Collection of Systematic Bacteriology

Cocci
The Staphylococci The Streptococci The genus Neisseria
Gram-positive Gram-negative
They are classified according to their They are classified according to: They are classified according to their
coagulase test into: 1. Hemolysis on blood agar pathogenicity into:
2. Serologic specificity
• Coagulase positive (most definitively) • Pathogenic Neisseria
only Staph. aureus. N. gonorrhea & N. meningitides
1. According to Hemolysis on blood agar
• Coagulase negative They are divided into three groups: • Non-pathogenic (commensal) Neisseria
Staph. epidermidis & • β-hemolytic streptococci Occurs in normal floral
Staph. saprophyticus “Complete hemolysis”
e.g. Strept. pyogenes & Strept. agalactiae.
• α-hemolytic streptococci
“Partial hemolysis”
e.g. Strept. viridans & Pneumococci.
- They are all catalase positive.
• γ-hemolytic streptococci
“No hemolysis”
e.g. Enterococcus.

2. According to Serologic specificity

Hemolytic streptococci are divided into 18
serologic groups (Lancefield groups A – U)
based on cell wall antigens Carbohydrates
antigens (C antigen)

- the capsular polysaccharide antigen is

used to classify streptococcus pneumonaie

- They are all coagulase & catalase

negative.

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 Collection of Systematic Bacteriology

Staphylococcus aureus Streptococcus pyogenes Streptococcus Neisseria

(Coagulase positive) (β-hemolytic & Group A) pneumoniae
Meningitidis Gonorrhea
“Pneumococci”
“Meningococci” “Gonocooci”
Stain Gram-positive Gram-negative
Shape Cocci
Pairs Pairs (diplococci)
Morphology

Arrangement Clusters Chains

(diplococci) with flat or concave adjacent sides
Polysaccharide
Microcapsule, Slime layer may be produced Polysaccharide
Capsule capsule of hyaluronic acid Capsule Capsulated
by other species. Capsule
“80 serotypes”
Motility Non-motile
Spore Non-spore forming
Culture characters

O2 Facultative anaerobes
CO2 Normal atmospheric Grow on normal, but 5-10% enhances growth 5-10%
Optimum 37C, but with narrow
Temperature Optimum 37C
range (30-38C)
- Ordinary media - Blood agar “β-hemolysis” - Blood agar - Chocolate agar
Golden yellow endopigment colonies “α-hemolysis” - Thayer-Martin media
Media - Blood agar “β-hemolysis” “selective media”
- Mannitol salt agar “selective medium”
- Skin lesions: Abscess, boils “furunculosis”,
carbuncle, styes.
- Spread: bacteremia & pyemia
Suppurative
leading to pneumonia, phlebitis, meningitis, UTI….etc. Cellulitis & Impetigo
“Pus forming” - Deep-seated infections: osteomyelitis,
endocarditis.
- A major cause of nosocomial infection.
- Pneumonia
- Scalded skin syndrome affects neonates. Scarlet fever “Erythrogenic toxin”
- Acute otitis - Acute - Gonorrhea
Diseases

- Food poisoning “Enterotoxins” - Usually affects children

media cerebrospinal - Opthalmia
Food: dairy milk products - Characterized by: fever, skin rash
Toxogenic Clinical picture: nausea, vomiting, and diarrhea, but no & stomatitis (strawberry tongue) - Sinusitis meningitis neonatorum
diseases fever. - Conjunctivitis
- The skin rash is due to the direct
Prognosis: Self-limited effect of the erythrogenic toxin on
- Toxic shock syndrome “TSST” the skin.
due to the systemic release of TSST or enterotoxins.
TSST Occurs as a sequel of any staphylococcal Begin after 1-3 weeks of infection
Post-infection infection. - Acute rheumatic fever
sequelae - Acute glomerulonephritis

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Staphylococcus aureus
(Coagulase positive)
Differs according to clinical disease
- Swabs from nose, throat,
armpits, groin or any
damaged skin.
- Pus
Sample - Sputum: pneumonia.
- C.S.F: meningitis.
Laboratory diagnosis
- Blood: bacteremia,
endocarditis…etc.
Direct Film
- Coagulase Positive
- Catalase positive
- DNase positive
- Mannitol fermentation
Biochemical
reaction
Antimicrobial - MRSA is resistant to all
clinically useful antibiotics
resistance
except vancomycin which
& may aid its survival in the
Prevention hospital environment.
& - The term MRSA refers
Treatment to methicillin resistance
- Most methicillin-
resistant strains are also
multiple-resistant.
Collection of Systematic Bacteriology
Streptococcus Streptococcus Neisseria
pyogenes pneumoniae (Pathogenic)
(β-hemolytic & Group A) “Pneumococci” Meningitidis Gonorrhea
(α-hemolytic) “Meningococci” “Gonocooci”
- Meningococci are found in • In acute disease
- Throat swab: - Rusty Sputum. C.S.F, blood skin rash & in Profuse pus & secretions from
Pharyngitis, scarlet the nasopharynx early in the urethra or cervix.
fever. the disease. • In chronic disease
- Blood: puerperal - C.S.F is obtained by - Scanty or no discharge
sepsis, erisypilas, lumbar puncture under - In males, the sample may be a
endocarditis, complete aseptic morning drop of the urethra,
bacteremia. precautions. centrifuged deposit of urine or by
- Sputum: pneumonia. - C.S.F is turbid & under prostatic massage.
tension due to presence - In females, cervical secretion is
of pus cells. obtained after cervical discharge.
Stained with gram stain for characteristic morphology
Catalase negative - Inulin fermentation - Oxidase positive - Oxidase positive
(Unlike staphylococci) - Bile solubility - Fermentation of glucose - Fermentation of glucose
- Sensitivity to optochin & maltose (with acid production)
- Pathogenicity to mice - Serology for diagnosis
- Quelling reaction • Definitive identification:
“Capsular swelling” by agglutination with
(These biochemical specific meningococcal
reactions are negative
with viridans antisera.
streptococci) • Rapid diagnosis:
Detection of meningococcal
antigen in C.S.F.
- No vaccine is available to - Capsular
combat S. pyogenes polysaccharide vaccines
infections. AC groups polysaccharide
vaccine
- Long-acting penicillin ACYW groups polysaccharide
can be used to prevent vaccine
recurrent pharyngitis - Chemoprophylaxis
and its immune- sulfonamides – Rifampicin
mediated sequelae.
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 Collection of Systematic Bacteriology

Commensal
staphylococci
Streptococcus Neisseria
(Coagulase negative)
(Non-pathogenic)

Staph. epidermidis Staph. saprophyticus Strept. agalactiae Strept. viridans

(β-hemolytic & Group B) (α-hemolytic)

- Is an inhabitant of the - Is free living, occasionally - They are part of the normal
skin. may be found on the skin. vaginal flora.
- May be found in oral &gastric
- Non-pathogenic - Is a common cause of flora.
urinary tract infection “UTI”
- Maybe a pathogen only in in young females. - Capsulated (five antigenic
the hospital environment, capsular types).
causing nosocomial, - produce large mucoid
bacteremia, endocarditis & colonies.
peritonitis.
- Are bacitracin resistant
- Hydrolyse Hippurate
- Positive for CAMP test.
-Diseases: Neonatal
septicemia, pneumonia &
meningitis which occur in
babies born to mothers with
Group B in the vagina.
- Present in the oral cavity and
incriminated in dental caries.
- Cause subacute bacterial
endocarditis after tooth
extraction in patients with heart
valve disease.
- Member of the normal flora.
- grow on the ordinary media.
- Do not grow on Thayer-
Martin medium.
- Can grow at room
temperature
- May give pigmented
colonies.
- Don’t react with specific
anti-meningococcal sera.

Notes
• Tests that can differentiate bt Strept. viridans & Pneumococci is (Inulin fermentation – Bile solubility - Sensitivity to optochi
- Pathogenicity to mice - Quelling reaction “Capsular swelling reaction”).
• The gold standard test in microbiology is Culture, especially Blood culture

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 Collection of Systematic Bacteriology

Bacilli

Gram-positive Gram-negative
Members Members

• Corynebacterium diphtheriae • Pseudomonas aeruginosa

• Clostridium • Proteus
(Proteus Mirabilis – Proteus Vulgaris)
• Lactobacillus • Hemophilus
• Anaerobic Bacilli
(Bacteroides fragilis – Prevotella melaningoenica –
Fusobacterium)

Hemophilus
- Important Species are (Hemophilus Influenza – Hemophilus parainfluenza – Hemophilus aegyptius –
Hemophilus ducreyi).
- Pharyngeal carriage of H. influenza type b is an important source of infection.

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 Collection of Systematic Bacteriology

Corynebacterium diphtheriae Pseudomonas aeruginosa Hemophilus influenzae

Stain Gram-positive Gram-negative
- Bacilli (with club-shaped end)
- Appear beaded due to presence of
Morphology

Shape metachromatic granules which can be Bacilli Coccobacilli

seen by the Nisser stain.
(Brown bacteria & blue granules)
Arrangement Palisade or Chinese letter Single
Capsule Non-capsulated Capsulated
Motility Non motile Motile by monotrichous flagella Non motile
Spore Non-spore forming
O2 Strict aerobic Facultative anaerobes
Culture characters

CO2 Normal atmospheric 5-10%

Temperature
- Sheep blood agar: Grow (with others).
- Loeffler serum slope: Grow (with
Media others) & give grayish-white colonies.
Optimum 37C
Grows well on most laboratory
media with production of two
soluble exopigments (Pyocyanine &
Pyoverdin)
- Chocolate agar
- Need two growth factors for growth
(Hemin “factor X” – NAD “factor V”), both
are present in the blood.

- Blood tellurite agar “Selective medium”

The disease is called diphtheria.
- Cause
Diphtherial exotoxin (Poly peptide in nature –
Heat labile – Converted to toxoid by
formaldehyde)
- Transmission: Droplet infection.
Diseases - Patients: commonly children but any age
may be affected.
- Source: Case or carrier
- Clinical picture
It is an upper respiratory tract illness
characterized by (Sore throat – An adherent
membrane on the tonsils, pharynx, or nose).
- P. aeruginosa is a commensal
pathogen (opportunistic pathogen).
Diseases: UTI – RTI – GIT infection
Soft tissue infections e.g. Dermatitis
Bacteremia – Bone joint infections.
All of these diseases occur mainly in
patients with severe burns & in
cancer & AIDS patients who are
immunosuppressed.
- The most important is Haemophilus
influenza type b (Hib)
- Causes: bacteremia & meningitis in
children less than 2 years old.
- Causes: epiglottitis, bacteremia,
cellulitis, otitis media, sinusitis,
tracheobronchitis & pneumonia in
all ages “infants – children – adults”.

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 Collection of Systematic Bacteriology

Pseudomonas Hemophilus
Corynebacterium diphtheriae
aeruginosa influenzae
A case A carrier Susceptibility
Swabs from the Nasopharyngeal Differs according to clinical disease
Sample pseudomembrane swabs

- Stained with gram stain for Stained with Gram stain for characteristic morphology.
Laboratory diagnosis

characteristic morphology.
Schick test
- May be stained with Methylene
Direct Film - Used to assess
blue.
immunity to diphtheria
toxin.
- Not used now.
Toxogencity (Virulence) tests to - Oxidase positive Detection of the capsule of
- Replaced by a
detect toxin production from - Ferment No sugars. bacteria in CSF by
serologic test for
C.diphtheria either Quelling reaction.
specific anti-diphtherial
• In vivo virulence tests toxin antibodies.
Biochemical • In vitro virulence tests
reaction - Elek immunoprecipitation test
- Tissue culture cytotoxicity
- ELISA
- PCR

• Antitoxin - Pseudomonas is naturally PRP “Poly Ribosylribitol

Should be given immediately if diphtheria is suspected clinically resistant to antibiotics. Phosphate” vaccine
Treatment as the antitoxin must react with the toxin before it becomes - This is due to the formed from capsular
& fixed into the cell. permeability barrier formed polysaccharides of
Prevention Dose: 20,000 – 100,000 units given intramuscular or by Lipopolysaccharides of its Haemophilus influenza
intravenous. outer membrane. type b.
& • Chemotherapeutic
Virulence

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 Collection of Systematic Bacteriology

In vivo virulence tests In vitro virulence test

• Two guinea pigs are selected, The test animal & Elek immunoprecipitation test
the control animal. In vitro detection of toxigenic strains of
• Control animal is injected with 1000 – 2000 units Use C.diphtheria.
of diphtheria antitoxin. Double diffusion of diphtheria toxin
Principle and antitoxin in agar.
• Wait for two hours.
• The two animals are injected (intradermal or Heavy inoculum of bacteria is streaked
subcutaneous) with .2 ml of 50 fold dilution of over the surface of agar medium.
broth culture filtrate of C.diphtheria.
A filter paper strip is impregnated with
• Observe for 48 hours. Method antitoxin and placed over the surface of
agar at right angles on inoculum.

Test animal Control animal Incubated at temp of 37 for 24 hours.

(Not protected)
- Necrosis at the site of injection
- Death after 2-5 days.
No pathological
- Post-mortem examination
shows Swelling and hemorrhage
of adrenal glands.
(Protected)
A positive test (Toxigenic bacteria)
Line of precipitation (toxin – antitoxin)
will form at 45 angle to the streak.
Interpretation
Negative test (Non toxigenic bacteria)
No line of precipitation
changes
Positive & Negative diphtheria strains
NB
should be inoculated on the same plate.

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 Collection of Systematic Bacteriology

Clostridium Lactobacillus Proteus

- The most important species
are Proteus mirabilis &
General Characters Proteus vulgaris.
- Opportunistic bacteria
Found in soil & normal - some species are sound as saprophytes in Normally found in soil, air & in
intestinal floral in humans & vegetables & animals. normal flora.
Location animals. - Others are present in oral gastrointestinal &
vaginal flora.
Stain Gram-positive Gram-negative
Morphology

Shape Bacilli Bacilli

Motility Non-motile Motile by peritrichious flagella
Spore-forming
Spore forming Its position is useful in Non-spore forming
species identification.
O2 Anaerobic
characters
Culture

Temperature Optimum 37C

- Shows optimum growth - Grow on tomato juice agar (PH 5)(Acidic Produce swarming on surface
Media
when plated on blood agar medium) off agar plates

Biochemical reaction
May lead to different
diseases according to
species
- C. tetani → Tetanus
(Drum stick appearance)
Diseases - C perfringens → Gas
gangrene
- C. botulinum → Botulism
- C. difficile →
Pseudomembranous cloitis
- Catalase positive
- Ferment glucose into lactic acid
- Lactobacillus is generally harmless to humans - Prostatitis
and rarely causes diseases. - Renal inflammation
(Pyelonephritis)
- Lactobacillus derives lactic from glucose & - Otitis media
creates an acidic environment that inhibits - Thick pus in bone
the growth of many bacterial species in vagina (osteomyelitis)
which can lead to urogenital infections. - Empyema
- Urinary bladder inflammation
(Cystitis)
- Spread in blood (Bacteremia)

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 Collection of Systematic Bacteriology

Anaerobic Gram -ve Bacilli

- Only Bacteroides, Prevotella, Fusobacterium, and Porphyromonas are of clinical importance.
- They present as members of the normal flora and may become involved in opportunistic infections throughout the body.
- The source of infection is endogenous when mucosal damage by surgery, trauma, or disease allows tissue penetration
by members of the normal flora.

Bacteroides fragilis Prevotella melaninogenica Fusobacterium

Stain Gram-negative
Morphology

shape Bacilli Bacilli (spindle)

Capsule Capsulated Non-capsulated
Motility Non-motile
Spore Non-spore forming
GIT GIT GIT
Habitat Genital tracts Oral cavity Oral cavity
Respiratory system Genital tracts
- Abdominal infection - Abdominal infection - Abdominal infection
- Genital infection - Oral infection - Genital infection
- Abscesses - Abscess (dental) - Abscesses
Infections - Endocarditis - Pulmonary infection - Gingival ulcers
- Pelvic infection - Vincent angina

Polysaccharide capsule (antiphagocytic) Release Release

Release - Collagenase - Hemolysin
- IgA protease - fibrinolysin - Endotoxin
Virulence - β-lactamase - Leucotoxin
- Collagenase
- DNase
Penicillin Resistant Sensitive
The most pediment cause of abdominal Produces brown to black Its most pathogenic member is
Other infection. pigments. Fusilbacterium nucleatum.

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 Collection of Systematic Bacteriology

Non-gram bacteria

Mycobacteria Spirochetes
Members of Non-
Leptospira Treponema
M.tuberculosis tuberculous M. lepra Borrelia
species pallidum
complex species

Cause tuberculosis
in different hosts.
Which causes
• M. tuberculosis Virtually all The causative Causes Lyme disease,
Causes syphilis
other species agent of leprosy leptospirosis and relapsing
• M. bovis
fever.
• M. africanum
• M. microtia

Important characters of mycobacteria General Characters

• The cell wall of mycobacteria contains high lipid content • Many are not cultivable
(40 – 60%) which is responsible for staining characters.
• Range from obligate anaerobe to aerobe
• Mycobacteria are facultative intracellular.
• Mycobacteria resist antibiotics, acids, and alkalies. • Not seen by ordinary light microscope because
• Mycobacteria are acid & alcohol fast (resist they are too small
decolorization with acid & alcohol).
• When cultured, Mycobacteria are very slowly growing as • Can be seen by a dark field microscope
they growing as they require 4 to 6 weeks to give visual
• Stained by silver salts
colonies

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 Collection of Systematic Bacteriology

Mycobacterium tuberculosis Treponema pallidum

- Not stained by Gram stain
- Not stained by Gram stain - Stained by Giemsa & Fontana stain
Stain
Morphology

- Stained by Zeihl-Nelsen stain (ZN) - Treponemes in tissues can be visualized by silver impregnation methods.
- Live treponemes can be seen unstained by dark-field microscopy.
Shape Bacilli (straight or slightly curved) Long slender organisms that appear as helical coils (spiral-shaped)
Arrangement Single, pairs, Groups, …….
Capsule Non-capsulated
Motility Non- motile Motile
Spore Non- spore forming
O2 Strict aerobic Microaerophilic
CO2 5-10%
characters

Optimum 37C (strict mesophile)

Culture

Temperature - Pathogenic treponemes have not yet been cultured in vitro.

NO growth below 30C or above 39C
Egg basal medium - The cells have a high lipid content (cardiolipin, cholesterol), which is unusual
- Lowenstein Jensen medium “selective media” for most bacteria.
Media - Dorest egg medium “not contaminated sample as C.S.F”
Agar based medium
- Middlebrook medium
Tuberculosis Syphilis
- Airborne disease - A strictly human disease transmitted by sexual contact.
- Characterized by: - Organisms penetrate mucous membranes or enter breaks in the skin.
• Chronic productive cough - Less than 10 Organisms can produce the disease.
• Low-grade fever - It is divided into 3 stages
• Night sweats 1) The primary stage
• Weight loss • The incubation period → 10: 90 days.
• Superficial painless ulcer with a firm base called a hard chancre, generally found on the
genitals.
• The lesion is filled with treponemes and is, therefore, highly contagious.
Diseases 2) The secondary stage
• Clinical manifestations include a mucocutaneous rash, mucous patches on mucous membranes &
wart-like lesions called condylomata in moist intertriginous areas.
• All of these lesions are highly contagious.
3) The tertiary stage
• Can affect all areas of the body and be fatal.
• Cardiovascular and neurological involvement is the most frequent causes of death
4) Congenital syphilis
• T. palladium can be transmitted through placenta to fetus which may die, miscarried, stillbirth at
term. Or others borne live with congenital syphilis manifestations e.g. Hutchinson’s teeth.

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 Collection of Systematic Bacteriology

Mycobacterium tuberculosis Treponema pallidum

• Sputum, bronchial or gastric washings. Exudates from chancre mucous patches, blood for
Samples • At least 3-5 morning samples of sputum are required serology.
for diagnosis.

1) Prepared from sputum

Either direct or after liquefaction by N-acetyl -L cysteine or by
other methods.
- Unstained fresh with a dark ground microscope for
2) Stained by motile treponemes
Direct
Carbol fuchsin (Ziehl Nelsen) or fluorochrome stain.
smear • By Ziehl-Nelsen (Z-N) stain - Stained with fluorescein-labeled anti-treponemal
Laboratory diagnosis

Acid-fast bacilli appear pink against blue background. antibodies and examined by fluorescent microscope.
• By fluorochrome stain
Bacilli appear as bright yellow fluorescent against a dark
background.

• The sputum sample is treated with NaOH, to kill other

contaminating bacteria (decontamination) but does not kill
the M.TB. because M.TB. is resistant to alkali.

• Sputum is inoculated in a selective medium containing

antimicrobial agents as (Lowenstein-Jensen).
Pathogenic treponemes have not yet been cultured in
• Cultures are incubated at 35°C to 37°C in an atmosphere of 5 to
Culture 10% CO2.
vitro

• All cultures should be examined weekly for 4- 8 weeks.

• Colonies on L-J medium

Raised, Rough, Confluent, Grayish & Dry (eugenic growth)
“Good offspring”.

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 Collection of Systematic Bacteriology

Intradermal skin test (Tuberculin test)

Principle:
- Individuals previously infected with TB will develop
hypersensitivity to proteins of TB bacilli (6-8 weeks) after
infection.
- Intradermal injection of PPD “Purified Protein Derivative” into a
previously infected person will result in the delayed (48-72 hr) Serological tests:
appearance of an indurated reaction. Serologic tests fall into two general categories:
Method: 1. Non treponemal antigen tests:
- Performed by using old tuberculin or PPD, injected either • Which measures antibodies directed against cardiolipin
intradermally or by multi-puncture. • Non-specific
- Mantoux test is the standard tuberculin test. • Reactive for a short period
- It requires the intradermal injection of (0.1 ml) containing (5
tuberculin units) of PPD. – Venereal Disease Research Laboratory (VDRL).
- The transverse diameter of induration is measured 48 to 72
– Rapid Plasma Reagin (RPR) test.
hours later.
Tests – Complement fixation test (Wasserman reaction).
Interpretation:
• Positive
- Diameter of the induration is 10 mm or greater. 2. Treponemal antigen tests:
- There is erythema, swelling & induration. • Which detect antibodies directed against protein of T.
- Positive reactions mean previous exposure to M.TB. pallidum.
• Negative • Specific
- Induration less than 5 mm. • Reactive for life

Recent methods for diagnosis – Fluorescent T. pallidum antibody-absorption.

DNA probes and gas-liquid chromatography: – Microhemagglutination for T. pallidum.
- Rapid, specific & sensitive methods for identification of mycobacteria – Treponema pallidum immobilization test.
after sufficient growth is present on the medium.
PCR:
- Useful for direct detection of mycobacteria in the clinical specimen
within 24 hours or less.
- It is rapid, specific & sensitive.

- 4 antibiotics are used for a duration of 6 to 9 months.

Treatment - Most commonly used antibiotics “RECIPES” Penicillin
Rifampin – Isoniazid – Pyrazinamide – Ethambutol – Streptomycin

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 Collection of Systematic Bacteriology
Meningitidis

BCG vaccine (Bacillus of Calmette and Guerin)

Type
- Live attenuated vaccine prepared from bovine strains.

Preparation
- Repeated subculture of bovine strain 250 times on Glycerol –
Potato – Bile medium.
Prevention
Administration
- Given by intradermal injection, single dose
- Given to
• All infants in the first year of life
• Tuberculin negative adults
- Should not be given to tuberculin-positive persons.
- BCG vaccinated person will be converted from the negative
reactor to positive reactors (tuberculin conversion).
NOTES
- Non-Capsulated: Prevotella melanogenic – Fusobacterium – TB – diphtheria except that is capsulated.

- Motile: P. aeruginosa by monotrichous flagella – Proteus by peritrichious flagella - Treponema pallidum.

- Spore-forming: Only Clostridium.

- Strict aerobic: diphtheria – P. aeruginosa – TB

- Strict anaerobic: Clostridium – Lactobacillus – Anaerobic Bacilli “Bacteroides fragilis - Prevotella melanogenic – Fusobacterium”

- Microaerophilic: Only Treponema pallidum

- Bacteria that grow on Ordinary media: Staphylococcus aureus – Neisseria commensal – P. aeruginosa.

- Bacteria that grow on chocolate media: Hemophilus influenzae – Neisseria meningitdis & Gonorrhea.

- Oxidase positive: Neisseria meningitdis and Gonorrhea - P. aeruginosa.

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