Chapter 19- Mycobacteria Flashcards
1
Q
General characteristics of mycobacteria
A
Slender, nonmotile, non-sporeforming, obligate aerobes- they require oxygen to survive. Mycobacteria are acid fast and are referred to as acid-fast bacilli. They grow slowly in comparison to other aerobic bacteria
2
Q
Are mycobacteria gram positive or gram negative?
A
Mycobacteria resist Gram staining because of lipids (mycolic acid, where their name comes from) in their cell wall that prevent penetration of crystal violet and safranin. Mycobacteria therefore do not develop a pink or purple color with gram staining and appear clear under the microscope
3
Q
Ziehl-Neelsen stain
A
Acid fast staining technique that requires heating during the staining step
4
Q
Kinyoun’s stain
A
Acid fast staining technique that does not require heating during the staining step
5
Q
Fluorochrome stain
A
Acid fast staining technique that stains with auramine/rhodamine as the primary stain. This technique requires a fluorescent microscope
6
Q
How many species of mycobacteria are there?
A
There are over 170 species, 14 of which are pathogenic to humans
7
Q
Which compounds are included in the cell wall of mycobacteria? (7)
A
From cell membrane to external cell surface:
1. PIM-phosphatidyinositol mannosides
2. Peptidoglycan
3. Lipoarabinomannan
4. Arabinogalactan
5. Mycolic acid
6. Phenolic lipids
7. Surface proteins
8
Q
Cell wall glycolipids (3)
A
The cell wall of mycobacteria contains multiple glycolipids. Examples are mycolic acid, lipoarabinomannan and phosphatidyinositol mannosides (PIM)
9
Q
Peptidoglycan function
A
Peptidoglycan prevents osmotic lysis
10
Q
Function of the arabinogalactan layer
A
The arabinogalactan layer is linked to both the peptidoglycan and to the mycolic acid outer membrane and probably provides additional strength to the cell wall.
11
Q
Glycolipids function
A
The mycolic acids and other glycolipids also impede the entry of chemicals causing the organisms to grow slowly and be more resistant to chemical agents and lysosomal components of phagocytes than most bacteria
12
Q
Functions of cell wall surface proteins
A
The surface proteins in the acid-fast cell wall, depending on the strain and species, carry out a variety of activities, including functioning as enzymes and serving as adhesins, which enable the bacterium to adhere intimately to host cells and other surfaces in order to colonize and resist flushing
13
Q
Porins
A
In the mycobacteria cell wall, porins extend from the cell wall surface down through the mycolic acid layer. Porins are required to transport small hydrophilic molecules through the outer membrane of the acid-fast cell wall. There are far fewer porins in the acid-fast cell wall compared to the gram-negative cell wall and the pores are much longer. This is thought to contribute significantly to the lower permeability of acid-fast bacteria.
14
Q
How does the cell wall of the mycobacteria influence acid-fast staining processes?
A
Because of its unique cell wall, when it is stained by the acid-fast procedure, it will resist decolorization with acid-alcohol and stain red, the color of the initial stain, carbol fuchsin. With the exception of a very few other acid-fast bacteria such as Nocardia, all other bacteria will be decolorized and stain blue, the color of the methylene blue counterstain.
15
Q
Ziehl-Neelsen staining procedure (5)
A
- Add carbol fuchsin
- Add acid alcohol (HCl and ethanol)
- The slide is heating and washed with acid alcohol again
- Methylene blue or malachite green are used as a counterstain
- Mycobacteria (acid fast bacteria) stain red against a blue background. Non-acid fast samples stain blue or green
16
Q
Kinyoun staining method (5)
A
- Add carbol fuchsin
- Add acid alcohol (HCl and ethanol)
- No heating is done with this method, the sample is washed with acid alcohol again
- Methylene blue or malachite green are used as a counterstain
- Mycobacteria (acid fast bacteria) stain red against a blue background. Non-acid fast samples stain blue or green
17
Q
Fluorochrome staining method
A
- Add auramine/rhodamine
- Add acid alcohol (HCl and ethanol)
- Add K permanganate
- The slide is viewed under a fluorescent microscope
- Mycobacteria/AFB stain yellow-orange against a dark background
18
Q
Advantage/disadvantage of a carbol fuchsin primary stain
A
Advantage- uses a light microscope
Disadvantage- 300 fields at 1000X magnification (oil immersion)
19
Q
Advantage/disadvantage of an auramine/rhodamine primary stain
A
Advantage- 30 fields at 250X magnification
Disadvantage- requires fluorescent microscope
20
Q
Which specimens are commonly received for AFB culture? (2)
A
- Lower respiratory tract specimens (sputum, bronchial washings)
- Urine, tissue and body fluids
21
Q
What is used for decontamination of a mycobacteria sample?
A
Mycobacteria are slightly more resistant to acids and alkalis than contaminating bacteria making up normal flora. Therefore mild treatments such as 2% NaOH can be used for decontamination. NaOH increases the pH to a level that is antibacterial
22
Q
What is used for digestion of a mycobacteria sample?
A
N-acetyl-L-cysteine (NALC) is a mucolytic agent that liquefies mucus in respiratory specimens, releasing the mycobacteria
23
Q
Digestion and contamination mycobacteria procedure (4)
A
- NaOH and NALC are added to the sputum sample in a tube
- The sample goes through the vortex and stands for 15 minutes
- PO4 (phosphate) buffer is added to the tube
- The tube is then put through the centrifuge for 15 minutes to produce a smear and culture
24
Q
Safety protocols when dealing with mycobacteria
A
Tuberculosis can be spread through aerosols caused by centrifuge or vortexing-respirators and other PPE are necessary. The laboratory is also under negative pressure- contaminated air leaves the lab and air is filtered. BSL3 protocols are required for labs dealing with tuberculosis or bovis
25
Types of solid culture media used for mycobacteria (3)
1. Lowenstein-Jensen (LJ)
2. Lowenstein-Jensen-Gruft
3. Middlebrook medium
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Agar
A gel derived from algae, can be mixed with various nutrients and used to grow microorganisms in a Petri dish.
27
Lowenstein-Jensen (LJ) culture media
Agar based and contains egg components for growth and malachite green to inhibit growth of normal flora. As specimens are generally from the respiratory tract or from other body fluids, the specimen may be contaminated by normal flora
28
Lowenstein-Jensen-Gruft culture media
LJ culture media that contains penicillin and naladixic acid
29
Middlebrook medium
Agar based and contains 2% glycerol to support better growth of some Mycobacterium species. Supports the growth of the MAC. This medium generally exhibits growth several days earlier than egg based media. Antimicrobials can be added to make the media selective for mycobacteria
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LJ agar slant
Egg based Lowenstein Jensen Media with malachite green, used to grow mycobacteria. The media is slanted inside the tube. This is done for safety and prevents agar from drying out over the 6 week growth period
31
Mycobacterium growth indicator tube (MGIT)
Liquid culture media- contains Middlebrook 7H9 broth. The large amount of oxygen in the broth quenches the fluorescence of a fluorochrome dye. As mycobacteria grow, they consume the oxygen (they are aerobes), and the fluorochrome dye will fluoresce when exposed to UV light
32
VersaTREK Myco
Liquid media- the sponges in the bottles provide a growth support matrix and increase the surface area exposed to headspace oxygen. The technology is based on the detection of headspace pressure changes within a sealed bottle. Changes in either gas production or gas consumption due to microbial growth will signal as positive.
33
How are mycobacteria classified by growth rate?
Mycobacteria producing growth within 7 days are termed rapid growers (Runyon Group IV). Those requiring longer are termed slow growers.
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How are mycobacteria classified by pigmentation/photoreactivity?
Slow growing mycobacteria (growth takes longer than 7 days) are classified into 3 groups based on the production of pigments: photochromogens, scotochromogens, and nonchromogens
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Photochromogens
Produce nonpigmented colonies when grown in the dark and pigmented colonies after exposure to light (Runyon Group I). The sample is orange when exposed to light, and produces a slight yellow color/barely any pigment when not exposed to light
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Scotochromogens
Produce deep-yellow-to orange pigmented colonies when grown in either light or darkness (Runyon Group II)
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Nonchromogens
Are nonpigmented in both the light and dark (Runyon Group III). The colonies exhibit a cream color
38
Preferred growth temperature of mycobacteria
30-42 degrees Celsius
39
Catalase
All mycobacteria typically produce catalase, however, there are different forms of catalase that can be differentiated in the laboratory. The enzyme can be detected by adding H2O2 (hydrogen peroxide) to a culture and looking for formation of bubbles. Different types of catalase include semiquantitative catalase and heat-sensitive catalase
40
Semiquantitative catalase
Detected when hydrogen peroxide is added to a culture of mycobacteria and after 5 minutes the height of the column of bubbles is measured < 45 mm or > 45 mm
41
Heat-sensitive catalase
Detected when a suspension of the mycobacterium is heated at 68 degrees C for 20 minutes. Then H2O2 is added and suspension is observed for bubbles
42
Niacin accumulation test
Niacin is a precursor in the synthesis of nicotinamide adenine dinucleotide (NAD). Although all mycobacteria produce niacin some species produce an excess amount that is excreted from the cell. Niacin accumulates in the medium and is detected by reacting with cyanogen halide. This test is used to identify different species of mycobacteria.The solution forms a yellow color as a positive result.
43
Niacin reduction test
In the test for nitrate reductase, NaNO3 (sodium nitrate) is added to a heavy suspension of mycobacteria and incubated. Then nitrate reagents (HCl, sulfanilamide, and N-naphylenediamine dihydrochloride) are added. Formation of a pink color is positive reaction, the suspension will remain colorless if negative. The test is used to identify different species of mycobacteria
44
Growth on MacConkey agar test
MacConkey agar without crystal violet is inoculated with test organism. Look for growth after 5 days. This agar will support the growth of rapidly growing mycobacteria only, therefore identifying whether rapidly growing mycobacteria species are present in the sample
45
Susceptibility to T2H test
Thiophene-2-carboxylic acid hydrazode (T2H) is added to 7H11 agar and used to look for growth and no growth. If the bacteria is resistant to T2H, it will grow in the tube. If it is susceptible to T2H, it will not grow. T2H selectively inhibits of growth of M. bovis. Other bacteria, like M. tuberculosis, can grow in a medium containing this compound. The test can be used to certain strains of M. bovis
46
Molecular tests for identification of mycobacteria (4)
1. Accuprobe (hybridization) assay – test growth from culture media
2. MALDI-TOF (mass spectrometry)– test growth from culture media
3. Nucleic acid amplification (NAAT) – test growth from culture media or directly from specimen
4. Gene sequencing – test growth from culture media or directly from specimen
47
Accuprobe (hybridization) assay
1. Ribosomal RNA target sequences are present in multiple copies per cell
2. A single stranded DNA probe is used, labeled with acridinium ester
3. Hybridization is allowed for 15 minutes at 60 degrees
4. A selection reagent is added
5. If the acridinium ester splits off, there is no chemiluminescent reaction. If the acridinium ester is protected by DNA-RNA hybrids, there is a positive chemiluminescent reaction
48
What is hybridization?
The process in which two complementary single-stranded DNA and/or RNA molecules bond together to form a double-stranded molecule. The bonding is dependent on the appropriate base-pairing across the two single-stranded molecules. To identify mycobacteria, target ribosomal RNA and a single stranded DNA probe bind
49
Chemiluminescent reaction
The emission of light that occurs as a result of certain chemical reactions that produce high amounts of energy lost in the form of photons when electronically excited product molecules relax to their stable ground state. Because CL does not involve initial absorption of light, measurements of CL emission are made against a lower background, thus allowing greater sensitivities of detection.
50
MALDI-TOF identification of mycobacteria
MALDI-TOF is a form of mass spectrometry that provides rapid identification and can differentiate between species for rapid growers and other mycobacteria groups and complexes.
51
How does MALDI-TOF work?
Charged particles are accelerated by a laser. Time of flight through the mass analyzer is proportional to the ion’s mass- a bigger ion will have a longer time of flight. Therefore, proteins and peptides are separated by increasing mass. A protein mass spectrum is produced of the whole cell, and 20-30 proteins directly match with the theoretical masses of ribosomal proteins in the sequence database
52
3 stages of PCR
1. Denaturation
2. Annealing
3. Elongation
53
How does PCR work?
During the denaturation phase (96 degrees Celsius), the DNA is heated so the two strands separate and one strand acts as a template for the next step. During the annealing phase (68 degrees), the reaction is cooled so that the primers can bind to the complementary regions of the single stranded DNA template. PCR uses short segments of DNA (primers) to select part of the genome to be amplified. During elongation (72 degrees), the reaction temperature is raised so an enzyme (Taq polymerase) can extend the primers and synthesize new strands of DNA. The reaction is repeated 25-35 times, exponentially increasing the number of DNA molecules. The DNA can be visualized by gel electrophoresis. PCR can detect different mycobacteria species
54
Slowly growing mycobacteria species (8)
1. Mycobacterium tuberculosis complex (Mtb complex)
2. Mycobacterium avium-intracellulare complex (MAC or MAI)
3. Mycobacterium haemophilum
4. Mycobacterium kansasii
5. Mycobacterium marinum
6. Mycobacterium gordonae
7. Mycobacterium scrofulaceum
8. Mycobacterium ulcerans
55
Species of the M. tuberculosis complex (7)
1. Mycobacterium tuberculosis- human tuberculosis
2. Mycobacterium bovis- tuberculosis in warm blooded animals
3. Mycobacterium bovis BCG
4. Mycobacterium africanum- human tuberculosis in Africa
5. Mycobacterium microti- tuberculosis in warm blooded animals
6. Mycobacterium canettii-human tuberculosis in Africa
7. Mycobacterium pinnipedii- tuberculosis in warm blooded mammals
56
Transmission of M. tuberculosis
Tuberculosis is a chronic lower respiratory tract disease that is spread by person to person contact, through infectious droplets. Only a few bacteria are necessary to cause disease. There are 2 stages- primary and secondary (active) tuberculosis- people are contagious during the active stage
57
Primary tuberculosis
Infection begins in lungs, when the bacteria is inhaled, and from there can spread to the lymphatic system and central nervous system. Macrophages phagocytize bacteria and form granulomatous lesions called tubercles. If the lesions calcify, they are called "gohn complexes". While bacteria are contained in granulomas, the patient is typically asymptomatic (latent infection). Primary TB may not lead to active TB in people with healthy immune systems.
58
Tuberculosis granuloma
Macrophages are cells of the immune system that phagocytose pathogens and cellular debris. With tuberculosis, they phagocytize bacteria and form granulomatous lesions called tubercles. The patient is asymptomatic and is not infectious when the bacteria is contained in granulomas. On a chest X-Ray, granulomas present as "vacant" darker areas in the lungs
59
Secondary tuberculosis
Also called reactivation- occurs in people who have had latent TB. Reactivation may occur because of alteration in the cell mediated immune response. It can be triggered by poor nutrition, alcoholism, or hormonal factors associated with pregnancy and diabetes.
60
Active tuberculosis symptoms
Symptoms include fever, fatigue, chronic cough, and production of sputum which may contain blood. Once people are symptomatic, they are contagious. Active tuberculosis requires long term antibiotic therapy using multiple antibiotics in combination. Treatment can last for up to 2 years
61
Disseminated (miliary) tuberculosis
With M. tuberculosis, extrapulmonary disease can also occur, impacting multiple organ systems. Complications include- cervical lymphadenitis, pleuritis, pericarditis, synovitis, meningitis, and infections of skin, joints, bones, and internal organs
62
Airborne precautions in the hospital for tuberculosis patients
Patients are placed in an airborne infection isolation room, which is a negative pressure room. Patients must wear a surgical mask when being transported outside their room. Healthcare professionals wear an N95 mask and must practice hand hygiene
63
Diagnosis techniques for latent tuberculosis (2)
1. Tuberculin skin test
2. Interferon Gamma Release Assays
64
Tuberculin skin test
Purified Protein Derivative (PPD)- injected intradermally and the zone of induration is examined 48 hours later for redness and swelling. The cellular immunity recognizes the person has been exposed and initiates an immune response. Positive indicates previous exposure but not necessarily active disease- a person will test positive on a PPD if they have received a tuberculosis vaccine.
65
Interferon Gamma Release Assays (IGRA)
Blood test that measures a person’s immune reactivity to M. tuberculosis. White blood cells from most persons that have been infected with M. tuberculosis will release interferon-gamma (IFN-g) when mixed with antigens derived from M. tuberculosis. Used to diagnose latent tuberculosis. This test is more specific than PPD, and less time consuming.
66
How is active tuberculosis diagnosed?
A culture and smear of the sputum specimen is obtained. An acid fast stain smear is done in order to obtain a rapid preliminary diagnosis. Direct molecular detection methods can be used if available. The growth of mycobacteria is then examined after 14-21 days of incubation at 37 degrees Celsius, and antimicrobial susceptibility testing can be done to determine the most effective treatment for the patient
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What is the next step if someone tests positive on PPD or IGRA?
X-Ray is done if someone tests positive on a PPD or IGRA, they may need a short course of antibiotics
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Identification of M. tuberculosis (4)
1. Staining- on an acid fast smear, the mycobacteria appears red with a blue background.
2. Solid media- forms nonpigmented (nonchromagen), dry, rough, colonies on LJ in 14-21 days.
3. Liquid media- there are ropelike formations (cording) from broth cultures.
4. Biochemical tests- the bacteria is niacin and nitrate positive, growth on T2H positive, and bubbles on the semiquantitative catalase test measure less than 45 mm
69
First line drugs for M. tuberculosis treatment (4)
1. Isoniazid
2. Rifampin
3. Ethambutol
4. Pyrazinamide
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Isoniazid mechanism
Inhibits synthesis of mycolic acid
71
Rifampin mechanism
Inhibits RNA synthesis by inhibiting RNA polymerase- can't make RNA from the DNA template. Used to treat M. tuberculosis and MAC
72
Ethambutol mechanism
Inhibits cell wall synthesis. Used to treat M. tuberculosis and MAC
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Pyrazinamide mechanism
Inhibits multiple targets such as energy production and translation of mRNA
74
M. tuberculosis antimicrobial susceptibility testing techniques (3)
1. Agar proportion method
2. Broth method
3. Molecular tests for resistance genes
75
Agar proportion method of susceptibility testing
Compares growth on solid agar media with and without one of the 4 primary antimicrobial drugs (on discs)
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Broth based method of antimicrobial susceptibility testing
Uses BACTEC or MGIT. Liquid broth is inoculated with each test drug. The growth of the bacteria in each vial indicates the bacteria's resistance to that drug
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Molecular tests for resistance genes susceptibility testing
This is the fastest method- whole genome sequencing can be used to check for genes that make the bacteria susceptible to certain drugs. Results can be obtained relatively quickly, within 5 days
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BACTEC broth method
A blood culture bottle that contains a lytic agent to release the mycobacteria that have been phagocytosed by white blood cells. The bottle is incubated and monitored. This method is used to culture bacteria and fungi that might be present in the bloodstream
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Multidrug Resistant M. tuberculosis (MDR-TB)
M. tuberculosis bacteria that exhibits simultaneous resistance to isoniazid and rifampin
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Extremely drug resistant M. tuberculosis (XDR-TB)
M. tuberculosis bacteria that exhibits resistance to isoniazid and rifampin, plus resistance to any fluoroquinolone, and at least one of the three second-line drugs (capreomycin, kanamycin, and amikacin)
81
Second line tuberculosis antimicrobials (3)
Capreomycin, kanamycin, and amikacin
82
Antimicrobial treatment of tuberculosis
Multiple drugs (at least 2) are always required, as tuberculosis becomes drug resistant quickly. Drug resistant strains of tuberculosis have resulted from tuberculosis patients only being prescribed one antimicrobial, which the bacteria developed resistance to. Antimicrobial therapy can last up to 2 years, and patients are often monitored by the state DOH
83
Mycobacterium bovis
Causes tuberculosis in warm blooded animals such as cattle, dogs, cats, pigs, parrots, badgers and some birds of prey. Human disease is a zoonosis (acquired from animals instead of humans) and is similar in presentation to pulmonary disease caused by M. tuberculosis. It is commonly acquired by drinking unpasteurized milk from an infected cow
84
Identification of M. bovis (4)
1. Nonpigmented (nonchromogen) rough colonies at 37 degrees C
2. Niacin and nitrate reductase negative
3. Susceptible to T2H
4. Semiquantitative catalase <45mm, 68oC negative
85
Mycobacterium bovis Bacillus Calmettte Guerin (BCG)
In many parts of the world, BCG is an attenuated strain used for vaccine purposes (it's very effective in children for preventing meningitis and disseminated disease). The vaccine is most efficacious in preventing tuberculosis meningitis in children, rather than preventing pulmonary tuberculosis. BCG is also used in immuno stimulation therapy against bladder cancer. The organism is instilled into the bladder and stimulates the immune system
86
Nontuberculous Mycobacterium (NTM)
This is an environmental mycobacteria. Its significance is determined by host factors, pathogenic potential and number of positive cultures. Diagnosed using a series of 3 sputum cultures in order to make sure the tuberculosis bacteria was not a contaminant. Usually best to collect in the morning because the sputum has accumulated in the lungs
87
Mycobacterium avium complex (MAC) species (4)
1. Mycobacterium avium- first found in poultry
2. Mycobacterium paratuberculosis
3. Mycobacterium intracellulare
4. Mycobacterium chimaera
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Mycobacterium avium complex (MAC)
This is the most significant species for causing disease, and is the most common environmental mycobacteria causing disease in humans. In general, it is isolated from water, soil, plants and other environmental sources. Important pathogens of poultry and swine. These animals excrete organisms in feces that remain viable in soil. No animal to human spread, and no human to human spread. Humans can be colonized with no apparent infection.
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Microorganism colonization of humans
When humans are colonized, the microorganism is present, but it does not cause infection
90
Effects of MAC in humans
In humans, MAC can cause pulmonary disease. Clinical presentation is similar to tuberculosis, but there is no person to person transmission. In AIDS patients, infection may become a disseminated disease, and the disease is more severe. Therefore, severe infection is often considered an “AIDS defining illness”. MAC has been isolated in up to 20% of cystic fibrosis patients, but its contribution to disease status is not established. CF patients have thick mucus that accumulates in their lungs, and they may just be colonized by this organism. MAC is also the leading cause of mycobacterial cervical lymphadenitis in children.
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Cervical lymphadenitis
Lymph node in the cervical area is infected with the microorganism- commonly MAC. The lymph node appears red and very swollen
92
Mycobacterium paratuberculosis
Causes Johne’s disease in cattle- this is a wasting disease, the intestinal tract of the animal can’t absorb nutrients. M. paratuberculosis may also contribute to Crohn's disease in humans
93
Mycobacterium chimaera
May be contracted from contaminated OR equipment. Patients who receive cardiothoracic surgery are put on a heart-lung machine that handles heating/cooling and oxygenating the blood before it is put into the patient. Patients who receive this type of surgery may develop systemic infection- the fan in the machine is difficult to decontaminate, it blows air contaminated with the bacteria into the OR and it lands in the surgical site. Hospitals now wall off the equipment to prevent the bacteria from being released into the OR
94
Identifying characteristics of MAC (5)
1. Nonpigmented (nonchromogenic) smooth to rough colonies at 37 degrees C
2. Niacin and nitrate reductase negative
3. Resistant to T2H (growth)
4. Semiquantitative catalase < 45 mm, 68oC catalase +/-
5. Molecular methods available for direct and culture identification
95
Decontamination of respiratory specimens from CF patients
Respiratory specimens from cystic fibrosis patients require an additional decontamination step to reduce levels of Pseudomonas- use 5% oxalic acid. Oxalic acid is added after centrifuging of the sample
96
Antimicrobial therapy of MAC
This is slightly different from tuberculosis treatment
1. Clarithromycin (primary)
2. Rifampin
3. Ethambutol
4. Rifabutin (if disseminated)
97
Clarithromycin mechanism
This is the primary drug of choice for MAC infection, and it may even be able to be used by itself. Clarithromycin is a protein synthesis inhibitor
98
Rifabutin
Used to treat disseminated MAC infection. It inhibits RNA synthesis
99
Mycobacterium kansasii
Isolated from tap water around the world, and was recognized in the beginning of the AIDS epidemic. Causes pulmonary infection resembling tuberculosis but no person to person spread. Rarely disseminates to other body sites except in patients with severe immunosuppression (e.g. AIDS)
100
Identifying characteristics of Mycobacterium kansasii (4)
1. Photochromogen with variable colony morphology at 37 degrees C- if grown in the light, it forms an orange colored colony
2. Niacin negative, nitrate reductase positive
3. Resistant to T2H (growth)
4. Semiquantitative catalase >45 mm and 68 degrees C catalase positive
100
Mycobacterium marinum
Found in fresh and saltwater environments (swimming pools, tropical fish aquariums, water cooling towers). Generally not an issue in well maintained swimming pools, unless chlorine levels are low. Typical infections involve the skin, usually resulting when traumatized skin comes in contact with water environments. Don’t usually cause pulmonary or disseminated infection
101
Mycobacterium marinum symptoms
Usually presents as a single nodular lesion confined to one extremity, usually elbow, knee, foot, toe or finger. Sometimes called “fish tank granuloma” or “swimming pool granuloma”.
102
Mycobacterium marinum treatment
6 weeks of doxycycline
103
M. marinum identifying characteristics (5)
1. Prefers growth at 30oC
2. Photochromogen with variable colony morphology
3. Niacin and nitrate reductase negative
4. Resistant to T2H (growth)
5. Semiquantitative catalase >45 mm, 68oC catalase negative
104
Mycobacterium gordonae
Found in freshwater, including tap water- this is one reason why drinking equipment must be sterilized or cultured regularly. It's rarely pathogenic; most commonly isolated as a contaminant in clinical laboratories (all water used for reagents must be sterile). Colonization of drinking water equipment and improperly sterilized laboratory reagents can lead to pseudo-outbreaks- if pulmonary patients consume contaminated water, their sputum may be contaminated by the bacteria, and it may seem like everyone is infected
105
Identifying characteristics of M. gordonae (4)
1. Scotochromogen producing smooth colonies at 37oC
2. Niacin and nitrate reductase negative
3. Resistant to T2H (growth)
4. Semiquantitative catalase >45mm, 68oC positive
106
Mycobacterium scrofulaceum
Found in environmental water sources, causes cervical lymphadenitis (scrofula) in children who put contaminated items in their mouth.
107
Identifying characteristics of M. scrofulaceum (4)
1. Scotochromogen producing smooth colonies at 37 degrees C- form orange colonies in the light or in the dark
2. Niacin and nitrate reductase negative
3. Resistant to T2H (growth)
4. Semiquantitative catalase >45 mm, 68oC positive
108
How can M. scrofulaceum be differentiated from M. gordonae?
By the urease hydrolysis test,
M. gordonae = negative while M. scrofulaceum = positive
109
Urease hydrolysis test
Urea is the product of decarboxylation of amino acids. Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive organisms turn the entire medium pink within 24 hours.
110
Mycobacterium xenopi
Found in hot water systems. It primarily causes pulmonary disease. Nosocomial (hospital acquired) and pseudo-infection from water storage tanks in hospitals- patients may become colonized with it
111
M. xenopi identifying characteristics (5)
1. Optimum growth temperature is 42oC
2. Scotochromogen producing smooth colonies
3. Niacin and nitrate reductase negative
4. Resistant to T2H (growth)
5. Semiquantitative catalase <45mm, 68oC catalase +/-
112
Mycobacterium haemophilum
Predominantly found in immunocompromised patients - classical clinical presentation- multiple painful subcutaneous nodules commonly involving extremities (the cooler areas of the body). Therefore, M. haemophilum prefers a low incubation temperature for growth- 30 degrees C. Also seen in immunocompetent children, causing lymphadenitis. Recently linked to infections after permanent eyebrow and tattoo procedures
113
How is M. haemophilum cultured?
Doesn’t grow on regular mycobacteria media, as ferric ammonium citrate or hemin is a growth requirement. Use chocolate agar slant if suspected- chocolate agar is required as it contains hemin
114
Identifying characteristics of M. haemophilum (4)
1. Nonpigmented (nonchromogenic) rough colonies
2. Niacin and Nitrate reductase negative
3. Resistant to T2H (growth)
4. Semiquantitative catalase <45mm, 68oC catalase negative
115
Mycobacterium ulcerans
Found in stagnant water- mainly in Central and West Africa, Malaysia, New Guinea, Guyana, Mexico and Australia, where people are exposed to rice fields or swampy environments. Causes painless lump under the skin, typically at the site of previous trauma. Later develops into a shallow non-healing ulcer. In Africa, it is called a “Buruli ulcer” and in Australia, it's called a “Bairnsdale ulcer”. Infection is generally preceded by a trauma that allows the bacteria to enter the body
116
Identifying characteristics of M. ulcerans (5)
1. Prefers low incubation temperature of 30 degrees C
2. Nonpigmented (nonchromogenic) rough colonies
3. Niacin and nitrate reductase negative
4. Resistant to T2H (growth)
5. Semiquantitative catalase <45mm, 68oC positive
117
Rapidly growing Mycobacteria
These mycobacteria take less than 7 days to grow. Their optimum growth temperature is 30 – 32oC, but they will grow at 37oC and will often grow on routine bacteriological media. 3 species are considered rapidly growing
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Which 3 mycobacteria species are considered rapidly growing?
1. Mycobacterium fortuitum
2. Mycobacterium chelonae
3. Mycobacterium abscessus
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Mycobacterium fortuitum
Environmental bacteria that can be found in water and dirt. Can occur with pedicures or in situations where other things are being put into the water. It can cause many complications- post traumatic wound infections, osteomyelitis (inflammation of the bone), joint infections and infections of the eye after trauma. Pulmonary infections of M. fortuitum are uncommon. Usually causes skin, soft tissue, and bone infections
120
Identifying characteristics of M. fortuitum (4)
1. Nonpigmented (nonchromogen)
2. Niacin negative, nitrate reductase positive
3. Iron uptake positive
4. Growth on MacConkey agar (without crystal violet)
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Iron uptake test
Uses LJ agar. Some mycobacteria have the ability to take up soluble iron salts from the culture medium and to produce a rusty brown appearance on addition of an aqueous solution of 20% ferric ammonium citrate. Production of a rusty brown color is a positive result
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Mycobacterium abscessus
An environmental bacteria that can also be found in water. It causes pulmonary infections in cystic fibrosis patients (it is not a colonizing bacteria). It may also cause chronic otitis media associated with ear tube placement
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Identifying characteristics of M. abscessus (4)
1. Nonpigmented (nonchromogen)
2. Niacin and nitrate reductase negative
3. Iron uptake negative
4. Growth on MacConkey agar (without crystal violet)
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Mycobacterium chelonae
Environmental bacteria that can also be found in water. It can cause disseminated cutaneous disease in immunosuppressed patients, and outbreaks have occurred after acupuncture therapy
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Identifying characteristics of M. chelonae
1. Nonpigmented (nonchromogen)
2. Niacin variable, nitrate reductase negative
3. Iron uptake negative
4. No growth on MacConkey agar (without crystal violet)
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Mycobacterium leprae
Causes leprosy (Hansen's disease). In the USA, about 150 people become infected each year (250,000 people worldwide). Majority of cases in south and southeast Asia, Africa and Latin America. Endemic in small pockets in the United States (Texas, California, Louisiana, Hawaii and Puerto Rico). This bacteria is not transmitted by touching people with leprosy, it is shed through the nose. Armadillos are also a reservoir of infection
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How is leprosy diagnosed?
Differs from all other mycobacteria in that it cannot be cultured using agar or liquid based culture media. Diagnosis is clinical with observation of acid fast bacteria from skin biopsy samples
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Leprosy (Hansen’s disease)
Anesthetic skin lesions and peripheral neuropathy with nerve thickening. Medical complications arise from nerve damage and immune reactions to disease. Patients may no longer have feeling in their hands and other parts of their body. They can be injured as they are unable to sense when things are hot
