MEDT 14 | CLIN. BACTERIOLOGY (LEC) LESSON 10 PART II.

FAMILY
Jonnel P. Andaya || 3rd Year
Transcribed by: Last Name, FN | Last Name, FN MYCOBACTERIACEAE

OUTLINE:
I. INTRODUCTION
II. GENERAL CHARACTERISTICS OF
MYCOBACTERIUM
III. MYCOBACTERIUM TUBERCULOSIS COMPLEX
A. RELATED INFECTION AND DISEASES ● AFB positive: contain N-glycolymuramic acid which is a
B. PREVENTION very high lipid content that resist decolorization with acid
IV. MULTIDRUG-RESISTANT MYCOBACTERIUM ethanol
TUBERCULOSIS (MDR-TB) ● Strictly aerobic and catalase positive, and produce Much’s
V. NON - TUBERCULOSIS MYCOBACTERIA granules (inclusion body)
A. NTM - SLOW GROWERS ● Most species are associated with disease that develop
B. NTM - RAPID GROWERS slowly (require two to six weeks of incubation) while the
VI. GROUPS OF MYCOBACTERIA rapidly growing mycobacteria species multiply in two to
three days)
VII. LABORATORY DIAGNOSIS
VIII. DECONTAMINATION AND DIGESTION ● Some species require 5% to 10% CO2
A. REAGENTS ● Microscopy: Slight curve or straight rods that have
IX. BIOCHEMICAL TESTS tendency to “clump”
X. STAINING ● Culture: Egg-based-agar- colonies are smooth and soft or
A. AFB STAINING METHODS have a rough and friable growth
B. CLASSIFICATION ● pH requirement: 6.5 to 6.8
XI. INTERPRETATION OF MICROSCOPY ● Generation time: >12 hrs
XII. CULTURE MEDIA ● 2 groups of mycobacteria, namely M. tuberculosis complex
A. SOLID MEADI (MTC) and non-tuberculous Mycobacteria.
B. LIQUID MEDIA
XIII. VIRULENCE TEST MYCOBACTERIUM TUBERCULOSIS COMPLEX
XIV. NUCLEIC ACID TEST ● M. Tuberculosis complex - Etiologic Agent of Human
XV. NON-CULTIVABLE NTM Tuberculosis
XVI. COMPARISON 1. Mycobacterium tuberculosis
2. Mycobacterium bovis
XVII. LABORATORY DIAGNOSIS
3. Mycobacterium bovis Bacille Calmette-Guerin strain
4. Mycobacterium caprae
5. Mycobacterium pinnipedii
6. Mycobacterium africanum
INTRODUCTION 7. Mycobacterium microtii
8. Mycobacterium canetii
● Worldwide, TB is the second largest contributor among
infectious disease to adult mortality. The world health Mycobacterium Tuberculosis (Koch’s Bacillus)
organization estimates that 2007, about 10 million cases of
TB and nearly 2 million deaths from TB were reported. ● It has the longest replication time among the Mycobacteria
That year, India ranked first in the number of incident species.
cases followed by china, Indonesia, Nigeria, and south ● Virulence factor: Cord factor
Africa. Among new cases of TB, approximately 5% (or ● Culture’s exhibit buff color; are raised and dry have a
500,000) worldwide are due to multi-drug resistant “Couliflower-like” appearance
organisms (MDR-TB), define as tubercle bacilli that are
resistant to rifampin and isoniazid. MDR-TB does not ● Rough colonies exhibit “cording” (curved strains of bacilli)
respond to the require up to 6 months’ treatment with drug ● Biochemical Test:
and that more toxics than the first line agents about 100x o (+) Niacin - yellow
times more costly. Mismanagement of drug use to treat o (+) Nitrate reduction - red
MDR –TB result “Extensively-drug resistant tuberculosis” o (-) T2H inhibition test
(XDR-TB), define as MDR-TB resistance plus resistance to o (-) 68°C catalase test - no bubbles
fluoroquinolones and any second line injectable anti o (+) Pyrazinamidase - red ring on top of the
tuberculous agent (e.g. Amikacin, Kanamycin, medium
Capreomycin) ● Causes Pulmonary Tuberculosis
o Pathogenesis: Infection can be acquired through
GENERAL CHARACTERISTICS OF MYCOBACTERIUM inhalation of contaminated aerosol or droplets
● Non-motile, non-spore forming, and non-encapsulated with pulmonary tuberculosis. Once bacteria are
inhaled, they will enter the respiratory tract and
● Gram-positive, but is described as gram-ghost under the about 5-10% will reach the alveoli. Inside the
microscope for they cannot absorb both Crystal violet and alveoli, bacteria encounter the macrophage and
Safranin shall be engulfed by the latter. Since the bacteria
are composed of cord factors, instead of
degradation, they will live inside the macrophage
because they inhibit the formation phagosome-
lysosome fusion. The macrophage will activate
the T-helper cells (1 and 2) and shall form small
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nodular lesions called tubercle in the lungs among
patients with pulmonary tuberculosis.
Mycobacterium bovis
● It produces tuberculosis in human and animals.
● Its attenuated strain is used for the vaccination (Bacillus-
Calmette-Guerin or BCG vaccine) of newborns against
tuberculosis
● Ingestion of contamination milk from infected cows or
exposure to infected animals and their carcasses
● Biochemical test: M. bovis - BCG strain
o (-) Niacin - no change in color
o (-) Nitrate reduction - light yellow color
o (+) T2H inhibition test
o (-) 68°C catalase test
o (-) Pyrazinamidase
● Causes: Extrapulmonary Tuberculosis specificially GI TB
(Gastrointestinal Tract Tuberculosis)
o Pathogenesis: Ingestion of contaminated milk
from cows which act as reservoirs of the
microorganism. Bleeding of the GI tract,
abdominal pain, weight loss, anemia (clinical
manifestations of GI - TB caused by M. bovis)
Mycobacterium africanum
● Associated with human cases of tuberculosis in tropical
Africa
● The detection of this organisms requires the use of
spoligotyping (Spacer oligotyping)
Mycobacterium canetii
● Smooth strain of M. tuberculosis
● Grows more than rapidly than M. tuberculosis (six day on
solid media)
● Isolated from an AIDS patient with mesenteric tuberculosis
Mycobacterium microtii
● It has been isolated from TB patient both
immunocompetent and immunocompromised individuals
RELATED INFECTIONS AND DISEASES
● Tuberculosis
o disease of the respiratory tract
o chronic granulomatous infection which is
transmitted through the inhalation of infected
droplets by means of coughing, sneezing or
speaking.
o Mode of acquisition: Inhalation of 1 to 5 um
airborne droplet of nuclei
o Signs and symptoms: Low-grade fever, night
sweats, fatigue, anorexia, and weight loss
● Pott’s Disease
o known as the tuberculosis spondylitis or skeletal
tuberculosis of the spine
o a serious form of the tuberculosis that is caused
by the invasion of M. tuberculosis into the spine
vertebrae
● Miliary Tuberculosis
o extrapulmonary tuberculosis that is caused by M.
tuberculosis that affects many organs through
hematogenous spread
o Common site of dissemination: spleen, liver, lungs,
bone marrow, kidneys, adrenal gland and eyes.
NOTES:
● M. tuberculosis cells are phagocytized by alveolar
macrophage and are able to replicate intracellularly
● Tubercle bacilli are not completely eradicated after the
recovery from tuberculosis. But can remain viable or
dormant for months or years, which signifies a possibility of
reactivations.
MYCOBACTERIUM TUBERCULOSIS COMPLEX
PREVENTION
● Creation of Safe Environment
o Providing autopsy suits to those practicing
cadavers
o Providing clinic waiting areas
o Providing negative pressure room for handling
potential infectious specimen
● Following Universal Precaution
o Avoiding contact with patient’s body fluids by
wearing PPE
● Attenuated BCG vaccination for preventing the M. bovis
Bacille-Calmette Guerin strain infection
o Vaccine is not recommended in US for risk of
infection is low and effectivity is variable among
adults
Treatment for Mycobacterium Tuberculosis
● Isoniazid
● Rifampin
● Ethambutol
● Pyrazinamide
MULTIDRUG-RESISTANT MYCOBACTERIUM
TUBERCULOSIS (MDR-TB)
● This strains develop when patient fails to take all the
prescribed drugs for tuberculosis treatment
● A result of the developed drug resistance of the M.
tuberculosis complex that is caused by either the infection
of originally drug-resistant tuberculosis strain or the lack of
compliance from the patient for the proper drug intake.
2 TYPES OF MDR-TB
● Primary MDR-TB
o without previous history of tuberculosis disease
and resistance to at least the two anti-TB drugs,
isoniazid and rifampin
● Extensively drug resistant TB (XDR-TB)
o resistance to isoniazid, rifampin, fluoroquinolones,
and at least one of the three parenteral second-
line anti-TB drugs (Aminoglycosides, Amikacin,
Kanamycin, or Capreomycin)
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NON-TUBERCULOUS MYCOBACTERIA
● These organisms are known as the typical mycobacteria or
mycobacteria other than tuberculosis (MOTT)
● These are found in the environment and they sometimes
colonize the skin, respiratory tract, and GI tracts of healthy
individuals
● Chronic pulmonary disease resembling tuberculosis is the
usual clinical presentation associated with these organisms
● Infections that are caused by these organisms are not
considered as transmissible from person to person.
NTM-SLOW GROWERS
Mycobacterium avium complex (MAC)
● most common cause of pulmonary infection
● includes M. avium; M. intracellulare
● pathogen in AIDS patient
● GI tract is the most common site of colonization and
dissemination in AIDS patient
● can be isolated from the sputum, blood and bone marrow
aspirates
● Habitat: Soil, Water, Contaminated Food
● Reservoir: Natural water
● Portal of entry: Respiratory and GIT (most common)
● Microscopy: Pleomorphic, short
● Biochemical test:
o (+) Heat-stable catalase
o (-) T2H inhibition Test
o Pleomorphic, short cocobacilli w/out beading with
periodic acid-Schiff staining
● Species: M. avium, M. intracellular, M. avium subsp
paratuberculosis, and M. avium subsp. Silvaticum (wood
pigeon bacillus)
● M. avium is the cause of disease in poultry and swine
● Diseases:
o Pulmonary Disease
o Cervical Lymphadenitis
o Note: Disseminated MAC (Systemic bacterial
infection) occurs almost exclusively in
immunocompromised individuals e.g. AIDS
patients
Mycobacterium kansasii (Yellow Bacillus)
● second to M. avium complex as the cause of NTM lung
disease (Chronic cavitary pulmonary lesions) in humans.
● not normally contagious from person to person
● Microscopy: long rods with distinct crossbanding
● Culture (Macroscopically)
o MB7H0: colonies are smooth to rough with dark
centers and waxy edges
o Photochromogenic colonies exhibit dark red
crystals/centers of 10-beta carotene
● Diseases:
o Chronic cavitary pulmonary lesions: usually
involves the upper lobes of the lungs
o Extrapulmonary manifestation: Cervical
lymphadenitis in children, cutaneous disease,
mucoskeletal involvement and genitourinary tract
disease
● Biochemical Test:
o (+) Tween 80 hydrolysis
o (+) Nitrate reduction test
o (-) Pyrazinamidase Test
Mycobacterium marinum
● causes diseases on fishes and can be isolated from
aquariums
● Causative agent of “Swimming pool granuloma” (fish tank
granuloma) which is a red or bluish red nodule on elbow,
knee, toe, or finger, that occurs when an open wound
comes in contact with contaminated chlorinated fresh water
or salt water - Mode of Transmission
● Microscopy: Long rods with cross-banding
● Culture: colonies are smooth to rough, wrinkled, and yellow
(Photochromogenic)
● Biochemical Test
o (+) Urease Test - pink color
o (+) Pyrazinamidase Test
Mycobacterium ulcerans
● third most common mycobacterium spp. after M.
tuberculosis and M. leprae
● Mode of Transmission: remains unknown
● Rare cause of Buruli ulcer (Uganda) or Bairnsdale ulcer
(Australia) which is a painless nodule/boils/subcutaneous
lumps under the skin after a previous trauma.
● Microscopy: Moderately long rods without cross-banding
● Culture: Colonies are smooth, rough and non-pigmented (6
to 12 weeks of incubation)
● Biochemical test: (+) Heat-stable catalase
Mycobacterium gordonae (Tap Water Bacillus)
● contaminates the tap water that is used by patients in
rinsing their mouths prior to the procedure for sputum
collection.
● Contaminant in the preparation of bacteriologic smears
(AFB)
● Rarely cause infection in humans
● Culture: colonies are smooth and yellowish-orange colored
● Biochemical test:
o (+) Tween 80-hydrolysis and heat-stable catalase
o (-) nitrate reduction
Mycobacterium xenopi
● Recovered from hot and cold water taps, especially from
water storage tanks of hospitals.
● Natural Reservoir: birds
● Recognized as pathogen to humans in 1965
● potential pathogen of pulmonary infection in adults
(reported in Canada) but is a rare mycobacterial infection
in the US
● can be either non-photochromogenic or scotochromogenic
● Microscopy: Long and filamentous rods
● Culture
o MB7H10: colonies are small with filamentous
edges
o Cornmeal glycerol agar: colonies are round with
branching filament
o Growth temp: 42°C
Mycobacterium terrae complex
● normally saphrophytic and rarely causes human infections
● Species: M. terrae, M. triviale, and M. nonchromogenicum
o Culture
⮚ M. triviale - colonies are rough and dry
⮚ M. terrae - colonies are smooth
⮚ M. nonchromogenicum - colonies are
smooth to rough and white to buff
● Biochemical Test:
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o (+) Tween Hydrolysis and heat stable catalase Mycobacterium abscessus subsp. abscessus
o (+) growth in 5% NaCl for M. triviale ● Reservoir: Tap water
Mycobacterium haemophilum ● Related infection and disease: chronic lung disease and
otitis media
● M. haemophillum: was first describe in 1978 but probably
was the nonculturable AFB ● Microscopy: Long and tapered rods with partial acid-
fastness; may be beaded or ovoid in form
● Causes painful swelling ulcer
● Biochemical test:
● Diseases: Recognize in skin ulcer in 1972 and 1974 o (-) 3-day arylsulfatase test
● Culture: Unique among mycobacteria in its growth o (+) nitrate reduction
requirement for Hemoglobin and Hemin
● Clinical manifestation: Multiple cutaneous nodules, ulcers, GROUPS OF MYCOBACTERIUM
or painful swelling, typically involving the extremities which ● Mycobacteria spp. are grouped depending on the duration
occasionally become abscess and open fistulas draining of growth and pigment production in the medium
purulent materials
Group I - Photochromogen
NTM-RAPID GROWERS
● mycobacterium that do not produce pigment unless
● organisms are considered as potentially pathogenic exposed to light
● found in dusts, soil, and water system
● enter their host through breaks in the skin and Group II - Scotochromogen
subscutaneous tissue by trauma, injection, surgery, or ● mycobacterium that produces pigment both in the dark and
animal contact under light exposure
● Can grow in routine bacteriologic media and mycobacterial
media Group III - Non-photochromogen
● Microscopy: ● mycobacterium that do not produce pigment with or without
o Weakly-staining gram-positive rods that resemble light
diptheriods
● Culture: Group IV - Rapid Growers
o Colonies appear in solid media seven (7) days or ● mycobacterium that varies in pigmentation
less
● Commonly isolated species: M. fortuitum, M. chelonae, M. Group I Photochromogen Group II Scotochromogen
abcessus
A. Grow in the dark - Pigmented both in the dark
B. Grow under light exposure and under light exposure
Mycobacterium fortuitum - not pigmented unless
exposed to light
● most common, rapidly growing mycobacteria that are
associated with localized cutaneous and soft tissue
infections
● Microscopy
o Pleomorphic – Long to short, Thick rods
o Old culture – Partially AFB Examples: Examples:
● Culture:
o MB7H11: colonies are branching and filamentous M. kansasii M. szulgai
with aerial hypae M. asiaticum M. gordonae
o MAC: colonies exhibit growth in a medium without M. marinum M. scrofolaceum
crystal violet M. simiae M. flavescens
M. xenopi
● Biochemical Test:
o (+) 3-day arylsulfatase Group III Non- Group IV Rapid Growers
o (+) nitrate reduction photochromogen
- Non-pigmented both in the - Varying pigmentation
dark ang light
Mycobacterium chelonae
● mostly associated with cutaneous infections in
immunocompromised persons
● exhibits greater resistance to antimicrobial agent than M.
fortuitum
● Microscopy: strongly acid-fast with pleomorphism in young
culture. Examples: Example:
● Culture: M. avium M. fortuitum
o Colonies are rough or smooth, non-pigmented, M. ulcerans M. chelonae
and have no filamentous branching. M. terrae complex M. smegmatis
o MAC- colonies exhibit growth in a medium without M. gastri M. phlei
crystal violet M. haemophilum M. abscessus
● Biochemical Test: M. xenopi (can be non- M. mucogenicum
o (+) 3-day arylsulfatase Photochromogenic also)
o (-) nitrate reduction
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LABORATORY DIAGNOSIS  These are used to identify patients who may be at risk
 Ideally, specimen for mycobacteria should be of developing M. aviumcomplex disease, especially
processed upon arrival in the laboratory to prevent false those with AIDS.
–negative results and multiplication of contaminants;  Stool specimens should be submitted without
otherwise, samples may also keep refrigerated preservative.
 Laboratory where specimens are processed should  It is utilized in automated equipment such as MGIT
have negative pressure in which air movement should 960 for identification of mycobacteria.
be from clean areas (Corridors) to less clean areas
(Mycobacteriology Lab) and without recirculation of the 6. Body Fluids
air.  Pleural, pericardial, and peritoneal fluids are
 The processing of specimens should be done in a collected in sterile tubes with anticoagulant such as
class II or class III biological safety cabinet Ethylenediaminetetraacetic acid (EDTA) or. Heparin
 If samples cannot be processed immediately, they Specimens - Pleural - joint aspirates
should be refrigerated. - Pericardial - CSF
- Peritoneal
1. Sputum
Specimen collection and processing: Required volume  CSF 2mL
Method of choice of spontaneously produced
collection sputum  Exudates, 3 to 5 mL
Preferred specimen early morning sputum (two pericardial, and
to three samples collected for synovial fluids
consecutive days)  Abdominal and 10 to 15 mL
chest fluids
Culture medium  BACTEC 13A vial
Advantage 7. Blood
 It is used for the isolation of M. avium
complex  It is collected to diagnosed individuals with AIDS
Required volume 5 to 10mL expectorated or
aerosol- induced sputum 8. Wound and skin lesion aspirate and tissue specimens
Microscopy prior to culture 10 epithelial cells and >25  For wound and skin aspirates, the specimen are
pus cells transferred to a lipid medium
 If the tissue specimens cannot be processed
If none or only one of the another specimen should be immediately, 10 to 15mL sterile saline should be
sputum samples is tested submitted for culture added to prevent dehydration
positive,
Pooled sputa unacceptable for culture DECONTAMINATION AND DIGESTION
because of the high
 Mycobacterium cultivation can be prone to overgrowth
probability of increase of contaminants. Especially when samples are
contamination collected from non-sterile body parts.
Aerosol-induced (use of done on ambulatory patients  Why do we need to dissolve the mucus? Simply
hypertonic saline) who are unable to follow because digestions allow the bacteria to utilize the
collection instructions. nutrients of the media.
 These procedures are performed to kill all
2. Gastric lavage contaminating organisms (non-mycobacteria) and to
Advantage This method is for senile, non-ambulatory dissolve mucous substances.
patient and children younger than 3 years-  The digestion of sputum prior to the inoculation into
old the culture medium allows the mycobacteria to utilize
Procedure Three specimen should be collected within the nutrients of the media
three days after an overnight fasting  How does mycobacterium protect themselves from the
Required 20 – 25mL of gastric secretions destruction of our digestants?
volume  The high lipid cell wall of mycobacteria protects the
cells from the disruptive action of the decontamination
3. Urine chemicals.
Preferred specimen  First morning midstream  After decontamination, specimens are concentrated,
urine (three consecutive days) usually through centrifugation, to enhance the
Required volume  15mL or the entire volume of recovery of mycobacteria in staining and culture.
voided urine  Concentration of the chemical agent, exposure or
Specimen Collection  The specimen may be collected contact time, and temperature affect the action of the
through an indwelling catheter decontaminating agent.
with a sterile needle and syringe  Specimens that do not require decontamination:
o CSF
4. Bronchoscopy specimen o synovial fluid
 These are the specimen of choice for detecting non- o biopsy from innermost organs
tuberculous mycobacteria.  If an insufficient quantity of CSF is received, the
 Specimens: following can be performed:
o Bronchoalveolar lavage (BAL) o The specimen should be used directly for
o bronchial washings smear and culture
o transbronchial biopsies o The CSF can also be concentrated by
centrifugation and can be directly inoculated.

5. Fecal specimens
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REAGENTS FOR DECONTAMINATION AND DIGESTION OF Niacin (nicotinic acid) Test

SPECIMEN

1. 2% to 4% NaOH
o It is the most common decontamination agent
(2mL- sputum + 2 mL NaOH)
o It serves as both a decontaminant and
digestant

2. 5% Oxalic acid
o It is used for the decontamination of sputum
specimens that contain Gram-negative rods
Pseudomonas.

3. Zephiran-trisodium PO4 (Z-TSP)

o It is a decontamination-digestion reagent
o Zephiran is an effective decontamination with
minimal bactericidal effect on M. tuberculosis
4. 1% Cetylpyridinium Chloride
o It is utilized to prolong the shelf life of
sputum up to 8 days
o It is ideal for the transport of specimen
5. N-acetyl- cysteine sodium hydroxide (NALC -
NaoH)
o is both a decontaminant and digestion agent
o is also known as dithiotreitol
o utilized in automated equipment such as
MGIT 960
 It is most commonly used biochemical test for the
identification of M. tuberculosis and other slow
growing mycobacteria
 This test detects the deficiency of the niacin-
connecting enzyme that converts free niacin into
niacin ribonucleotide
 It should be performed with cultures in a Lowenstein-
Jensen medium that are three-weeks-old and show
50 colonies
Reagents Cyanogen bromide and aniline reagent
(+) Result Yellow color – M. tuberculosis
Nitrate reduction test (Broth method)

BIOCHEMICAL TESTS
Heat-stable catalase Test

 The enzyme, nitroreductase, converts nitrates into

nitrites
 To determine if the bacteria is capable of producing
nitroreductase
Reagent  1mL of 30% H2O2
Medium  Tween 80 ( Polyoxyethylene Reagents Sulfanilamide, N-(1-
sorbitan monooleate) Naphthyl) ethylenediamine
Temperature  68 degrees Celsius dihydrochloride, and HCL
Time of exposure  20 minutes Indicator reagent Zinc (detects nitrate)
(+) Result  >45-mm height/column of bubbles (+) Result Red color:
 M. tuberculosis
 M. kansasii
 The reagent and medium are added to a two-week
 M. szulgai
old subculture into a Lowenstein-Jensen and placed
upright for five minutes  M. fortuitum

High-level, heat-stable, Arylsulfatase Test

 M. asiaticum
catalase-positive:  M. scrofulaceum
 M. simiae
 M. kansasii
 M. flavescens
 M. terrae complex
Heat-stable, catalase  M. szulgai
positive:  M. xenopi
 M. malmoense
 M. avium complex
Heat-stable catalase  M. tuberculosis complex
negative:  M. gastri
 M. haemophilum
 M. marinum
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 This enzyme breaks down phenolphthalein disulfate

into free phenolphthalein
 to detect if the bacteria is capable of converting
phenolphthalein disulfate into free phenolphthalein

Reagents Potassium phenolphthalein Reagent Ferrous ammonium sulfate

SO4 and sodium (+) Result Red pigment
bicarbonate  M. marinum and M. tuberculosis
(+) Result Pink color
(+) 3-day and 14-day  M. fortuitum Iron Uptake
arylsulfatase test  M. chelonae
 M. xenopi  It detects the ability of mycobacterium to convert ferric
(+) 14 day arylsulfatase test  M. fortuitum ammonium citrate into iron oxide
 M. chelonae
 M. xenopi Reagents 20% ferric ammonium citrate
(+) Result Rusty brown colonies in an egg-based
Tween 80 hydrolysis test medium - M. smegmatis group
 It is useful for separating species of non-
photochromogens and scotochromogens

pH indicator Neutral red (amber color) bound to

Tween 80

End product Oleic acid and polyoxyethylated sorbitol

(+) Result  M. asiaticum
 M. gastri
 M. marinum
 M. malmoense
 M. scrofolaceum
 M. flavescens,
 M. terrae complex; exhibit the color
in three days for M. kansasii

Tellurite Reduction
Urease Test
 All NTM rapid growers reduce tellurite in three days  It is helpful in distinguishing M. scrofulaceum
(positive) from M. gordonae (negative)
Reagent Potassium tellurite (colorless)
(+) Result Black metallic color (tellurite)

Growth Inhibition by Thiophene - 2 carboxylic acid

hydrazide (TCH) Reagents - mL urea broth incubated
 It is used to distinguish M. bovis from M. at 35 degrees Celsius
tuberculosis
 (+) Result: M. bovis exhibits no growth at 10 mg/mL (+) Result Pink color in three days – M.
TCH tuberculosis, M. furtuitum,
M. kansasii and M. marinum
Pyrazinamidase Test
NAP Inhibition Test
 P-nitroacetylamino-B-hydroxypropiophenone (NAP)
is a precursor to the synthesis of chloramphenicol

Specimen Colonies from agar or broth like a BACTEC

medium
(+) Result 20% increase in growth index –
M. tuberculosis complex
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 The tubes media are incubated at a slanted position

Sodium Chloride Tolerance Test with the screw caps loose for one week to allow the
evaporation of excess fluid and entry of CO2
Reagent Egg-based media with 5%  Cultures are examined weekly for growth
NaCl  Slow-growing mycobacteria appear between three
(+) Result Growth on the media – to six weeks
M. triviale, M. flavescens  The malachite green that is added to the media is an
and most NTM rapid inhibitory agent for non-mycobacteria
growers
2 TYPES OF SOLID MEDIUM
STAINING Egg-based media
 Smears can be prepared from specimens with or  These are mainly composed of fresh whole egg,
without the decontamination and digestion procedures potato, glycerol, and malachite green
 5,000 to 10, 000 organisms/mL is needed to obtain a  The egg yolk (lipid source) promotes the growth of
positive AFB staining mycobacteria.
 10^4 AFB/mL is the amount that is needed for  Shelf-life: One year
detection with concentrated specimens o Lowenstein-Jensen medium
 Acid-fastness is affected by colonial age, culture  is the most commonly used egg-based
media and exposure to UV light medium that contains lipids (egg yolk) for
the growth of mycobacteria
AFB STAINING METHODS
1. Ziehl- Neelsen/ Hot stain procedure
2. Kinyoun/Cold stain procedure
3. Auramine- rhodamine fluorochrome staining
 a sensitive, reliable, and specific method
 Auramine is sensitive than carbol
fuchsin
 Acid Fast bacteria (AFB) are examined
at 250X and 400X magnification using
a fluorescence microscope
 (+) Result: Bright yellow-orange bacilli
against a dark background
 It is recommended that all positive
fluorescent smears are confirmed with
Ziehl_Neelsen stain or examined by
another technologists

CLASSIFICATION OF AFB AFTER ZIEHL-NEELSEN OR

KINYOUN STAINING
1. Distinct AFB
 Mycobacterium o American Thoracic Society (ATS) medium
2. Partially AFB  is used for sterile specimens like CSF and
 Nocardia bone marrow
 Gordonia o Petragani medium
 Rhodococcus  It is used for the recovery of mycobacteria
 Tsukamurella from heavily contaminated specimen.
 It contains 0.052 g/dL of malachite green
 Legionella micdadei
which acts as the inhibitory agent
o Wallenstein medium
INTERPRETATION OF MICROSCOPY
 is used for M. avium complex
1. If none or only of three submitted samples is positive,
additional specimens are needed for culture confirmation.

Ziehl- Neelsen Fluorochrome Quantitative

Kinyoun method stain (450X) Report
(1000X)
0 0 No AFB observed
1-2/300 fields -2/70 fields Doubtful AFB
observed; request
for another
specimen
1-9/100 fields 1-2/70 fields 1+
1-9/10 field 2-18/50 fields 2+
1-9 / field 4-36/fields 3+
>9 / field >36/fields 4+

CULTURE MEDIA
SOLID MEDIA
Serum Agar-based medium (Transparent media)
 Cultures are incubated at 35 degrees Celsius in the
dark with 5% to 10% CO2  These are mainly composed of fresh whole egg,
potato, glycerol, and malachite green
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VIRULENCE TEST
Serpentine cord Formation

Tuberculin test-purified protein derivatives (PPD) skin

 They can be examined using a dissecting microscope
for early detection of growth
o Middlebrook 7H10-/7H11
 It contains 0.1% casein hydrolysate (MB
7H11) that improves the recovery of
Isoniazid-resistant strains of M. tuberculosis
 It can produce cultures in three to four weeks
o Middlebrook 7H10-/7H11
 It contains polymyxin V, amphotericin B,
carbenicillin and trimethoprim lactate
 It use PPD antigen which is a protein extracted and
purified from the cell wall of culture- grown M.
tuberculosis
 It does not differentiate active diseases from infection
 Interpretation: A reactive or positive result indicates
past exposure to M. tuberculosis
 Mantoux test (0.1 Ml)
o (+) Result: Erythema and induration or
hardening around and site of injection
o Size of induration or swelling: 10mm
o <10 mm induration: Exposure to other
Mycobacterium species
o A positive result indicates exposure to M.
tuberculosis
b. von Pirquet’s test (scratching method)
c. Vollmer tuberculin patch test ( for infants)
NUCLEIC ACID TEST FOR MYCOBACTERIUM
TUBERCULOSIS
1. Restriction enzyme analysis
2. Automated DNA sequencing

LIQUID MEDIA 3. Direct nucleic acid amplification test - is designed to

BACTEC 12B (MB 7H12) and BACTEC 13A (MB7H13)
 These liquid media are part of the BACTEC system,
which is an automated method for detecting
mycobacteria
 It requires daily agitation to enhance the growth and
should be read within four days of inoculation
 Antimicrobial agents may be added to the medium
such as polymyxin B, amphotericin B, nalidixic acid,
Trimethoprim, azlocillin
 14C-Labeled substrate: Palmitic acid (metabolized
by mycobacteria;produces radioactive CO2)
 Growth enhancer: Polyoxyethylene stearate
 Growth indicator: Release of CO2
 Advantage of 12B and 13B: Compared to conventional
media, it reduces the processing time for the isolation
of AFB and it also be used for the antimicrobial
susceptibility testing of M. tuberculosis
 Growth detection in BACTEC culture bottles:
a. M. tuberculosis -It is detected in 9 to 14 days
b. NTM – These are generally detected in less than
seven days
detect M. tuberculosis complex directly from patient’s
specimens
4. Nucleic acid hybridization test - is the most rapid test for
identification of common mycobacterial species
NON-CULTIVABLE NTM - MYCOBACTERIUM LEPRAE
(HANSEN’S BACILLUS)
 It is cultivated in vitro in any synthetic
mycobacterial media
 It invades peripheral nerves and skin cells and
becomes an obligately intracellular parasite
 Microscopy: Rod shaped and exhibits “cigar-pocket”
or pocket-fence” arrangement
 Culture: colonies exhibit growth in living tissues of the
footpads of mice and armadillos
 Optimal growth: 30 degrees Celsius
 Skin Test: Fernandez and Mitzuda reaction

BBL mycobacteria growth indicator tubes

Middlebrook 7H9 broth and Dubos Tween albumin broth

 These are used as non-selective liquid media
 They are also used for subculturing stock strains

Septi-Chek AFB
 It is biphasic medium that is the used for rapid growth
and identification of Mycobacteria

PIGMENT PRODUCTION
 It is used differentiate mycobacteria as
photochromogen, non-photochromogen and
scotochromogen (According to Runyon’s
classification).
 M. szulgai is scotochromogenic at 35 degrees
Celsius and non-pigmented at 25 degrees Celsius
to 30 degrees Celsius
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LEPROSY (HANSEN’S DISEASE) ANTIMICROBIAL AGENTS RECOMMENDED FOR PRIMARY

 chronic disease of the skin, mucous membranes, and TREATMENT OF COMMON MYCOBACTERIAL INFECTIONS
nerve tissues From: Henry’s clinical diagnosis and management by laboratory
 not considered a highly contagious disease methods. 22nd ed
 Modes of transmission: Person to person contact
through inhalation (nasal secretion), contact with Mycobacteriu Site of Antimicrobial Agents
infected skin, arthropod bites, and ingestion of breast m spp. Infection
milk and transplacental transmission for infants 1. MTBC Any Isoniazid, Rifampin,
 Forms of leprosy: Tuberculoid and lepromatous ethambutol, pyrazinamide
leprosy 2. MAC Pulmonary Clarithromycin, rifampin
and ethambutol

Clarithromycin, rifabutin,
and ethambutol

Disseminated
None

COMPARISON BETWEEN TUBERCULOID LEPROSY AND Lymphadenitis

LEPROMATOUS LEPROSY 3. M. kansasii Pulmonary Isoniazid, rifampin,
Indicator Tuberculoid Lepromatous ethambutol
Leprosy Leprosy 4. M. Non-pulmonary Clarithromycin (if
Distribution Localized and Dessiminated abscessus, susceptible) and one or
Benign and Malignant M. more additional agents
Cell-mediated Effective Ineffective chelonae,
immune response M. fortuitum
Presence in skin Rare and often Abundant and 5. M. marinum Skin/ Soft Clarithromycin,
scrapings and negative positive tissue doxycycline/minocycline,
biopsy specimen trimethoprimsulfamethoxa
Symptoms Skin lesions and Facial and nasal zole, or rifampin and
damage nerves deformities ethambutol
Lepromin skin Positive Negative
test

LABORATORY DIAGNOSIS
 Specimens: Nasal mucosal smears and skin snips from
eyebrows and other sites

Acid-fast staining - biopsy specimen

 The number of organisms per immersion filed is reported
as the bacteriologic index (BI)
 The number of solid-staining cells per 100 total bacilli is
reported as the morphologic Index (MI)
 The BI and MI aid in identifying the progress of the
disease

Culture-inoculation of specimen into the footpads of mice

 It is the definitive test for M. leprae
 Specimen: Biopsy material from the infected individual
 (+) Result: The development of the Hansen’s disease in
the animal

Serological Test
 Fluorescent leprosy antibody absorption test
 DNA amplification
 Enzyme-linked immunosorbent assay (ELISA)

Biochemical test-heat stable catalase test
 Result: Negative