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Lecture PowerPoint to accompany

Foundations in
Microbiology
Sixth Edition

Talaro
Chapter 19
The Gram-Positive
Bacilli of Medical
Importance
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Medically Important Gram-Positive
Bacilli
Three general groups:
1. Endospore-formers
Bacillus, Clostridium
2. Non-endospore-formers
Listeria, Erysipelothrix
3. Irregular shaped and staining properties
Corynebacterium, Proprionibacterium,
Mycobacterium, Actinomyces, Nocardia
2
3
Spore-forming Bacilli
Genus Bacillus
Genus Clostridium

4
General Characteristics of the Genus
Bacillus
• Gram-positive, endospore-forming, motile rods
• Mostly saprobic
• Aerobic and catalase positive
• Versatile in degrading complex macromolecules
• Primary habitat is soil
• 2 species of medical importance:
– Bacillus anthracis
– Bacillus cereus (hay bacillus)

5
Bacillus anthracis
• Large, block-shaped rods
• Central spores that develop under all conditions
except in the living body
• Virulence factors – glutamic acid capsule and 3
component exotoxins
• Anthrax toxins consists of three proteins:
each of which is nontoxic but together act
synergistically to produce damaging effect
a) Protective antigen- binding molecule for EF and LF
b) Edema factor (EF)- causes edema
c) Lethal factor (LF) – causes cell death
6
Clinical Infections- Anthrax
1. cutaneous / malignant pustule – spores enter
through skin,(skin cuts, abrasions) black sore-
eschar; least dangerous, most common form of
anthrax

2. pulmonary/ Woolsorter’s disease –inhalation


of spores, initial phase resembles flu that lasts 2-3 days
followed by a sudden severe phase in which respiratory
distress is common (dyspnea, cyanosis, pleural
effusion) mortality rate is high

3. gastrointestinal – ingested spores, abdominal pain,


nausea, anorexia, and vomiting, accounts for less than
1% of anthrax cases, fatality rate is high because of
difficulty to diagnose 7
Cutaneous anthrax

8
Laboratory Diagnosis
• 1. Microscopy- large, square-ended, gram-
positive or gram variable singly or in chains
(bamboo rods) in clinical samples
encapsulated
• 2. Cultural characteristics- non-hemolytic,
gray, flat with irregular margins (Medussa
heads/lion head) Sticky (tenacious) consistency.
When teased with loop, will stand up like beaten egg
white.
• 3. Identification- catalase (+) non-motile,
ferments glucose, lecithinase (+) susceptible9
to penicillin
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11
12
Lecithinase test
• Note opaque zone of hemolysis around
colonies on egg yolk agar

13
String of Pearls Test

• This test reflects susceptibility of a strain to


penicillin
• Organism is streaked on MHA with a 10 u
peniciliin disk placed over the streak and
incubated for 3-6 hrs at 37C
• The cells become large and spherical
occurring in chains as seen on the surface of
agar, resembling a string of pearls (+) result
14
Ascoli Test
• Diagnostic precipitin test for B. anthracis
• Extracts of infected tissues show a ring of
precipitate when layered over immune
serum

15
Control and Treatment
• Treated with penicillin, gentamicin,
erythromycin, tetracycline,
chloramphenicol, ciprofloxacin
• Vaccines
– live spores and toxoid to protect livestock
– purified toxoid; for high risk occupations and
military personnel; toxoid 6X over 1.5 years;
annual boosters

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Bacillus cereus
• Common airborne and dustborne; usual
methods of disinfection and antisepsis are
ineffective
• Spores grow in food and survive cooking and
reheating
• Ingestion of toxin-containing food causes food
poisoning
• Food is the specimen of choice for proof of
food poisoning caused by B. cereus

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Bacillus cereus – Clinical
Infection
• Food poisoning two forms
a) diarrheal – ingestion of contaminated
meat and poultry , IP- 8-16 hrs.
b) emetic- ingestion of fried rice

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Differentiation of Bacillus anthracis and
Bacillus cereus
Characteristic B. anthracis B. cereus
Hemolysis on SBA - +
Motility - +
Penicillin susceptibility S R
Lecithinase Production + +
Fermentation of salicin - -/+
Growth on PEA - +
String of Pearls reaction + -
Gelatin Hydrolysis - +

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Treatment
• Most food poisoning cases caused by B.
cereus do not require antimicrobial
treatment
• B. cereus is resistant to penicillin
• Vancomycin and clindamycin

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The Genus Clostridium
• Gram-positive, spore-forming rods
• Anaerobic and catalase negative
• 120 species
• Oval or spherical spores produced only under
anaerobic conditions
• Synthesize organic acids, alcohols, and exotoxins
• Cause wound infections, tissue infections, and
food intoxications

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Clostridium
 Clostridium form endospores under adverse environmental
condition
 Spores are characterized on the basis of position, size and shape
 Most Clostridium spp., including C. perfringens and C.
botulinum, have ovoid subterminal (OST) spores
 C. tetani have round terminal (RT) spores
 All are motile with peritrichous flagella except C.perfringens.
C.ramosum
 All are non-encapsulated except C. perfringens
 All have swollen sporangia except C.perfringens, C.bifermentans
 All are Nagler negative except C.perfringens, C. sordelii,
C.bifermetans, C. baratti
Virulence Factors
• Virulence factors
– Toxins/exoenzymes
– Cytotoxins
– Collagenase
– Enterotoxin
– hyaluronidase
– Dnase
– Neurotoxins
– Phospholipases
– Proteases
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Clostridium Associated Human
Disease

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Clostridium spp. Anaerobic Gram-Positive Spore-Forming Bacilli
Four broad types of pathogenesis:
1. Histotoxic group — tissue infections
(C. perfringens type A, exogenously acquired more commonly than endogenously)
(C. septicum; endogenously-acquired)
a. cellulilitis
b. myonecrosis
c. gas gangrene
d. fasciitis
2. Enterotoxigenic group — gastrointestinal disease
a. clostridial foodbome disease (8-24h after ingestion of large numbers of organisms on con-taminated
meat products, spores germinate, enterotoxin produced (C. perfringens type A)
b. necrotizing enteritis (beta toxin-producing C.perfringens type C)
(C. difficile endogenously-acquired or exogenously-acquired person-to-person in hospital)
c. antibiotic-associated diarrhea
d. antibiotic-associated pseudomembrane colitis

3. Tetanus (exogenously acquired) — C. tetani neurotoxin


a. generalized (most common)
b. cephalic(primary infection in head, comnnonly ear)
c. localized
e. neonatal (contaminated umbilical stump)

4. Botulism (exogenously acquired) — C. botulinum neurotoxin


a. foodborne (intoxication,1-2days incubation period)
b. infant (ingestion of spores in honey)
c. wound (symptoms similar to foodborne, but 4 or more days incubation)
Gas Gangrene
• Clostridium perfringens (Welch-Franke’s
bacillus, Bacillus aerogenes capsulatus, Gas
gangrene bacillus) most frequent clostridia
involved in soft tissue and wound infections -
myonecrosis
• Spores found in soil, human skin, intestine, and
vagina
• Predisposing factors – surgical incisions,
compound fractures, diabetic ulcers, septic
abortions, puncture wounds, gunshot wounds 27
Pathology
• Not highly invasive; requires damaged and
dead tissue and anaerobic conditions
• Conditions stimulate spore germination,
vegetative growth and release of exotoxins,
and other virulence factors.
• Fermentation of muscle carbohydrates
results in the formation of gas and further
destruction of tissue.

28
29
Treatment and Prevention
• Immediate cleansing of dirty wounds, deep
wounds, decubitus ulcers, compound fractures,
and infected incisions
• Debridement of disease tissue
• Large doses of cephalosporin or penicillin
• Hyperbaric oxygen therapy
• No vaccines available

30
Clostridium difficile-Associated
Disease (CDAD)
• Normal resident of colon, in low numbers
• Causes antibiotic-associated colitis
(pseudomembranous colitis)
– relatively non-invasive; treatment with broad-spectrum
antibiotics kills the other bacteria, allowing C. difficile to
overgrow
• Produces enterotoxins that damage intestines
• Major cause of diarrhea in hospitals
• Increasingly more common in community acquired
diarrhea
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C. dificile Virulence Factors
Treatment and Prevention
• Mild uncomplicated cases respond to fluid and
electrolyte replacement and withdrawal of
antimicrobials.
• Severe infections treated with oral vancomycin
or metronidazole and replacement cultures
• Increased precautions to prevent spread

33
Tetanus
• Clostridium tetani (tackhead bacillus)
• Common resident of soil and GI tracts of
animals
• Causes tetanus or lockjaw, a neuromuscular
disease
• Most commonly among geriatric patients and IV
drug abusers; neonates in developing countries

34
Pathology
• Spores usually enter through accidental puncture
wounds, burns, umbilical stumps, frostbite, and crushed
body parts.
• Anaerobic environment is ideal for vegetative cells to
grow and release toxin.
• Tetanospasmin – neurotoxin causes paralysis by
binding to motor nerve endings; blocking the release of
neurotransmitter for muscular contraction inhibition;
muscles contract uncontrollably
• Death most often due to paralysis of respiratory
muscles

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36
Treatment and Prevention
• Treatment aimed at deterring degree of toxemia
and infection and maintaining homeostasis
• Antitoxin therapy with human tetanus immune
globulin; inactivates circulating toxin but does
not counteract that which is already bound
• Control infection with penicillin or
tetracycline; and muscle relaxants
• Vaccine available; booster needed every 10
years
37
Clostridial Food Poisoning
• Clostridium botulinum – rare but severe
intoxication usually from home canned food
• Clostridium perfringens – mild intestinal
illness; second most common form of food
poisoning worldwide

38
Botulinum Food Poisoning
• Botulism – intoxication associated with
inadequate food preservation
• Clostridium botulinum (canned good
bacillus, Van Ermengem bacillus) – spore-
forming anaerobe; commonly inhabits soil
and water
• Justinius Kerner (1786–1862), deduced that a substance in spoiled
sausages, which he called wurstgift (German for sausage poison),
caused botulism. The toxin's origin and identity remained vague until
Emile van Ermengem (1851–1932), a Belgian professor,
isolated Clostridium botulinum in 1895 and identified it as the source
of food poisoning .
39
Pathogenesis
• Spores are present on food when gathered and
processed.
• If reliable temperature and pressure are not achieved air
will be evacuated but spores will remain.
• Anaerobic conditions favor spore germination and
vegetative growth.
• Potent toxin, botulin, is released.
• Toxin is carried to neuromuscular junctions and blocks
the release of acetylcholine, necessary for muscle
contraction to occur.
• Double or blurred vision, difficulty swallowing,
neuromuscular symptoms
40
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Infant and Wound Botulism
• Infant botulism – caused by ingested
spores that germinate and release toxin;
flaccid paralysis
• Wound botulism – spores enter wound and
cause food poisoning symptoms

42
Treatment and Prevention
• Determine presence of toxin in food,
intestinal contents or feces
• Administer antitoxin; cardiac and
respiratory support
• Infectious botulism treated with penicillin
• Practice proper methods of preserving and
handling canned foods; addition of
preservatives.

43
Clostridial Gastroenteritis
• Clostrium perfringens
• Spores contaminate food that has not been
cooked thoroughly enough to destroy
spores.
• Spores germinate and multiply (especially if
unrefrigerated).
• When consumed, toxin is produced in the
intestine; acts on epithelial cells, acute
abdominal pain, diarrhea, and nausea
• Rapid recovery
44
Laboratory Diagnosis
• 1. Gram stain/Microscopy- gram positive
bacilli
C.tetani- swollen terminal spores
C. perfringens- box-car large square rods,
large central to subterminal spores
C. septicum, C.sordelii, C. sporogens– thin
rods, subterminal spores

45
Clostridium tetani Gram Stain

NOTE: Round terminal spores give cells a


“drumstick” or “tennis racket” appearance.
REVIEW
Laboratory Diagnosis
• 2. Cultivation
Media- Anaerobic blood agar or any of the following
media: CNA blood agar, anaerobic PEA, KVLB blood
agar, thioglycollate broth, or chopped meat broth
- C. difficile- cycloserine cefoxitin fructose agar
(CCFA), white to yellow opaque non-hemolytic,
horse stable odor on BA it fluoresce under UV
light
- C. perfringens- gray to grayish yellow,
translucent with double zone of beta-hemolysis
- C.tetani- gray, irregular margin, narrow zone of
beta-hemolysis, may swarm over agar surface 47
Micro & Macroscopic C. perfringens
NOTE: Large rectangular NOTE: Double zone of hemolysis
gram-positive bacilli

Inner beta-hemolysis = θ toxin Outer


REVIEW
alpha-hemolysis = α toxin
Laboratory Diagnosis
• 3. Nagler test- based on the presence of
lecithinase (alpha toxin)
• (+) result opaque zone of hemolysis around
the colony
Nagler positive – C.perfringens, C.sordelii
Nagler negative- C.difficile, C. septicum
• 4. Urease test- C. sordelii only Clostridia
that has urease activity
• 5. Spot indole test- C.pefringes negative
C.sordelii: positive 49
C. perfringens Nagler Reaction

NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes


phospholipids in egg-yolk agar around streak on right. Antibody
against α-toxin inhibits activity around left streak.
REVIEW
Gram-Positive Regular Non-Spore-
Forming Bacilli
Medically important:
• Listeria monocytogenes
• Erysipelothrix rhusiopathiae

51
Listeria monocytogenes
• Non-spore-forming Gram-positive
• Ranging from coccobacilli to long filaments
• 1-4 flagella
• No capsules
• Resistant to cold, heat, salt, pH extremes and
bile
• Virulence attributed to ability to replicate in the
cytoplasm of cells after inducing phagocytosis;
avoids humoral immune system
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53
Epidemiology and Pathology
• Primary reservoir is soil and water; animal intestines
• Can contaminate foods and grow during refrigeration
• Listeriosis - most cases associated with dairy
products, poultry, and meat
• Often mild or subclinical in normal adults
• Immunocompromised patients, fetuses and neonates;
affects brain and meninges
– 20 – 50% death rate
In Pregnant women Listeriosis causes spontaneous abortion
and stillborn neonates
54
Laboratory Diagnosis
• 1. Microscopy – Gram (+) coccobacillus,
singly, short chains or palisades
• 2. Cultural Characteristics – small, round
smooth and translucent, narrow zone of beta
hemolysis
• Culture media: SBA, McBride agar,
Tryptose agar, PEA (for contaminated
specimen) nalidixic acid selective medium
• Cold enrichment technique – used to
isolate the organism from clinical specimen
• specimen are inoculated into broth and 55
Laboratory Diagnosis
• 3. Identification-
- catalase (+)
- motile at room temperature
- tumbling motility on wet mount
- in motility medium, characteristic umbrella
pattern is seen when the organism is incubated at
room temperature but not at 35C
-ferments glucose, trehalose
- esculin hydrolysis and CAMP (+) but block
hemolysis in contrast to arrowhead of S. agalactiae
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Virulence Test: Anton’s Test
• Development of purulent conjunctivitis in
rabbit’s eye inoculated with
L.monocytogenes suspension

58
Diagnosis and Control
• Culture requires lengthy cold enrichment
process.
• Rapid diagnostic tests using ELISA available
• Ampicillin and trimethoprimsulfamethoxazole
• Prevention – pasteurization and cooking

59
Erysipelothrix rhusiopathiae
• Gram-positive rod widely distributed in
animals and the environment
• Catalase negative, pleomorphic that has
tendency to form long filaments
• Commensal or pathogen of domestic swine,
birds and fish
• Enters through skin abrasion, multiples to
produce erysipeloid, dark red lesion

60
Clinical Infections
• 1. Erysipeloid- localized skin disease,
resembles streptococcal erysipelas, sharply
defined, slightly elevated purplish-red zone
that spreads peripherally
• 2. Septicemia – often associated with
endocarditis, 38% mortality rate
• 3. Diffuse cutaneous infection -
exacerbation of erysipeloid

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Erysipeloid

62
Laboratory Diagnosis
• 1. Microscopy – gram positive that may
form long filaments, arranged singly, in
short chains or V shape.
• 2. Culture characteristics- two colony
types
- small, smooth form is transparent,
glistening and convex
- large, rough colonies are flat and has
irregular edges
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Laboratory Diagnosis
• 3. Identification
- catalase negative
- non-motile
- H2S positive
- test tube brush like pattern in gelatin stab
culture at 22C

65
Gardnerella vaginalis
• Short pleomorphic, gram-positive rod or
coccobacillus that often stain gram variable
or gram negative
• Clinical infections –
1. bacterial vaginosis; characterized by
malodorous discharge and vaginal pH
greater than 4.5
2. UTI

66
Laboratory Diagnosis
• 1. Microscopy
-pleomorphic, gram variable coccobacillus
- wet mount of vaginal fluid will reveal clue
cells; large squamous epithelial cells with
gram positive and gram variable bacilli and
coccobacilli clustered on the edges

67
Laboratory Diagnosis
• 2. Culture characteristics
- pinpoint, nonhemolytic colonies
- medium of choice human blood bilayer
tween (HBT) agar, when cultured on blood
agar colonies are small, gray, opaque and
beta-hemolytic
• 3. Identification
- catalase negative
- oxidase negative
- hippurate hydrolysis positive 68
Gram-Positive Irregular Non-Spore-
Forming Bacilli

Medically important genera:


• Corynebacterium
• Proprionibacterium
• Mycobacterium
• Actinomyces
• Nocardia

69
• Pleomorphic; stain unevenly
• 20 genera; Corynebacterium, Mycobacterium,
and Nocardia greatest clinical significance
• All produce catalase, possess mycolic acids,
and a unique peptidoglycan.

70
Corynbacterium diptheriae
• Gram-positive irregular bacilli
• Virulence factors assist in attachment and
growth.
– diphtherotoxin – exotoxin
• 2 part toxin – part B binds and induces endocytosis;
part A arrests protein synthesis

71
72
Epidemiology and Pathology
• Reservoir of healthy carriers; potential for
diphtheria is always present
• Most cases occur in non-immunized children
living in crowded, unsanitary conditions.
• Acquired via respiratory droplets from carriers
or actively infected individuals

73
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Epidemiology and Pathology
2 stages of disease:
1. Local infection –upper respiratory tract
inflammation
– sore throat, nausea, vomiting, swollen lymph nodes;
pseudomembrane formation can cause asphyxiation
2. Diptherotoxin production and toxemia
– target organs primarily heart and nerves

75
Diagnostic Methods
• Pseudomembrane and swelling indicative
• Stains
• Conditions, history
• Serological assay

76
Treatment and Prevention
• Antitoxin
• Penicillin or erythromycin
• Prevented by toxoid vaccine series and
boosters

77
Genus Proprionibacterium

• Propionibacterium acnes most common


• Gram-positive rods
• Aerotolerant or anaerobic
• Nontoxigenic
• Common resident of sebaceous glands
• Causes acne

78
Mycobacteria: Acid-Fast Bacilli
• Mycobacterium tuberculosis
• M. leprae
• M. avium complex
• M. fortuitum
• M. marinum
• M. scrofulaceum
• M. paratuberculosis

79
Genus Mycobacterium
• Gram-positive irregular bacilli
• Acid-fast staining
• Strict aerobes
• Produce catalase
• Possess mycolic acids and a unique type of
peptidoglycan
• Do not form capsules, flagella or spores
• Grow slowly
80
81
Mycobacterium tuberculosis
• Tubercle bacillus
• Produces no exotoxins or enzymes that
contribute to infectiousness
• Virulence factors - contain complex waxes
and cord factor that prevent destruction by
lysosomes or macrophages

82
Epidemiology of Tuberculosis
• Predisposing factors include: inadequate nutrition,
debilitation of the immune system, poor access to
medical care, lung damage, and genetics.
• Estimate 1/3rd of world population and 15 million
in U.S. carry tubercle bacillus; highest rate in U.S.
occurring in recent immigrants
• Bacillus very resistant; transmitted by airborne
respiratory droplets

83
Course of Infection and Disease
• Only 5% infected people develop clinical
disease
• Untreated, the disease progresses slowly;
majority of TB cases contained in lungs
• Clinical tuberculosis divided into:
– primary tuberculosis
– secondary tuberculosis (reactivation or
reinfection)
– disseminated tuberculosis
84
Primary TB
• Infectious dose 10 cells
• Phagocytosed by alveolar macrophages and
multiply intracellularly
• After 3-4 weeks immune system attacks,
forming tubercles, granulomas consisting of a
central core containing bacilli surrounded by
WBCs – tubercle
• If center of tubercle breaks down into necrotic
caseous lesions, they gradually heal by
calcification.
85
86
Secondary TB
• If patient doesn’t recover from primary
tuberculosis, reactivation of bacilli can occur.
• Tubercles expand and drain into the bronchial
tubes and upper respiratory tract.
• Gradually the patient experiences more severe
symptoms.
– violent coughing, greenish or bloody sputum, fever,
anorexia, weight loss, fatigue
• Untreated, 60% mortality rate
87
Extrapulmonary TB
• During secondary TB, bacilli disseminate to
regional lymph nodes, kidneys, long bones,
genital tract, brain, and meninges.
• These complications are grave.

88
Diagnosis
1. In vivo or tuberculin testing
Mantoux test – local intradermal injection of
purified protein derivative (PPD); look for red
wheal to form in 48-72 hours- induration;
established guidelines to indicate interpretation of
result based on size of wheal and specific
population factors
2. X rays
3. Direct identification of acid-fast bacilli in
specimen
4. Cultural isolation and biochemical testing
89
90
Management and Prevention of TB

• 6-24 months of at least 2 drugs from a list


of 11
• One pill regimen called Rifater (isoniazid,
rifampin, pyrazinamide)
• Vaccine based on attenuated bacilli Calmet-
Guerin strain of M. bovis used in other
countries

91
Mycobacterium leprae: The Leprosy
Bacillus
• Hansen’s bacillus/Hansen’s Disease
• Strict parasite – has not been grown on artificial
media or tissue culture
• Slowest growing of all species
• Multiplies within host cells in large packets called
globi
• Causes leprosy, a chronic disease that begins in
the skin and mucous membranes and progresses
into nerves

92
Epidemiology and Transmission of
Leprosy
• Endemic regions throughout the world
• Spread through direct inoculation from
leprotics
• Not highly virulent; appears that health and
living conditions influence susceptibility
and the course of the disease
• May be associated with specific genetic
marker

93
Course of Infection and Disease
• Macrophages phagocytize the bacilli, but a
weakened macrophage or slow T cell response
may not kill bacillus.
• Incubation from 2-5 years; if untreated, bacilli
grow slowly in the skin macrophages and
Schwann cells of peripheral nerves
• 2 forms possible:
– tuberculoid – superficial infection without skin
disfigurement which damages nerves and causes loss of
pain perception
– lepromatous – a deeply nodular infection that causes
severe disfigurement of the face and extremities
94
Diagnosing
• Combination of symptomology, microscopic
examination of lesions, and patient history
• Numbness in hands and feet, loss of heat and
cold sensitivity, muscle weakness, thickened
earlobes, chronic stuffy nose
• Detection of acid-fast bacilli in skin lesions,
nasal discharges, and tissue samples

95
Treatment and Prevention
• Treatment by long-term combined therapy
• Prevention requires constant surveillance of
high risk populations.
• WHO sponsoring a trial vaccine

96
Infections by Non-Tuberculosis
Mycobacteria (NTM)
• M. avium complex – third most common cause of
death in AIDS patients
• M. kansaii – pulmonary infections in adult white
males with emphysema or bronchitis
• M. marinum – water inhabitant; lesions develop after
scraping on swimming pool concrete
• M. scrofulaceum – infects cervical lymph nodes
• M. paratuberculosis – raw cow’s milk; recovered
from 65% of individuals diagnosed with Crohn’s
disease
97
Non-Spore Forming, Branching
Aerobic Actinomycetes

98
Nocardia
• Aerobic, branched, beaded gram-positive
bacilli
• May not stain by gram stain
• Weakly acid fast
• Colony and microscopic morphology, and
types of infections resemble those of fungi
• Commonly found in soil
• Infections mostly occur in
immunocompromised patients
• Most common species are N.asteroides, N.
99
brasiliensis, N.nova, N. farcinica
Virulence factors
• Toxins
• Extracelluar proteins
• Superoxide dismutase and catalase provide
resistance to oxidative killing of phagocytes
• Nocobactin – iron chelating compound

100
Clinical Infections
• 1. Pulmonary infections
- most commonly caused by N.asteroides
- present as bronchopneumoia that is
usually chronic but maybe acute and
relapsing
- progresses rapidly than tuberculosis
- dissemination to other organs may occur

101
Clinical Infections
• 2. Cutaneous lesions
- most commonly caused by N.brasiliensis
- localized subcutaneous abscess that is
invasive and quite destructive of the tissues
and underlying bones
- actinomycotic mycetomas- swelling,
draining sinuses and sulfur granules

102
Laboratory Diagnosis
• 1. Microscopy
- filamentous, branching weakly acid
fast/gram positive bacilli
- granules in specimens from cutaneous
infections
• 2. Culture characteristics
- colonies might have chalky, matte or velvety
appearance or dry crumbly appearance
- examination of colonies with dissecting
microscope may reveal presence of hyphae
103
Nocardia Gram stain morphology

104
Nocardia colony morphology

105
Laboratory Diagnosis
• 3. Identification
a) substrate hydrolysis – casein, tyrosine,
xanthine
b) other substrate and carbohydrate
utilization- arylsulfatase, gelatin
liquefaction, carbohydrate utilization
c) fatty acid analysis by HPLC
d) antimicrobial susceptibility profile

106
Other Actinomycetes-
Actinomadura
• Clinically important species Actinomadura
madurae and A. pelletieri
• Etiologic agent of mycetoma similar to those
caused by Nocardia
• Microscopic and colony morphology similar to
that of Nicardia
• Differentiation can be made by using metabolic
variations; A.madurae is cellobiose and xylose
positive whereas Nocardia is negative
107
Streptomyces
• Primarily saprophytes found as soil
inhabitants and resemble other
actinomycetes
• Most species are positive for nitrate and
urease

108