COLLEGE OF MEDICAL LABORATORY SCIENCE

BACTERIOLOGY
01/04/24 I DEAN PINES LIMPIADO I 1ST SEM I LEC

LESSON NO. 11: MYCOBACTERIA

TOPIC OUTLINE Mode of - Contact with patient shedding the

1 Family Mycobacteriaceae Transmission bacteria in the nasal secretions
A. Mycobacterium Leprae (Inhalation) or ulcers exudates.
B. Mycobacterium Tuberculosis Incubation - 2 to 4 years or up to 40 years (3
2. Actinomyces period months the earliest)
3 Clostridia group
4 Nocardia LEPROSY (TYPE OF LESION)

OVERVIEW Table No. 2 Comparison of the two types of lesion

In this lesson, we will continue our discussion on gram-positive Lepromatous Tuberculoid
bacilli. We will be discussing Mycobacteria, which has two (Nodular Type) (Anesthetic Type)
important species, namely Mycobacterium leprae and - Progressive and - Non-Progressive/
Mycobacterium tuberculosis. We will also have a brief malignant (severe Benign
discussion of Actinomyces. The last topic will be on the and extensive form) - Few organisms in skin
Clostridia, and it has four important species, the C. tetani, C. with tumor outgrowth lesions
perfringens, C. botulinum and C. difficile, which is considered on the skin. Anesthetic type means a loss
to be the normal flora of our gut. Manifest multiple organism in of sensation in the affected
the lesions. areas (inability to sense pain
etc.) thus making this type
GENERAL CHARACTERESTICS more infectious.
− Family Mycobacteriaceae, Genus Mycobacteria - With numerous - Selectively colonize
⮩ Gram positive organisms in the the Schwann cells of
When subjected to gram staining lesions CNS
⮩ Acid-fast bacilli - Lepromin test (-) - Lepromin test (+)
One of the very important characteristics of mycobacterium which is
considered to be one of the methods that we are using in identifying
the organism is the acid-fast staining. LEPROMIN TEST (GOOGLE)
- The test is based on the principle that individuals
Bailey & Scott’s Diagnostic Microbiology 15th Edition. infected with M. leprae will exhibit a specific cellular
Mycobacterium spp. have an unusual cell wall; it contains N- immune response when injected with a preparation
glycolylmuramic acid instead of N-acetylmuramic acid and has containing components of the bacterium.
a very high lipid content, which creates a hydrophobic Lepromatous- poor cell-mediated immune response (-)
permeability barrier. This important property of mycobacteria Tuberculoid- strong cell-mediated immune response (+)
which derives from their cell wall structure, is referred to as acid
fastness; this characteristic distinguishes mycobacteria from
other genera. Figure 1. Lepromatous Leprosy

⮩ Aerobic, non-motile, non-spore former, non-

capsulated
You can notice that those different factors that will contribute to
pathogenicity are not inherited by the Mycobacterium species.
However, the presence of lipases in the cell wall of the
mycobacterium makes them pathogenic, which is responsible for
their virulence.
CLINICALLY SIGNIFICANT SPECIES
A. Mycobacterium tuberculosis
B. Mycobacterium leprae
Figure 2. Tuberculoid Leprosy
C.
MYCOBACTERIUM LEPRAE
Table No. 1 Mycobacterium Leprae
Characteristics - obligate intracellular parasite that
multiply very slowly within
mononuclear phagocytes.
- straight, slightly curve rods,
packed in a cell in an
arrangement as “packets of
cigar”
- Non-motile, non-spore former
Other Name - Hansen’s bacillus
Host - Human is the only natural host
Pathogenicity - Causative agent of leprosy or
Hansen’s Disease
- Leprosy is a chronic
communicable disease that
involves the skin, mucous
membrane, and nerves.

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LABORATORY DIAGNOSIS
Table No. 3 Lab Diagnosis of Leprosy
.
Lepromin Test - A skin test using sterile extract CULTIVATION, CONTROL AND TREATMENT
from Lepromatous nodule
- Two types: Table No. 4 Cultivation, Control and treatment of
⮩ Early (Fernandez rection, Leprosy
24-48 hours) Cultivation - Non- culturable in artificial
A positive reaction may manifest as medium
erythema (redness) and induration - Grow in the foot pods of mice and
(swelling) at the injection site armadillo
⮩ Late (Mitsuda, 3-4 weeks) Control - Pre-immunization with BCG
AFB Staining - Sample from nasopharynx Bacillus Calmette-Guerin
- Bacilli appears as parallel - Isolation and treatment of cases
bundles or packets of cigar Treatment - MDT for 6 months
- Acid- Fast bacilli Multi-drug therapy
If observed microscopically, it is already an
indication that the patient is suffering from
leprosy

MYCOBACTERIUM TUBERCULOSIS
Table No. 5 Mycobacterium Tuberculosis
Characteristics - Slender beaded rods, non- motile, non-spore former, no capsulated, produce “much-granules”
In Corynebacterium diphtheria is also capable of producing polluted granules called Babes & Ernst’s granules.
- could be stained with fluoro-chromatic dye, carbolfuchsin.
- Do not produce toxin but protected by intracellular environment making them resistant to heat drying and
chemical agents.
Other Name - Koch’s bacillus
⮩ First discovered by Robert Koch in 1882
- Human tubercle bacillus
Lipase - Responsible for:
Important ⮩ Hydrophobic nature
constituent of M. Hydrophobic Nature means that they have no affinity to water. Plasma is composed 91% of water but due to their
TB hydrophobic barrier, it helps the bacteria to resist the host’s immune response and contributes to their ability to persist
within host cells. It can also affect the penetration of certain antibiotics, making the bacteria more resistant to treatment.
⮩ Formation of cord factor (virulence factor), responsible for “serpentine cords in the culture
- Mycolic acid (acid fastness)
Epidemiology - Tuberculosis is endemic worldwide, less frequent in developed countries
Also correlated with environmental sanitation and overcrowding of the population.
- Seen from a worldwide perspective, tuberculosis is still a major medical problem
- Estimated that every year approximately 15 million persons contract tuberculosis and that three million
dies of the disease.
Mode of - Main source of infection is the human carrier.
transmission - Generally direct, in most cases by droplet infection (through Inhalation)
- Indirect transmission (ingestion) via milk (udder tuberculosis in cattle) or milk product.
- Direct contact through the skin (Ulceration at the site of invasion).
Incubation - Four days to 1 to 2 weeks
period
Drug Resistant A. Multi-drug resistant tuberculosis
Strains (MDR-TB)
⮩ Indicates resistance to both Isoniazid and Rifampin
B. Extensively drug-resistant TB
(XDR-TB)
⮩ Indicates resistance to Isoniazid, Rifampin, Fluroquinolone and second line injectable drugs.
- Transmission of MDR-TB and XDR-TB are primary concern.
Pathogenicity - The bacilli neither produce exotoxin or endotoxin, virulence is due to the cord factor
- Initial droplet of infection results in primary tuberculosis, localized mainly in the apices of the lungs
- Primary disease develops with the Ghon focus (Ghon’s complex or primary complex), the hilar lymph
nodes are involved as well
- 10% with primary tuberculosis progresses to the secondary stage (reactivation or organ tuberculosis) after
a few months or even years, characterized by extensive tissue necrosis.
- 90% of primary infection foci remain clinically silent
- Ingestion may cause primary lesion in the mouth or tonsils with the enlargement of the lymph node of the
neck called “cervical adenitis” or “scrofula”
- Penetration of organism in Intestinal mucosa will cause lesion on the wall of Intestine
- Extrapulmonary tuberculosis may occur (e.g. TB meningitis)
Immunology - Age influences likelihood and pattern of the disease
- Infection requires a cellular immune response
- Specific immunity and allergy that develop in the course of an infection reflect T lymphocyte functions
- Allergy is measured in terms of the tuberculin reaction to check for clinically in apparent infections with TB
- Granulomas form when antigen load is small and tissue hypersensitivity is high.

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Figure 3. Possible Courses of Pulmonary Tuberculosis
PRE-DISPOSING FACTORS OF PULMONARY
TUBERCULOSIS
1. Susceptibility of an individual to the infection
Something to do with our immune system; Individual immunity.
2. Low level of general health and resistance
3. Malnutrition
COMMON SIGNS AND SYMPTOMS
− Low grade fever, night sweating, malaise, weakness,
dry and productive cough.
Similar to flu like symptoms but the duration is much longer.
− Haemoptysis
Coughing of blood
LABORATORY DIAGNOSIS
Table No. 6 Lab Diagnosis of M. Tuberculosis
Specimens - Sputum (early morning sputum)
More concentrated in the morning
- CSF (inoculated in Middle Brock 7H)
If we are suspecting for TB meningitis.
- Biopsy Material
- Pleural fluid
- Synovial fluid
- Gastric content
For intestinal tuberculosis
1. Direct Smear - AFB staining
- AFB (+) is due to mycolic acid or
hydroxymetoxy acid
- Methods:
1. Ziehl-Nelseen
⮩ Carbolfuchsin (with
steaming)- primary stain (5
minutes)
⮩ Acid-Alcohol- decolorizer (30
seconds to 1 minute)
⮩ Methylene blue / Malachite
green - counter stain (1 minute
① Prepare smear
② Air dry
③ Flame Sterilized/ Heat Fixed
④ Add Carbolfuchsin and the same time,
perform steaming. (5minutes)
⑤ After 5 minutes, wash with running
water and then add acid alcohol as
decolorizer. (1 minute or 30 seconds)
The purpose of heating or steaming is to soften
the mycolic acid and when liquified, it allows
carbolfuchsin to enter the cell wall.
⑥ After 1minute or 30 seconds, wash
with water then add Methylene blue or
Malachite Green.
⑦ Observe under microscope
Indicator = slender bacilli (Red color)
2. Kinyouns (cold method)
For parasites
3. Pappenheims
4. Baugartins
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2.Concentration - use of digesting substance: 4%
Technique NaOH, 6% oxalic acid, 4% sulfuric
acid)
3. Niacin or - Mycobacterium tuberculosis is
Nicotinic Niacin test (+)
Acid Test - Principle: All mycobacterium species
produce niacin ribonucleotide
enzymes which converts niacin to
nicotinamide adenine dinucleotide
(NAD) of which M. tuberculosis
lacks the said enzyme, thus
accumulation of niacin in the culture
medium which can be extracted with
physiologic saline and can
be determined using Niacin detection
kit (positive test is indicated by yellow
color)
4. Virulence A. Serpentine cord formation
test - a glycolipid present in the cell wall of
three Mycobacteria that contribute
virulence and promotes growth
5.Tuberculin - based on characteristic reaction to
test several soluble components of the
cell (tuberculin)
a. OT (old original tuberculin)
b. PPD (Purified Protein Derivative)
- Positive test: past infection, close
association with infected patient
Only good if the individual if newly exposed to
the pathogen.
Methods (tuberculin skin test)
1.) Mantoux (injected intracutaneously)
2.) Von Pirquet (scratching the skin)
3.) Vollmer Patch Test (PPD soaked in a
cloth then placed over the skin), less
sensitive
4.) Mono-percutaneous Test
5.) Tuberculin Time (multiple puncture
technique)
6.) Jet injection method (using a jet gun,
done intracutaneously at high pressure)
6. Culture - Culture is considered as gold
standard
- Requires increase Co2 (8-12%)
- Requires complex media
⮩ Agar Base Media (Middle
Brooke 7H-10)
⮩ Egg Base Medium (Petragnani,
LJM)
- Growth is relatively slow (3-8 weeks
incubation in solid Medium)
- Additives are used to suppress the
growth of other microorganisms.
- Colonies are dry, rough, granular
(Cauliflower like)
7.Molecular - NAAT for TB (Nucleic Acid
Diagnostics Amplification Test)
- Molecular test used to detect DNA of
M. tuberculosis complex (MTBC) in
sputum or other respiratory samples
⮩ Example: GeneXpert, a rapid
test that can simultaneously
identify:
1.Mycobacterium tuberculosis
2. Resistance to Rifampin
⮩ Consist of integrated DNA
extraction (using sputum
sample) and real time PCR
system that is safe and simple to
use
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8.Animal OTHER MYCOBACTERIUM SPECIES

Inoculation Test
Table 7. Other Mycobacterium species
Figure 4. Colonies of M. Tuberculosis in diff. complex media
Mycobacterium - Bovine tubercle bacilli
bovis - shorter and plumper in
morphology
- grow slowly in the culture
producing smaller colonies
- common source of the disease is
drinking heavily infected milk with
bovine tubercle bacilli.
- most useful test that differentiate
from M. tuberculosis is Niacin
test based on the difference in
the amount of free nicotinic acid
produced by the two strains
Mycobacterium - grow very slowly in cultures (3
Lowenstein-Jensen Ulcerans months)
Middlebrook 7H11 - small, dried, smooth, green to
Medium
(Cauliflower like) yellow colonies.
- produces a destructive tropical
skin disease which later
Figure 5. Scheme for Chemotherapy of Tuberculosis
develops a chronic ulcer with
necrotic center.
Mycobacterium - acquired from bodies of water
marinum causing skin and soft tissue
infection.
Non-tuberculous - Mycobacteria that are neither
Mycobacteria tuberculosis nor leprosy bacteria
(NTM) - categorized as atypical
mycobacteria (old designation),
nontuberculous mycobacteria
(NTM) or mycobacteria other
than tubercle bacilli (MOTT)

Figure 6. Infections Caused by Nontuberculous Mycobacteria

DISCUSSION.
TB patient is treated for 6 months (Philippines). During the first
2 months you will be given isoniazid, rifampicin, ethambutol
and pyrazinamide. After 2 months, only isoniazid and
rifampicin will be taken up and this is the reason why it become
resistant to those drugs, due to the constant exposure of the
microorganism to it.

− Direct Observe Therapy, one of treatment approaches

introduced.
TB DOTS is an innovation of the DOH to address multidrug-resistant
TB (MDR-TB) cases. Healthcare workers or trained community health
workers directly observe and ensure that TB patients take their
prescribed medications.
− With appropriate therapy, the patients become non-
infectious within 2 weeks. ACTINOMYCETES
− Susceptibility testing is important guide to therapy
− Gram-positive bacteria
− Bedaquiline is a recently approved Drug for MDR-TB
Fungi appearance
− Treatment of XDR-TB is usually associated with poor
outcomes. − tend to grow in the form of branched filaments (mycelial
masses, however, not observed in older cultures)
PREVENTION
OCCURRENCE
− Prophylactic Bacillus Calmette-Guerin (BCG)
vaccination in high-burden countries proved to decrease − Part of the normal mucosal flora in humans and animals,
incident of tuberculosis. colonize mainly the oral cavity.
Vaccination with BCG only guarantee 65% immunity. We still have − Ninety percent of actinomycetes infections in humans
are caused by A. israelii, with far fewer cases caused
35% chance to acquire TB.
by A. naeslundii and other species.
− Avoid contact with infected person.
− Case finding, Isolation and treatment of cases is the
most effective method of tuberculosis control.

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A. ISRAELII Figure 7. Actinomyces Israeli

Table No. 8 A. Israelii

Characteristics - May show branching (showed,
lobate colonies resembling molar
tooth, some with sulfur granules.
- Whitish macro-colonies often with
a rough surface, begin to appear
after two weeks.
- Requires enriched mediums and
an anaerobic milieu containing
5–10% CO2.
- Ferments sugars, major
metabolic products are Acetic and
succinic acid.
Pathogenesis - Genuine actinomycoses are Actinomycosis,
always polymicrobial (mixed flora) Cervicofacial
⮩ Cervicofacial “lumpy jaw”
actinomycosis the most
frequent form of
actinomycetes infection
(>90%)
⮩ Thoracic actinomycosis.
sometimes this type also
develops from an
actinomycosis in the throat
or hematogenous spread CLOSTRIDIA GROUP
⮩ Abdominal actinomycosis - Most species are motile with peritrichous flagella
results from injuries to the - Hemolytic and non-capsulated
intestine or female genitals - Found in soil, feces of horses and other animals
⮩ Genital actinomycosis.
May result from use of Figure 8.
intrauterine contraceptive
devices. (IUD)
⮩ Canaliculitis, an
inflammation of the lacrimal
canaliculi
⮩ Caries and possible
contribution to periodontitis
Diagnosis - Microscopy and culturing of pus,
fistula secretion, granulation Clostridium perfringens
tissue or bronchial secretion.
- Microscopic detection of
branched rods, mycelial micro-
colonies on enriched nutrient
mediums after one to two weeks.
- Final identification: direct
immunofluorescence, cell wall
analysis, and metabolic analysis
(several weeks) CLASSIFICATION OF GROUP
Treatment - Includes both surgical and anti-
biotic measures. A. Gas gangrene group/Histotoxic clostridia
- antibiotic of choice is an 1. C. perfringens/ C. welchii
Aminopenicillin 2. C. histolyticum
Epidemiology - Occur sporadically worldwide. 3. C. novyi
Average morbidity (incidence) 4. C. septicum
levels are between 2.5 5. C. sporogenes
and 5 cases per 100 000 pop.
/year. B. Toxigenic group
- Men are infected twice as often as 1. Clostridium tetani
women 2. Clostridium botulinum

NOTE: One distinctive characteristic of the different Clostridia spp.is

based on the location of their spores
Clostridium perfringens – Centrally located spore
Clostridium tetani- Terminally located spore
Clostridium botulinum- Sub-terminally located spore

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CLOSTRIDIUM PERFRINGENS
Table No. 9 Clostridium perfringens
Characteristics - Centrally located spore
- Produce five types of toxins (type
A- infect humans type B to E –
domestic animals)
Enzymes produced:
a. Collagenase (Kappa Antigen)
b. Deoxyribonuclease (NU antigen)
c. Hyaluronidase (MU antigen)
Other Name - Gangrene bacillus
Foul smell of the wound/lesion caused by
the gas produced coming from the
fermentation of sugar in the muscle.
Pathogenicity 1. Causes anaerobic cellulitis
⮩ fermentation of muscle
sugar causing gas formation
(causing crepitus) in the
tissues which causes
necrosis.
⮩ Common among soldier’s
wound infection
2. Gangrenous foot infection
(Myonecrosis)
⮩ common among diabetic
patient
3. Causative agent of food poisoning
in dried food
⮩ due to exotoxin
4. Necrotic enteritis
Laboratory 1. Gram staining
diagnosis Centrally located spore
2. Culture
⮩ BAP – double zone of
hemolysis, circular
smooth colonies
⮩ Chopped meat glucose
medium (abundant growth
with gas formation)
⮩ Fluid Thioglycolate medium
⮩ Milk medium- “stormy”
fermentation producing large
amount of acid from lactose
causing casein in milk to
coagulate
3. Naglers reaction
⮩ Neutralization
determination of the type of
toxin produce
⮩ Bacteria is streaked in agar
plate containing egg yolk
(lecithin), half of the plate is
covered with anti-toxin, the
other half, no antitoxin
⮩ Positive is indicated by
inhibition of opalescence in
the anti-toxin treated media.
⮩ Opacity is due
degradation of licithin by
alpha toxin produced by C.
perfringens.
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MYCOBACTERIA
CLOSTRIDIUM TETANI
Table No. 10 Clostridium Tetani
Morphology - Long slender with swollen
terminally located spore giving a
characteristic drumstick or tennis
rocket appearance.
- motile with peritrichous flagella
Transmission - Common source of infection: dirty
pointed objects, injured area with
spores deposited on it, then
absorbed by the blood stream
Incubation - Incubation period: 4 to 10 days
Period
Cultural/ and - Obligate anaerobes, moderately
other fastidious, does not ferment CHO
Characteristics and liquefy gelatin.
- BAP: produce swarming growth with
translucent edge of the colonies with
a faint beta-hemolysis.
- Resistant to various disinfectant,
survive boiling, for practical purpose,
autoclaving is the best method of
sterilizing
Pathogenicity - Responsible for tetanus infection
due to toxins produced
✓ Tetanus lysin (Hemolysin)
⮩ Responsible for the Beta-
hemolytic activity
✓ Tetanospasmin (Neurotoxin)
⮩ Responsible for all the
symptoms of tetanus
⮩ Action is inhibition of GABA
causing spasm of masseter
muscles resulting to “locked
and jaw” or Trismus
✓ Also causes Tetanus neonatorum
⮩ common among newborn due
to cutting of umbilical cord with
unsterilized material.
Open lower-leg Fully manifest case
and fracture following a of tetanus in a patient
traffic accident; the “locked jaw”
portal of entry of C.
tetani.
Laboratory - Specimen: swab from wound
diagnosis infection.
Table No. 9 1. Gram-staining,
2. Anaerobic culture
a. Thioglycolate medium
- reducing agent: Na thioglycolate
- indicator: Resazurin (for redox
to potential)
b. Cooked Meat medium
sealed with petrolatum
3. Clinical picture and history of injury
of the patient
4. Toxin neutralization in vivo (use of
mice. indicated by death of mice due to
tetanic spasm.
Prevention - Vaccination (toxoids)
- Prophylaxis (use of anti-toxins)
- Cleaning of wound with soap and
water or H202
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CLOSTRIDIUM BOTULINUM CLOSTRIDIUM DIFFICILE

− commonly found in colon of some individuals.
Table No. 11 Clostridium Botulinum − causes Pseudo-membranous colitis.
Characteristics - Common in smoked food, left C. difficile is a normal flora of the lung, but when its population
over foods and other processed becomes greater than normal , it can cause infection.
food, contaminated
vegetable/fruits and widely TYPE OF TOXIN PRODUCED
distributed in soil. A. Toxin A
- Straight to slightly curved rods ⮩ enterotoxin, causes increase secretions of
with swollen sub-terminally electrolytes and fluids.
located spores, motile with Common manifestation is nausea and vomiting when having
peritrichous flagella. electrolyte imbalance.
Other Name - Canned goods bacillus causing B. Toxin B
fatal food poisoning. ⮩ cytotoxin, causes damage to mucosa of colon.
Pathogenicity - Causative agent of botulism with
incubation period of 18 – 96 CLINICAL COURSE
hours
- Pathogenicity is due to the • Fever
botulinum toxin (a neurotoxin), a • Diarrhea
very potent toxin (100x than • Spasmodic Abdominal pains
cobra venom)
- Acquired through ingestion of LABORATORY DIAGNOSIS
food containing the preformed − Involves culturing the pathogen from patient stool.
toxins − Detection of the cytotoxin in bacteria-free stool filtrates
- Toxin is absorbed in the intestine, on the basis of a cytopathic effect (CPE) observed in cell
to the lymphatics and blood cultures.
stream then gain access
to peripheral nervous system NOCARDIA
blocking the release of acetyl
choline − Includes species with morphology similar with
- Effect of toxin: flaccid paralysis actinomycetes.
⮩ blocked the excitatory
− Obligate aerobes found in the soil and damp biotopes.
neurotransmitter,
acetylcholine synapses and
− Gram-positive, fine pleomorphic rods that sometimes
neuromuscular junction.
show branching.
Symptoms - Double vision, speech difficulty,
inability to swallow and
constipation − Pathogens known for involvement in nocardioses, (rare
typ of infection), include:
Cause of Death - Cardiac arrest
- Respiratory paralysis ⮩ N. asteroids
Types of 1. Botulinal food poisoning ⮩ N. brasiliensis
Botulism ⮩ ingestion of food containing ⮩ N. farcinia
pre-formed toxins ⮩ N. otitidiscaviarum
2. Wound botulism (rare) ⮩ N. nova
3. Infant botulism − Can be cultured on standard nutrient Medium,
⮩ ingestion of spores
⮩ causes SIDS (Sudden Infant PATHOGENESIS
Death Syndrome) − penetrate from the environment into the host via the
Laboratory 1. Stained smear respiratory tract or dermal wounds infection
Diagnosis 2. Culture of contaminated food\
3. Serological test − Nocardiosis show clinical features of invasive pulmonary
- there are 8 serological types, human infection, disseminated disease, or brain abscess; 20%
botulism is due to type A, B and E show cellulitis.
Prevention -Boil food for 15 minutes/ heat at least
80°C
-Proper canning of food and
preservation

Nocardia asteroides Nocardia brasiliensis

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