Bacteriology

Gram Negative
Bacteria

Enterobacteriaceae:

large family of Gram-negative bacteria

Salmonella, Escherichia coli, Yersinia
pestis, Klebsiella , ShigellaProteus, Enteroba
cter, Serratia, and Citrobacter.
They are so called because they inhabit the
intestines of humans and animals .

Enterobacteriaceae family has four major

features:
All ferment glucose (dextrose)
All reduce nitrates to nitrites
All are oxidase negative
All except Klebsiella, Shigella and Yersinia
are motile

A- Escherichia coli

is a Gram-negative, rod-shapedbacterium of the genus Escherichia that

is commonly found in the lower intestine of warm-blooded organisms.
Most E. coli strains do not cause disease, but virulent strains can
cause gastroenteritis, urinary tract infections

Diarrheal diseases are diagnosed by examining stool microscopically

(rod shaped bacteria).
Urinary tract infections are diagnosed by testing and culturing a
midstream urine sample then biochemical test.

A- Escherichia coli

Lactose Fermenters, on MacConkeys agar gives pink

color, due to fermentation of lactose into lactic acid which
change the color of medium (indicator neutral red) into pink

B- Klebsiella

Laboratory diagnosis
perform large polysaccharide capsules.

Lactose Fermentor.

Klebsiella pneumonia causes pneumonia in individuals

comprised by alcoholism, diabetes and also causes urinary
tract infections and bacteremia

C- Shigella
All species cause bacillary dysentery (infection in large intestine,
causing diarrhea with blood, mucus and painful abdominal
cramping).
non-motile .
Laboratory diagnosis:
Taken sample from Stool, not lactose fermenters gives creamy
color on MacConkey agar.

D- Proteus
-Urinary tract infections and also wound infections, pneumonias.
Laboratory diagnosis:
characteristic swarming motility.
Inability to metabolize lactose, gives creamy colonies on
MacConkeys agar
distinct fishy odour, Proteus produce Urease enzyme catalyzes
urea to ammonia

E- Salmonella species:

rods motile with peritrichous flagella

cause illnesses such as typhoid fever

(smallintestine)(Salmonella typhi) (diarrhea, loss of appetite and
nausea)

paratyphoid fever (Salmonella paratyphi) and food

poisoning (Salmonella enteritidis).

Laboratory diagnosis of typhoid fever:

by cultivation of the organism from samples of blood, feces or urine on
macCkoney agar gives creamy colonies. (Stool culture).
CBC( complete blood count) will show a high number of white blood cells
due to the infection.

Other tests
ELISA
Widal test:
In the Widal test, patients with typhoid exhibit a 4
fold or greater rise in antibody titer.

Sandwish ELISA=Enzyme linked immunosorbant assay)

Widal test

Neisseria species
They are diplococci shaped like 2 coffee beans joined
longitudinally by their flat sides.

1-Neisseria gonorrhoeae
Clinical picture:
1-causative agent of gonorrhea
infection of the genitals (mainly in
females).
2-Gonococcal Ophthalmia Neonatorum
3- Disseminated gonococcal infections
endocarditis, meningitis or
gonococcal dermatitis-arthritis
syndrome

Laboratory diagnosis
Microscopical examination reveals the presence of many PMNs
(polymorphonuclear leukocytes )with intracellular gram-negative
diplococci.

Samples are taken from blood, skin lesions and joints using
Dacron swabs as cotton swabs may contain materials toxic for
gonococci.
Due to its a weak organism so Cultivation on chocolate and
Thayer-Martin media ( contains antibiotics and nutrients to prevents other
bacterial growth).
They give positive superoxol ( catalase )test.

2-Neisseria meningitidis
commonly called meningococcus. It is the cause of meningitis
(meningococcal meningitis)& other forms of meningococcal
disease such as meningococcemia, a life-threatening sepsis.
Clinical picture:
- Meningitis is an inflammation of the membranes covering
the brain and spinal cord known as the meninges.
symptoms
fever, headache, photophobia and stiff neck

-Meningococcal septicaemia typically causes

a purpuric rash (Petechial rash )which aids in

diagnosis of the disease due to endotoxin release.

Laboratory diagnosis:
They are cultivated on blood agar ,chocolate agar or
modified T-M agar.
Blood, urine or CSF samples from patients should not be
refrigerated before being cultured because refrigeration
kills meningococci.
CSF samples are taken by lumbar puncture from lumber
vertebrae.
Blood samples should be obtained before antibiotics are
initiated.

Cystine trypticase agar test: (CTA test)

It is a Sugar Fermentation Media
supplemented with glucose, maltose or sucrose;
test for utilization of carbohydrates.
Neisseria meningitidis detected by its ability to
convert glucose and maltose into acetate (acid)
which changes the color of the indicator (phenol
red) from red to yellow.
While Neisseria gonorrhaeae ferment only glucose

Haemophilus influenzae
There are six generally recognized types of encapsulated H.
influenzae :a, b, c, d, e, and f.

It is the major pathogen that causes life-threatening

diseases like meningitis and epiglottitis in young children
Clinical picture:

Laboratory diagnosis of Hib meningitis

It is based on:
Direct visualization of PMNs and gram-negative
coccobacilli in CSF samples.
Demonstration of low glucose and high protein levels in
CSF samples.
Immunologic demonstration of Hib capsular material in
blood, CSF or urine samples
e.g.: Latex agglutination test (LA) and
Staphylococcal protein A coagulation test (COA).
A-Both tests involve the use of anti PRP (polyribose
phosphate) antibodies. These are attached either to latex
beads in LA test or to staphylococci in COA test.

Latex agglutination test

Laboratory diagnosis of Hib meningitis (cont)

B- A small sample of blood, CSF, urine is added to the

conjugate.
If PRP is present, the beads or Staphylococci will
agglutinate within seconds or minutes.
LA is the most specific test. The samples should be
collected before antibiotics are administered and should be
transported quickly to the laboratory and must not be
refrigerated.

Bordetella Pertussis
Pertussis (whooping cough) is associated with cough
paroxysms by the end of which cyanosis, apnoea and
seizures may occur.

Laboratory diagnosis:
It must be cultivated on a special medium containing
starch or charcoal to remove fatty acids and other toxic
substances that are released by the organism as it grows.
e.g.: Bordet Gengou agar (contains potato starch)
Regan-Lowe agar (contains charcoal).
It grows after 2-7 days.
Samples must be taken early after the onset of
symptoms.
Dacron swabs are used as cotton is toxic to B. pertussis

Laboratory diagnosis: (cont)

If the medium does not contain antibiotics as in
Bordet-Gengou, the swab is passed through a drop
of Penicillin. G. on the plate to inhibit the growth of
other respiratory organisms.
The samples are incubated at 35oc for up to 7 days in
7.5% CO2.
The isolated organism is identified by agglutination
with specific antiserum
Also PCR : polymerase chain reaction to detect genes is very
sensitive & highly specific.

Helicobacter pylori
H. pylori is a slightly curved or spiral-shaped gram-

negative rod.
It causes gastric ulcers and may
cause stomach cancer.

Laboratory diagnosis:
- It can be cultured on Brucella agar, BHI (brain Heart
Infusion) medium or trypticase soy agar containing 5%
defibrinated sheep or horse blood. It is incubated for 3-5
days at 37 C in an atmosphere containing 10% CO2, 5%
O2 and 85% N2.
-It is oxidase, catalase and urease positive.

-ELISA to detect the presence of H. pylori in serum

samples.
-Also microhemagglutination and latex agglutination
tests can be used.

Laboratory diagnosis (cont)

-Urea

breath test
To test for the presence of H.pylori in the stomach:
The patient must be fasting for six hours & must stop
antibiotics two days before the test. Let him breath in a
special bag, then put the bag in an apparatus which
measures the amount of labelled CO2initial value.
Give him a labeled urea solution(C13),after 30` let him
breath in another bag &measure the amount of labeled
CO2positive result.

Campylobacter

Campylobacters "CAMP "are thin, Gram-negative rodshaped organisms. They are microaerophilic or anaerobic
and move by means of a single polar flagellum.
Campylobacter jejuni is a major cause of enteritis in
humans and is mainly transmitted by contaminated food

Laboratory diagnosis :

Campylobacter is grown on specially selective

"CAMP" agar plates at 42C rather than at 37C.
They are oxidase positive.
A selective blood agar medium (Skirrow's medium) is
used;
its selectivity is due to an infusion of a cocktail of
antibiotics: vancomycin, polymixin B and trimethoprim,
and growth under microaerophilic conditions at 42C.

Toxigenic bacteria
Vibrio cholerae
-They are gram-negative rods that are shaped like commas.
- -Motile by means of a polar flagellum.

Laboratory diagnosis:
Samples of stools are examined by darkfield :- darting
motility.
-V. cholerae can be cultured rapidly in alkaline peptone
water: this is done on food specimens in which the
number of organisms is much lower than in stool so
peptone water is an enrichment medium.
Also, the organism can be isolated on TCBS ( Thiosulfate
citrate bile salt).
On TCBS agar yellow sucrose fermenting colonies.

Then further identification of colonies is done biochemically

(cholera red test).
Cholera red test:
Inoculate the vibrios in nitrate-peptone broth. Add few
drops of H2SO4red colour (due to nitroso-indole).
This test is used to differentiate between cholera &
clinically similar disease caused by enterotoxin producing
strains of E.coli.

Serologically :with O1 antiserum coagulation of O1

strains.
.NB. the darting motility will stop immediately if O1
antiserum is added to the organism on a slide.

Pseudomonas aeruginosa
The family Pseudomonadaceae consists of a large group of
gram-negative rods.
Ps. aeruginosa typically infects the pulmonary tract, urinary
tract, burns, wounds, and also causes other blood and
Outer_ear"Otitis_externa" infections.

Laboratory diagnosis:

Ps. aeruginosa infections are often suspected when blue-green

pus is present in the lesions.
For specific identification of Ps. aeruginosa, the sample is
cultured on a selective cetrimide agar medium that enhances
the production of pyocyanin. This medium contains acetamide
as the only C&N source.
positive oxidase, the diagnosis can be confirmed by API 20
NE.

Zoonotic bacteria
Brucella species

They are Gram-negative coccobacilli.Causes

brucellosis also called Bang's disease, Malta fever,

Mediterranean fever or rock fever, is a highly
contagious caused by ingestion
of unsterilized milk or meat from infected animals or
close contact with their secretions.
Transmission from human to human,
through sexual contact or from mother to child, is rare but
possible.

Laboratory diagnosis:

Antibodies to Brucella can be detected by tube

agglutination test.
Brucellae can be cultured from blood and bone
marrow of patients with bacteremic brucellosis.
Clinical isolates grow slowly, they take 5-10 days,
but sometimes they may require as long as 4-6 weeks.
Individual species of Brucella are identified by
fermentation reactions, urease and H2S production,
ability to grow on media containing thionine and
basic fuchsin and agglutination with specific antisera.

The spirochetes

They are motile, slender, helically coiled, flexible

organisms.

Treponema pallidum

Appears as a helical coil with the ends finely tapered,

causing syphilis.

Lab diagnosis
2Treponemal tests
Non-treponemal
tests
Serological tests

detect
antibodies
treponemalorantigens.
MeasureThey
the so
called
reaginicto
antibodies
Patients with syphilis
develop 2(APA)
typeswhich
of antibodies:
antiphospholipid
antibodies
also appears in
a.Non-specific
antibodies
against
phospholipids
T. who
pallidum
immobilization
test
: malaria and
people
had certain
infections
such
as(TPI)
leprosy,
(cardiolipin)
which
can be
detected
by non-treponemal
In which
the serum
sample
is incubated
with
rheumatoid
arthritis.
tests.
living
T. pallidum on a slide to determine if it contains
which
wouldantigen
inhibit used
organisms
motility.
This
The
complement
fixing
is a preparation
antibodies
Specific
antibodies
against
treponemal
surface
test
is difficult
andcan
replaced.
containing
cardiolipin
(an
from
beef heart muscle)
antigens
which
be extract
detected
by treponemal
tests.
and lecithin in cholesterol to increase the sensitivity to reagin.
e.g. of non-treponemal tests are:
Venereal disease research laboratory (VDRL).
Rapid plasma reagin (RPR).
Wassermann test.

Unusual bacteria
Chlamydiae
Chlamydiae are non-motile, they have no flagella or pili.
1. Elementary body (E.B.)
A small, dense, spherical body.
It is the infectious form of the organism which is responsible for the
attachment to host cell and promoting its entry.
When it enters the host cell, it is converted to R.B.
2. Reticulate body (R.B.)
It is the intracellular, metabolically active form.
It divides by binary fission.
It is larger in size than E.B.

Gram-negative bacteria
Picture only

Proteus
swarming motility.

Salmonella species:

typhoid fever ( Salmonella typhi)

paratyphoid fever (Salmonella paratyphi)
food poisoning (Salmonella enteritidis).

Neisseria gonorrhoeae
Gonococcal Ophthalmia Neonatorum

Other diseases:
gonorrhea
Disseminated gonococcal infections:
endocarditis, meningitis or
gonococcal dermatitis-arthritis
syndrome

Neisseria meningitidis
Meningitis

- purpuric rash (Petechial rash )

septicaemia

Meningococcal

Neisseria meningitidis

Cystine trypticase agar test: (CTA test)

detect
Neisseria meningitides

Haemophilus influenzae
meningitis and epiglottitis in young children

Bordetella Pertussis
Pertussis (whooping cough)

Helicobacter pylori
gastric ulcers and may
cause stomach cancer.

Pseudomonas aeruginosa

selective cetrimide agar medium

pulmonary tract, urinary tract, burns, wounds, and also

causes other blood and Outer_ear"Otitis_externa"
infections.

Treponema pallidum

causing syphilis.

Gram positive
Bacteria

I- Staphylococcus aureus
Members of the genus Staphylococcus appear as mass of
Gram-positive cocci with grape like arrangement (clusters).

-Catalase positive.

Catalase

2 H2O2

2 H2O + O2
in the form of bubbles

Classification of Staphylococcus aureus

The most important is phage typing. They are divided into
four groups based on their patterns of sensitivity to various
phages (group I, II, III, IV).

Test: A seeded agar with the unknown strain of

Staphylococcus aureus is prepared, then 4 drops of the
different phage groups are dropped on the surface of the
agar.
Clear plaques of lysis appear around the specific phage after
24hr of incubation at 37C

Clinical pictures of some infections caused by S. aureus

A- primary infections
Skin Infections:
1) Furuncles and carbuncle.

2) Impetigo

3- Abscess

4- Cellulitis

B-Deep lesions
A wide variety of infections of deep tissues by bacteremic
spread from a skin lesion.
These include infections of bones, joints, lungs, deep
organs and soft tissues.

C-Toxin mediated S. aureus infections

(toxigenic infections)
1. Staphylococcal scalded skin
syndrome ( SSSS ).

2- Toxic shock syndrome

(TSS).

3-Food poisoning
It is not the bacteria that cause illness,
however, they produce a family of toxins
called enterotoxins, which are potent emetic
agents.

Laboratory diagnosis
Isolate the organism from infected lesions or from blood
samples by cultivation on blood agar plate ---> observe
the characteristic golden color & shape of colonies.
Gram stain ---> Gram-positive spherical clusters.

- Cultivate on Mannitol Salt agar, result in yellow color.

Laboratory diagnosis
Coagulase Test
-Coagulase

Test ---> to differentiate

Staphylococcus aureus from other species
of Staphylococci (opportunistic
pathogens).
1-Tube Test

2-Slide Test

1)Tube test:
detects free coagulase
Citrated rabbit plasma is inoculated with
the organism and incubated at 37C. The
plasma contains fibrinogen. So if the
staphylococci produce free coagulase
(which is secreted outside the cell) the
rabbit plasma will be changed to jelly
shaped within 1-4 h

2) Slide test:
detects bound coagulase (clumping
factor).
The organism is mixed on a slide with
a loopful of human plasma. If coagulase
positive clumping of the cells occur in
few seconds.

2-Streptococci
The streptococci are Gram-positive spherical bacteria that form
pairs or chains.

Classification of streptococci
A) Hemolytic classification

B) Lancefield's Group Classification

A) Hemolytic classification
Based on their ability to lyse erythrocytes when grown on blood
agar.
3 groups were identified:
1-Alpha-hemolytic (): produce partial hemolysis resulting in
a greenish-brown discoloration around the colonies. e.g.
Streptococcus pneumoniae
2-Beta-hemolytic (): produce complete hemolysis due to
extracellular streptococcal enzymes (streptolysins) causing
production of clear zones around colonies e.g. Streptococcus
pyogenes.
3-Gamma-hemolytic (): cause no hemolysis.

A) Hemolytic classification

1-Alphahemolytic ()
2-Beta-hemolytic
()
3-Gammahemolytic ()

B) Lancefield's Group Classification

Streptococci are subdivided into groups by
using type-specific antibodies that
recognize carbohydrate surface antigens.
21 groups of carbohydrates are identified
and are given the letters from A to u

Some infections caused by

Streptococcus pyogenes (GABHS):
1- Pharyngitis ( Strep throat)
2- Erysipelas: Inflamed area of swelling with an advancing
bright red margin.

3- Necrotizing fasciitis
4- Scarlet fever ( toxigenic disease )
A- Erythema
B- Circumoral pallor
C- Strawberry tongue

5-Other toxigenic S. pyogenes infections may

lead to streptococcal toxic shock syndrome,
which can be life threatening.
6- Rheumatic fever is characterised by
inflammation of the joints and/or heart following
an episode of streptococcal pharyngitis and acute
glomerulonephritis, inflammation of the renal
glomerulus, can follow streptococcal pharyngitis
or skin infection.

Laboratory diagnosis of GABHS infections:

*Diagnosis is based largely on clinical grounds:

1-Throat swabs, materials from the lesions, pus, biopsy

materials and blood samples can be Gram stained.
2-Different samples are cultured on blood agar to see
beta hemolytic bacterial colonies which are catalase
negative.
3-ELISA or agglutination technology for antigens.

4-If results are uncertain: serum should be tested for

ASO (antistreptolysin o) Ag or Ab against DNAase,
NADase and hyaluronidases

Streptococcus
pneumonia

Non lancefield, -hemolytic.

1- Pneumococcal pneumonia.
2- Other invasive pneumococcal
diseases:
acute sinusitis, otitis media,
meningitis,bacteremia,
sepsis,osteomyelitis, septic arthritis,
endocarditis, peritonitis, pericarditis,
cellulitis, and brain abscess.

pneumonia

Otitis media:

Laboratory diagnosis of Streptococcus

pneumoniae:
X-rays help to determine the nature of the disease:
1-Sputum and blood samples should be cultured under CO2
on blood agar containing gentamicin then Gram stain.
2-ELISA or agglutination test can be used to detect Ag in blood
or urine.

3-Test depending on its high bile solubility (other streptococci

are bile insol.).
4-Using commercial available optochin disks (pneumococcus
is inhibited by optochin but not other streptococci).
5-Highly virulent to mice so inject sputum or spinal fluid
intraperitoneally into a white mouse or rabbit ---> death within
1 or 2 days.

4-Corynebacterium diphtheriae:
Gram-positive, nonspore forming, non
motile pleomorphic rods
Chinese letter shaped

Diphtheria desease
Upper respiratory tract illness with sore throat.
An adherent, dense, grey pseudomembrane
covering the posterior aspect of the pharynx. In
severe cases, it can extend to obstruct the air
way.

Peudomembrane

Bull Neck

Laboratory diagnosis:
1-Throat swabs or tiny pieces of pseudomembrane
should be obtained and stained with Methylene blue
(club shaped bacteria, showing Chinese letter
shapes).
2-Selective Media:
- Loeffler agar coagulated blood serum.
- Blood-tellurite agar potassium tellurite is added
to inhibit the growth of other respiratory isolates.
Colonies of diphtheria acquire typical black
colour due to precipitation of tellurium metal inside
the cell.

3-Test for toxigenicity:

Elek test:
A strip of filter paper impregnated with
diphtheria antitoxin is placed on the surface of
serum agar plate. The plate is then streaked with
the test organism at right angle to the paper strip,
and incubated. If the toxin is produced it will
diffuse into the agar and a precipitation arc will
be seen when it meets the antitoxin diffusing
from the strip in optimal conc.

5- Propionibacterium acnes:
This organism is an anerobic
diphtheroid commonly found on
the skin.

Cystic Acne

6-Bacillus anthracis:
Malignant
pustule

7- Clostridium tetani:
Gram-positive

anaerobic spore
forming bacillus showing a drumstick appearance.
The spores remain viable for years
and can be found in soil and in
animal faeces.

Localized or generalized tetanus

Risus sardonicus

lockjaw

Neonatal tetanus

Generalized tetanus

Laboratory diagnosis:
(serological tests are difficult)

A clinical picture of tetanus with a history of

injury is usually sufficient for diagnosis.
1- Swab from wound examined microscopically.
2- Animal inoculation: S.C. injections of guinea
pigs or mice with the organism develop tetanus
convulsions compared with control animals
receiving tetanus antitoxins.

8- Clostridium botulinum:
Laboratory diagnosis:

Samples of food and samples of

the patients serum, faeces and
gastric contents are examined for
the botulinum toxin.
I.P. injection of toxin in mice 4
days death, compared with control
mice receiving sp. antitoxins.

Infant botulism

Wound botulism

9- Clostridium perfringens :
Gram-positive boxcar shape rods
forming spores, obligate anaerobic,
non motile and form capsule.

Gas gangrene

Laboratory diagnosis:
1-Gram stain film from wound exudates.
2-Cultivation on cooked meat broth or blood
agar and incubate anaerobically.
3-Stormy fermentation of lactose in milk
i.e. the coagulated milk is blown apart by gas
formed during the fermentation process.
4- toxin production: detected by opaque
area formed around colonies grown on egg
yolk media due to its hydrolysis of the lipid
emulsion.

 toxin production Stormy fermentation

10- Clostridium difficile:

Similar to clostridium perfringens.
Laboratory diagnosis:

1-Stool samples are cultured on a

selective agar CCF agar (contains
cycloserine, cefoxitin, fructose).
2-Toxin B is measured by its ability to
kill culture fibroblasts

11- Mycobacterium tuberculosis:

-Straight or slightly curved bacilli, don't form spores or
capsules, when stained appear beaded.
-Acid and alcohol fast (non pathogenic sp. are acid fast
only).
-An obligate aerobe. Growth is very slowly, takes as long
as 6 weeks. Colonies show typical parallel growth
(serpentine cords).
-The organism is killed by heat but is quite resistant to
dryness and chemical agents.

Lungs of a TB patient

Consumption disease

Laboratory diagnosis:
1- Direct microscopical examination:
By observing acid-fast rods in smears of sputum, gastric
washing or urine by Ziehl-Neelson technique or by
fluorescent dyes (rhodamine - auramine dye) using
U.V. light.
2- Culture:
a) Lowenstein Jensen's media: composed of aspargine,
potato flour and malachite green. The latter for
inhibiting other organisms. Glycerin is also added to
prevent the dryness of the media due to the very long
incubation period (3-5 weeks).

Laboratory diagnosis: (cont.)

3- Rapid DNA probe technology can identify culture
isolates with in 2-8 hrs and the use of PCR.
4- Tuberculin Skin Test: (Mantoux Test).
I.D. injection of PPD on the forearm. A positive Rx is
shown as an area of edema after 48-72 hr (which
can be measured).
5-Chest X-rays:
May reveal Ghon complex or cavities in the lung.

Tuberculin skin test

12- Mycobacterium leprae:

-Morphologically similar to M. tuberculosis.
-Intracellular bacterium.
-It has the longest generation time.
-Grows best in cool tissues, never been cultivated in
artificial medium or cell cultures.

Lepromatous leprosy

Laboratory diagnosis:
1- Acid fast stained films from scraping of the nasal
septum or skin.
2- Cultivation in the foot pad of mice or armadillo
(body temperature 30-35C) and also serve as a
good source of lepromin.
3- Lepromin Test: analogous to tuberculin test.

THE END