MICROBIOLOGY PPT LECTURE NOTES | Karya Tulis Ilmiah

Brucella
• The genus Brucella consists of six species,
four of which cause human brucellosis 布布
布布布 Brucella melitensis 布布布布 , Brucella
suis 布布布布 , Brucella abortus 布布布布 , and
Brucella canis 布布布布
• Are all intracellular organisms
• B. neotomae; B. ovis

• Brucella are small (0.4 ～ 0.8
×0.5 ～ 1.5μm), non-motile,
non-capasulate, gram-negati
ve coccobacilli.
• The organism is aerobic, an
d their nutritional requirem
ents are complex.
• All strains grow best in a me
dium enrich with animal ser
um and glucose
• 5-10% carbon dioxide

Antigenic Structure and
classification
• Two main antigen: A and M
• The three main Brucella differ from one another
in the amount or the two main antigen they have
in common :
B.abortus : A:M=20:1
B.melitensis: A:M=1:20
B.suis:
A:M=2:1

B. abortus
• Bacteria is excreted in genital secretions (in
cluding semen), milk, colostrum.
• Survival time:
Cheese at 4oC: 180 days !!!
Water at 25oC: 50 days
Meat and salted meat: 65 days
Manure at 12oC: 250 days !!!!

• Widespread:

Cattle, Bison, Elk, Deer, Moose, Horse, S
heep, Goat, Swine, Donkey, Dogs, Birds, Hares, Fox, Rats, m
ice, Camels and Human.

B. abortus
• Bacteria is excreted in genital secretions
(including semen), milk, colostrum.
• Survival time:
Cheese at 4oC: 180 days !!!
Water at 25oC: 50 days
Meat and salted meat: 65 days
Manure at 12oC: 250 days !!!!

• Widespread:

Cattle, Bison, Elk, Deer, Moose, Horse,
Sheep, Goat, Swine, Donkey, Dogs, Birds, Hares, Fox, Rats,
mice, Camels and Human.

B. abortus
• Sources of Human Infection:
Raw milk and products /Direct contact

• Portal of entry: oral mucosa, nasopharynx and

conjunctivae, genital then X in regional lymph
node and spread to RES (nodes of udder, uterus,
erythritol...). Placentitis with endometritis. Fetus
die with edema /congestion of lung, dissimenated
hemorrhages of epicardium and splenic capsule.
Bacteria in lung and digestive tract of the fetus.

B. suis

B. melitensis
• Goat (1886), Sheep,
Cow (1905 in
Malta), Swine,

Hares, Camels,
Buffalo, Impala.

• Wild pigs, Rats, Swine.
• Abortion,metritis,
bursitis, spondylitis
(Lumbar and sacral),
arthritis, orchitis,
paralysis.

Brucella canis
• Brucella canis was first described as a cause of
abortion in beagles in the USA
• It was subsequently shown to infect dogs in many
other countries, irrespective of breed
• An occasional cause of brucellosis in humans

Spread of Brucella in the body

Incubation period

• Acute or subacute disease follows an incubation per
iod which can vary from 1 week to 6 or more mon
ths.
• In most patients for whom the time of exposure ca
n be identified, the incubation period is between 2 a
nd 6 weeks
• The length of the incubation period may be influen
ced by many factors
– virulence of the infecting strain
– size of the inoculum
– route of infection
– resistance of the host

Portals of entry
• Oral entry - most common route
– Ingestion of contaminated animal products (ofte
n raw milk or its derivatives)
– contact with contaminated fingers

• Aerosols

– Inhalation of bacteria
– Contamination of the conjunctivae

• Percutaneous infection through skin abrasi
ons or by accidental inoculation

Clinical Manifestations
• The presentation of brucellosis is characteristically
variable
• The onset may be insidious or abrupt
• Influenza-like with fever reaching 38 to 40oC
– Limb and back pains are unusually severe, nigh
t sweating and fatigue are marked.
– Anorexia, weakness, severe fatigue and loss of w
eight, depression
– Headache
• The leukocyte count tends to be normal or reduced,
with a relative lymphocytosis
– Relative leukopenia
• On physical examination, splenomegaly may be the

only finding.

COURSE OF BRUCELLOSIS
• If the disease is not treated, the symptoms
may continue for 2 to 4 weeks
– Many patients will then recover spontaneously
– Others may suffer a series of exacerbations

• May produce an undulant fever in
which the intensity of fever and
symptoms recur and recede at about 10
day intervals.

Brucellosis
• Cyprus fever/Gibraltar fever/Malta
fever/Rock fever/Undulant fever

• Most affected persons
recover entirely within
3 to 12 months

• Some will develop
complications
– involvement of various
organs,
– a few may enter an ill-defined
chronic syndrome.

Undulant fever

39.5

37.0

COMPLICATIONS
• Arthritis, often sacroiliitis, and spondylitis (i
n about 10 percent of cases)
• central nervous system involvement including
meningitis (in about 5%)
• Uveitis, epididymo-orchitis
• Endocarditis very rare

• In contrast to animals, abortion is not a fea
ture of brucellosis in pregnant women.

LARGE JOINTS

SPONDYLITIS 布布布

SACROILIITIS 布布布布布

Chronic Brucellosis- Depression

Population risk
• The main source of infection for the general popula
tion is dairy produce prepared from infected milk.
• B. melitensis presents the greatest hazard.
• The milk of infected sheep and goats may contain l
arge numbers of viable organisms, which become c
oncentrated in products such as soft cheeses.
• Indeed, soft cheese has been recognized as a major
vehicle of infection in the Mediterranean region, th

e Middle East and Latin America

Occupational hazard
• Infection arises from occupational or domestic
contact with infected animals or with an
environment contaminated by their discharges
• Farmers and their families, abattoir workers,
butchers and veterinarians are particularly at
risk
• Infected animals that have recently aborted or
given birth present the greatest hazard

Extending spectrum of zoonosis
• The recent isolation of distinctive Brucella strains, t
entatively named Brucella maris, from marine anim
als in the United Kingdom and the United States ext
ends the ecologic range of the genus and, potentially
, its scope as a zoonosis
– seals, sea otters, dolphins and porpoises
• An incident of laboratory-acquired infection suggest

s that this type is pathogenic for humans
• Infection could result from occupational contact wit
h infected seals or cetaceans.

CLINICAL DIAGNOSIS

Sanitary
• Pasteurization of dairy products and use of
protective clothing prevent human infection
. More importantly, systematic identificatio
n and elimination of infected animals and v
accination of animals reduces the reservoir.

Prevention
• Eradication of brucellosis in cattle can be attempted by t
est and slaughter,active immunization of heifers with avi
rulent live strain 19,combined testing,segregation, and i
mmunization.Cattle are examined by means of agglutin
ation tests
• Active immunization of humans against brucella infectio
n is experimental.Control rests on limitation of spread a
nd possible eradication of animal infection,pasteurizatio
n of milk and milk products, and reduction of occupatio
nal hazards wherever possible.

Treatment
• Brucella may be susceptible to tetracyclines or ampicill
in.
• Symptomatic relief may occur within a few days after t
reatment with these drugs is begun.
• However ,because of their intracellular location,the org
anisms are not readily eradicated completely from the
host.
• For best results,treatment must be prolonged.Combined
treatment with streptomycin and a tetracycline may be
considered

YERSINIA

Enterobacteriaceae
Genus Escherichia

Genus Yersinia
• Y. pestis
• Y. enterocolitica
• Y. pseudotuberculosis

Biological Features
– Small, 0.5-0.8 μm in width
1.0-2.0 μm in length.
– Gram-negative rods.
– Sometimes appearing as
cocco bacilli.
– Bipolar Staining:Retaining
stain at the ends of cells.

Biological Features
• Cultural Features
– Facultative anaerobes.
– Optimal growth temperature range form 28˚C
to 30˚C.
– Optimal growth pH: 6.9 布 7.2.
– Growth is more rapid in media containing
blood or tissue fluids.
– Nonmotile when grow at temperatures above 30
˚C.

Pathogenicity
Transmission:
Flea

Bite

Respiratory Tract

Antigenic Structure
•
•
•
•

F1 Antigen:
V,W Antigen:
Yersinia Outer membrane Protein (Yop)
Murine Toxin (MT)
0.3%-0.4% formaldehyde Toxoid
• Endotoxin (LPS)

Ca2+ Dependent
V-W Gene
Gene

Plasmid

W Antigen
V Antigen

F1 Gene

F1 Antigen

Plasmid

LPS

Y. Pestis Virulence factors schematic diagram

Pathogenesis
Invade

Enter

In Groin and
Axilla

Phagocyte

Y. pestis

Lymph Nodes

Pneumonic Plague

Bubonic Plague

Respiratory System

Septicemic Plague

Invade Blood Stream

meningitis

Pathogenicity
• Clinical Forms :
– Bubonic Plague: High fever, Swelling,
Bleeding, Necrosis of lymph nodes
– Pneumonic Plague: chills, cough, respiratory
failure, circulatory collapse ——Black Death
– Septicemic Plague: Fever (39-40 ˚C) , Shock ,
DIC

Y.Enterocolitica & Y.Pseudotuberculosis
• Gram negative, No capsule, No spore, Facultative a
naerobes
• V 布 W antigen
• More than 50 serotypes of Y.Enterocolitica
6 serotypes of Y.Pseudotuberculosis
• Diseases:
–
–
–
–

Gastroenteritis
terminal ileitis, appendicitis, mesenteric lymphadenitis,
dermatitis contusiformia, arthritis
Septicemia

• Sanitary precautions, Antibiotic

Epidemiology
• Plague
– Probably originated in Asia or central Africa.
– One of the earliest record pandemics occurred i
n 542 B.C.
– Three pandemics in the history.
– 1989 布 1998 布 5440 cases, 681 dead.

Immunity
Humoral Immuni
ty
Antibody To:
1) F1 Ag
2) V,W Ag

Cellular
Immunity

Phagocytose

Promote phagocytose , agglutinate and kill bacteria

• A. Specimens:
–
–
–
–
 B.

Diagnosis

Aspirates of lymph nodes
Cerebrospinal fluid
Blood
Sputum

Smears:

 Giemsa’s

stain
 immunofluorescent stain

• C. Culture:

Diagnosis

– All materials Cultured on blood agar and
MacConkey’s agar and in infusion broth
– Positive in 24 hours
– Tentatively identified by biochemical reations
Definite identified by immunofluorescence
CAUTION: All cultures are highly infectious and must be
handled with extreme caution

Diagnosis
• D. Serology:
In patients who have not been previously
vaccinated, a convalescent serum antibody
titer of 1:16 or greater is presumptive
evidence of Y.pestis infection.A titer rise in
two sequential specimens confirms the
serologic diagnosis.

Treatment
• Streptomycin
• Tetracycline:
alternative drug
combination with streptomycin
essential for control early in disease
• Sulfonamides

Summary of Yersinia infections

Bacillus

Spore-Forming Gram-Positive Bacilli:

Bacillus Species
• At least 48 species are known but only
• B. anthracis and B. cereus cause defined
diseases in humans.
 B. anthracis is responsible for the disease anthrax.
 This is a disease primarily of animals (zoonosis) but humans can
acquire via handling, inhaling or ingesting contaminated animal
products.

 B. cereus is predominantly responsible for food
poisoning in humans.
 Bacitracin and polymyxin are two well-known
antibiotics obtained from Bacillus species.


Spores of many Bacillus species are resistant to heat, radiation, disinfectants
and desiccation

• It was from studies on anthrax that Koch
established his famous postulates in 1876
• Pasteur (1881) developed a vaccine against
anthrax

B. anthracis Gram stain
demonstrating spores

B. anthracis,
Colony on SBA

“STICKY” Consistency of
B. anthracis’ Colony on SBA

Anthrax infections are classified
by route of entry
• Cutaneous
• Gastrointestinal
• Respiratory

Cutaneous Anthrax
•
•

•
•
•

> 95% of naturally occurring cases
Spores enter breaks in skin after contact with
contaminated animal products
Papule 布布 - Vesicle 布布 - Ulcer - Eschar 布布
Up to 20% case fatality rate if untreated
Mortality with treatment < 1%

• After a 2- to 3-day incubation period, a small
pimple or papule appears at the inoculation
site.
• A surrounding ring of vesicles develops
• Over the next few days, the central papule
ulcerates, dries, and blackens to form the
eschar

Vesicles & Black Eschar

Painless & Edema
• The lesion is painless and is surrounded by marked edema that
may extend for some distance
• Pus and pain appear only if the lesion becomes infected by a
pyogenic organism
• Similarly, marked lymphangitis 布布布布 and fever usually point
to a secondary infection.

Evolution of an anthrax eschar in
a 4-year-old boy
DAY 6

DAY 10 - 15

•

Evolution of an anthrax eschar in a 4-year-old boy.
(A&B) the lesion when first seen (day 0).Note the arm swollen from the characteristic
edema.
(C) Day 6. (D) Day 10. (E) Day 15.
Although penicillin treatment was begun immediately and the lesion was sterile by a
bout 24 hours,
it continued to evolve and resolve as seen.

Cutaneous anthrax
Differential diagnosis
Ecthyma gangrenosum
Rat-bite fever

Pseudomonas aeruginosa
Streptobacillus moniliformis,
Spirillum minor
Ulceroglandular tularemia
Francisella tularensis
Plague
Yersinia pestis
Glanders
Pseudomonas pseudomallei
Rickettsialpox
Rickettsia akari
Orf
Parapoxvirus
Staphylococcal lymphadenitis Staphylococcus aureus
Cutaneous tuberculosis
Myocbacterium tuberculosis
Leprosy
Mycobacterium leprae
Buruli ulcer
Mycobacterium ulcerans

Cutaneous anthrax

For cutaneous and gastrointestina
anthrax, low-level germination
occurs at the primary site, leading
to local edema and necrosis

Inhalation
• Bacillus spores are inhaled and ingested
by alveolar macrophages 布布布布布布
• These cells carry the bacteria to the
regional lymph nodes, causing necrotic
hemorrhaging which leads to death

Gastrointestinal
• Ingestion of contaminated meat produces
systemic symptoms which can lead to death
• Mortality by gastrointestinal anthrax may
be 50%

Gastrointestinal and pulmonary
anthrax are both more dangerous
than the cutaneous form
because they are usually
identified too late for treatment
to be effective

PATHOGENESIS
• Anthrax infections result only if the bacteria
produce a
– i) capsule (poly-y-D-glutamic acid polypeptide)
– ii) exotoxins
– both encoded on plasmids

•
•
•
•

three proteins
protective antigen (PA) (82. 7 kDa)
lethal factor (LF) (90.2 kDa)
edema factor (EF) (88.9 kDa)

ANTHRAX
ANTHRAX TOXINS
TOXINS
kDa 20

PA
PA

LF
EF

Host
Protease
PA
HOST CELL

The complex (PA+LF or
PA+EF) is internalized by
endocytosis
acidification of the endosome

LF

the LF or EF cross the
membrane into the cytosol via
PA-mediated ion-conductive
channels

Effects of anthrax
exotoxins on macrophages
• Edema toxin is a
calmodulin 布布布布布 dependent adenylate
cyclase that increases
intracellular levels of
cyclic AMP (cAMP) on
entry into most types
of cell
• This is believed to
alter water
homeostasis
– resulting in massive
edema

Effects of anthrax
exotoxins on macrophages
• Lethal toxin is a zinc
metallo-protease that
causes a hyperinflammatory
condition in macrophages
– activating the oxidative
burst pathway
• release of reactive
oxygen intermediates
– production of
proinflammatory cytokines
• responsible for shock
and death.

•

MAPKK denotes mitogenactivated protein kinase
kinase

•Endospores
are
phagocytose
d by
macrophage
s and
germinate
•Macrophage
s containing
bacilli
detach and

•Vegetative anthrax bacilli grow in the lymph node,
creating regional hemorrhagic lymphadenitis
•Bacteria spread through the blood and lymph and
increase to high numbers, causing severe septicemia
•High levels of exotoxins are produced that are
responsible for overt symptoms and death.

•In a small number of cases, systemic anthrax can lead to
meningeal involvement by means of lymphatic or
hematogenous spread
•In pulmonary anthrax, peribronchial hemorrhagic
lymphadenitis blocks pulmonary lymphatic drainage
• leading to pulmonary edema

Once they have been released from the
macrophages, there is no evidence
that an immune response is initiated
against vegetative bacilli

Protective immunity
• Antibodies against protective antigen
• Both the noncellular human vaccines and
live-spore animal vaccines confer protection
by eliciting antibodies to protective antigen
• The poly-g-D-glutamic acid capsule of B
anthracis is poorly immunogenic, and
antibodies to the polysaccharide and other
components of the cell wall are not
protective.

Species differences
• Anthrax has been documented in a wide
variety of warm-blooded animals
• Some species, such as rats, chickens, and
dogs, are quite resistant to the disease
• Others (notably herbivores such as cattle,
sheep, and horses) are very susceptible
• Humans have intermediate susceptibility.

reservoir of B anthracis is
contaminated soil
• Spores remain viable for long periods
• Herbivores, the primary hosts, become infected
when foraging in a contaminated region
• Because the organism does not depend on an
animal reservoir, it cannot readily be eradicated
from a region
– anthrax remains endemic in many countries
• Humans become infected almost exclusively
through contact with infected animals or animal
products

Cycle of infection in nature
• As a susceptible animal with anthrax approaches death, its
blood contains as many as 109 bacilli/ml
• Necrosis of the walls of small blood vessels during the
acute phase of the illness leads to hemorrhages and to
characteristic bloody exudations from the mouth, nose,
and anus, a highly diagnostic sign
• These exudates carry vast numbers of the bacilli
– sporulate on exposure to air
– produce a heavily contaminated environmental site
– potentially capable of infecting other animals for
many years

Handling of carcasses 布布布布
• Sporulation of B anthracis requires oxygen
– therefore does not occur inside a closed carcass
– regulations in most countries forbid
postmortem examination of animals when
anthrax is suspected
– The vegetative cells in the carcass are killed
in a few days by the process of putrefaction.

• In endemic areas, animals that die suddenly should
be handled cautiously
• Livestock should be vaccinated annually.

Do I look
that I am going
?to die

Non-Industrial vs Industrial
Anthrax
• Nonindustrial anthrax
• usually affects people who work with animals or animal
carcasses
– farmers, veterinarians, butchers
– almost always cutaneous

• Industrial anthrax
• acquired from handling contaminated hair, hides, wool,
bone meal, or other animal products
– higher chance of being pulmonary as a result of the inhalation of
spore-laden dust

Transmission

Clinical syndrome

Who is at risk

Cutaneous a

Injured skin or mucous
membranes inoculated by
spores from the soil or a
contaminated animal or
carcass

Skin infection begins as a
raised itchy bump and within
1-2 days develops into a
vesicle and then a painless
ulcer, usually 1-3 cm in
diameter, with a
characteristic black necrotic
area in the center. Lymph
glands in the adjacent area
may swell.

People in endemic areas in
contact with infected anim
als of contaminated soils;
people who work with ani
mals materials (hides, fur,
wool, hair) imported from
endemic area, such as far
mers, veterinarians, knack
ers, butchers and laborator
ians.

Intestinal
anthrax

The ingestion of poorly
Initial signs of nausea, loss of
cooked meat or milk from appetite, vomiting, and fever
infected animals
are followed by abdominal
pain,
vomiting of blood, and
severe diarrhea.

Pulmonary
anthrax

Inhalation of sporecontaining dust where
animal hair or hides are
being handled

nthrax

begins abruptly with high
fever and chest pain,
progresses rapidly to a
systemic hemorrhagic
pathology

Bacillus Cereus 布布布布布布
• B. cereus food poisoning results from the ingesti
on of preformed enterotoxins, producing predo
minantly vomiting and diarrhea.
• The vomiting form is most often associated wit
h ingestion of a heat stable toxin from contamin
ated rice, while the diarrheal form is most often
associated with ingestion of a heat labile toxin f
rom contaminated meat or vegetables

B cereus virulence factors
• A 38 to 46-kDa protein complex has been shown in animal models:
– to cause necrosis of the skin or intestinal mucosa
– to induce fluid accumulation in the intestine
– a lethal toxin
• Responsible for the necrotic and toxemic nature of severe B cereus
infections and for the diarrheal form of food poisoning
Bacillus cereus also produces two hemolysins
Phospholipases produced by B cereus may act as exacerbating
factors
by degrading host cell membranes following exposure of their
phospholipid substrates in wounds or other infections

Bacillus Food Poisoning
Two
Distincttype
Types
• Diarrheal
• diarrhea and abdominal pain
• 8 to 16 hours after consumption of the contaminated food
• Associated with a variety of foods, including meat and
vegetable dishes, sauces, pastas, desserts, and dairy
products

• Emetic ～～ disease
• nausea and vomiting begin 1 to 5 hours after the
contaminated food is eaten
• Boiled rice that is held for prolonged periods at ambient
temperature and then quick-fried before serving is the
usual offender, although dairy products or other foods are
occasionally responsible.