COCCI

10/30/16

GRAM POSITIVE COCCI

Genus

staphylococci
Genus streptococci

Genus staphylococci
General characteristic
gram-positive cocci
Non-sporulating non-motile
grape-like clusters
facultative anaerobe
round colony in media
Some of them are normal flora of the skin and
mucus membrane of human
It can produce catalase, which differentiate it
from the streptococcus
10/30/16

The

genus Staphylococcus includes

over 30 species and subspecies, but

only three are human pathogen:
1. Staphylococcus aureus
2. Staphylococcus epidermides
3. Staphylococcus saprophytics

10/30/16

10/30/16

10/30/16

Staphylococcus aureus
Gram-positive spherical bacteria
Clusters resembling grapes
Produce yellow colony in culture
Facultative anaerobes
Catalase-positive
Oxidase-negative

Coagulase positive and

Can grow in temperature ranging from 15C to

45C
10/30/16

Habitat
S.

aureus is normal inhabitant of human oropharynx with

a carrier rate of 15% to 25%

Transmission

Contact with skin lesions, hands of carrier

Ingestion of contaminated food with enterotoxins

10/30/16

virulence factors:

surface proteins that promote colonization of

host tissues

invasins that promote bacterial spread in tissues

(leukocidin, kinases, hyaluronidase)

surface factors that inhibit phagocytic engulfment

(capsule, Protein A)

biochemical

properties

that

enhance

their

survival in phagocytes (carotenoids, catalase

production);

immunological mask (Protein A, coagulase,

clotting factor)
10/30/16

 Membrane-damaging

membranes

toxins

that

(hemolysins,

lyse

cell

leukotoxin,

leukocidin)
Exotoxins

that

damage

host

tissues

or

otherwise provoke symptoms of disease

Inherent

and

acquired

antimicrobial

resistance

to

agents

10/30/16

10/30/16

10

Pathogenesis
Adherence

to Host Cell Proteins

S.

cells express on their

aureus

surface

proteins that promote attachment such as:

laminin and fibronectin
fibrin/fibrinogen
adhesin

10/30/16

11

Invasion

The

invasion of host tissues by staphylococci

apparently involves the production of a huge

array of extracellular proteins
1. Membrane-damaging toxins
1.1 a-toxin (a-hemolysin)
Most potent membrane-damaging toxin of
S. aureus is a-toxin
platelets and monocytes are particularly
sensitive to a-toxin
Susceptible cells have a specific receptor
for a-toxin which10/30/16
allows the toxin to bind12

1.2-toxin
sphingomyelinase which damages membranes

rich in this lipid

The majority of human isolates of S. aureus do

not express -toxin

1.3 d-toxin
very small peptide toxin produced by most

strains of S. aureus
It is also produced by S. epidermidis.
The role of d-toxin in disease is unknown
10/30/16

13

1.4 Leukocidin
damage membranes
is hemolytic, but less so than alpha hemolysin
1.5 Coagulase and clumping factor
Coagulase is an extracellular protein which
binds to prothrombin in the host to form a
complex called staphylothrombin
it is reasonable to speculate that the bacteria
could protect themselves from phagocytic and
immune defenses by causing localized clotting
10/30/16

14

1.6 Staphylokinase
This factor lyses fibrin
localized fibrinolysis might aid in bacterial spreading
1.7 Other extracellular enzymes
proteases
lipase
Deoxyribonuclease (DNase)
fatty acid modifying enzyme (FAME).
The

first

three

provide

nutrients

for

the

bacteria, and it is unlikely that they have

anything but a minor role in pathogenesis
10/30/16

15

 The FAME enzyme important in abscesses

Modify anti-bacterial lipids and prolong
bacterial survival

10/30/16

16

Clinical manifestation
It

causes superficial skin lesions such as boils

and furunculosis
more

serious infections such as:

Pneumonia
mastitis,
meningitis, and
urinary tract infections; and

Deep-seated infections, such as:

Osteomyelitis and endocarditis
10/30/16

17

S. aureus is a major cause of hospital

acquired (nosocomial) infection of surgical
wounds

and

infections

associated

with

indwelling medical devices

Causes

food

poisoning

enterotoxins into food,

by

releasing

and toxic shock

syndrome by release of superantigens into

the blood stream

10/30/16

18

2.

S.

Avoidance

of

Host

Defenses

aureus expresses a number of factors that

have the potential to interfere with host defense

mechanisms
2.1 Capsular Polysaccharide

The majority of clinical isolates of S aureus

express

surface

polysaccharide

of

either

serotype 5 or 8

Although it does impede phagocytosis in the

absence

of

complement,

it

also

impede

colonization of damaged heart valves, perhaps by

masking adhesins

10/30/16

19

2.2 Protein A
Protein

A is a surface protein of S. aureus

which binds IgG molecules by their Fc region

The

bacteria will bind IgG molecules in the

wrong

orientation

on

their

surface

which

disrupts opsonization and phagocytosis.

Mutants

of S. aureus lacking protein A are

more efficiently phagocytosed in vitro, and

mutants in infection models have diminished
virulence
10/30/16

20

2.2 Leukocidin

S. aureus can express a toxin that specifically

acts on polymorphonuclear leukocytes

Phagocytosis is an important defense against

staphylococcal infection so leukocidin should
be a virulence factor

Leukocidin

causes

membrane

damage

to

leukocytes

10/30/16

21

2.3 Exotoxins
Systemic

release of a-toxin causes septic

shock, while enterotoxins and TSST-1 are

superantigens that may cause toxic shock
Staphylococcal

enterotoxins

cause

emesis

(vomiting) when ingested and the bacterium is

a leading cause of food poisoning
exfoliatin

syndrome

toxin
in

causes

neonates,

the

scalded

which

results

skin
in

widespread blistering and loss of the epidermis

10/30/16

22

 There

are two antigenically distinct forms of the

toxin, ETA and ETB

The

and

toxins have esterase and protease activity

apparently

target

protein

which

is

involved in maintaining the integrity of the

epidermis

10/30/16

23


Superantigens:

S.

enterotoxins

and

toxic

shock

syndrome

toxin

aureus secretes two types of toxin with

superantigen activity, enterotoxins, of which

there are six antigenic types (named SE-A, B, C,
D, E and G), and toxic shock syndrome toxin
(TSST-1)
Enterotoxins

cause diarrhea and vomiting when

ingested and are responsible for staphylococcal

food poisoning
TSST-1

is expressed systemically and is the

cause of toxic shock syndrome (TSS)

10/30/16

24

 When

expressed

systemically,

enterotoxins

can also cause toxic shock syndrome.

TSST-1

is weakly related to enterotoxins, but it

does not have emetic activity

TSST-1

is responsible for 75% of TSS, including

all menstrual cases

Superantigens

specifically

stimulate
without

normal

cells

non-

antigenic

recognition
10/30/16

25

 Up

to one in five T cells may be activated,

whereas only 1 in 10,000 are stimulated

during a usual antigen presentation
Cytokines

are released in large amounts,

causing the symptoms of TSS

Cytokines

causing

are released in large amounts,

the

symptoms

10/30/16

of

toxic

shock

26

Exfoliatin toxin (ET)

The

exfoliatin

toxin,

associated

with

scalded skin syndrome, causes separation

within the epidermis, between the living
layers and the superficial dead layers.
This

is probably why healing occurs with

little scarring although the risks of fluid

loss

and

secondary

infections

are

increased
10/30/16

27

Clinical Feature /diseases

Skin infections, folliculities, wound infections
Food poisoning due to entertoxins
Toxic shock syndrome

10/30/16

28

Clinical significance
S. aureus causes disease
by infecting tissues
typically creating
abscesses and/or by
producing toxins.
The localized host
response to staphylococcal
infection is inflammation,
characterized by swelling,
accumulation of pus, and
necrosis of tissue.

10/30/16

29

1. Localized skin infections:

The most common S. aureus infections are
small,
superficial abscesses involving hair
follicles
(folliculitis) or sweat or sebaceous
glands
Subcutaneous abscesses called furuncles
(boils) often
form around foreign bodies, such as
splinters.

10/30/16

30

Carbuncles are larger, deeper,

multiloculated skin infections that can
lead to bacteremia and require antibiotic
therapy
Impetigo is usually a localized,
superficial, spreading crusty skin lesion
generally seen in children. It can be
caused by S. aureus, although more
commonly by Streptococcus pyogenes, or
10/30/16

31

2. Deep, localized infections:

These may be metastatic from superficial infections or skin
carriage, or may result from trauma.
S. aureus is the most common cause of acute and chronic
infection of bone marrow.
S. aureus is also the most common cause of acute infection
of joint space in children (septic joint).
[Note: Septic joints are medical emergencies because pus
can rapidly cause irreparable cartilage damage. They must
be treated promptly with drainage and an antibiotic.]

10/30/16

32

3. Acute endocarditis, generally associated with

intravenous drug abuse
caused by injection of contaminated preparations or by
needles contaminated with S. aureus.
S. aureus also colonizes the skin around the injection site,
and if the skin is not sterilized before injection, the bacteria
can be introduced into soft tissues and the bloodstream, even
when a sterilized needle is used.

10/30/16

33

4.

Septicemia- is a generalized infection with sepsis

5.Pneumonia: S. aureus is a cause of severe,

necrotizing pneumonia.
6. Nosocomial infections: S. aureus is one of the most
common causes of hospital-acquired infections, often of
wounds (surgical, decubital) or bacteremia associated
with catheters

10/30/16

34

7.Toxinoses- are diseases caused by the action

of a toxin, frequently when the organism that
secreted the toxin is undetectable. Toxinoses
caused by S. aureus include:
A- Toxic shock syndrome, which results in
high fever, rash (resembling a sunburn, with
diffuse erythema followed by desquamation)

10/30/16

35

B.

Staphylococcal gastroenteritis -is caused by ingestion of

food contaminated with enterotoxin-producing S. aureus.

Often contaminated by a food-handler, these foods tend to
be protein-rich and improperly refrigerated.
Symptoms, such as
Nausea
vomiting, and
diarrhea

10/30/16

36

10/30/16

37

C. Scalded skin syndrome- involves the appearance of superficial bullae

resulting from the action of an exfoliative toxin that attacks the
intercellular adhesive of the stratum granulosum, causing marked
epithelial desquamation. The bullae may be infected or may result from
toxin produced by organisms infecting a different site.

10/30/16

38

Diseases
Clinical symptoms
Pathogenicity factors
Gastroenteritis (food 2-6 hours after ingesting toxin: Enterotoxins A-E preformed in
poisoning) - toxin
nausea, abdominal pain,
food
ingested preformed
vomiting, followed by
in food
diarrhea
Infective
endocarditis

Fever, malaise, leukocytosis, Fibrin-platelet mesh, cytolytic

heart murmur (may be absent
toxins
initially)
Abscesses/furuncle Subcutaneous tenderness,
Coagulase, probably the
s/carbuncles
redness and swelling; hot
cytolysins
Toxic Shock
Fever, hypotension,
TSST-1
Syndrome
scarlatiniform rash which
desquamates (particularly on
palms and soles, multiorgan
failure
Impetigo
Pneumonia

Erythematous papules to
bullae
Productive pneumonia with
rapid onset, high rate of
necrosis and high fatality;
nosocomial, ventilator,
postinfluenza, IV drug abuse,
CF, CGD, etc.
10/30/16

Coagulase, exfoliatins
?, all

39

10/30/16

40

10/30/16

41

Resistance of Staphylococci to Antimicrobial Drugs

Hospital

strains of S. aureus are usually

resistant

to

variety

of

different

antibiotics
A

few strains are resistant to all clinically

useful antibiotics except vancomycin

But,

vancomycin-resistant strains are

increasingly-reported
10/30/16

42

 The

term

MRSA

refers

to

Methicillin

resistant Staphylococcus aureus

It

is resistant to all currently available -

lactam antibiotics (penicillins, cephalosporins)

A

plasmid

associated

with

vancomycin

resistance has been detected

in

Enterococcus

faecalis

which

can

be

transferred to S. aureus in the laboratory

it is speculated that this transfer may occur
naturally (e.g. in the gastrointestinal tract)
10/30/16

43

Resistance

is due to:

Mutation
followed

in
by

chromosomal
selection

of

genes
resistant

strains and
Acquisition

of

resistance

genes

as

extrachromosomal plasmids

10/30/16

44

Coagulase-Negative Staphylococci
Of twelve coagulase-negative staphylococcal species that
have been recovered as normal commensals of human skin
and anterior nares, the most abundant and important is S.
epidermidis.
The

second

most

important

coagulase-negative

staphylococcus is S. saprophyticus.
This species are important agents of hospital-acquired
infections associated with the use of implanted prosthetic
devices and catheters.

10/30/16

45

A. Staphylococcus epidermidis
Despite its low virulence, it is a common cause of infection
of implants such as heart valves and catheters.
B. Staphylococcus saprophyticus
Is a frequent cause of cystitis in women, probably related to
its occurrence as part of normal vaginal flora.
It tends to be sensitive to most antibiotics, even penicillin
G.
S.saprophyticus can be distinguished from S. epidermidis
by its natural resistance to novobiocin
10/30/16

46

Laboratory DX
Specimen- surface swabs of wound, pus, blood
sputum, CSF, Faces
Gram stains- Gram positive cocci in cluster
Culture Grow well aerobically and in CO2 enriched
media at 37 oC
Colony appearance

S.aureus Golden colony

S. epidermids- White colony
S. saprophytics-white or yellow colony
10/30/16

47

Biochemical Test
Catalase test
Stapyiococici- positive
Streptococci -negative
Coagulase test S. aureus - coagulase positive
S. epidermids- coagulase negative
S. saprophytics - coagulase negative

10/30/16

48

Prevention and Treatment

- Prevention of contact with S-aureus is almost
impossible
Treatment= penicillin, Ampicillin- for penicillin
sensitive staphylococci. Cloxacillin,
Floxacillin- for penicillin resistance
staphylococci

10/30/16

49

Species

Coagulase/hemolysis

Additional virulence
factors and Lab ID

Common diseases

Staphylococcus
aureus

Coagulase Positive/
-hemolysis

Protein
ATSST-1
Enterotoxins
Exfoliatins
Cytolysins

Infective endocarditis
(dominant cause in IV
drug abusers)
Abscesses
Toxic shock syndrome
Osteomyelitis
Gastroenteritis
Suppurative lesions

Staphylococcus
epidermidis

Coagulase
Negative/
No hemolysis

Susceptible to
novobiocin
Normal skin flora
Plastic adherence:
biofilm

Catheter & prosthetic

device infections
Endocarditis in IV
drug abusers

Staphylococcus
saprophyticus

Coagulase
Negative/
No hemolysis

Resistant to
novobiocin

Urinary tract
infections in newly
active adolescent
women

10/30/16

50

Catalse test- a positive test

10/30/16

51

Staphylococcus aureus
Distinguishing Characteristics
-hemolytic, yellow colonies of Gram+
cocci (clusters)
on blood agar (BA)
Catalase-positive, coagulase-positive,
Salt tolerant; ferments mannitol on
mannitol salt agar

10/30/16

52

Streptococcus
oGram positive
o Facultatively-anaerobic
oCatalase negative, no spores, nonmotile
oLancefield Grouping
Species-specific CHO cell wall antigens
Groups designated A-H, K-V
Some not groupable

10/30/16

53

Classification of streptococcus Based on

1. Hemolytic reaction
2. Lance field carbohydrate group
3. Biochemical reaction

10/30/16

54

2. Classification based on lance field

system
Many streptococci possess a polysaccharide
in their cell wall which is chemically and
antigenically distinct form the cell wall
polysaccharide in other bacteria in their genus
This discovered by Rebecca Lance field
enable the use of specific antisera to identify
specific organisms

10/30/16

55

It is designated A-H and K-V

Clinically important are A,B,C,D,F,G
The main species and groups of Medical
importance
1. S- Pyogens ----------- Lance field group
A
2. S agalactial ----------- Lance field group
B
3. Enterococci ----------- Lance field group
D
NB. Viridan streptococci and anaerobic
10/30/16

56

Streptococcus pyogenes
S. pyogen:
Gram-positive Group A streptococcus
Nonmotile
nonsporeforming coccus
chains
catalase-negative
facultative anaerobe
have a capsule composed of hyaluronic
acid????
10/30/16

57

 Streptococcus

pyogenes.

Left.

Gram

stain

of

Streptococcus pyogenes in a clinical specimen. Right.

Colonies

of

Streptococcus

pyogenes

on

blood

agar

exhibiting beta (clear) hemolysis

10/30/16

58

Pathogenesis
Virulance

factor

M protein fibronectin-binding protein (Protein F)

and lipoteichoic acid for adherence

M-protein to inhibit phagocytosis

Hyaluronic acid capsule as an immunological

cover and to inhibit phagocytosis

Invasins such as streptokinase, hyaluronidase, and

streptolysins O & S
exotoxins, such as pyrogenic (erythrogenic) toxin

which causes the rash of scarlet fever and systemic

toxic

shock
10/30/16

syndrome
59

Extracellular toxins and enzymes

The most important in the context of

pathogenicity are:
Streptolysin

O,

streptolysin

S:

Destroy

the

membranes of erythrocytes and other cells

Pyrogenic streptococcal exotoxins (PSE) A, B, C

Responsible for fever

Streptokinase: Dissolves fibrin
DNases. Breakdown of DNA, producing runny pus
Hyaluronidase. Breaks down a substance that
cements tissues together
10/30/16

60

Laboratory diagnosis
Detection

of the bacteria from clinical specimen

Gram stain
Culture
Catalase test negative

Identification from non pyogen B-hemolytic is by

bacitracin test
S. pyogen is more sensitive
Serology

Group A antigen can be detected using particles

coated with antibodies (latex agglutination)
ASO

10/30/16

61

Clinical significance
1.

Acute pharyngitis or pharyngotonsilitis

2.

Impetigo

3.

Erysipelas

4.

Puerperal sepsis

5.

Acute rheumatic fever

6.

Acute glomerulonephritis

7.

Streptococcal

toxic
10/30/16

shock
62

Treatment
Penicillin

is

still

uniformly

effective

in

treatment of Group A streptococcal disease

No

effective vaccine has been produced, but

specific M-protein vaccines are being tested

Prevention
Rheumatic

fever

is

prevented

by

rapid

eradication of the infecting organism

Prolonged

prophylactic antibiotic therapy is

indicated after an episode of rheumatic fever

10/30/16

63

Streptococcus agalactiae
General characteristics

Approximately

25%

of

healthy

women

harbor

S.

agalactiae in their vaginal flora and suffer no resulting ill

effects

But immunosuppressed patients and new bornes are

susceptible to S. agalactiae infection

Transmission occurs from an infected mother to her

infant at birth and venereally (propagated by sexual
contact) among adults

Group B streptococci are a leading cause of meningitis

and septicemia in neonates, with a high mortality rate
10/30/16

64

 They

are

infections

also
in

an

occasional
postpartum

cause

of

women

(endometritis) and individuals with impaired

immune systems, in whom the organism may
cause septicemia or pneumonia.

10/30/16

65

Laboratory diagnosis
Smear- Non motile gram positive cocci in
chains
Culture- grow in aerobic and anaerobic
environment at temp 35-37%
Grow in ordinary media with shiny or dry
colonies with gray white or colorless
appearance
Shows clear zone of hemolysis on blood Agar
Biochemical Test and Sensitivity Test
Catalase Negative
Bile solubility test Negative
CAMP test positive
Bacitracin Negative
Treatment -Penicillin
10/30/16
66

Streptococcus
(Pneumococcus)

Pneumoniae

Encapsulated, spherical

Anaerobic

Orientation: Pairs or Chains

Lancet-shaped cocci

Catalase negative

Fastidious

(having

requirements

complex

nutritional

that grow only in specially

fortified artificial culture media)

10/30/16

67

Ferment glucose to lactic acid

Gram-stain: POSITIVE

Old cultures: NEGATIVE

Alpha hemolytic

Transmission

Aerosols are major means of dissemination

10/30/16

68

10/30/16

69

Virulance
Capsule
Choline

Binding Proteins

PspA

(protective

antigen):

inhibit

complement-mediated opsonization
Autolysin

LytA:

responsible

lysis

in

stationary phase as well as in the presence

of antibiotics
Autolysin

LytB

is

glucosaminidase

involved in cell separation and

LytC exhibits lysozyme-like activity
10/30/16

70

 Hemolysins

Pneumolysin is stored intracellularly and is

released upon lysis of pneumococci by
autolysin
IgA

protease

10/30/16

71

Clinical significance
Acute
Otitis

bacterial pneumonia

media

Bacteremia/sepsis
Meningitis

10/30/16

72

Laboratory diagnosis
Clinical

sample, gram staining and

culture

1. CATALASE
NEGATIVE
2 H202 2
H20 + 02

10/30/16

73

2. OPTOCHIN TEST (ethylhydrocupreine HCl)

Inhibits growth of pneumococci but not viridans

Optochin positive

Optochin negative
10/30/16

74

3.

Laboratory Identification
BILE SOLUBILITY TEST
Bile or bile salts (surface-active agents)
activate

an

autolytic

AMIDASE

cleaves the bond between

muramic

acid

in

the

which

alanine &

peptidoglycan

resulting in lysis of microorganism

Amidase is present in pneumococcus but

not in viridans streptococci

10/30/16

75

4. QUELLUNG (capsular precipitation)

REACTION
Most rapid & most useful

Pneumococcal

specimen

mixed

with

antipneumococcal serum & methylene

blue

Positive result: refractile & swollen

capsules on oil immersion

10/30/16

76

5. Laboratory Identification

ANIMAL INOCULATION

Fatal
hours

infection
to

within

mice

pneumococci

16-48
injected
antigen

intraperitoneally

For experimental purposes

10/30/16

77

Serologic Diagnosis
Detection

of

pneumococcal

antibodies

Radioimmunoassay
Detection

of

capsular

polysaccharide

10/30/16

78

Treatment
Multiple

antibiotic

resistant

strains

of

S.

pneumoniae that emerged in the early 1970s

Increases

in penicillin resistance have been

followed by resistance to cephalosporins and

multidrug resistance
Antibiotic

susceptibility testing is important

10/30/16

79

Prevention

There

are two types of pneumococcal vaccine:

pneumococcal polysaccharide vaccine (PPV)

and
pneumococcal conjugate vaccine (PCV7)
PPV:

immunizes against 23 serotypes of S.

pneumoniae and is indicated for the protection of

high-risk individuals older than two years
PCV7:

is effective in infants and toddlers (six

weeks to five years of age)

It is made up of seven pneumococcal antigens conjugated
to CRM197 a mutant nontoxic diphtheria toxin
10/30/16

80

Gram negative diplococci

Include

Neisseria

Neisseria

are Gram-negative cocci that typically a kidney

bean appearance
They
The

are nonmotile, non-spore forming, and non-acid fast.

genus contains two pathogenic and many commensal

species, most of which are harmless inhabitants of the

upper respiratory and alimentary tracts
The

pathogenic

(meningococcus),

species
a

are

major

Neisseria

cause

of

meningitidis

meningitis

and

bacteremia, and Neisseria gonorrhoeae (gonococcus), the

cause of gonorrhea

10/30/16

81

NEISSERIA
The

structural elements of N. meningitidis

and N. gonorrhoeae are the same, except

that the meningococcus has polysaccharide
capsule external to the cell wall
Gonococci

are

more

fastidious

than

meningococci
All

Neisseria are oxidase positive

10/30/16

82

Neisseria gonorrhoeae

N.Gonorrhoeae

is

polymorphonuclear

frequently

observed

leukocytes

of

inside
clinical

samples obtained from infected patients

usually

transmitted during sexual contact or,

more rarely, during the passage of a baby

through an infected birth canal
It

does not survive long outside the human body

because it is highly sensitive to dehydration

Gonococci

are unencapsulated
10/30/16

83

1.

Structure

Pili

enhance

attachment

and

resistance

to

phagocytosis

among the most important virulence factors

At least twenty gonococcal genes code for pilin

By shuffling and recombining chromosomal regions

of these genes, a single strain of N. gonorrhoeae can,
at different times, synthesize (express) multiple
pilins that have different amino acid sequences

This process, known as gene conversion, allows

the organism to produce antigenically different
pilin molecules over time
10/30/16

84

2. Lipooligosaccharide:
Gonococcal

lipooligosaccharides (LOS) have

shorter, more highly branched, nonrepeat Oantigenic

side

lipopolysaccharides

chains
found

in

than

do

other

gram-

negative bacteria (LPS).

The

bactericidal antibodies in normal human

serum are IgM molecules directed against LOS

antigens???

10/30/16

85

3. Outer membrane proteins (OMPs):

OMP I, functioning as a porin in complex with OMP
III, is antigenically diverse in different strains of
gonococci
OMP II (opacity protein), along with pili, mediates
attachment of the organism to a host cell
Because of OMP II's ability to undergo extensive
antigenic variation, it also contributes significantly
to the ability of the organism to evade the
immune response and cause repeated infections
10/30/16

86

Classification and Antigenic Types

There

are at least 70 different strains,

characterized

by

the

absence

or

presence of pili, opacity of colonies,

auxotyping (nutritional requirements),
serotyping and genotyping

10/30/16

87

Pathogenesis

associated with the acute onset of symptoms and

purulent mucosal drainage due to the organisms
ability

to

recruit

polymorphonuclear

leukocytes

(PMNs)

Pili and OMP II facilitate adhesion of the gonococcus

to epithelial cells of the urethra, rectum, cervix,
pharynx,

or

conjunctiva

and,

therefore,

make

colonization possible

Pili also enable the bacterium to resist phagocytosis

gonococci produce an IgA protease that cleaves IgA,

helping the pathogen to evade immunoglobulins
10/30/16

88

 After

gonococci

attach

to

the

nonciliated

epithelial cells of the fallopian tube, they are

surrounded by the microvilli, which draw them
to the surface of the mucosal cell
The

gonococci appear to enter the epithelial

cells by a process called parasite-directed

endocytosis??
This

process seems to be initiated by microbial

factors because it does not occur unless the

gonococci are viable and because it involves
host cells that are not normally phagocytic
10/30/16

89

 Gonococci

are

not

destroyed

within

the

phagocytic vacuole; it is not clear whether they

replicate in the vacuoles
The

major porin protein of the gonococcal

outer membrane, Por (protein I), has been

proposed as a candidate invasin (a substance
that helps mediate invasion into a host cell)
Gonococci

membrane

can produce one or several outer

proteins

called

Opa

proteins

(proteins II)
10/30/16

90

These proteins are subject to phase variation and are

usually found on cells from colonies possessing an
opaque phenotype (O+).

At any one time, a gonococcus may express zero, one,

or several different Opa proteins, though each strain
has 10 or more genes for different Opas.

Trypsin-like proteases present in cervical mucus may

help select for protease-resistant transparent (O-)
colony phenotypes

O+

colony

phenotypes

(protease

sensitive)

predominate in cultures taken during the middle

portion of the menstrual cycle?
10/30/16

91

Cervical proteases increase during the second half of

the

cycle,

resulting

in

an

increase

in

the

O-

phenotype

Rmp (Protein III) is an outer membrane protein found

in all strains of N gonorrhoeae

It does not undergo phase variation and is found in a

complex with Por and LOS

It shares partial homology with the Omp A protein of

Escherichia coli. Antibodies to Rmp, induced either by
a neisserial infection or by colonization with E coli,
block bactericidal antibodies directed against Por and
LOS.
10/30/16

92

LOS has a profound effect on the virulence and

pathogenesis of N gonorrhoeae.

Gonococcal LOS produces mucosal damage in fallopian

tube organ cultures and brings about the release of
enzymes, such as proteases and phospholipases, that
may be important in pathogenesis

More recent evidence suggests that gonococcal LOS

stimulates the production of tumor necrosis factor
(TNF) in fallopian tube organ cultures; inhibition of
tumor necrosis factor with specific antiserum prevents
tissue damage
10/30/16

93

 Thus,

gonococcal LOS appears to have an

indirect role in mediating tissue damage

Gonococcal

LOS

is

also

involved

in

the

resistance of N gonorrhoeae to the bactericidal

activity of normal human serum
Oligosaccharides

by

specific

containing epitopes defined

monoclonal

antibodies

are

associated with a serum-resistant phenotype

10/30/16

94

Gonococci

release

are highly autolytic and

peptidoglycan

fragments

during growth
These

fragments, released by bacterial

and/or host peptidoglycan hydrolases,

are toxic for fallopian tube mucosa and
may

contribute

to

the

intense

inflammatory reactions characteristic

of gonococcal disease
10/30/16

95

 Gonococci

(and

meningococci)

bind

only

human transferrin and lactoferrin

This

specificity is thought to be the reason

these

organisms

are

exclusively

human

pathogens
Nevertheless,

the role of transferrin- and

lactoferrin-bound iron in in vivo growth is

unknown

10/30/16

96

Host Defenses

Not everyone exposed to N gonorrhoeae acquires the

disease

This may be due to variations in the size or virulence of the

inoculum, to nonspecific resistance, or to specific immunity

Nonspecific

factors

have

been

implicated

in

natural

resistance to gonococcal infection

In women, changes in the genital pH and hormones may

increase resistance to infection at certain times of the
menstrual cycle

The variability in the susceptibility of gonococcal strains to

the bactericidal and bacteriostatic properties of urine is
thought to be one of the reasons some men do not develop
a gonococcal infection when exposed
10/30/16

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Most uninfected individuals have serum antibodies that

react with gonococcal antigens????

Infection with N gonorrhoeae stimulates both mucosal and

systemic antibodies to a variety of gonococcal antigens

Mucosal antibodies are primarily IgA and IgG

In genital secretions, antibodies have been identified that

react with Por, Opa and LOS, and some of the ironregulated proteins

Gonococcal antigens such as pili, Por, Opa, Rmp, and LOS

elicit a serum antibody response during an infection

Antipilus antibody levels tend to be higher in women than

in men??

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98

 The

predominant IgG subclass that reacts with

a variety of gonococcal antigens is IgG3,

followed by IgG1 and IgG4.
IgG2

is

minimal,

polysaccharides

are

not

suggesting
important

that
in

the

immune response to gonococcal infection

10/30/16

99

ANTIGENIC VARIATION
It

is among several microorganisms

whose surface structures are known to

change antigenically from generation to
generation during growth of a single
strain
The

antigenic

structures

of

major

interest are pili, Opa proteins and LOS,

for which there is evidence of antigenic
variation both in vitro and in vivo
10/30/16

10

10/30/16

10

10/30/16

10

Epidemiology

The only natural host for N gonorrhoeae is the human

Gonorrhea is transmitted almost exclusively by sexual

contact

The highest rates occur in women between the ages of

15 and 19 years and in men 20 and 24 years of age

Gonorrhea is usually contracted from a sex partner who

is either asymptomatic or has only minimal symptoms

It is estimated that the efficiency of transmission after

one exposure is about 35 percent from an infected
woman to an uninfected man and 50 to 60 percent from
an infected man to an uninfected woman
10/30/16

10

Clinical significance

The organisms may cause a localized infection with

the production of pus, or may lead to tissue invasion,
chronic inflammation, and fibrosis.

A higher proportion of females than males are

generally asymptomatic; these individuals act as the
reservoir

for

maintaining

and

transmitting

gonococcal infections.

Genitourinary tract infections: presents with a yellow,

purulent urethral discharge and painful urination. A
greenish-yellow cervical discharge is most common,
often accompanied by intermenstrual bleeding
10/30/16

10

Clinical significance.

The disease may progress to the uterus, causing

salpingitis (inflammation of the fallopian tubes), pelvic
inflammatory disease (PID), and fibrosis.

Infertility occurs in approximately twenty percent of

women with gonococcal salpingitis, as a result of tubal
scarring.

Rectal infections, prevalent in male homosexuals, are

characterized by constipation, painful defecation, and
purulent discharge.

Pharyngitis

is

contracted

by

oral-genital

contact.

Infected individuals may show a purulent exudate, and

the condition may mimic a mild viral or a streptococcal
sore throat

10/30/16

10

Clinical significance

Ophthalmia

neonatorum

is

an

infection

of

the

conjunctival sac that is acquired by a newborn during

passage through the birth canal of a mother infected
with gonococcus.

If untreated, acute conjunctivitis may lead to blindness.

Treatment is with erythromycin because the antibiotic
also eradicates Chlamydia trachomatis, if present.

Disseminated infection: Most strains of gonococci have

a limited ability to multiply in the bloodstream.
Therefore, bacteremia with gonococci is rare. (Note: In
contrast, meningococci multiply rapidly in blood )

10/30/16

10

Diagnosis

Specimens for the laboratory diagnosis of gonorrhea

should be collected before treating the patient

N. gonorrhoeae grows best under aerobic conditions,

and most strains require enhanced CO2

N. gonorrhoeae ferments glucose but not maltose,

lactose, or sucrose.

oxidase-positive

Thayer-Martin medium (chocolate agar supplemented

with several antibiotics that suppress the growth of
nonpathogenic

neisseriae

and

other

normal

and

abnormal flora) is typically used to isolate gonococcus

10/30/16

10

Control

There is no effective vaccine to prevent gonorrhea

Candidate vaccines consisting of pilus protein or Por

are of little benefit

The development of an effective vaccine has been

hampered by the lack of a suitable animal model and
the fact that an effective immune response has never
been demonstrated

Condoms are effective in preventing the transmission

of gonorrhea

The

evolution

of

antimicrobial

resistance

in

gonorrhoeae may ultimately affect the control of

gonorrhea
10/30/16

10

 Strains

with multiple chromosomal resistance to

penicillin,
cefoxitin

tetracycline,
have

been

erythromycin

identified

in

and

different

geographical areas
Sporadic

high-level resistance to spectinomycin

and fluoroquinolones have been reported

10/30/16

10

NEISSERIA MENINGITIDIS

Twelve serogroups have been defined on the basis of

the antigenic specificity of a polysaccharide capsule

The most important disease-producing serogroups are

A, B, C, W-135, and Y

In addition to the group polysaccharides, individual N.

meningitidis strains may contain two distinct classes
of pili and multiple classes of OMPs

Nasopharyngeal carrier rate is10%

Spreading is by respiratory droplets

Sero -groups C and A are associated with epidemic

disease
10/30/16

11

Virulence factors
Meningoccal

endotoxin

(LOS):

is

responsible for many toxic effects

Capsule

Protects

bacteria

from

antibody mediated phagocytosis

Pili

Allow

to

colonization

of

nasopharynx

10/30/16

11

PATHOGENESIS

The

meningococcus

is

an

exclusively

human parasite
it

can either exist as an apparently

harmless member of the normal flora or

produce acute disease
Meningococci

range from carrier state to

bacteremia
Pili

attach to microvilli as introduction to

invasion

10/30/16

11

PATHOGENESIS.
Spread

to blood and CNS produce

systemic endotoxemia
LPS

and peptidoglycan trigger

cytokine release

10/30/16

11

MANIFESTATIONS
Meningitis

is most frequent infection

Meningococcemia

(presence

of

meningococci in the blood) and rash may

progress

to

disseminated

intravascular

coagulation /DIC/
Meningococcal encephalitis
Pneumonia
endocardiatis
Urethritis
10/30/16

11

10/30/16

11

DIAGNOSIS
Laboratory diagnosis

Specimen: Cerebrospinal fluid, blood

Smear: Gram-negative

Culture:

Transparent

or

grey,

shiny,

mucoid

colonies in chocolate agar after incubation at 3537Oc in a CO2 enriched atmosphere. intracellular
diplococci

Serology:
polysacharides

Antibodies
can

be

to

meningo-

measured

using:

coccal
latex

agglutination or hemagglutination tests

10/30/16

11

Biochemical reactions
Species

Glucose

Lactose

Maltose

Sucrose

Neisseria
gonorrhoeae

Positive

Negative

Negative

Negative

Neisseria
meningitidis

Positive

Negative

Positive

Negative

10/30/16

11

TREATMENT
Penicillin

is

the

meningococcal

treatment

infections

antimeningococcal

activity

of

choice

because
and

of

good

for
its
CSF

penetration
Third

generation

cefotaxime

are

cephalosporins
effective

such

as

alternatives

to

penicillin

10/30/16

11

PREVENTION
Rifampin

is

now

the

primary

chemoprophylactic agent, but ciprofloxacin

has also been effective
A,

C,Y, and W-135 polysaccharide vaccines

are useful in high-risk populations

Protein

conjugate

vaccines may enhance

immunogenicity in children
Nonimmunogenic

serogroup B polysaccharide

remains a problem
10/30/16

11

Enterobacteriaceae
Morphology

and General Characteristics

Gram-negative, non-sporing, rod shaped bacteria

Oxidase
Ferment glucose and may or may not produce
gas
Reduce nitrate to nitrite (there are a few
exceptions)

10/30/16

12

 Are facultative anaerobes

If motile, motility is by peritrichous flagella
Many

are

normal

inhabitants

of

the

intestinal tract of man and other animals

Some are enteric pathogens and others
are urinary or respiratory tract pathogens.
Differentiation is based on biochemical
reactions

and

differences

in

antigenic

structure
10/30/16

12

 The

antigenic

structure

is

used

to

differentiate organisms within a genus or

species
Three major classes of antigens are found:
Somatic O antigens these are the heat stable
polysaccharide part of the LPS

Variation from smooth to rough colonial

forms is accompanied by progressive
loss of smooth O antigen

10/30/16

12

Flagellar
Envelope

H antigens are heat labile

or capsule K antigens

overlay the surface O antigen and may

block

agglutination

by

specific

antisera
Boiling for 15 minutes will destroy the K
antigen and unmask O antigens
The K antigen is called the Vi (virulence)
antigen in Salmonella typhi
10/30/16

12

10/30/16

12

Escherichia coli (E.coli)

E. coli is in the bacterial family Enterobacteriaceae
Characteristics of E. coli
Gram-negative
Non-spore forming
Rod-shaped
Lactose fermenter
Motile by means of flagella
Oxidase negative
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12

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12

Grow well on the usual laboratory media in both the presence and
absence of oxygen
Facultative anaerobe
Normal flora of the mouth and intestine:-

Protects the intestinal tract from bacterial infection

Produces small amounts of vitamins B12 and K
Lives symbiotically with us (we help them to live, and they
help us to live)

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12

 There

are more than 700 different serotypes of E. coli

Distinguished

by different surface proteins and

polysaccharides

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12

Transmission

Ingestion of contaminated food or water

Main

sources of E.coli include:Undercooked meat

Unpasteurized milk, apple juice and orange juice
Swimming in or drinking water contaminated with
sewage
Unwashed vegetables and fruits
Bacteria in the diarrhea of infected persons can be
passed on if their hands are not washed well

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Epidemiology
E.coli

Many

have world wide distribution

countries around the world are constantly

suffering from E. coli contamination

Is

mostly common cause of diarrhea in tourists

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Antigens of E.coli
O

antigen

Somatic (on LPS)

171 antigens
H

antigen( protein)

Flagella
56 antigens
K

antigen

Capsule and
(polysaccharides)
80 antigens
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13

Virulence factors of pathogenic strains of E. coli

Fimbriae (Pili)
Hemolysins
Flagella
Exotoxins(Thermolabile toxin (LT), Thermostable toxin (ST)
Endotoxin LPS
Capsules
K antigens
Drug resistance plasmids

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13

Infections Caused by Pathogenic E. coli

While most E. coli are good for us, there are a few strains of E.
coli that are harmful to humans
E. coli is responsible primarily for three types of infections in
humans
urinary tract infections
neonatal meningitis and
intestinal diseases

These conditions depend on a specific array of pathogenic

(virulence) determinants possessed by the organism

13

E. coli is best known for its ability to cause

intestinal diseases
Five classes of E. coli that can cause diarrheal diseases
are recognized:
Enterotoxigenic E. coli (ETEC)
Enteroinvasive E. coli (EIEC)
Enterohemorrhagic E. coli (EHEC)
Enteropathogenic E. coli (EPEC) and
Enteroaggregative E. coli (EAggEC)
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13

Intestinal Diseases..
1. Enterotoxigenic E. coli (ETEC)
Is an important cause of diarrhea in infants and
travelers in underdeveloped countries or regions of
poor sanitation

Travelers diarrhea

Diseases vary from minor discomfort to a severe

cholera-like syndrome
The disease requires colonization and elaboration
of one or more enterotoxins
13

Pathogenesis of ETEC
ETEC strains express.
Heat-labile (LT) enterotoxins: an A-B toxin which increases intracellular
levels of cAMP. Subunit A causes intense and prolonged hyper-secretion of
chloride ions and inhibits the reabsorption of sodium and chloride
The gut lumen is distended with fluid and hypermotility and secretory
diarrhea occur, lasting for several days
It stimulates the production of neutralizing antibodies, and cross-reacts
with cholera toxin

13

ETEC count

The pathogenesis of LT is initiated by binding of LT-B

subunits to the ganglioside receptor GM1 found in
caveolae on the host cell surface

After binding, the LT holotoxin is internalized in vesicles

through the Golgi and the endoplasmic reticulum (ER)

In the Golgi, the holotoxin is disassembled

The A subunit is transported to the ER

Subunit A1 is the active molecule whereas subunit A2

anchors the subunit A to the B pentamer

The A1 subunit translocates through the intracellular

membrane to interact with ADP-ribosylating factors
(ARFs) in the cytoplasm
13

ETEC count

The

activated A1 subunit then ADP-ribosylates

the subunit of a regulatory Gs, an intracellular

guanine nucleotide protein
The

inhibition of Gs GTPase activity results in

the irreversible activation of the transmembranar

adenylate cyclase complex leading to cAMP
accumulation in the cell
Elevated

levels

of

cAMP

activate

cAMP-

dependent protein kinase A (PKA), inducing

phosphorylation

of

the

cystic

fibrosis

transmembrane regulator (CFTR)

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13

ETEC count
Activation

of CFTR provokes the secretion of Cl-

and HCO3
PKA

is also responsible for inhibition Na+ re-

absorption by the Na+/H+-exchanger 3 (NHE3).

Overall,

the result is an osmotically-driven

increased

in

penetration

of

water

and

electrolytes resulting in fluid accumulation in

the intestine.

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13

ETEC count

ST (heat-stable) toxin - causes an increase in cyclic GMP in the

host cytoplasm - leads to secretion of fluid and electrolytes
resulting in diarrhea.

binds to the extracellular domain of guanylate

cyclase C (GC-C) found on the brush border of the
intestinal epithelium

Activation of the GC-C intracellular catalytic domain

leads to the conversion of GTP to cGMP and its
intracellular accumulation

This increase activates cGMP-dependent protein

kinase II (cGMPKII) leading to the phosphorylation of
the CFTR

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14

ETEC count
Also,

elevated

cGMP

levels

inhibit

phosphodiesterase 3 leading to cAMP increase

and activation of PKA.
Then,

PKA act on the CFTR and also inhibits

the re-absorption of sodium.

ST

of

produces secretory diarrhea via stimulation

the

CFTR

channel

and

causing

over-

secretion of Cl- and HCO3 which is followed by

an

osmosis-driven

electrolytes

and

water

release by the cells.

14

Colonization factors (CFAs): facilitate the attachment of

E. coli strains to intestinal epithelium. Usually are pili in
nature

Preventing their rapid removal by intestinal

peristalsis
Epidemiology :World-wide; both adults and children
Incubation 1-2 days; persists 3-4 days

Mild

symptoms,

including

cramps,

nausea,

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14

vomiting, watery diarrhea

2.

Enteroinvassive

E.

coli

(EIEC)

EIEC closely resemble Shigella in their pathogenic

mechanisms and the kind of clinical illness they
produce

These bacteria can penetrate into the

colonic

mucosa,

where

they

cause

ulcerous, inflammatory lesions

Like Shigella, EIEC are invasive organisms
not produce LT or ST toxin and, unlike Shigella,
they do not produce the shiga toxin

14

Pathogenesis
In EIEC the Plasmid encode a gene for a K surface
antigen which enables to: penetrate and multiply within epithelial cells of the
colon causing widespread cell destruction

lyses the phagosomal vacuole, spread through the

cytoplasm and infect adjacent cell

Invade and destroy colonic epithelium

14

clinical syndrome is identical to Shigella dysentery and

includes a dysentery-like diarrhea with fever
Cramps with blood and leukocytes in stool

Uncommon; often food-borne

14

3.

Enteropathogenic

Induce

E.

coli

(EPEC)

a watery diarrhea similar to ETEC, but they do

not possess the same colonization factors and do not

produce ST or LT toxins
Produce a non fimbrial adhesin designated intimin, an
outer membrane protein that mediates the final stages of
adherence

Adhesiveness is mediated by plasmid-encoded

intimin

14

Pathogenesis
diarrhea and other symptoms of EPEC infections
probably are caused by bacterial invasion of host
cells and interference with normal cellular signal
transduction, rather than by production of toxins
Cause childhood diarrhea(Infantile diarrhea)
Infants < 1 year affected
watery diarrhea

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4. Enteroaggregative E. coli (EAggEC)

Have the ability to attach to tissue culture cells in an
aggregative manner

Cause persistent diarrhea in young children

Resemble ETEC strains in that the bacteria adhere to

the intestinal mucosa and cause non-bloody diarrhea
without invading or causing inflammation
14

Pathogenesis of EAggEC
Bacterial cells autoagglutinate
o

Stick to one another

Then produce a hemolysin related to the hemolysin

produced by E. coli strains involved in urinary tract
infections.
Watery diarrhea, with vomiting, dehydration and low
grade fever are significant features

Infants < 6 months

AIDS patients

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5. Enterohemorrhagic E. coli (EHEC)

EHEC are represented by a single strain (serotype
O157:H7), which causes a diarrheal syndrome distinct
from EIEC (and Shigella) in that there is copious
(abundant) bloody discharge and no fever

A frequent life-threatening situation is its toxic

effects on the kidneys (hemolytic uremia).
e.g. Pediatric diarrhea caused by this strain can be fatal
due to acute kidney failure (hemolytic uremic syndrome
[HUS]).
15

Pathogenesis of EHEC
do not invade mucosal cells as readily as Shigella, but EHEC
strains produce a toxin that is virtually identical to the Shiga
toxin.

The shiga toxin / cytotoxin plays a role in the intense

inflammatory response produced by EHEC strains and
may explain the ability of EHEC strains to cause HUS
The toxin is phage encoded

Results in Severe abdominal pain, bloody diarrhea, no

fever
affects children < 5 years
15

Diagnosis of E. coli

Clinical
Laboratory
Serology
culture
Microscopy

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15

Treatment,

prevention

and

Control

Treatment

Antibiotic

therapy must take into consideration the

resistance pattern of the pathogen

Aminopenicillins
ureidopenicillins
cephalosporins,
4-quinolones, or
cotrimoxazole
are useful agents

Severe diarrhea necessitates oral replacement of fluid and

electrolyte losses
15

Prevention and control

Sanitary: Diligent adherence to hygiene is a
paramount
Prophylactic antibiotics. eg cotrimoxazole

10/30/16

15

Shigella
Enteric rods

Non-motile
anaerobes

gram-negative

facultative

Four species
Shigella sonnei (most common in industrial
world)
Shigella

flexneri

(most

common

in

developing countries)
Shigella boydii and
Shigella dysenteriae
15

Shigella count

Characteristics of shigella:
Gram-negative
non-motile
non-spore forming
rod-shaped bacteria
Possess O and some have K antigens
Do not produce gas from glucose
Do not produce H2S on TSI
Non-lactose fermenters
Oxidase negative

15

Habitat and Transmission

Shigella species are found only in the human intestinal

tract
Humans are the only reservoir

Transmitted by the fecal-oral route

Mode of spread: person-to-person, contaminated fingers,
food, flies, fomites etc.

Infective dose: 10-100 viable bacilli

Incubation period: 1 to 7 days

15

Habitat and Transmission

Carriers

of pathogenic strains can excrete the

organism up to two weeks after infection and

occasionally for longer periods.

Duration

of illness:

untreated: severe symptoms for about two weeks

Antibiotic treatment shortens illness and prevent
spread to others
15

Pathogenesis of Shigella

Shigellosis
Two-stage disease:
Early stage:

Watery diarrhea attributed to the enterotoxic activity of

Shiga

toxin

colonization,

following

ingestion

multiplication,

and

and

noninvasive

production

of

enterotoxin in the small intestine

Fever attributed to neurotoxic activity of toxin

Second stage:

Adherence to and tissue invasion of large intestine with

typical symptoms of dysentery

Cytotoxic activity of Shiga toxin increases severity

15

Pathogenesis of Shigella.
Virulence attributable to:
Invasiveness

Attachment
internalization

(adherence)
with

complex

and
genetic

control
Large

multi-gene

virulence

plasmid

regulated by multiple chromosomal genes

Exotoxin (Shiga toxin)

Intracellular survival & multiplication

16

Pathogenesis of Shigella.
Invasiveness in Shigella-Associated Dysentery
Penetrate through mucosal surface of colon and
invade and multiply in the colonic epithelium
Preferentially attach to and invade into M cells in
Peyers patches (lymphoid tissue, i.e., lymphatic
system) of small intestine

16

Pathogenesis of Shigella.

M cells typically transport foreign antigens from

the intestine to underlying macrophages, but
Shigella can lyse the phagocytic vacuole and
replicate in the cytoplasm
Note: This contrasts with Salmonella which
multiplies in the phagocytic vacuole

Actin filaments propel the bacteria through the

cytoplasm and into adjacent epithelial cells with
cell-to-cell passage
effectively

avoiding

antibody-mediated

humoral

immunity
16

Pathogenesis of Shigella.
Characteristics of Shiga Toxin
Enterotoxic, neurotoxic and cytotoxic
Encoded by chromosomal genes
Similar to the Shiga-like toxin
enterohemorrhagic E. coli (EHEC)

of

Note: except that Shiga-like toxin is

encoded by lysogenic bacteriophage

16

Clinical Features of Shigellosis

Persistent diarrhea with frequent and painful passage
of stools consisting mostly of blood, mucus and pus
accompanied

by fever and stomach cramps

Cause breaks (ulcers) in mucous membrane lining of

intestine:Inflammation and tissue damage
Causes painful straining to pass stools; can lead to
rectal prolapse
Ulcers commonly in the rectum
results in increased production of mucus
loss of blood and serum proteins into intestinal
cavity

16

Clinical Features.
Physical signs are those of dehydration beside
fever, lower abdominal tenderness & normal or
increased bowel sounds.
The severity of the disease depends upon the
species one is infected

S. dysenteria is the most pathogenic followed by S. flexneri,

S. sonnei and S. boydii.

Case fatality rates of 5-15%

16

Shigellosis - Complications
Severe anorexia (lack of appetite for food)
Hypoproteinaemia
Dilation of the large intestine
Seizures
Anaemia
Hemolytic uremic syndrome(HUS)
Disseminated intravascular coagulation (DIC)
Reiter syndrome
Persistent diarrhoea
Rectal prolapse
Weight loss and malnutrition
Arthritis, conjunctivitis & urethritis
Myocarditis
16

Laboratory diagnosis
1.
2.
3.
4.

Direct microscopy
Culture
Slide agglutination test
Macroscopy
Bright red stool
Adherent to container
Odorless
small quantity

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16

Treatment, Control and Prevention

Treatment:

Replace fluids: Oral or IV rehydration with fluids and

electrolytes

Treat with antibiotics: trimethoprim/sulfamethoxazole,

Ampicillin, Ciprofloxacin,
Prevention and Control:

Hand washing, especially after defecation

Improved sanitation and hygiene

improve water, waste treatment/disposal and food sanitation
reduce overcrowding, etc.

No effective vaccine

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16

Salmonella
ubiquitous

human

and

animal

humans

usually

pathogens
Salmonellosis

in

takes the form of a self-limiting food

poisoning (gastroenteritis)
but

occasionally manifests as a

serious systemic infection (enteric

fever)
10/30/16

16

Salmonella count

Characterstics of Salmonellae
Gram-negative
Motile (Peritrichous flagella)
Non-sporulated
Produce gas from glucose
Produce H2S on TSI media
Oxidase negative
Non lactose fermenter
facultatively anaerobic
Possessing three major antigens
H or flagellar antigen
O or somatic antigen and
Vi antigen

Vi antigen is a superficial antigen overlying the O antigen;it is

present in a few serovars, the most important being S typhi.
17

Salmonella count

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17

Habitat
The

principal habitat of the salmonellae is the

intestinal tract of humans and animals

Typhi

and Paratyphi A are strictly human

serovars that may cause grave diseases often

associated with invasion of the bloodstream

10/30/16

17

Salmonella count
Salmonella

classification

has

undergone

numerous revisions; currently, all strains are

grouped

in

two

species:

S.

enterica

and

Salmonella bongori
2500 different serotypes
S.

Typhi, S. Choleraesuis, and perhaps

S.

Paratyphi A and S. Paratyphi B are primarily

infective for humans, and infection with these
organisms implies acquisition from a human
source

17

Salmonella count
S. enterica is further divided into 6 subspecies and
contains different serotypes differentiated on the O
and H- Antigens. Examples include:Salmonella serotype (serovar) Typhimurium
Salmonella serotype Enteritidis
Salmonella serotype Typhi
Salmonella serotype Paratyphi

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17

Patogenesis

Salmonella infections in humans vary with

the strain
the infectious dose
the nature of the contaminated food and
the host status

An oral dose of at least 105Salmonella Typhi cells

are needed to cause typhoid in 50% of human
volunteers

whereas at least 109 S. Typhimurium cells (oral

dose) are needed to cause symptoms of a toxic
infection

10/30/16

17

 Infants,

immunosuppressed

patients,

and

those affected with blood disease are more

susceptible

to

Salmonella

infection

than

healthy adults

10/30/16

17

Salmonella pathogenesis..
In humans, Salmonella are the cause of two diseases
called salmonellosis:
enteric fever (typhoid), resulting from bacterial invasion of the
bloodstream E.g S. typhi and S. paratyphi
acute gastroenteritis (Non-typhoidal), resulting from a food
borne infection/intoxication. E.g S. typhimurium, S. enteriditis

Salmonella

invade epithelial cells of the small

intestine

Disease

may remain localized or become systemic,

sometimes with disseminated foci

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17

Salmonella count

1. Gastroenteritis
This

localized disease is caused primarily by

serotypes

enteriditis

and

typhimurium

(nontyphoidal salmonella)
Transmission

is via consumption of contaminated food

Infectious dose:
Typically about 1,000,000 bacteria
But much lower if the stomach pH is raised and if the
vehicle for infection is chocolate (about 100 bacteria)
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17

Salmonella count

Mechanisms of Pathogenicity
Virulence factors:

Fimbriae or pili: attach the bacteria to cells lining the intestinal lumen

The Vi antigen is a capsule that affords salmonellae some protection

from phagocytosis

Endotoxic lipopolysaccharide, which is responsible for septic shock in

patients with bacteriemia

Enterotoxins: responsible for many of the clinical signs of Gastro

enteritis

binds to GM1 gangliosides and cause hypersecretion of fluids and

electrolytes by elevating levels of cAMP.
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17

Salmonella count
Pathogenesis Gastroenteritis
Pathogenic salmonellae ingested in food survive passage
through the gastric acid barrier
invade intestinal mucosa
invasion of epithelial cells stimulates the release of
proinflammatory cytokines

induces an inflammatory reaction

causes diarrhoea and may lead to ulceration and
destruction of the mucosa
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18

Salmonella count

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18

Salmonella count
Clinical Features of Gastroenteritis
Symptoms usually begin within 6 to 48 hours

Nausea and Vomiting

Diarrhoea
Abdominal pain
headache
Fever

duration varies, usually 2 to 7 days

Seldom fatal, elderly or immunocompromised
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18

Typhoid salmonellosis
caused

by the serovars typhi and

paratyphi A, B and C
salmonellae are taken up orally
enter lymphatic tissue
First spreading lymphogenously, then
hematogenously
A generalized septic clinical picture
results
Human carriers are the only source of
infection
10/30/16

18

Salmonella count
Asymptomatic

Carriage

Chronic carriage in 1-5% of cases

following S. typhi or S. paratyphi
infection
Gall bladder usually the reservoir
Chronic
carriage
with
other
Salmonella spp occurs in <<1% of
cases and does not play a role in
human disease transmission
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18

Laboratory
Specimen:

Blood, stool, urine, serum for typhoid fever

Blood

=80% positive in the first week

Stool=70-80%

positive in the second and 3rd

week
Urine

=20% positive in the third and fourth week

Serum

for widal test Positive after second week

of illness

Culture and biochemical tests

10/30/16

18

Serology
Widal test
The

patient serum is tasted for O and H

antibodies

against

following

antigen

suspension
Results
Serum agglutinins rise sharply during the
second and third weeks
High or rising titer of O ( 1:160) suggests
that active infection is present
High titer of H ( 1:160) suggests past
immunization or past infection
High titer of antibody to the Vi antigen
occurs in some carriers
10/30/16
18

Treatment
Chloramphenicol

(CAF)

Norfloxaclin

Prevention

????

10/30/16

18

Vibrio cholerae
Vibrios

are

curved,

Gram-negative

rods

commonly found in saltwater

Cells

may be link end to end, forming S shapes

and spirals
They

are rapidly motile by means of a single

polar flagellum
Pathogenic

vibrios include:

1) V. cholerae, serogroup O1 strains

-epidemic cholera;
2) non-O1 V. cholerae and related strains
18

V.cholera.
There

are over 150 O antigen serotypes, only

two of which (O1 and O139) cause cholera

Cholera

is a life-threatening secretory diarrhea

induced by an enterotoxin secreted by

V.

cholerae
An

O1 variant, V. cholerae biogroup El Tor is

distinguished by biochemical reactions

O139

strains resemble O1 El Tor strains but

possess a unique O

antigen

and have a

polysaccharide capsule
18

Structure, Classification, and Antigenic

Types
The

vibrios that caused epidemic cholera is

subdivided into two biotypes: classical and El

Tor
Both

biotypes (El Tor and classical) contain two

major serotypes, Inaba and Ogawa

These

serotypes

are

differentiated

in

agglutination and vibriocidal antibody tests on

the

basis

of

their

dominant

heat-stable

lipopolysaccharide somatic antigens

19

Structure.
The

cholera group has a common antigen, A,

and the serotypes are differentiated by the

type-specific antigens, B (Ogawa) and C
(Inaba)
An

additional serotype, Hikojima, which has

both specific antigens (rare)

V

cholerae O139 appears to have been

derived from the pandemic El Tor biotype but

has lost the characteristic O1 somatic antigen
19

Structure..

19

EPIDEMIOLOGY
Transmission

is through feco oral

Incubation period is hours-2 days

19

PATHOGENESIS
To

produce disease, V. cholerae must reach the

small intestine in sufficient numbers to multiply

and colonize
Large
Pili

doses required to pass stomach acid barrier

mediate epithelial adherence

CT-stimulated

intestinal hyper secretion; causes

diarrhea
Small intestine loses liters of fluid
K

and

bicarbonate

losses

cause

hypokalemia and acidosis

19

Cholera Toxin
CT
B

is an A-B type ADP-ribosylating toxin

subunit receptor is a ganglioside on cell surface

Its

molecule

polypeptide

is

chains

an

aggregate

organized

into

of

multiple
two

toxic

subunits (A1, A2) and five binding (B) units.

A1

enters

cytoplasm

and

ADP

ribosylates

regulatory G protein
Adenylate
The

cyclase becomes locked in active state

cAMP causes the active secretion of Na, Cl, K,

HCO3 and water out of the cell into the intestinal

lumen
19

MANIFESTATIONS

Extreme watery diarrhea causes large fluid loss

Dehydration and electrolyte imbalance are the major problems

DIAGNOSIS

The initial suspicion of cholera depends on recognition of the

typical

clinical features

in

an

appropriate

epidemiologic

setting.

bacteriologic diagnosis is accomplished by isolation of V.

cholerae from the stool.

The organism grows on common clinical laboratory media such

as blood agar and MacConkey agar

but its isolation is enhanced by the use of a selective medium

(thiosulfate-citrate-bile salt-sucrose agar).
19

TREATMENT
Oral

or

intravenous

fluid

and

electrolyte

replacement is crucial
Antimicrobic

therapy can reduce duration and

severity
Tetracyclines
Trimethoprim and
Erythromycin

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19

PREVENTION
Water

sanitation and cooking shellfish prevent

infection
Vaccines

prepared

from

whole

cells,

lipopolysaccharide, and CT B subunit have

been disappointing, providing protection that
is not long-lasting
Other

Vibrios ?????

19

Pseudomonas

Pseudomonas

is

widely

distributed in soil

and water
P.

seudomonas

colonizes humans

aeruginosa

sometimes

and is the major human

pathogen of the group.

P.

aeruginosa

produces

is

invasive

and

toxigenic,

infections in patients with abnormal

host defenses, and is an important nosocomial

pathogen
19

p. auroginosa
Characteristics

of p. auroginosa

gram-negative
Motile
aerobic rods
some produce water-soluble pigments
Pseudomonas

aeruginosa

is

frequently

present in small numbers in the normal

intestinal flora and on the skin of humans
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20

Antigenic Structure & Toxins

Pili

(fimbriae) extend from the cell surface and

promote attachment to host epithelial cells

The

lipopolysaccharide, which exists in multiple

immunotypes, is responsible for many of the

endotoxic properties of the organism
Most

aeruginosa

isolates

from

infections

produce

extracellular

including

elastases,

proteases,

clinical

enzymes,
and

two

hemolysins: a heat-labile phospholipase C and a

heat-stable glycolipid
20

Antigenic Structure & Toxins

Many

strains

of

aeruginosa

produce

exotoxin A, which causes tissue necrosis and

is lethal for animals when injected in purified
form
The

toxin blocks protein synthesis

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20

Pathogenesis
P

aeruginosa is pathogenic only when introduced

into areas devoid of normal defenses

The

bacterium attaches to and colonizes the

mucous membranes or skin, invades locally, and

produces systemic disease.
promoted

by pili, enzymes, and toxins

Lipopolysaccharide

fever,

shock,

leukopenia,
coagulation,

plays a direct role in causing

oliguria,

leukocytosis

disseminated
and

adult

and

intravascular

respiratory

distress

syndrome.
20

Clinical Findings
P

aeruginosa produces wounds and burns

infection,

giving

rise

to

blue-green

pus

(major)
meningitis
urinary

tract infection

necrotizing
fatal

pneumonia

sepsis

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20

Diagnostic Laboratory Tests

Specimens

Skin lesions, pus, urine, blood, spinal fluid,

sputum, and other material
Smears

Gram-negative rods
Culture

blood agar
P. aeruginosa does not ferment lactose
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20

Treatment
Clinically

significant infections with P aeruginosa

should not be treated with single-drug therapy

success rate is low with such therapy and

can rapidly develop resistance when single

drugs are employed
penicillin

active against P aeruginosaticarcillin

or piperacillinis used in combination with an

aminoglycoside, usually tobramycin

20

Other

drugs

include

aztreonam,

imipenem, and the newer quinolones,

including ciprofloxacin

The

susceptibility

patterns

of

aeruginosa vary geographically

susceptibility tests should be done as an
adjunct

to

selection
10/30/16

of

antimicrobial
20

Helicobacter Pylori

Helicobacter

pylori is a small, curved, gram-

negative, rod-shaped bacterium.

H.

pylori causes more than 90% of duodenal

ulcers and up to 80% of gastric ulcers

Growth

requires

microaerophilic

atmosphere and is slow (3 to 5 days)

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Epidemiology
pylori

infection is one of the most common

bacterial infections in the world

Humans

are the only known reservoir

Transmission

by fecal-oral pathway

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20

pathogenesis

Virulance factor
Urease ammonia production neutralizes acid
flagella allows the organisms to swim to the less
acid pH locale beneath the gastric mucus
surface proteins (Cag protein)
neutrophil-activating protein (NAP)

H. pylori colonization is virtually always accompanied

by

cellular

infiltrate

ranging

from

minimal

mononuclear infiltration of the lamina propria to

extensive

inflammation

with

neutrophils,

lymphocytes, and microabscess formation

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21

H.pylori,
This

inflammation may be due to toxic

effects of the urease or the VacA

The

Cag

protein

may

contribute

by

stimulation of cytokines (interleukin-8), and

a neutrophil-activating protein (NAP) has
been shown to recruit neutrophils to the
gastric mucosa
Added

together

urease,

Cag,

and

NAP

provide ample explanation for the gastritis

that is universal in H. pylori
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infection.

21

H.pylori,
A

prolonged and aggressive inflammatory

response could lead to epithelial cell death

and ulcers, but other virulence factors play a
more direct role.
The

chief

of

these

is

VacA,

which

is

responsible for much of the epithelial cell

erosion seen in human infection.

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21

Clinical manifestation
Primary

infection with H. pylori is either silent or causes

an illness with nausea and upper abdominal pain

lasting up to 2 weeks
Years

later, the findings of gastritis and peptic ulcer

disease include nausea, anorexia, vomiting, epigastric

pain, and even less specific symptoms such as belching
Many

patients are asymptomatic for decades, even up

to perforation of an ulcer
Perforation

can

lead

to

extensive

bleeding

and

peritonitis due to the leakage of gastric contents into

the peritoneal cavity
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21

Diagnosis
The

most

sensitive

means

of

diagnosis

is

endoscopic examination, with biopsy and culture of

the gastric mucosa
Active

or

14

infection can also be diagnosed with a

13

C-

C-labeled urea breath test or with a fecal

antigen detection assay

H.

pylorispecific IgG (serum or salivary antibody)

is a useful marker for epidemiologic studies of past

or current infection, but its sensitivity is suboptimal

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21

Treatment
H.

pylori is susceptible to a wide variety of

antimicrobial agents

Requires

combination therapy with two or

more antibiotics
A

typical regimen includes amoxicillin plus

clarithromycin plus a proton pump inhibitor,

such as omeprazole

21

Genus Campylobacter
General characteristics

Campylobacters cause both diarrhoeal and

systemic diseases and are among the most
widespread causes of infection in the world

Campylobacters are motile, curved, oxidasepositive,

Gram-negative

morphology to vibrios

rods

similar

in

The cells have polar flagella and are often are

attached at their ends giving pairs S shapes

More than a dozen Campylobacter species

have been associated with human disease

Of these C. jejuni, and C. coli are by far the

most common and similar enough to be
considered as one

Some

other

Campylobacter

potential causes of diarrhea

species

are

The primary reservoir is in animals

Transmitted

to

humans

by

ingestion

of

contaminated food or by direct contact with pets

Campylobacters are commonly found in the

normal gastrointestinal and genitourinary flora
of

warm-blooded

animals,

including

sheep,

cattle, chickens, wild birds, and many others.

Domestic animals such as dogs may also carry

the organisms and probably play a significant
role in transmission to humans.

The most common source of human infection is

undercooked poultry

Outbreaks have been caused by contaminated

rural water supplies and unpasteurized milk often
consumed as a natural food

Species of medical importance

- Campylobacter jejuni
- Campylobacter coli

Virulence factors

Lipopolysaccharides with endotoxic activity

Cytopathic extra cellular toxins

Enterotoxins

Pathogenesis and clinical manifestations

Campylobacter jejuni accounts for 90 to 95% of

human campylobacter infections in most parts of
the world.

Campylobacter jejuni and Campylobacter coli

cause enteritis which may take the form of
toxigenic watery diarrhoea or dysentery

In developing countries, C. jejuni and C. coli

cause disease mainly in children under 2 years

The jejunum and ileum are the firs sites to be

come colonized followed by the colon and
rectum

In well developed infections, mesenteric lymph

nodes are enlarged

Diarrhoea is likely to result from disruption of

intestinal mucosa due to cell invasion by
Campylobacters and the production of toxins

The toxin blocks the cell cycle of host cells, but

its precise role is not yet clear

Laboratory diagnosis
Specimen:

Fresh diarrhoeal or dysenteric

specimens containing blood, pus and mucus

Microscopy:

Typical

gull-wing

shaped

gram-negative rods
Typical

darting motility of the bacteria under

dark field microscopy or phase contrast

microscopy

Culture

Campylobacter

species

are

strictly

micro-

aerophilic

C. jejuni and C. coli are thermophilic, i.e. they

will grow at 42-43 C and 3637 C but not at
25 C

Blood agar: C. jejuni and C. coli produce nonhaemolytic

spreading, droplet-like colonies on blood agar.
Butzllers medium and Charcoal - based
blood -free agar containing antimicrobial
agent such as vancomycin, polymyxin B, and
trimethoprims are selective culture media for
campylobacter species

Biochemical tests

Campylobacter

species

are

oxidase

and

catalase positive
Hippurate hydrolysis:

If required, this test (e.g. using a Rosco

diagnostic tablet) can be used to differentiate
C. jejuni from C. coli. Hippurate is hydrolyzed
by C. jejuni and not hydrolyzed by C. coli.

C jejuni .. hydrolyzes hippurate.

C. coli .. does not hydrolyze
hippurate.

Treatment
1. Erythromycin or ciprofloxacin are drugs of
choice for C. jejuni enterocolitis.
2.

C.

jejuni

is

typically

susceptible

macrolides and
fluoroquinolones but resistant to -lactams

to

Prevention and control

1. Proper

cooking of foods.

2. Avoiding

contact with infected human or

animals and their excreta

3. Pasteurizing
4. No

of milk and milk products

vaccine available

Mycobacterium
Mycobacterium

genus contain over 17,000 different strains

TB is a disease of humans and humans are the only

reservoir for the bacterium

Human pathogens under mycobacterium genus include:
M.tuberculosis
M.bovis------consumption of unpasteurized milk
M. leprae
M.aviumTB like disease (HIV Pts)

10/30/16

22

Myco
Classification
Family-Mycobacteriaceae
Order

_Actinomycetales

Genus Four

Mycobacterium

species under this genus:

M. tuberculosis complex
The non-tuberculosis mycobacteria
M. leprae and
M.ulcerance
10/30/16

22

Tube

10/30/16

23

M.tuberculosis

Characteristics

of M. tuberculosis :

Rod-shaped
Neither Gram-positive nor Gram-negative
Non-motile and non-sporulated
Obligate aerobe
Facultative intracellular parasite
Slow generation time (15-20hrs)
Lack exotoxins or endotoxins
Classified as acid-fast bacteria
10/30/16

23

Tube

10/30/16

23

Tube

The disease
tuberculosis
TB

infection means that M.TB is in the body

but the

immune

system

is keeping the

bacteria under control

People

who have TB infection but not TB

disease are NOT infectious

The

immune system does this by producing

macrophage
TB

infection is not considered

a case of TB
10/30/16

23

Tube

Tuberculosis: Infection vs
Disease
TB Infection

TB disease in lungs

M.TB. present

M.TB. present

Tuberculin skin test positive

Tuberculin skin test positive

Chest X-ray normal

Chest X-ray usually reveals lesion

Sputum smears and cultures negative Sputum smears and cultures positive

No symptoms

Symptoms such as cough, fever, weight lo

Not infectious

Often infectious before treatment

Not defined as a case of TB

Defined
10/30/16 as a case of TB

23

Tube
Predisposing factors for TB
infection
Close

contact with large populations of

people, i.e., schools, nursing homes,

dormitories, prisons, etc
Poor

nutrition

HIV

infection is the #1 predisposing

factors for M.TB

10/30/16

23

virulance
M.

tuberculosis do not produce any toxins

Its

ability

to

survive

and

multiply

within

macrophage is its main virulence factor

The

organism is slow growing and tolerates the

intracellular environment
It

is known to prevent the acidification of the

phagosome that is needed for effective killing of

microbes by lysosomal enzymes
Lipoarabinomannan

is a heteropolysaccharide

that inhibits macrophage

activation by IFN-gamma 23
10/30/16

Pathogenesis

Mycobacterium tuberculosis is spread by small

airborne droplets generated by:
coughing
Sneezing
talking or
singing

leads to infection of the respiratory system

The

infection

may

not

be

progress

in

to

disease( immunity, strain and infectious dose)

The majority of the bacilli are trapped in the upper

parts of the airways

10/30/16

23

Patho.
Stages
In

an M.TB sensitive host, the disease tuberculosis will manifest in five stages.

only

a small percent of M.TB infections progress to disease and even a smaller

percent progress all the way to stage 5.

Stage-1
Droplet nuclei are inhaled
After droplet inhaled ,the nuclei are non specifically taken up by
alevioular macrophage

Stage 2
Begins 7-21 days after initial infection
TB multiplies within a group of phagocytic cells called macrophages
Other macrophages migrate to the site from the peripheral blood, and they
begin to engulf the M.TB by phagocytosis
The result is that the macrophages themselves burst

10/30/16

23

Patho.
Stage 3
At

this stage lymphocytes begin to infiltrate

T-cells,

bacteria and respond by liberating cytokines

including IFN that activate the macrophages to

become capable of destroying M.TB
Individual

becomes tuberculin-positive

Activated

macrophages release soluble substances

that

contribute

to

the

pathology,

including

Interleukin 1 ( IL-l) and tumor necrosis factor (TNF)

It

is at this stage that tubercle formation begins

10/30/16

23

Patho.

The center of the tubercle is characterized by "caseation

necrosis" meaning semi-solid or "cheesy" consistency

M.TB cannot multiply within these tubercles (low pH and

anoxic environment)

M.TB can, however, persist within these tubercles for

extended periods of time

Stage 4

Many other macrophages present remain unactivated or

poorly activated

M.TB uses these macrophages to replicate and hence the

tubercle grows

The tubercle is a granulomatous lesion resulting from the

accumulation of macrophages and granulocytes
10/30/16

24

Patho.
Stage 5
For

unknown reasons, the caseous centers of the

tubercles liquefy
This

liquid is very conducive to M.TB growth and

hence

the

orgnism

begins

to

rapidly

multiply

extracellularly
The

walls of nearby bronchi become necrotic and

rupture, this results in cavity formation

This

also allows M.TB to spill into other airways and

rapidly spread to other parts of the lung

10/30/16

EPT
24

Diagnosis of Pulmonary Tuberculosis

1.

Clinical diagnosis

The medical history includes obtaining the

symptoms of pulmonary PTB:
prolonged cough
chest pain
Hemoptysis
low grade fever
night sweating??
appetite loss and

weight loss
It cannot be used to confirm or rule out PTB

10/30/16

24

Diagngnosis.

2.
Laboratory
Tuberculosis
I. Microscopy

Diagnosis

of

Most successful tests for the diagnosis

of mycobacterial disease

Two main staining methods:

ZiehlNeelsen and

Fluorochrome techniques
10/30/16

24

Diagngnosis.

1.

AFB staining procedure:

Flood entire slide with filter 1 % Carbol-fuchsin and
Heat slowly, leave for 5min

2.

Rinse the slide in a gentle stream of running water

3.

Flood the slide with 3% acid alcohol for 2-3 minutes

4.

Rinse the slide thoroughly with water

5.

Flood the slide with 0. 1% Methylene blue for 30

seconds

6.

Rinse the slide thoroughly with water and let it air

dry
10/30/16

24

Diagngnosis.
Grading

system of AFB /WHO/

AFB

Grading report

No AFB/100 oil immersion filed

No AFB seen

1-9/100 oil immersion fields

exact count

10-99/100 immersion fields

1+

1-10/ immersion field

2+

> 10/ immersion field

3+
10/30/16

24

Diagngnosis.

II. Culture
Culture

of mycobacteria remains the

cornerstone

of

microbiological

diagnosis of tuberculosis
Isolation

media available for primary

diagnosis:
Egg based such as LowensteinJenssen
Bactec system(radio labled palmitate)
10/30/16

24

Diagngnosis.

10/30/16

24

Diagngnosis.
III. Molecular Diagnosis
Enable

rapid

detection

of

Mycobacterium

tuberculosis in clinical specimens

Detection

of drug resistance, and typing for

epidemiological investigation
Allow

for detection of mycobacterial DNA or RNA

directly from the specimens before the culture

results are available

10/30/16

24

Diagngnosis.
IV. Tuberculin Skin Test
is

used globally in screening for MTB infection

The

skin test works by injecting Purified Protein

Derivative (PPD) into the skin

PPD

test

measures

hypersensitivity

to

tuberculoprotein
PPD

test interpreted by area of Induration

Positive

PPD indicates past or current infection

10/30/16

24

Diagngnosis.
Interpretation
Read
An

48 to 72 hours later

area of measured induration of 10 mm or

more constitutes a positive reaction

No
A

induration indicates a negative reaction

positive PPD test indicates that the individual

has been infected at some time with M.

tuberculosis or with a strongly cross-reacting
mycobacterium of another species
10/30/16

25

Diagngnosis.
A

negative PPD test in a healthy individual

indicates that he or she has not been infected
with M. tuberculosis

10/30/16

25

Diagngnosis.

Radiographic Procedures

The initial suspicion of pulmonary TB is often based on

abnormal chest radiographic findings in a patient with
respiratory symptoms

Treatment of M. tuberculosis

M. tuberculosis is susceptible to
antimicrobics
Rifampicin
Isoniazide
Bactericidal
Pyrazinamide
Streptomycin
Ethambutol
Ethionamide
Thiacetazone
Bacteriostatic
Paraminosalicylic acid
10/30/16

several

effective

25

Treatment
Ionized

and rifampin are active against both

intra- and extracellular organisms

Pyrazinamide

acts at the acidic pH found within

cells
Streptomycin

does not penetrate into cells and is

thus active only against extracellular organisms

Rifampin

(RIF)(inhibit transcription)

Isoniazid

(INH)( mycolic acids synthesis inhibitor)

10/30/16

25

Treatment

Pyrazinamide

(PZA

)(

bactericidal

drug

active)
Ethambutol

(EMB)(affects the biosynthesis of

the cell wall, it contributes towards increasing

the susceptibility of M. tuberculosis to other
drugs)
Streptomycin

(SM)(inhibits the initiation of

mRNA translation)

10/30/16

25

Treatment
Antimicrobics

Commonly Used in Treatment of

Tuberculosis
FIRST-LINE DRUG

SECOND-LINE DRUG

Isoniazid
Ethambutol
Rifampin
Pyrazinamide
Streptomycin

Aminosalicylic acid
Ethionamide
Cycloserine
Fluoroquinolones
Kanamycin

10/30/16

25

Treatment.
Drug resistant MBT
Drug

resistance in tuberculosis (TB) is a matter

of great concern for TB control programs

Resistance

is usually acquired by alteration of

the drug target through mutation or by titration

of the drug through overproduction of the target
Mono

Resistance: Resistance to single drug

10/30/16

25

Treatment.
Poly-Resistance:

Resistance to more than one

drug other than Rifampicin and Isoniazid together

Multi-Drug

Resistance (MDR): Resistance to at

least Isoniazid and Rifampicin

Extensive

Drug Resistance (XDR-TB): MDR plus

Resistance to:
A

fluoroquinolone

Ciprofloxacin,Ofloxacin,

Levofloxacin, Moxifloxacin etc. and

One or more of injectable agents: Kanamycin,
Amikacin, capreomycin, Viomycin
PREVENTION?????
10/30/16

25

M.leprea
Typical

acid-fast bacillisingly, in parallel

bundles, or in globular masses

are

regularly found in scrapings from skin or

mucous membranes (particularly the nasal

septum) in lepromatous leprosy
The

bacilli

are

endothelial

cells

often
of

found

blood

within

vessels

or

the
in

mononuclear cells

10/30/16

25

Pathogenicity
M.

leprae is transmitted from human to human through

prolonged contact

Obligate intracellular parasite of macrophages

Clinical significance
Leprosy

is a chronic granulomatous condition of peripheral

nerves and mucocutaneous tissues, particularly the nasal

mucosa.
It

occurs between two clinical extremes: tuberculoid and

lepromatous leprosy
In

tuberculoid leprosy, the lesions occur as large maculae

(spots) in cooler body tissues such as skin (especially the nose,

outer ears, and testicles), and in superficial nerve endings
Neuritis

leads to patches of anesthesia in the skin

10/30/16

25

patho.

The lesions are heavily infiltrated by lymphocytes and

giant and epithelioid cells, but caseation does not occur.

The patient mounts a strong cell-mediated immune

response and develops delayed hypersensitivity

which can be shown by skin test with lepromin, a

tuberculin-like extract of lepromatous tissue.

The

course

of

lepromatous

leprosy

is

slow

but

progressive.

Large numbers of organisms are present in the lesions

and

reticuloendothelial

system,

and

immunity

is

severely depressed
10/30/16

26

10/30/16

26

10/30/16

26

Laboratory identification
M.

leprae is an acid-fast bacillus. It has not

been

successfully

maintained

in

artificial

culture, but can be grown in the footpads of

mice, which may also be a natural host
although playing no role in human disease.
Laboratory

diagnosis of lepromatous leprosy,

where organisms are numerous, involves acidfast stains of specimens from nasal mucosa or
other infected areas.
In

tuberculoid

leprosy,
10/30/16

organisms

are
26

Treatment and prevention

Several

drugs are effective in the treatment of

leprosy, including sulfones such as dapsone,

rifampin, and clofazamine.
Treatment

is prolonged, and combined therapy

is necessary to ensure the suppression of

resistant mutants.
The

fact that vaccination with BCG has shown

some

protective

encouraged

effect

further

in

interest

leprosy
in

has

vaccine

development.
10/30/16

26

Spirochetes
The

spiral

Many

are difficult to see by routine microscopy

Many

are thin and take stains poorly

Only

darkfield

immunofluorescence,
techniques

that

or

microscopy,
special

effectively

staining

increase

their

diameter can demonstrate these spirochetes.

26

 Some

are strict anaerobes, others require low

concentrations of oxygen and still others are
aerobic
Some have not been isolated in culture
Spirochetes

that

are

important

human

pathogens are confined to three genera:

Treponema (T. pallidum causes syphilis)
Borrelia (B.burgdorferi causes Lyme disease,
B. recurrentis causes relapsing fever) and
Leptospira

(L.

interrogans

causes

leptospirosis)
26

Treponema
Nonpathogenic

treponemes may be part of the

normal flora of
intestinal tract
oral cavity or
genital tract

Some of the oral treponemes have been

associated

with

gingivitis

and

periodontal

disease

26

Treponema pallidum, subsp. pallidum

(Syphilis)
fastidious

organism
has not been successfully cultured in vitro
(Growth is limited and slow in cell Culture)
The

lipid composition of T pallidum is complex,

consisting of several phospholipids, including

cardiolipin,
glycolipid

and
which

immunologically

poorly
is

characterized

biochemically
distinct

and
from

lipopolysaccharide

26

EPIDEMIOLOGY

is an exclusively human pathogen

Transmission is by contact with mucosal surfaces or blood

In most cases, infection is acquired from direct sexual

contact with an individual who has an active primary or
secondary syphilitic lesion

Less commonly, the disease may be spread by non

genital contact with a lesion (eg, of the lip), sharing of
needles by intravenous drug users, or transplacental
transmission to the fetus within approximately the first 3

years of the maternal infection

Modern screening procedures have essentially eliminated
blood transfusion as a source of the disease

26

PATHOGENESIS

The spirochete reaches the subepithelial tissues through in

apparent breaks in the skin /passage between the
epithelial cells
where it multiplies slowly with little initial tissue
reaction
May be due to the relative paucity of exposed
antigens on the surface of the organism, but no
specific reasons are known
As lesions develop, the basic pathologic finding is an
endarteritis
The small arterioles show swelling and proliferation of their
endothelial cells
This reduces or obstructs local blood supply, probably
accounting for the necrotic ulceration of the primary lesion
and subsequent destruction at other sites
Slow multiplication produces endarteritis, granulomas
Ulcer heals but spirochetes disseminate

27

 syphilis

is then silent/unknown reason/ until

the disseminated secondary stage develops

and then silent again with entry into latency
Although

evasion of host defenses is clearly

taking place, the mechanisms involved are

unknown.
Latent periods may be due to surface
binding of host components

27

The inflammatory response to:

immune complexes

spirochetal lipoproteins and

complement in arteriolar walls accounts for

some of the injury in syphilitic lesions.

The granulomatous nature of the lesions in late

syphilis

is

consistent

with

injury

caused

by

delayed-type hypersensitivity responses

no toxins, virulence factors, or other molecules

can yet be linked with specific features of syphilis

10/30/16

27

MANIFESTATIONS

I.

Primary stage

Spirochetes

multiply locally at the site of entry,

and some spread to nearby lymph nodes and

then reach the bloodstream
In

210

weeks

after

infection,

papule

develops at the site of infection and breaks

down to form an ulcer with a clean, hard base
("hard chancre")
The

inflammation

is

characterized

by

predominance of lymphocytes and plasma cells

27

Clinical significance

This "primary lesion" always heals spontaneously,

but 210 weeks later the "secondary" lesions appear

II. Secondary stage

Manifest with generalized Maculopapular rash and

white patches in the mouth

There may also be syphilitic meningitis, hepatitis,

nephritis

The secondary lesions also subside spontaneously

Both primary and secondary lesions are rich in

spirochetes and highly infectious
27

Clinical significance
III. Latent stage

In about 30% of cases, early syphilitic infection

progresses spontaneously to complete cure without
treatment

In another, the untreated infection remains latent

(principally evident by positive serologic tests)

Patients are symptom free but relapse can occur

In early stage patient is infectious but in late stage

of latent syphilis patients are none infectious

27

Clinical significance

In the remainder, the disease progresses to the

"tertiary stage

IV. Tertiary stage

characterized by the development of granulomatous

lesions

(gummas)

in

skin,

bones,

and

liver;

degenerative changes in the central nervous system

(meningovascular syphilis); or cardiovascular lesions

In all tertiary lesions, treponemes are very rare, and the

exaggerated tissue response must be attributed to
hypersensitivity to the organisms

However, treponemes can occasionally be found in the

eye or central nervous system in late syphilis
27

DIAGNOSIS
Specimens

include tissue fluid expressed from

early surface lesions for demonstration of

spirochetes; blood serum for serologic tests
1. Darkfield Examination
A

drop of tissue fluid or exudate is placed on a

slide and a coverslip pressed over it to make a

thin layer.
The

preparation is then examined under oil

immersion

with

darkfield

illumination

for

typical motile spirochetes

27

2. Serologic Tests
These

tests

use

either

nontreponemal

or

treponemal antigens

Nontreponemal includes:
VDRL (Venereal Disease Research Laboratory) and
RPR (rapid plasma reagin)

Treponemal

tests includes

Fluorescent Treponemal Antibody (FTA-ABS) Test

Treponema

pallidum-Particle

Agglutination

(TP-PA)

Test(T pallidum hemagglutination (TPHA))

27

TREATMENT
Doxycycline

and amoxicillin are the first-line

antimicrobics for the treatment of early Lyme

disease and arthritis
Prevantion?????

27

Rickettsiae
General Characteristics:

Obligate

intracellular

gram-negative

coccobacilli

occurring in single, pairs, short rods and filaments

Poorly stained in gram reaction

Grow in yolk sac of embryonated eggs, cell culture

and laboratory animals

Destroyed by heat, drying and bactericidal chemicals

With the exception of C. burnetii (Coxiella . burnetii) ,

the organisms are transmitted by arthropods

C. burnetii is transmitted exclusively by inhalation of

dust containing the pathogens
10/30/16

28

Rickettsiae.
Clinical Features:
Clinical

illness is due to the invasion and multiplication of

rickettsiae in the endothelial cells of small blood vessels

It

manifests with fever, headache, malaise skin rash and

enlargement of liver and spleen

The

genus rickettsia has three main groups based on

their antigenic structure

These

are:

Typhus group
Scrub typhus group
Spotted fever group
10/30/16

28

Rickettsiae.
Hosts

and vectors of the medically important

rickettsiae
Organism
Vector
Disease
Host
Typhus group
R. prowazeckii Louse-borne
typhus
R. typhi
Murine typhus
Scrub typhus
Scrub typhus
R.
tsutsugamushi
Spotted fever
Rocky Mountain
group
Spotted fever
R. conorii
Rickettsial pox
R. rickettis
R. akaris

Man
Rat

Body
louse
Rat flea

Rodents

Mite

rodents, dogs
Rodents, dogs
Mice

Tick
Tick
Mite

10/30/16

28

Rickettsiae.
R.

prowazeckii and R. typhi are common in Ethiopia

1. Rickettisia Prowazeckii

It causes epidemic or louse-borne typhus

Clinical Features:

It is transmitted by self-inoculation of the organism by

scratching after bite by infected louse
The illness manifests with sudden onset of fever, headache,
malaise and skin rash.
Epidemics of the disease are associated with overcrowding
,cold weather ,lack of washing facilities ,famine and war
10/30/16

28

Rickettsia Typhi

It causes endemic or flea-borne typhus

Clinical Features:

It is transmitted to man when bitten by an infected ratflea

The disease is milder than louse-borne typhus and

occurs in those individuals living or working in highly ratinfested area.

Laboratory Diagnosis:

Specimen: Serum for serological test

The serological tests to diagnose typhus are

Weil-felix
10/30/16

28

Treatment

Tetracycline
Chloramphenicol

10/30/16

28

Chlamydia

Chlamydiae

are

obligate

intracellular

parasites
Cell

with

envelope consists of two lipid bilayers

associated

cell

wall

material

that

resembles a gram-negative envelope

Contains

no

peptidoglycan,

and

muramic acid is not present

Two

stages of reproductive cycle:

Elementary bodies (EB) (outsideof host cells)

initial bodies (IB) (reproduce inside the

host cells)

10/30/16

28

Chlamydia.

The

EB

is

taken

up

by

phagocytosis

into

the

elementary

body

susceptible host cells

Once

inside

the

cell,

prevents fusion of the phagosome and lysosome,

protecting itself from enzymatic destruction
The

particle reorganizes over the next eight hours

into a larger, noninfectious reticulate body,

which becomes metabolically active and divides
repeatedly by binary fission within the cytoplasm
of the host cell
10/30/16

28

Chlamydia.

As the reticulate body divides, it fills the endosome

with its progeny, forming an inclusion body

After 48 hours, multiplication ceases and reticulate

bodies

condense

to

become

new

infectious

elementary bodies

The elementary bodies are then released from the cell

by cytolysis, ending in host cell death

The three human pathogen species of chlamydiae are:

C. psittaci
C. trachomatis and
C. pneumoniae
10/30/16

28

Chlamydia psittaci
The
This

natural hosts of C. psittaci are birds

species

causes

infections

of

the

respiratory organs, the intestinal tract, the

genital tract and the conjunctiva of parrots
and other birds
Humans

are infected by

inhalation

of

dust

(from

bird

excreta

containing the pathogens)

10/30/16

28

Chlamydia psittaci..
After

an incubation period of one to three

weeks, psittacosis /ornithosis presents with

fever, headache, and a pneumonia that often
takes an atypical clinical course
The

infection may, however, also show no

more than the symptoms of a common cold, or

even remain clinically silent

10/30/16

29

Diagnosis
Diagnosis

is

primarily

serologic
The pathogen can be grown
from sputum in special cell
cultures
Therapy:
Tetracyclines
(doxycycline)

10/30/16

29

Chlamydia trachomatis

C.

trachomatis is a pathogen that infects only

humans
General characteristics
There

are 15 serotypes of C trachomatis

C. trachomatis serotype A, B, Ba, C causes

trachoma
C. trachomatis serotype D-K causes genital
infection
C.

trachomatis

serotype

L1-L3

causes

lymphogranuloma venereum (LGV)

10/30/16

29

C. trachomatis serotype A, B, Ba, C

It

causes trachoma

Trachoma

is a follicular keratoconjunctivitis

Incubation

period is 3-10 days

Transmission:

- is though direct contact like

eye-to eye by infected fingers

10/30/16

29

Clinical feature

The

disease

occurs

in

all

climatic

zones,

although it is more frequent in warmer, lessdeveloped countries

It

is associated with a low standard of living and

poor personal hygiene

It

manifests as a chronic keratoconjunctivitis

producing scarring and deformity of the eyelids,

corneal vascularization and opacities which may
lead to blindness
10/30/16

29

Laboratory Diagnosis
involves

detection

conjunctival

of

C.

smears

trachomatis
using

in

direct

immunofluorescence microscopy
The

fluorochrome- marked monoclonal antibodies

are directed against the MOMP (major outer

membrane protein) of C. trachomatis
The
The

pathogen be grown in cell cultures

therapeutic method of choice is systemic

and local application of tetracyclines over a period

of several weeks
10/30/16

29

prevention
Improving hygienic standard
Treatment
Surgical

of cases with antibiotics

correction of eyelid deformities

10/30/16

29

C. trachomatis serotype D-K

responsible

for
3060%
of
cases
nongonococcal urethritis (NGU) in men

The

of

pathogens are communicated by venereal

transmission.
The

source of infection is the female sexual

partner, who often shows no clinical symptoms

Femal

----- urihritis, Cervicits

and Pelvic

inflammatory
If

complicated in female, it cause infertility and

ectopic pregnancy
10/30/16

29

C. trachomatis serotype D-K..

Inclusion

conjunctivitis

resembling

trachoma (TRIC)
Transmission is by self-inoculation of the
eye with infected genital secretion

Neonatal inclusion conjunctivitis and

neonatal pneumonia
Transmission is during passage through
the infected birth canal
10/30/16

29

C. trachomatis serotype D-K..

The

relevant diagnostic tools include:

Detection under the microscope in smear

material using direct immunofluorescence
Direct

identification

by

means

of

amplification of a specific DNA sequence in

smear material and urine
Growing in special cell cultures

10/30/16

29

C. trachomatis serotype L1-L3

Causes ymphogranuloma venereum (LGV)

A herpetiform primary lesion develops at the site of

invasion in the genital area, which then becomes an
ulcus (soft chancre) with accompanying lymphadenitis

Complication : Elephantiasis of penis ,scrotum or

vulva due to lymphatic obstruction

Laboratory

diagnosis

is

based

on

isolating

the

proliferating pathogen in cell cultures from purulent

material obtained from the ulcus or from matted
lymph nodes
10/30/16

30

Treatment
Tetracyclines

and macrolides are the potentially

useful antibiotic types

Chlamydia pneumoniae
This

new chlamydial species causes infections

of the respiratory organs in humans that usually
run a mild course:
Influenza like infections
Sinusitis
Pharyngitis
Bronchitis
pneumonias (atypical)
10/30/16

30

C. pneumoniae..

Clinically
The

silent infections are frequent

pathogen is transmitted by aerosol

droplets
These

infections are probably among the

most frequent human chlamydial infections

The

antibiotics of choice are tetracyclines or

macrolides

10/30/16

30

Mycoplasma
General

Characteristics:

Formerly

named

as

pleuropneumonia-like

organism (PPLO)
The smallest living micro-organism capable of

independent existence
Highly pleomorphic due to absence of rigid cell

wall, instead bounded by a unit membranes

Completely

resistant

to

penicillin

and

vancomycine
Have an affinity to mammalian cell membrane
10/30/16

30

Mycoplasma..
Human

family

pathogen species are found in the

Mycoplasmataceae,

genus

Mycoplasma and Ureaplasma

Species

of medical importance

Mycoplasma pneumoniae
Mycoplasma hominis
Ureaplasma urealyticum
Other species are part of the apathogenic
normal flora
10/30/16

30

Mycoplasma pneumoniae
It

is wide spread in nature being found in soil

,plant ,mucous surface of animal

Transmission:-infected
The

respiratory secretion

cells attach themselves to the epithelia of the

trachea, bronchi, and bronchioles.

The

mechanisms that finally result in destruction of

the epithelial cells are yet unknown

The

infection develops into pneumonia with an

inflammatory exudate in the lumens of the bronchi

and bronchioles
10/30/16

30

Clinical features
The

incubation period is 1020 days

The

infection manifests with fever, headache

and a persistent cough

The

clinical pictures of the infection course is

frequently

atypical,

i.e.,

the

pneumonia

cannot be confirmed by percussion

Complication

may result hemolytic anemia

10/30/16

30

Clinical features.
Laboratory Diagnosis:
Specimen: Sputum
Culture
Serology

Treatment:
Tetracycline
Erythromycin

10/30/16

30

Infections of the urogenital tract

Caused by M. hominis and Ureaplasma urealyticum

M.urealyticum is considered responsible for 1020%

of cases of nongonococcal urethritis and prostatitis
in men

Both agents can be cultured.

They grow more rapidly than M. pneumoniae, and

can be distinguished by their carbon utilization
patterns

M. hominis degrades arginine, U. urealyticum

hydrolyses urea
10/30/16

30

Infections of the urogenital tract.

A

number of serotypes of M. hominis have been

described. It is important to note that M.

hominis isolates are uniformly resistant to
erythromycin, in contrast to other mycoplasmas
U.

urealyticum is a common cause of urethritis

when neither gonococcus nor chlamydia can be

demonstrated, particularly in men
Tetracycline is effective for specific treatment

10/30/16

30

Assignment

Enterbacter
Citrobacter

Group one

Bacillus spp.
Clostridium spp
Corynebacterium
Listeria
Pseudomonas spp.
Campylobacter spp.

Group two

Group three

Group four

Haemophilus spp.
Brucella spp.
Bordetella spp.

Group five

Rickettsiae
Mycoplasma species

Group six
10/30/16

31

THANK YOU
10/30/16

31