vortexed/mixed rigorously and allowed to sit for 15 minutes to digest/decontaminate and to prevent dispersion of fine aerosols generated during the mixing
iii. oxalic acid-5% (EXTRA added step IN SOME LABORATORIES for extra thick mucous secretions from cystic fibrosis patients OR KNOWN PSEUDOMONAS/HX OF PSEUDOMONAS)
- This is used to treat specimens that contain known GNR's , particularly Pseudomonas aeruginosa and Proteus (these contaminants are difficult to get rid of)
- This is a sputolysin and is very acidic, a reducing agent, and a chelater of metal ions
- The specimen is first decontaminated-digested with the NALC-NaOH method
- After the centrifugation/decanting steps, add equal volumes of 5% oxalic acid to the sediment button and mix/vortex
- Let this solution sit for 30 minutes, vortexing every 10 minutes
- Add phosphate to the 50 mL line again and centrifuge again @ 3,000 rpm for 15 minutes
- Decant and resuspend with serum-albumin buffer as in the NALC-NaOH method to a pH of 7.0 (test with pH paper), which brings the NaOH to 4%
- Proceed with the steps as in the NALC-NaOH method
iv. STEP 2: ADDITION OF potassium phosphate buffer, ph 6.8, 0.67 m: neutralization
- Buffer and aid in maintaining a constant pH
- Neutralizes the NaOH and dilutes the toxic substances contained in the NaOH
- Decreases specific gravity for sedimentation during the centrifugation process
- After the digestion-decontamination process (15 min.), this buffer is then added to the 50 mL line of the conical tube, which is then capped tightly and inverted to mix and centrifuged
v. STEP 3: centrifugation: sedimentation and decanting
- Spinning the solution down at 3,200-3,600 rpm for 15-20 minutes enables a button of sediment to form at the bottom (my lab spins the solution down for 17 minutes)
- The specimen is decanted into a container with a microbicidal agent and the supernatant is poured off into this container containing cavicide or bleach
vi. STEP 4: resuspension AND MEDIA INOCULATION
- The sediment button is resuspended (preferably in 1 mL of bovine serum albumin or MB7H9 broth) and mixed/vortexed
- Bovine serum albumin is also a buffer and an adhesive that helps the cells to stick to the slide and to the media
- This mixture is used to inoculate the slides for fluorescent smear and acid-fast stain and the solid media
- The rest of the albumin or broth is then added to the remaining solution, mixed and used to inoculate the liquid broth medium containing 0.5-0.8 mL of antibiotics (PANTA) (my lab uses 0.8 mL put into MGIT broth ahead of time and at this step, uses 0.5 mL of resuspended sediment into the MGIT broth containing the antibiotics), the LJ slant or slants (0.2 mL), and any other media required if the MGIT was positive and this was an extra digestion (CHOC, MB7H11, MB7H9, 2 LJ's)
NEXT STEP: The auramine fluorescent/fluorochrome stain:
- This smear is prepared with Auramine M, Auramine O, or Auramine-Rhodamine, which should be stored in the dark, protected from light, moisture or variances in pH
- Special slides are used that are to be protected from light (cover with a paper towel after making them and view in the dark)
- This stain is a fluorochrome stain meant to be used with UV microscopy (filter)
- This stain is considered to be more sensitive than the carbolfuchsin stains
- Fluorescent bacilli stand out brightly yellow or orange against the dark black background on 20x or 40x objective lenses (it depends upon which kit and filter your lab uses) (my lab uses a kit in which the mycobacterium fluoresce bright orange against a black background and our fluorescence microscope uses a blue filter)
- The positive stain can be restained with Kinyoun or Ziehl-Neelsen acid-fast stain
- Drawback: rapid-growers may not appear fluorescent (ex: M. fortuitum)
- Confirm positives with the acid-fast stain and if need be, perform an extra digestion and reinoculate another MGIT so it has longer to grow and incubate
- Fluorochrome dyes form a complex with the mycolic acids in the cell wall
- DIRECT SMEAR: made directly from the sample prior to digestion-decontamination and stained
- CONCENTRATED SMEAR: made from digested-decontaminated sediment and stained
- DIRECT SMEAR: made directly from the sample prior to digestion-decontamination and stained
2) Decolorize with 0.5% acid-alcohol (70% ethanol/0.5% hydrochloric acid) for 2-3 minutes and rinse with sterile water
3) Counterstain with 0.5% potassium permanganate for 2-4 minutes and rinse. Dry in slide protector sleeves or paper towels protected from light. View in the dark under fluorescence microscopy.
Allow the slide to sit on a heat block at 80 degrees C for 15 minutes after making the smear prior to staining it, or flame it to heat fix it. Use care and caution, as the smear and stain process may not kill ALL Mycobacterium that may be present.
acid-fast stain: 20-80% sensitivity
the kInyoun stain method:
- This is referred to as the COLD METHOD, because you do not need to heat the stain due to an increased concentration of carbolfuchsin stain and phenol
- Flood the slide with KINYOUN carbolfuchsin stain for 4-5 minutes; Rinse with sterile water
- Decolorize the slide with 3% acid-alcohol (70% ethanol/3% hydrochloric acid) for 2 minutes; Rinse with sterile water
- Flood the slide with methylene blue or brilliant green stain for 1-3 minutes; Rinse with sterile water
- Allow slides to dry at room temperature
- After you make the smear, before staining with this method, allow the slide to sit on a slide warmer at 80 degrees Celcius for 15 minutes
- View under oil immersion
- Mycobacterium will stain purple-to-red or red-pink against the blue background and appear as short or long rods (2-8 uM), slightly curved, possibly beaded, banded or corded, pleomorphic bacilli (some strains appear as coccobacilli)
- Other bacteria types will stain blue against the background
- Scan at least 300 fields (3 full sweeps)
the ziehl-neelsen stain method:
- This is referred to as the HOT METHOD, since heating is required to allow greater penetration of the carbolfuchsin into the cell wall
- Mycolic acids and waxes complex with the basic dye, retaining the stain
- Flood the slide with carbolfuchsin and steam the slide for 1 minute, without boiling or allowing the slide to dry out
- Allow the stain to remain on for 4-5 minutes longer without heat; Rinse with sterile water
- Decolorize with 3% acid-alcohol (95% ethanol/3% hydrochloric acid) for 2 minutes; Rinse with sterile water
- Flood the slide with methylene blue for 1 minute; Rinse with sterile water
- Let slide air dry
- View under oil immersion and scan at least 300 oif, 3 full sweeps
dna probe testing:
The Gen-Probe is a luminometer that measures the amount of light that is emitted from chemiluminescent reactions. A photomultiplier tube detects these reactions and converts it into a digital signal that can be measured. The final measurable signal is directly proportional to the number of photons released by the reaction itself. Luminescent molecules are coupled with genetic probes called DNA or RNA probes, which detect hybridization reactions, or else they are coupled with antibodies that detect antibody-antigen reactions/interactions. The reader is a microprocessor. Detection reagents basically initiate a chemical reaction that results in the emission of photons. A photon counter signal is converted to RLU's, or relative light units, which are processed by the microprocessor. The instrument consists of reagent reservoirs and pumps (a pair), a photomultiplier tube, and a microprocessor/microcomputer convertor.
Samples:
I. Sputum and Respiratory Secretions (Bronchial Aspirates, Washes)
- If 2 out of 3 sputum smears are positive, that is typically enough to confirm the diagnosis
- Sputum: need 5-10 mL produced by a deep cough or expectorated sputum induced by inhalation of an aerosol of hypertonic saline
- Bronchoscopy:general washing or bronchoalveolar lavage (BAL) or brushing
- Used to recover mycobacterium that has been swallowed overnight
- Used when patients cannot produce sputum by aerosol induction
- Used for children <3 years of age
- Used for nonambulatory patients
- Collection: 3 specimens in 3 days, obtained in the morning after an overnight fast collected utilizing 30-60 mL of sterile water
- Specimens are neutralized by buffer to 7.0 pH
- First morning urine preferred
- Need 15 mL of urine in sterile container
- Refrigerate
- Process as soon as possible
- Clean container, unpreserved
- Analyzed within a few hours or frozen at -20 degrees Celsius
- Wampole isolator lysis-centrifuge system
- Culture into BACTEC bottles for fluids is useful
- Tissue should be collected aseptically and placed in a sterile container and sterile saline (10-15 mL) can be added to it and homogenized
- Fluids: 2 mL CSF, 3-5 mL exudates, pericardial and synovial fluids, 10-15 mL abdominal or chest fluids
NALC-Sodium hydroxide digestion-decontamination procedure:
How It Works:
NaOH (Sodium hydroxide) is a decontaminating agent and a digestant. NaOH is toxic to mycobacterium, so a low concentration is needed. NALC (N-acetyl L-cysteine) is a mucolytic agent containing enzymes that allow for a lower concentration of NaOH to be used. These two together optimize recovery of mycobacterium.
To Make NALC-NaOH Digestant:
NaOH (Sodium hydroxide) is a decontaminating agent and a digestant. NaOH is toxic to mycobacterium, so a low concentration is needed. NALC (N-acetyl L-cysteine) is a mucolytic agent containing enzymes that allow for a lower concentration of NaOH to be used. These two together optimize recovery of mycobacterium.
To Make NALC-NaOH Digestant:
- Combine equal volumes of 2.94% sodium citrate dehydrate and 4% sodium hydroxide (NaOH)
- Add 0.5 g of powdered NALC per 100 mL of solution just before use
- Let mixture sit for 30 min.
- Refrigerate when not in use
- Discard after 24 hours
- Phosphate Buffer (0.067 M, pH 6.8)
- Sterile Distilled Water (30-40 mL/specimen)
- Bovine Albumin (0.2%)
- Don appropriate PPE
- Work under the biological safety hood
- Transfer 5-10 mL of sample to a 50-mL conical screw-cap centrifuge tube
- Note: If the specimen is >10 mL, transfer the most purulent material into the tube
- Add an equal amount of NALC-NaOH digestant to the sample
- Tighten the cap and vortex until it is liquefied (about 15 seconds)
- Invert the tube to make sure the solution contacts all particles in the tube
- Let tubes stand at room temperature for 15 minutes
- If additional decontamination is needed, increase the concentration of the NaOH
- Dilute the now digested-decontaminated specimen to the 50-mL mark with sterile water or sterile phosphate buffer
- Tighten cap and invert and swirl to mix
- Centrifuge at 3000 rpm for 15 min using aerosol-free safety cups
- Hold the tube so the sediment is on the upper side of the tube, pour off supernatant into a splashproof biohazard container or disinfectant under the biological safety hood
- Keep the tube in a horizontal position and use a sterile applicator stick to remove a bit of the sediment to place on a microscopic slide and make a smear (1-2 cm)
- Resuspend the sediment in 1-2 mL of sterile 0.2% bovine albumin solution, sterile water or sterile saline
- You can also prepare a 1:10 dilution using 0.5 mL of resuspended sediment in 4.5 mL sterile water to decrease the concentration of possible toxins that might inhibit growth of mycobacterium
- Inoculate diluted and undiluted specimens to solid media
- Ziehl-Neelsen Stain
- Involves application of heat with the carbolfuchsin stain
- Kinyoun Stain
- Is a cold stain (no heat involved)
- Examination: 100x oil immersion on light microscope for 15 min., viewing at least 300 fields before calling a slide negative
- Auramine or Auramine-Rhodamine Fluorochrome Stains
- More sensitive than ZN or Kinyoun stains
- May be positive when traditional stains are negative
- Smear may be screened at a lower objective (250x or 400x)
- Fluorescence microscopy is used, which is equipped with a special filter system and a mercury vapor light with a strong blue filtered light in a dark room
- Positive stains: bright red or yellow-orange bacilli against a dark background
- Make a smear and air dry, protected from light
- Stain with Auramine-Phenol for 20 mins, protected from light
- Rinse with water
- Decolorize in acid alcohol (HCl acid + denatured ethanol/methanol)
- Rinse with water
- Counterstain with 0.1% potassium permanganate for 30 seconds (or 2-3 minutes depending on kit)
- Rinse and air dry, protected from light
- Read in the dark under special immunofluorescence microscopy
- Niacin Accumulation (Niacin +)
- Useful of M. tuberculosis recovery
- Nitrate Reduction (Nitrate +)
- Useful for recovery of M. kansasii, M. szulgai, M. fortuitum, M. tuberculosis
- Catalase
- Mycobacteria are catalase positive, however, not all strains produce a positive reaction when cultures are heated to 68 degrees Celsius for 20 min.
- Hydrolysis of Tween 80
- Iron Uptake
- Useful for recovery of M. chelonei
- Arylsulfatase
- Useful for recovery of M. fortuitum-chelonei complex and M. triviale, M. marinum and M. szulgai
- Pyrazinamidase
- Useful for recovery of M. marinum and M. bovis
- Urease
- Useful for recovery of M. scrofulaceum
- Inhibitory Tests
- M. fortuitum-chelonei complex will grow on MacConkey agar without crystal violet
- Slow Versus Rapid Growth
- Chromatography
- DNA Probe
- PCR
- Mass Spec.
Ziehl-neelsen stain:
kinyoun stain:
Auramine o phenol stain AND POTASSIUM PERMANGANATE IN WATER:
Precautions:
Use proper PPE and handle all stains under the biological safety hood and protect solutions and slides from light exposure.
Auramine O Stain:
Acridine Orange Stain:
Potassium Permanganate:
Use proper PPE and handle all stains under the biological safety hood and protect solutions and slides from light exposure.
Auramine O Stain:
- Auramine O, Auramine Phenol Stain and Auramine-Rhodamine Stains are immunofluorescent stains utilized to stain mycobacterium (acid-fast bacilli)
- Bacteria stain reddish-yellow
- Affordable and sensitive
- Used as a screening tool
- Protect from light
- Carcinogenic
Acridine Orange Stain:
- Organic compound
- Nucleic acid selective fluorescent cationic dye
Potassium Permanganate:
- Oxidizing agent
LABORATORY WORKFLOW:
- Clean and stock for the day.
- Take temperatures and record QC of things such as incubators and refrigerators.
- Make sure the negative pressure is working.
- Gather PPE for the day.
- Direct Smears are made straight from the original sample and are stained with fluorescence stain and viewed under LED microscopy. Sometimes these slides can be 18% more sensitive than Carbolfuschin.
- If positive, a Direct Smear should be reported to the physician or nurse.
- If the patient is in the hospital, additionally, an infection control MD or nurse should be contacted and informed that an AFB has been discovered by Direct Smear. They will work with the patient and family to educate them about what to expect and what to do. Make sure they are informed that the patient should be in a negative pressure room and on isolation and other precautions.
- Keep the MD's and RN's informed of progress, ID and susceptibility patterns when the results return.
- Concentrate/Digest/Decontaminate the sample and perform a Concentrated Smear, AFS and GS, and inoculate all media for growth (MGIT, LJ's, CHOC, MB7h9, MB7H11).
- Digestion/Decontamination/Concentration is performed daily, concentrated smears are made, stained by FAFS, and read
- MGIT broth is positive:
- Concentrate the sample by vortexing followed by centrifugation for 15-20 minutes
- Make 2 smears and heat fix: AFS, GS
- If AFS is positive:
- Subculture to 2 LJ's, MBhH9 broth, MB7H11 plate, CHOC
- Document that AFS was positive and morphology seen
- If AFS is negative:
- Perform an extra digestion (the same process as the digestion/decontamination/concentration method)
- Some places perform the oxalic acid (5%) extra dig
- Some places perform up to one extra dig
- Some places perform up to two extra dig's
- Perform an extra digestion (the same process as the digestion/decontamination/concentration method)
- If GS is positive:
- Save the slides
- Document morphology and any other bacteria seen, including on the media itself
- Some places add an LJ Gruft formulation for subculture at this point, since Gruft contains antibiotics that help prevent overgrowth of normal flora
- If GS is negative:
- No need to do anything or write anything or document anything but no organisms seen
- If there is growth on any of the media (LJ, broth, MB7H11 plate, CHOC agar):
- Make smears from the media and perform AFS or MAFS and GS
- Document all morphology, positive or negative
- When there is enough growth, perform MALDI-TOF MS, DNA probe testing, or send out for ID and susceptibility testing
- Make smears from the media and perform AFS or MAFS and GS
- Don't final anything for 6-8 weeks, depending upon your laboratory
- If waiting for ID, confirmation or susceptibility testing, wait until these return before finaling
- Some places hold LJ's and broths and MB7H11 plates for up to 2 months after in case susceptibility is added on
- Subculture your QC organisms and perform regular slide QC as required
- Autoclave all biohazard materials within an AFB room prior to discarding it. Use autoclave tape and the spore ATTEST. Save a piece of tape and document autoclave. Clean the autoclave. Incubate the ATTEST and check it 48 hours later. Discard ATTEST spore tests. Document results.
- Clean and bleach surfaces daily at minimum
- Use tuberculocide or bleach in the bottom of containers that loops, syringes and other materials are discarded into
- On your work surface area, use bleach towels or tuberculocide soaked paper towels that stay wet at all times while you are working under the biological hood
- Clean up any spills immediately
- Make sure you wear all proper PPE during the decontamination/digestion process. Put UV light on in biological hoods at the end of the day to kill any AFB.
- Document results. Save/discard slides.
- Clean the microscope.
- Restock supplies that are low or needed.
Nocardia species:
Nocardia spp are partially acid fast and will stain pink-red in a modified acid-fast stain. These GPR's are also beaded, filamentous and branched and resemble Mycobacterium in Gram stains, fluorescent stains and even sometimes in acid-fast stains. The plate morphology is different, however, and resembles mold. It also grows on LJ agar. Nocardia species are weak Gram-positive rods that are catalase positive. There are close to 100 species, many of which are nonpathogenic, however, some are pathogenic and cause nocardiosis. Inhalation of the bacteria or introduction of the bacterium through traumatic inoculation, such as during a wound infection, results in infection. It is found worldwide and is a soil bacterium. Most infections are opportunistic and immunocompromised patients are at risk for this. Here are some facts:
- The most common species encountered in the laboratory are N. asteroides, N. brasiliensis, N. nova, N. farcinica and N. cyriacigeorgica
- Noninvasive cutaneous disease is most often the result of infection with N. asteroides
- Cutaneous nocardiosis is most often caused by N. brasiliensis
- Cutaenous nocardiosis may present as actinomycetoma, cellulitis, lymphatic disease, or an abscess
- Pneumonia (slow onset) is the most common form of nocardiosis in humans
- Nocardiosis may also become systemic and may present as acute, subacute or chronic
- Nocardiosis may present as endocarditis
- Nocardiosis may present as encephalitis and may be linked to some brain abscesses
- Nocardiosis may present as lung effusions, infiltrates, lung abscess, and/or cavitations
- BCYE agar may be used to help isolate the Nocardia from mixed cultures
- Nocardia quad plates are available to provide nutrients that help Nocaria spp grow
- The modified acid-fast stain (modified Kinyoun) uses weak sulfuric acid followed by ethanol as decolorizers
- The weak sulfuric acid (1-5%) as opposed to the HCl acid in the regular Kinyoun stain enables the Nocardia spp to retain the carbolfuschin stain and will aid in differentiation between Nocardia spp and Streptomyces spp since Nocardia will stain positive (red) and Streptomyces will not
Nocardia species can cause significant infections and should be reported to the patient's physician and sent out for ID confirmation if not in the MALDI-TOF MS database and susceptibility testing.