MYCOBACTERIUM TUBERCULOSIS

CORYNEBACTERIUM DIPHTHERIAE
DISAMPAIKAN OLEH:
PROF. DR.dr. NOORHAMDANI AS, DMM, SpMK (K)
KaLab/KaSMF MIKROBIOLOGI KLINIK FKUB/RSUD DR SAIFUL ANWAR (RSSA)
MALANG
Ketua Tim Pencegahan dan Pengendalian Infeksi RSSA
Ketua Tim Program Pengendalian Resistensi Antimikroba RSSA

Types of Microorganisms
Principles of Infection
Transmission
Host resistance
Virulence and pathogenicity
Control of transmission and infection

Development of Infection
Onset and course
Clinical signs and symptoms
Diagnostic tests

Antimicrobial Drugs

Bacteria
Viruses
Chlamydiae, Rickettsiae, Mycoplasmas
Fungi
Protozoa
Parasites (not microorganisms)
Helminths

Cocci
Diplocci
Streptococci
Staphylococci

Staphylococcus aureus

Bacilli
Bacillus

anthracis, Clostridium

tetani

Spirals
Borrelia

sp.
Treponema pallidum

Respiratory tract
Gastrointestinal Tract
Genitourinary tract
Unnatural routes opened up
by breaks in mucous
membranes or skin
Different levels of host
defense mechanisms are
enlisted depending on the
number of organisms
entering and their
virulence.

Extracellular Bacteria
Humoral

immune response
Humoral antibodies produced by plasma cells in
regional lymph nodes and submucosa of respiratory
and gastrointestinal tracts
The antibodies remove the bacteria and inactivate
bacterial toxins to protect the host cell from
invading organisms.

Extracellular Bacteria

Intracellular Bacteria

Antibody neutralizes
bacterial toxins
Complement activation
Antibody and complement
split product C3b bind to
bacteria, serving as
opsonins to increase
phagocytosis.
C3a and C5a induce local
mast cell degranulation
Other complement split
products are chemotactic
for neutrophils and
macrophages.

Intracellular Bacteria
Cell-mediated

immune response (Delayed-type

hypersensitivity)
Activate Natural Killer (NK) cells provide early
defense against bacteria.

In delayed type
hypersensitivity,
cytokines secreted by
CD4+ T cells, such as
IFN gamma, activate
macrophages to kill
ingested pathogens
more effectively.

Four Steps in Bacterial Infection

Attachment

to host cells
Proliferation
Invasion of host tissue
Toxin-induced damage to host cell
Many bacteria have developed ways to overcome some
of these host defense mechanisms

Disease can also be caused by the immune response to

the pathogen.

Pathogen-stimulated overproduction of cytokines can

lead to symptoms of bacterial septic shock, food
poisoning, and toxic shock syndrome.

Bacteria that can survive intracellularly

within infected cells can result in chronic
antigenic activation of CD4+ T-cells, leading
to tissue destruction by a cell-mediated
response with characteristics of a delayed
type hypersensitivity reaction
Cytokines secreted by CD4+ cells can
accumulate, leading to the formation of
granulomas. The concentrations of lysosomal
enzymes in the granulomas can cause tissue
necrosis.

Gram positive, rod-like organism

Bacterial disease caused by a
secreted exotoxin.
Spread via airborne respiratory
droplets
Exotoxin destroys underlying
tissue, forming a tough, fibrous
membrane compose of fibrin,
white blood cells and dead
respiratory cells
Also responsible for systemic
manifestations.

> metachromatic granule

> Club Shaped

C.diphtheriae
(Neisser stain)

Loefflers serum agar slant

Damage to different organs such

as the heart, liver, kidneys and
nervous system.
Choking layer of bacteria and
dead cells in the respiratory
system, accompanied by an
unworldly stench
Difficulty swallowing and
breathing
Pus and blood discharge through
nostrils following death from
asphyxiation

The exotoxin is encoded by the tox gene carried by

phage B (beta)
Some strains can exist in the state of lysogeny.
Exotoxin has two disulfide linked chains, a binding
chain and a toxin chain. The binding chain interacts
with ganglioside receptors on susceptible cells,
facilitating internalization of the exotoxin.
Inhibitory effect of toxin chain on protein synthesis
leads to toxicity.
Removal of the binding chain prevents exotoxin from
entering the cell.

Elek test

Toxoid prepared by treating diphtheria toxin with

formaldehyde.
Reaction with formaldehyde cross-links the toxin,
resulting in loss of toxicity and enhancement in its
antigenicity.
Usually administered with tetanus toxoid and
inactivated Bordetelal pertussis in a combined
vaccine that is given to children 6-8 weeks of age.
Immunization with toxoid induces production of
antibodies which bind to the toxin and neutralize its
activity.

1.
2.
3.
4.
5.

Intracellular Pathogen
Lifelong Infection
Pulmonary Infection
Arrives at Alveoli
Phagocytized by Alveolar Macrophages
M. tuberculosis Blocks Phagosome From Fusing With
lysosome (not nutrient containing vesicles, though)
Other Phagocytes Are Attracted
Forms Multinucleated Giant Cells (Langhans Cells)

Bacilli shaped organism

Pulmonary infection by inhalation of
small droplets of respiratory
secretions containing a few bacilli
Inhaled bacilli are ingested by
alveolar macrophages and multiply
intracellulary by inhibiting formation
of phagolysomes.
Macrophages lyse and large numbers
of bacilli are released.
Cell mediated response by CD4+ T
cells may be responsible for much of
the tissue damage of the disease.
Most common infection of
tuberculosis is pulmonary
tuberculosis.

X-ray :
Pulmonary TB

Direct Smear :
> Sputum
> Ziehl-Neelsen
Staining
> Acid Fast Bacilli
(AFB)

Cause tuberculosis, very important human pathogen

The WHO estimate there are 8 million new cases and
3 million deaths directly attributable to disease/year
tuberculosis is the leading cause of death in many poor and
developing countries.
Factors:
Drug abuse
HIV AIDS
Malnutrition
Poor socio-economic
Dirty environment
Industrial area:

air pollution
over populated

Robert

Koch (1882): establish the cause of tuberculosis

M. tuberculosis;
in tissue: thin straight rods,
in artificial media: coccoid, filamentous.
Size: 0.2 to 0.6 by 1.0 to 4.0m
Non sporeforming
Non motile, capsule (-)

The cell wall are complex, contain large amount of

lipid that are composed of fraction A, B, C, and D.

Mycobacteria are difficult to stain (the cell wall has

a very high lipid content)
However after absorbing the dye, these organisms
resist decolorization by acid alcohol

Acid Fast Bacilli (AFB)

Staining Methodes:
1. Z.N. (Ziehl Neelsen)
Basic stain: carbol fuchsin
Counter stain: methylene blue
red bacilli with blue background
2. Kinyoun carbol fuchsin
3. TTH (Tan Thiam Hok/ H.O.K.Tanzil)
4. Auramine rhodamine (fluorescense dyes)
5. Gram staining : Gram positive

Resistance
Physical effect :
M.tuberculosis are highly resistant to drying
When exposed to direct sunlight, organisms from
culture are killed within 2 hours, in sputum
require an exposure for 20 30 hours.
In dry sputum protected from direct sunlight: 6
8 months
In dust (as droplets) remain infectious for 8 10
days

Resistance against chemical agents and

antibiotic:
Mycobacterium tuberculosis tend to be more
resistant to chemical agent because of the
hydrophobic nature of the cell surface (malachite
green, penicilin incorporated into media without
inhibiting the growth M.tuberculosis)
Sensitive to :
INH
Pyrazinamid
Streptomycin
Rifampin

Resistance rapidly to PAS (para-aminosalicylic acid)

Culture
For growth, Mycobacterium need:
Fatty acid
Amino acid
Nitrogen compound
Glycerol as carbon source
Optimum growth temperature: 35 370C.
Aerobic atmosphere
(M. tuberculosis obligate aerobe),
grow better in atmosphere of 5 10 % CO2
Incubation time : 1014 days , up to 4-8 weeks.
For rapid growers: 7 days.

Media for primary culture:

I.Solid media
Lowenstein-Jensen (L-J) medium-inspissated
egg media agar
Kudoh agar
Petragnani agar
Middlebrook 7H10 agar, 7H11-semisynthetic agar
media
II.Liquid media
Dubos broth
Middlebrook 7H9
Middlebrook 7H12
Liquid media contain Tween 80 and albumin

Morphology of colonies on LJ medium

Human strain:
Non pigmented, rough
Nodular surface with irregular thin periphery
Dry, creamy color
Bovine strain:
Pyramid-shaped colonies
Smooth
Colorless

Can take 2 to 4 weeks to visualize colonies

Lowenstein-Jensens media

Antigenic structure
The component of bacterial cell wall which have
antigenic properties :
Polysaccharides
Peptides
Cell

wall of Mycobacterium contain much

lipid/wax hydrophobic properties

Difficult to be stained/
Impermeable to staining substances.
Resistant to acid and alkali.
Resist phagocytosis and intracellular
killing mechanisms.
Slow growth/long generation time

Lipid Fractions:

Wax A
Wax B
Wax C (serpentine cord virulence)
Wax D (delayed type hypersensitivity)

According to Seibert :
M. tuberculosis composed of 5 fractions:
4 fractions of protein A, B, C & D
1 fraction of polysaccharides

Immunity:

Cellular immunity
Dead M. tuberculosis gives low grade immunity
Live attenuated M. tuberculosis (bovis) BCG
vaccine gives better immunity

Virulence factors
(Determinants of Pathogenicity)

Produce serpentine cord correlated with

the presence of glycolipid : trehalose-6,6dimycolate.
Produce enzyme catalase.
Neutral red test (+).
Mycobacterium do not produce toxin

CLINICAL MANIFESTATIONS
Primary Tuberculosis
Inhaled bacilli alveoli and multiplies in
peripheral lung tissue (tubercles, necrosis)
break spread via blood flow and lymph
miliary tuberculosis
Ingestion (food & beverage) mouth & tonsil
enlarged of cervical lymph nodes cervical
adenitis/scrofula intestines (mesenterial
adenitis) peritonitis
Skin direct contact ulceration
adenitis of regional lymph nodes

Pulmonary TB occurs in the lungs

85%

of all TB cases are pulmonary

Extrapulmonary TB occurs in places other than the

lungs, including the:
Larynx
Lymph nodes
Brain and spine
Kidneys
Bones and joints

Miliary TB occurs when tubercle bacilli enter the

bloodstream and are carried to all parts of the body

46

Reactivation Tuberculosis :

Spreading focus - caseous necrosis from primary

infection
Usually occurs in lung
Reactivation type is characterized by:
Chronic tissue lession
Formation of tubercles
Caseation
Fibrosis

Persons more likely to progress from LTBI to TB disease

include
HIV infected persons
Those with history of prior, untreated TB
Underweight or malnourished persons
Injection drug use
Those receiving TNF- antagonists for treatment of
rheumatoid arthritis or Crohns disease
Certain medical conditions

48

Symptoms of TB depend on where in the

body the TB bacteria are growing. TB
bacteria usually grow in the lungs. TB in
the lungs may cause
a bad cough that lasts longer than 2
weeks
pain in the chest
coughing up blood or sputum (phlegm
from deep inside the lungs)
Other symptoms of TB disease are
weakness or fatigue
weight loss
no appetite
chills
fever
sweating at night

Cytokines produced by CD4+ T cells activate

macrophages, which kill the bacilli or inhibits their
growth.
High levels of interleukin-2 (IL-2), produced by
macrophages, stimulates Th 1-mediated responses.
IL-2 may also contribute to resistance by inducing
production of chemokines that attract macrophages
to the site of infection.
CD4+ T cell mediated response mounted by those
exposed to M. tuberculosis controls the infection and
protects against later infection.

Tuberculosis is treated with several

drugs including isoniazid, rifampin,
streptomycin, pyrazinamide, and
ethambutol.

Drug therapy must continue for at

least 9 months to get rid of the
bacteria since the intracellular
growth of M. tuberculosis makes it
difficult for the drugs to reach the
bacilli.

Vaccine : attenuated strain of M.

bovis called BCG (Bacillus CalmetteGuerin) Most effective against
extrapulmonary tuberculosis.

Biofilms are multicellular aggregates of bacteria

and yeast that congregate on surfaces.
Biofilm may form on any surface exposed to
biofilm-forming bacteria and some amount of
water.
Biofilms are formed to protect the bacteria from
host defenses, antibiotics, and from harsh
environmental conditions.

Bacterial biofilms can form almost anywhere,

even on your teeth if you don't brush for a
day or two.
When they accumulate in hard to reach
places such as the insides of food
processing machines or medical catheters
however, they become persistent sources of
infection.

Biofilms are found almost

everywhere in nature,
including rivers, lakes, soil,
water pipes, and even inside
the human body.
A common type of bacterial
biofilm-responsible for plaque.

Bacterial biofilms are often a cause of

infections associated with medical implants
such as catheters and IV lines and other
medical devices.

Due to the morphology of biofilms, bacteria

capable of forming them are highly resistant to
antibiotics, making treatment very difficult.
In the US alone, one million nosocomial (hospital
acquired) infections each year are caused by
bacterial biofilms, leading to longer
hospitalization, surgery, and even death.

Biofilm fouling of fiber filter

Placque on teeth
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