Medical Bacteriology.pptx

Medical
Bacteriology
[Bacteria causing medically important diseases]
8/11/2023 Medical Bacteriology Module 2 1

Table of contents
Gram-negative rods
Enterobacteriaceae
. Lactose-fermenters
Escherichia spp.
Klebsiella spp.
Enterobacter spp.
Citrobacter spp.
b. Non-lactose fermenters
Salmonella spp.
Shigella spp.
Proteus spp.
• Staphylococcus
species
• Streptococcus species
Gram-
positive
cocci
• Neisseria species
Gram-
negative
cocci
• Bacillus species
• Clostridium species
Gram
positive
spore
forming
rods

• Mycoplasma
• Ureaplasma
• Rickettsiaceae,
Bartonellaceae and
Coxiella
• EHRLICHIA,
• ANAPLASMA AND
• NEORICKETTSIA
• Bartonella and
• Grahamella
Gram negative
rods
Oxidase
Positive
Pseudomonas
spp.
Vibrio
Campylobacter
Helicobacter
Borelia species
Leptospira

Chlamydia and
Chlamydophila
Yersinia,
Pasteurella,
Francisella

1.GRAM
POSITIVE COCCI
1. Staphyloccocus

• Gram-positive spherical cells (0.5-1.5 mm) in singles,
pairs, and clusters
• Appear as “bunches of grapes”
Gram-stained smear of
staphylococci from colony
Scanning electron micrograph of
staphylococci
Morphology

General characteristics
• Non motile
• Non–spore-forming
• Non encapsulated
• Catalase-producing
• Oxidase: negative
• Glucose fermenters
• Primarily aerobic, some facultatively
anaerobic
• Bacitracin resistant
• Some are ß-hemolytic
• Colony morphology: buttery looking, cream
or white colored

Staphylococcus aureus
Primary pathogen of the genus
Habitat:
• Anterior nares (carriers)
• Colonization: axilla, vagina, pharynx
Produce superficial to systemic infections
• Skin
• Bacterial sepsis
• Hospital acquired infections

• Mode of transmission
oTraumatic introduction
oDirect contact with infected person
oInanimate objects
• Predisposing conditions
oChronic infections
oIndwelling devices
oSkin injuries
oImmune response defects
• Infection will elaborate
inflammatory response with
accumulating pus
• Pus: mix of active and
inactive neutrophils, bacterial
cells and extravascular fluid
Staphylococcus aureus con’t

Virulence Factors of S. aureus
Cell associated virulence factors
Enterotoxins
Cytolytic toxins
Enzymes

Capsule or slime layer (glycocalyx)
Peptidoglycan (PG)
Teichoic acid is covalently linked to PG and is species specific:
• S. aureus ribitol teichoic acid (polysaccharide A)
• S. epidermidis glycerol teichoic acid (polysaccharide B)
Protein A is covalently linked to PG
Clumping factor (bound coagulase)
Cell-Associated Virulence Factors

• Exotoxin: protein produced by a
bacteria and released into
environment
• Heat stable @ 100o C for 30 minutes
Heat-stable
exotoxins that
cause diarrhea
and vomiting
• Food poisoning
• Toxic shock syndrome
Implications
Enterotoxins

Types of enterotoxin
• Exfoliatin
o Epidermolytic toxin
• TSST-1: Toxic shock syndrome toxin-1
o Multisystem disease
o Stimulates T cell production & cytokines
• Cytolytic Toxins
oAffects RBCs and WBCs
o Hemolytic toxins: alpha, beta, gamma, delta
o Panton-Valentine leukocin, lethal to WBCs

• Hydrolyzes hyaluronic acid in connective
tissue allowing spread of infection
Hyaluronidase:
• Fibrinolysin which allows spread of
infection
Staphylokinase:
• Virulence marker
Coagulase:
• Lipase:
oAllows colonization by acting on
lipids present on the surface of
the skin.
• Penicillinase:
oConfers resistance
• DNase:
oDegrades DNA
• Beta-lactamase:
oCuts the beta-lactam wall of
certain antibiotics
Extracellular Enzymes

• Pass skin – first line of defense
• Benign infection
• Phagocytosis
• Antibody
• Inflammatory response
• Chronic infections
• Delayed hypersensitivity
•Clinical manifestation
• SKIN
• folliculitis
• boils (furuncles)
• carbuncles
Pathogenicity

Pathogenicity

Diagnosis
1. Laboratory Diagnosis: Specimen Collection and
Handling
• Samples must be taken from the actual site of infection
• Prevent delay in the transport of collected material from
infected sites
• Transport in an appropriate collection device that would
prevent drying and minimize the growth of contaminating
organisms
2. Direct Smear Examination
• Microscopic Examination
• Gram reaction
o Gram-positive cocci
• Cell arrangement
o Pairs and clusters
• Presence/Absence of PMNs
o Numerous polymorphonuclear cells (PMNs)

3.Cultural Characteristics
• Staphylococcus aureus
• Colony morphology
• Smooth, butyrous, white to yellow, creamy
• Grow well @ 18-24 hours
• S. aureus may produce hemolysis on blood agar
4.Identification Tests: Catalase
• Principle: tests for enzyme catalase
2 H2O2 2 H2O + O2
• Procedure
o Smear a colony of the organism to a slide
o Drop H2O2 onto smear
Interpretation
oPresence of bubbles
• Positive in contrast is the negative
Diagnosis

Diagnosis con’t
5.Identification test: slide coagulase test
• Differentiates members within the Staphylococci
• Detects clumping factor found in S. aureus
• Procedure
oPlace a drop of sterile water on a slide and emulsify a colony
oAdd a drop of rabbit plasma to the suspension
oObserve
• Agglutination = Positive
• No agglutination= Negative

Susceptibility
• Deep/metastatic infections
o semi-synthetic penicillins
o cephalosporins
o erythromycin
o clindamycin
• Endocarditis
o semi-synthetic penicillin +
an aminoglycoside
Note:
The treatment is guided by
the result of the antibiotic
sensitivity test.

Medically important Staphylococcus Sp
* CNS: coagulase-negative staphylococci

2. Streptococcus species

General Characteristics
of Streptococci
• Gram-positive spherical/ovoid cocci arranged in
long chains; commonly in pairs
• Non-spore-forming, nonmotile
• Can form capsules and slime layers
• Facultative anaerobes
• Do not form catalase, but have a peroxidase
system
• Most parasitic forms are fastidious and require
enriched media.
• Small, nonpigmented colonies
• Sensitive to drying, heat and disinfectants
• 25 species

Classification

Lancefield classification
• Group A - Streptococcus pyogenes
• Group B - Streptococcus agalactiae
• Group C - Streptococcus equisimilis,
Streptococcus equi, Streptococcus
zooepidemicus, Streptococcus
dysgalactiae
• Group D - Enterococci,
Streptococcus bovis
• Group E - Streptococcus milleri and
mutans
• Group F - Streptococcus anginosus
• Group G - Streptococcus canis and
Streptococcus dysgalactiae
• Group H - Streptococcus sanguinis
• Group L - Streptococcus dysgalactiae
• Group M: Streptococcus fryi sp. nov
• Group N - Lactococcus lactis
• Group R&S - Streptococcus suis

Alpha hemolysis
• Alpha-hemolysis. Colonies
on blood agar are
surrounded by a green
zone. This “greening” is
caused by H2O2, which
converts hemoglobin into
methemoglobin.

Beta hemolysis
• Beta-hemolysis.
Colonies on blood agar
are surrounded by a large,
yellowish hemolytic zone
in which no more intact
erythrocytes are present
and the hemoglobin is
decomposed.

Gamma hemolysis
• Gamma hemolysis is a lack of
hemolysis in the area around a
bacterial colony. A blood agar
plate displaying gamma
hemolysis actually appears
brownish. This is a normal
reaction of the blood to the
growth conditions used (37° C
in the presence of carbon
dioxide).

Streptococcus
pyogenes (A
Streptococci)
 Virulence factors
Capsule
• Antiphagocytic; Nonspecific adherence
• Hyaluronic acid (polysaccharide) mimics animal tissue
Lipoteichoic Acid
• Cytotoxic for wide variety of cells
• Adherence: Complexes with M protein (LTA-M) and
binds to fibronectin on epithelial cells
M-Protein
• LTA-M protein is adhesin
• Antiphagocytic
• Inhibits alternate C’ pathway and opsonization
M-like Proteins: bind IgM and IgG
F Protein: mediates adherence

Virulence
factors
• Exotoxins:
• Streptolysin O (SLO):
• Hemolytic and Cytolytic
• Prototype of oxygen-labile and thiol-
activated cytolytic exotoxins (e.g.,
Streptococcus, Bacillus, Clostridium,
Listeria)
• Lytic for variety of cells: bind to
cholesterol-containing membranes
and form arc- or ring- shaped
oligomers that make cell leaky (RBC's,
WBC’s, PMN's, platelets, etc.)

Virulence
factors
• Streptolysin S (SLS):
• Hemolytic and Cytolytic
• Oxygen stable, non-antigenic
• Lytic for red and white blood
cells and wall-less forms
(protoplast, L- forms)
• Pyrogenic (Erythrogenic)
Exotoxins (Types A, B &C)
• Produced by more than 90% of
Grp A strep
• Cardiohepatic toxin

Virulence
factors
• Nucleases: Four antigenic types (A,B,C,D)
• Facilitate liquefication of pus generating
growth substrates
• Nucleases A, C have DNase activity
producing runny pus
• Nucleases B, D also have RNase activity
• Streptokinases: Two different forms
• Lyse blood clots: catalyze conversion of
plasminogen to plasmin, leading to
digestion of fibrin
• C5a Peptidase: destroys C’ chemotactic
signals (C5a)
• Hyaluronidase: hydrolyzes hyaluronic acid
• Others: Proteinase, NADase, ATPase,
phosphatase, etc
Enzymes:

Pathogenicity
• Diverse group of acute suppurative (pus-forming) &
nonsuppurative diseases
Group A Streptococcus (S. pyogenes):
• Pharyngitis (& tonsilitis):
• Cutaneous & Soft Tissue Infxns.
• Pyoderma (Impetigo: contagious pyoderma with
superficial yellow weeping lesions)
Suppurative Streptococcal Diseases
Suppurative Streptococcal Diseases
• Cellulitis: Involvement of deeper subcutaneous
tissues; Deeper invasion with systemic symptoms
• Wound Infections
Cutaneous & Soft Tissue Infxns(cont.)

Pathogenicity
• Systemic Disease
• Streptococcal Toxic Shock
• Syndrome (TSS): Multisystem toxicity
following soft tissue infection progressing to
shock and organ failure (not to be confused
with
• Staphylococcal Toxic Shock Syndrome
where hyperabsorbent tampons have been
identified as an important risk factor)
• Bacteremia
• Nonsuppurative Sequelae
• Post-infection complications of Group A
streptococcal disease; Serious complications
in pre-antibiotic era; still important in
developing countries

• Acute rheumatic fever (ARF):
• Inflammation of heart, joints, blood
vessels, sub-cutaneous tissues
• Rheumatic heart disease (RHD):
• Chronic, progressive heart valve
damage
• Acute glomerulonephritis (AG):
• Acute inflammation of renal
(kidney) glomeruli
• Foodborne Disease

Laboratory diagnosis
1.Direct Gram stain from sample
2.Culturing
3.Biochemical test:
• Catalase Negative: Differentiates from Staphylococcus
• Bacitracin test positive
• Treatment is based on Antibiotic sensitivity test result.
 Penicillin G or V
Alternatives are oral cephalosporins or macrolide antibiotics
• In treatment of septic shock, a polyvalent immunoglobulin is
used

Pathogenesis
and clinical
pictures of S.
pyogenes
infections

Erysipelas caused by
S. pyogenes

Streptococcus
pneumoniae
• Gram positive diplococci.
• Possess a capsule of polysaccharide that permits
typing with specific antisera.
• Found as a normal flora in the upper respiratory
tract.

Antigenic
structure
• Capsular polysaccharide:
Pathogenicity determinant with anti-
phagocytic property
• C substance: Cell wall associated
antigen
• Protein M antigen
• IgA1 protease: Enzyme which
cleaves IgA1

Clinical
features
Lobar
Pneumonia
Otitis media
Sinusitis
Bacteremia:
Meningitis
Endocarditis
Septic
arthritis

Laboratory Diagnosis
Specimen: Sputum, blood, cerebrospinal fluid,
ear discharge and drainage.
Smears: gram positive diplococci
Culture: Grow best in chocolate agar media in
CO2 enriched atmosphere.
• Shows α-hemolytic,
• Greater sensitivity to optochin (ethyl hydrocuprein
hydrochloride) in the disk test or their bile solubility.

Antibacterial Susceptibility, & Prevention
Antibacterial Susceptibility
• Amoxicillin
• Chloramphenicol
• Third generation Cephalosporins
Prevention
• Pneumococcal conjugate vaccine: Immunization of individuals with type
specific polysaccharide vaccine that is effective in children under two years
of age.

Differentiation of streptococcus
species
Species Catalase Bacitracin Optochin Litmus
milk
reduction
CAMP
(Christie–
Atkins–
Munch-
Peterson)
S. pyogenes - + - - -
S. agalaciae - - - - +
Enterococci - - - + -
Viridans
streptococci
- - - - -
S.
pneumoniae
- - + - -

The Most Important Human Pathogen
Streptococci and Enterococci

The Most Important
Human Pathogen
Streptococci and
Enterococci

Oral Streptococci
 Most of the oral streptococci of the type often known as the
viridans group have no group antigen.
 They usually cause a-hemolysis, some c-hemolysis as well.
 Oral streptococci are responsible for 50–70% of all cases of
bacterial endocarditis
 Predisposing factors include congenital heart defects, acute
rheumatic fever, cardiac surgery, and scarred heart valves
 Laboratory diagnosis of endocarditis involves isolation of
the pathogen from blood cultures
 Drug therapy of endocarditis is carried out with either
penicillin G alone or combined with an aminoglycoside
(mostly gentamicin). Bactericidal activity is the decisive
parameter.

Pronounced Dental Caries
• Certain oral streptococci
(S. mutans) are the main
culprits in tooth decay.
 S. mutans, S. sanguis, and S. mitis are, besides
Actinomyces viscosus and A. naeslundii, responsible
for dental caries

GRAM NEGATIVE COCCI
3.Neisseria species

Neisseria
Introduction
The
Neisseria
species are
gram
negative
cocci.
They occur in
pairs.
Neisseria
gonorrhea
“gonococci”
and Neisseria
meningitidis
“meningococci”
are pathogenic
for human
Die rapidly
outside
the
human
organism

General
characteristics
• Aerobic, Nonmotile
• Gram-negative cocci , in pairs
(diplococci) with kidney-shaped.
• Oxidase positive
• Most catalase positive
• Intracellular diplococci
• Ferment carbohydrate producing acid but
not gas
• The main species of medical importance
are:
• N. meningitidis
• N. gonorrhoea

N. gonorrhea
& N.
meningitidis
• Gonococci :
1) do not have
capsules
2) They have plasmids
3) They cause genital
infection
4) Ferment glucose
only
• Meningococci:
1) have
polysaccharide
capsules
2) Rarely have
plasmids
3) They cause a
disease called
meningitis
4) Ferment glucose
and maltose

Neisseria gonorrhoeae
(gonococcus)
• Readily transmitted by sexual contact
• Gram-negative diplococci
• Susceptible to cool temperatures,
drying and fatty acids
• Produce acid from glucose, but not from
other sugars

Virulent factors
1. Pili: Hair-like appendages and enhance attachment to host cells and
evade human defense.
2. Por (Protein I)
Pores on the surface of bacteria through which nutrients enter the cell.
3. Opa (Protein II)
Important for attachment of bacteria to host cells.
4. Protein III: Reduction-modifiable protein. It is associated with por in the
formation of pores in the cell.
5. Lipooligosaccharide(LOS): Responsible to damage epithelial cells.
Toxicity in gonococcal infection is largely due to the endotoxic effects of
LOS
6. IgA1 protease: Splits and inactivates major mucosal IgA(IgA1)

Clinical
manifestation
• Route of infection: Sexual contact
Male:
• Gonococcal urethritis
• Gonococcal epididymitis
• Gonococcal epididymo-orchitis (inflammation of
testis)
• Infertility
• Gonococcal suppurative arthritis
Female:
• Gonococcal cervicitis
• Gonococcal salpingitis (infection and
inflammation in fallopian tubes)
• If complicated: Gonococcal tubo-ovarian abscess

Females Males
50% risk of infection after single exposure 20% risk of infection after single exposure
Asymptomatic infections frequently not diagnosed Most initially symptomatic (95% acute)
Major reservoir is asymptomatic carriage in females Major reservoir is asymptomatic carriage in females
Genital infection primary site is cervix (cervicitis), but vagina,
urethra, rectum can be colonized
Genital infection generally restricted to urethra (urethritis)
with purulent discharge and dysuria
Ascending infections in 10-20% including salpingitis, tubo-
ovarian abscesses, pelvic inflammatory disease (PID) ,
chronic infections can lead to sterility
Rare complications may include epididymitis, prostatitis,
and periurethral abscesses
Disseminated infections more common, including septicemia,
infection of skin and joints (1-3%)
Disseminated infections are very rare
Can infect infant at delivery (conjunctivitis, opthalmia
neonatorum)
More common in homosexual/bisexual men than in
heterosexual population

D) Throat infection occur in abnormal person’s
suffering from Gonococal pharyngitis. It’s common
in homosexual person.
C) Eye infection: occur in new born babies and it
will lead to blindness.
B) Rectal involvement: infection caused by Neisseria
gonorrhoeae is called proctitis and characterized by
purulent discharge from anus.

Laboratory
diagnosis
a) Specimens:
Pus and secretions are taken from the urethra,
cervix, rectum, conjunctiva, throat or synovial fluid
for culture and smear.
b) Smears:
Gram stained smears of urethral or endocervical
exudate
reveal many diplococci within pus cells.
c) Culture:
Immediately after collection pus or mucous is
streaked on
enriched selective medium.
d) Serology:
Serum and genital fluid contain IgG and IgA
antibodies by
ELISA

Prevention &
Susceptibility
• Penicillin no longer drug of choice due to:
• Continuing rise in the MIC
• Plasmid-encoded beta-lactamase
production
• Chromosomally-mediated resistance
• Uncomplicated infection: ceftriaxone,
cefixime or fluoroquinolone
• Combined with doxycycline or azithromycin
for dual infections with Chlamydia
• Treatment of newborns with opthalmia
neonatorum with ceftriaxone
• Measures to limit epidemic include
education, aggressive detection, and follow-
up screening of sexual partners, use of
condoms or spermicides.

Neisseria
meningitidis
(meningococcus)
Encapsulated small
Gram-negative diplococci
Second most common cause (behind S.
pneumoniae) of community-acquired
meningitis in previously healthy adults; swift
progression from good health to life-
threatening disease
Virulence factors
• Pili-mediated, receptor-specific
colonization of nonciliated cells of
nasopharynx
• Antiphagocytic polysaccharide capsule
allows systemic spread in absence of
specific immunity
• Toxic effects mediated by hyperproduction
of lipooligosaccharide

Pathogenicity
Following dissemination of virulent
organisms from the nasopharynx:
Meningitis
Septicemia (meningococcemia)
with or without meningitis
Meningoencephalitis
Pneumonia
Arthritis

Laboratory
diagnosis
a) Specimens:
Specimens of blood are taken for culture and specimes of
Cerebrospinal fluid (CSF) are taken for smear, culture
and chemical determination and masopharyngeal swab
are taken for carrier survey’s.
b) Smears:
Gram stain smear of CSF show typical Neisseria within
polymorphnuclear leukocytes.
c) Culture:
CSF specimens are placed on heated blood agar
(chocolate)
agar and Thyer martin media) and incubated at 37c.
d) Serology:
Antibodies to meningococcal polysaccharides can be
measured by latex agglutination or by immuno
electrophoresis.
e) PCR

Laboratory
diagnosis
a) Specimens:
Specimens of blood are taken for culture and specimes of
Cerebrospinal fluid (CSF) are taken for smear, culture and
chemical determination and masopharyngeal swab are taken
for carrier survey’s.
b) Smears:
Gram stain smear of CSF show typical Neisseria within
polymorphnuclear leukocytes.
c) Culture:
CSF specimens are placed on heated blood agar (chocolate
agar and Thyer martin media) and incubated at 37c.
d) Serology:
Antibodies to meningococcal polysaccharides can be
measured by latex agglutination or by immuno
electrophoresis.
e) PCR

Susceptibility
and
Prevention
Susceptibility
Pencillin G is the drug of choice for treating meningococcal disease
and third-generation cephalosporins.
In patient allergic to penicillin, chloramphenicol and ceforamime (or
ceftriaxone) can be used.
Prevention
1) Irradiation of the carrier states (major source).
2) Isolation of the patient.
3) Chemoprophylaxis for contact people.
4) Vaccination.

Neisseria gonorrhoeae and
Neisseria meningitidis
N. meningitidis: gram
staining of a preparation of
cerebrospinal fluid sediment.
Clinical diagnosis: acute
purulent meningitis.
N. gonorrheae: gram staining of a
preparation of urethral secretion:
coffee-bean-shaped diplococci,
grouped within a granulocyte.
Clinical diagnosis: gonorrhea

GRAM NEGATIVE COCCOBACILLI
4. GENUS: HAEMOPHILUS
This is a group of small gram-negative, non-spore forming, non-motile,
pleomorphic bacteria that require enriched media for growth.
• Growth is enhanced in CO2 enriched atmosphere.
• Present in upper respiratory tract as a normal microbial flora in healthy
people.
• The group is fastidious requiring growth factors for isolation.
The growth factors are X-factor(Hematin) and V-factor (Diphosphopyridine
nucleotide).

Cont
GENUS:
HAEMOPHILUS
The main species of
medical importance
are: H. influenza, H.
ducreyii H. aegyptius
Growth factor
required
Haemophilus
species:
X and V factor for H.
influenzae, H.
aegyptius, H.
hemolyticus.
X factor for H.
ducreyii .
V factor for H.
parainfluenzae, H.
parahemolyticus.
The main species of
medical importance
are:
H. influenza, H.
ducreyii and H.
aegyptius

Haemophilus
influenzae
Characteristics:
• Gram-negative cocobacilli.
• Fastidious bacteria requiring growth factors for isolation.
• .Found in upper respiratory tract as normal flora in
healthy people.
• Antigenic structure
• Capsular polysaccharide .
• There are six serotypes of H. influenzae, A-F. . Capsular
antigen type b is composed of polyribose ribitol
phosphate.
• H. influenzae type b is the most common cause of disease
in humans.
• It is the main virulence factor which provides anti-
phagocytic property.
• Outer membrane protein
• . Lipo-oligosaccharide

Cont
Clinical features: The bacteria causes disease most
commonly in young children.
• Acute pyogenic meningitis, Acute epiglottis ,Pneumonia , Otitis
media , Sinusitis . Cellulitis , Acute pyogenic arthritis
• Laboratory diagnosis:
• Specimen: Cerebrospinal fluid, sputum, blood, pus Smear: Gram-
negative short rods.
• Serology: Quellung reaction (using specific antisera)
Immunofluorescence stain
• Culture: Chocolate agar contain both X and V factor; blood agar
contain only X factor.
• Satellitism test is used to identify H. influenzae in blood agar.

Satellitism
test
• Mix a loopful of haemophilus growth in 2ml
of sterile saline.
• Inoculate the bacteria suspension on a plate
of blood agar using a sterile swab.
• Streak a pure culture of S. aureus across the
inoculated plate which provides V-factor for
H. influenzae.
• incubate the plate over night in a CO2 -
enriched environment at 35- 37 Oc.
• . Look for growth and satellite colonies in
next morning. NB: Colonies are largest
nearest to the S. aureus column of growth.

H.ducreyii
Slender, gram-negative, ovoid bacilli, slightly larger
than H. influenzae.
It causes chancroid (tender genital ulcer).
• Cultured in special enriched media (20-30% rabbit
blood agar) with colonic morphology of small grey
glistening colonies surrounded by zone of hemolysis.
• It is treated by erythromycin, cotrimoxazole and
third generation cephalosporins
3. H. aegyptius It causes contagious conjunctivitis.

5. GENUS:
BORDETELLA
Characteristics: Minute strictly aerobic, non-
motile gram-negative rods.
Bordetella species of medical importance: B.
pertussis.
Antigenic structure:
Pili: Adheres to ciliated epithelial cells of the
respiratory tract. . Filamentous haemagglutinin:
Adheres to the ciliated respiratory tract.

BORDETELLA
Pertussis toxin: . Lymphocytosis promoting factor .
Histamine sensitizing factor
. Insulin secretion enhancing factor
. Adenylyl cyclase toxin
. Dermonecrotic toxin
. Hemolysin .
Tracheal cytotoxin: Inhibits DNA synthesis in ciliated
respiratory epithelial cells.

BORDETELLA
Clinical features:
Incubation period: 2 weeks
Route of transmission is respiratory from
early cases and possibly carries.
It has three stages: 1. Catarrhal stage 2.
Paroxysmal stage 3. Convalescence stage.

BORDETELLA
During catarrhal stage, the patient is highly infectious but not very
ill manifesting with mild coughing and sneezing.
During paroxysmal stage, the patient presents with explosive
repetitive cough with characteristic ‘whoop’ upon inhalation leading
to exhaustion, vomiting, cyanosis and convulsion.
During convalescence stage, the patient presents with prolonged cough

BORDETELLA
oLaboratory diagnosis:
• Specimen: Saline nasal wash (Preferred
specimen) Nasopharyngeal swab or cough
droplets on cough plate.
• Smear: Small, non-motile, capsulated,
gram-negative cocobacilli singly or in pair,
and may show bipolar staining.
• Culture: Inoculate the primary specimen on
Bordet-Gengue agar medium and incubate
for 2-6 days at 37 o c in a moist aerobic
atmosphere which produces small, raised,
shiny, mucoid colonies.
• Biochemical reaction:
• No growth on blood agar
• . Oxidase positive .
• Most of them are catalase positive

GRAM POSITIVE SPORES FORMING
RODS
4. Bacillus and 5. Clostridium
species

6.BACILLUS
Characteristics
• Aerobic, non-motile, spore-
forming, gram-positive chain
forming rods.
• Bacillus species are saprophytes
• Important human pathogens
• B. anthracis
• B. cereus

Bacillus
anthracis
• Major agent of bioterrorism and biological
warfare
• Major pathogen of domestic herbivores that
come in contact with humans
• Spores can survive in soil for years
• Capsule: Enables the organism to evade
phagocytosis
• Polysaccharide somatic antigen
• Protein somatic antigen
• Anthrax toxin- Protein toxin: Complex of 3
protein factors
• Edema factor + Protective antigen = Edema
toxin
• Lethal factor + Protective antigen = Lehtal toxin

Pathogenesis and Clinical feature
• Acquired by the entry of spores through injured skin in
cutaneous anthrax, or mucus membrane in intestinal anthrax, or
inhalation of spores in the lung while handling skin and hides
There are four forms of anthrax
1. Cutaneous anthrax (Malignant pustule): 95 % of anthrax
presentation
• Characterized by a black necrotic lesion with edematous margin
on hands, arms, face or neck with regional lymphadenitis
associated systemic symptoms.
2. Pulmonary anthrax: 5% of anthrax presentation
• Presents with pain, cough with haemorrhagic mediastinitis

Pathogenesis and Clinical feature
3. Bacteremic anthrax: presents with
clinical features of sepsis
4. Intestinal anthrax: Presents with
abdominal pain, vomiting, and bloody
diarrhea
Bacteremic and intestinal anthrax are rare
to occur

Laboratory
diagnosis
• Specimen: Fluid or pus from skin
lesion, Blood, sputum
• Smear: Non-capsulated gram-
positive rods
• Culture: Grows aerobically in
ordinary media: Non-hemolytic,
large, dense, grey-white irregular
colonies
• Serology: ELISA has been
developed to measure antibodies
to edema toxin and lethal toxin

Susceptibility & Prevention
Susceptibility
• Ciprofloxacin
• Penicillin+ gentamicin or streptomycin
Prevention and control:
• Disposal of animal carcasses by deep burial or
burning
• Decontamination (autoclaving) of animal product
• Protective clothing and gloves for handling
potentially infected materials
• Active immunization of domestic
• Immunize high occupation risk persons with anthrax
toxoid

Bacillus cereus
• General characteristics:
• Exhibit motility by swarming in
semisolid media
• Produce β lactamase, so not sensitive
to penicillin

Clinical features
1. Food poisoning
Pathogenicity
determinant: Exotoxin
a. Emetic type food
poisoning
Characterized by nausea,
vomiting, abdominal
cramps, and self-limited
with in 24 hrs
b. Diarrheal type food
poisoning
Characterized by profuse
diarrhea and abdominal
cramps.
Lab. Diagnosis: Isolation
of B.cereus in stool is not
diagnostic since it is
present in normal stool
specimen
Treatment: Fluid
replacement
Antibiotics not required

2. Ocular infection
Ocular disease following
trauma from non-surgical
penetrating objects
Susceptibility: Clindamycin
+ Aminoglycosides

7.Clostridium
Characteristics
• Clostridia are anaerobic, spore-forming motile, gram-
positive rods
• Most species are soil saprophytes but a few are
pathogens to human
• They inhabit human and animal intestine, soil, water,
decaying animal and plant matter
• Spores of clostridia are wider than the diameter of
organism and located centrally, subterminally and
terminally
• Species of medical importance:
• C. perfringens
• C. tetani
• C. botulinum
• C. difficile

Clostridium
perfringens
Characteristics:
Capsulated, non-motile, short gram-positive
rods in which spores are hardly seen
There are five toxigenic groups : A-E
Human disease is caused by C. perfringens
type A and C

Clostridium perfringens
• Clinical diagnosis: gas
gangrene in a gunshot
wound.
• C. perfringens: gram
staining of a preparation
of wound pus. Large,
thick, gram-positive rods.

Pathogenicity determinant
1. Enzymes: Digest collagen of
subcutaneous tissue and muscle.
• Collagenase
• Proteinase
• Hyaluronidase
• Dnase
2. Toxins
• PhospholipaseC (α toxin)
• It has lethal, necrotizing and
hemolytic effect on tissue.
• Theta toxin
• It has hemolytic and necrotic effect
on tissue
• Enterotoxin

Clinical
manifestation:
1. Clostridial myonecrosis: Gas
gangrene
• IP(Incubation period) =1-3 days
• Colonization of devitalized traumatized
wound by C. perfringens spores, and
organism germination and release of
toxins
• Presentation: Muscle and subcutaneous
tissue necrosis and crepitation
• Foul smelling wound discharge
• Fever, toxaemia, hemolytic anemia, shock
2. Clostridial food poisoning
• It causes secretory diarrhea due to
release of enterotoxin in the intestine

Laboratory
diagnosis
Specimen: Infected
tissue, pus, vomitus,
left over food, serum
Smear: Non-motile,
capsulated, thick brick-
shaped gram positive
rods in smears from
tissue; spores are
rarely seen.
1. Blood agar medium
• β-hemolytic colonies
are seen anaerobic
atmosphere.
2. Cooked meat
medium(Chopped meat-
glucose medium)
• Thioglycolate medium
• Nagler reaction:
Lecithinase C activity-
Opacity in the egg-
yolk medium due to
lecithin break down

Identification of C. perfringens rests on colony form,
hemolysis pattern, biochemical reaction, and toxin
production and neutralization by specific antisera.
Treatment: Penicillin
• Prompt and extensive wound debridement
• Polyvalent antitoxin
Prevention and control
• Early adequate contaminated wound cleansing and
debridement

Clostridium
difficile
General characteristics:
• Not frequently found in the healthy
adult, but is found often in the hospital
environment
• Produce cytotoxins ( A and B)
• Human feces are the expected source of
the organism
Pathogenesis and clinical
features:
• Clinically presents with
pseudomembraneous colitis and
manifests with fever, abdominal cramps,
watery or bloody diarrhea leading to
dehydration, septicemia and shock

Lab. Diagnosis :
• Identification of toxin A and B in feces by
latex agglutination test
Susceptibility
• Discontinuation of offending drugs
• Administration of metronidazole or
vancomycin
• Administration of antibiotics like
ampicillin, clindamycin and
cephalosporins results in killing of colonic
normal flora and proliferation of drug
resistant C.difficile and release of
cytoxins

Clostridium tetani
• World wide in distribution in the soil and in
animal feces
• Gram -positive rods with round terminal spores
• There are ten antigenic types of C. tetani but all
produce the same neurotoxin.
• The toxin has two components:
• 1. Tetanospasmin: Neurotoxic property
• 2. Tetanolysin: Hemolytic property

Pathogenesis
and Clinical
manifestation
• Infection of devitalized tissue (wound,
burn, injury, umblical stamp, surgical
suture) by spores of C. tetani →
Germination of the spore and
development of vegetative organism→
Neurotoxin release from vegetative cells
→ The toxin binds to receptors on the
presynaptic membrane of motor neuron
the retrograde axonal transport to the
spinalcord and brain stem → Inhibition
of inhibitory glycinergic and GABAergic
secreting neurons → Spatic paralysis,
muscle spasms and hyperreflexia

Tetanus
Open lower-leg fracture following a
traffic accident; the portal of entry
of C. tetani.
Risus sardonicus: fully manifest
case of tetanus in a patient with
lower-leg fracture. Patient was
not vaccinated.

• IP= 4-5 days to several weeks
Tetanus classical presentation:
• Arm flexion and of leg extension
• Fever and sweating
• Muscle spasm and rigidity
Laboratory diagnosis:
• The bacteria can be cultured in a media with anaerobic
atmosphere.
• Proof of isolation of C. tetani must rest on production of
toxin and its neutralization by specific antitoxin
• Diagnosis is by clinical picture and history of injury

Susceptibility
• Administration of penicillin
• Provision of tetanus antitoxin (TAT)
• Avoid traditional application of mud or
ash over the umbilical stump
• Proper wound handling
• Immunization with tetanus toxoid
• NB: Since treatment of tetanus is not
satisfactory, prevention is all important

Clostridium
botulinum
Spores of C. botulinum are widely distributed in
soil, they often contaminate vegetables, fruits and
other materials.
Produce a neurotoxin which is the most active
known poison, and is considered to be the major
agent of bioterrorism and biological warfare
There are seven serotypes(A-G) of which A,B and
E are the principal causes of human illness.

Pathogenesis
and Clinical
manifestation
• Food botulism
• IP = 18-24 hrs
• Route of entry is under cooked
consumption of C. botulinum toxin food
• The toxin is absorbed from the gut and
acts by blocking the release of
acetylcholine at synapses and
neuromuscular junction and manifests
with flaccid paralysis and visual
disturbance, inability to swallow, and
speech difficulty
• Death is secondary to respiratory failure
or cardiac arrest

Clostridium
botulinum
2. Infantile botulism
• C. botulinum type A or B is usually implicated and
affects infants when mixed feeding starts (after fourth
month of life).
• Ingestion and colonization of the gut with C. botulinum,
and production of toxin and adsorption of toxin leads
to poor feeding, paralysis (floppy baby), and cranial
nerve palsy.
• Diagnosed by demonstration of the organism or toxin
from the stool
3. Wound botulism
• C. botulinum type A is usually implicated and caused by
the production of toxin by C. botulinum in wounds.
• The symptoms are the same as those in food poisoning.

Clostridium botulinum
• Laboratory diagnosis:
• Demonstration of toxin in patient’s serum and leftover food.
• .Death of mice after intra-peritoneal injection of toxin.
Susceptibility
• Administration of intravenous trivalent antitoxin ( A,B,E)
• Mechanical ventilator for respiratory support
• Prevention and control:
• Sufficient heating of canned foods before consumption
• Strict regulation of commercial canning
• Proper home canning methods

8.LISTERIA
MONOCYTOGENES
• Organisms of the genus Listeria are nonsporing gram-
positive bacilli. The genus contains eight species, but
almost all cases of human listeriosis are caused by L.
monocytogenes.
• Listeria monocytogenes is a small, coccoid, Gram-positive
bacillus measuring approximately 0.5 × 2–3 µm
• Cultural Characters
• Listeriae are aerobes and facultative anaerobes. They can
grow over a temperature range of 2–43°C, the optimum
temperature for the growth is 35–37°C.
• They can grow on ordinary media containing fermentable
carbohydrate, but growth is better on blood agar or
tryptose phosphate agar. After 24 hours incubation at 37°C,
colonies are 0.5–1.5 mm in diameter, smooth, translucent
and emulsifiable and non-pigmented.

Pathogenisis
• Human infection is believed to result from
contact with infected animals, inhalation of
contaminated dust or ingestion of contaminated
milk or food.
• Hospital acquired infections have also been
reported.
• L.monocytogenes produces a hemolysin known
as listeriolysin-O, which is a virulence factor
antigenically related to streptolysin-O and
pneumolysin.
• Clinical Features
• Intrauterine and neonatal infection:
Intrauterine infection of the fetus may result in
abortion, premature delivery, or acute-onset
disseminated infection in the newborn infant

Pathogenisis
Adult and juvenile infection: It may cause
meningitis or meningoencephalitis, particularly
in neonates and in the elderly.
Disease in healthy adults: Most Listeria
infections in healthy adults are asymptomatic or
occur in the form of a mild influenza-like illness.
Several foodborne outbreaks of acute
gastroenteritis with fever .
Other infections: Listeriosis may also present as
abscesses, conjunctivitis, pharyngitis, urethritis,
pneumonia, infectious mononucleosis like
syndrome, endocarditis or septicemia.

Laboratory
diagnosis
1. Specimens
Blood, CSF, amniotic fluid, placenta, pus and biopsy
material from the organs involved may be collected.
Specimens may also be collected from neonate, stillbirth
or products of conception.
2. Microscopy
If the Gram stain shows organisms, they are intracellular
and extracellular gram-positive coccobacilli.
3.Culture
Specimens should be inoculated on blood agar,
chocolate agar and tryptose phosphate agar, and
incubated at 35–37°C for 1–3 days. Greater success in
isolation is achieved if the materials are stored in
tryptose phosphate or thioglycollate broth at 4°C and
subcultures are done at weekly intervals for 1–6 months

9.Corynebacterium
• Corynebacteria are gram-positive, nonacid fast, nonmotile rods with
irregularly stained segments, and sometimes granules.
• They frequently show club-shaped swellings and hence the name
Corynebacteria (from coryne, meaning club).
• Diseases:
• The major disease caused by C. diphtheriae is diphtheria (greek,
diphtheria, “leathery skin,” referring to the pseudomembrane that initially
forms on the pharynx).

Corynebacterium diphtheriae
Morphology
• They are, thin, slender gram-positive bacilli but are decolorized
easily, particularly in old cultures, measuring approximately 3–6
μm × 0.6–0.8 μm.
• They have a tendency to clubbing at one or both ends. They are
highly pleomorphic. They are nonmotile, non-spore-forming,
and nonacid-fast.
• They can be found as groups or as individual cells lying at sharp
angles to one another, resembling the letters V or L.
• This particular arrangement with C. diphtheriae has been called
the Chinese letter

Toxin
• Toxigenic strains of C. diphtheriae produce a a very
powerful exotoxin. The toxicity observed in diphtheria is
directly attributed to the toxin secreted by the bacteria at
the site of infection.
• Mode of Action
• The diphtheria toxin acts by inhibiting protein synthesis.
It inhibits polypeptide chain elongation in the presence of
nicotinamide adenosine dinucleotide (NAD) by
inactivating elongation factor 2 (EF-2), an enzyme
required for elongation of polypeptide chains on
ribosomes.
• Inhibition of protein synthesis is probably responsible for
both the necrotic and neurotoxic effects of the toxin.
• Antigenic Structure Diphtheria bacilli possess three
distinct antigens:
• 1. A deep-seated antigen found in all Corynebacterial
species
• 2. A heat-labile protein (K-antigen)
• 3. A heat-stable polysaccharide (O-antigen).

nicotinamide adenine dinucleotide
Toxins:
Exotoxin:
diptheria
toxin

Pathogenesis
• The organism is carried in the upper respiratory tract and spread by
droplet infection or hand-to-mouth contact.
• The incubation period of diphtheria is 2–5 days, with a range of 1–10
days. Diphtheria, which occurs in two forms (respiratory and cutaneous),
is found worldwide.
• Respiratory Diphtheria
• The illness begins gradually and is characterized by low-grade fever,
malaise, and a mild sore throat. The most common site of infection is the
tonsils or pharynx.
• The organisms rapidly multiply on the epithelial cells, and the toxigenic
strains of C. diphtheriae produce toxin locally, causing tissue necrosis and
exudate formation triggering an inflammatory reaction.

Pathogenesis
B.Systemic effects
The toxin also is absorbed and can produce a variety of systemic effects
involving the kidneys, heart, and nervous system, although all tissues
possess the receptor for the toxin and may be affected. Intoxication
takes the form of myocarditis and peripheral neuritis, and may be
associated with thrombocytopenia.
Complications
The common complications are as follows:
1. Asphyxia due to mechanical obstruction of the respiratory passage
by the pseudomembrane for which an emergency tracheostomy
may become necessary.
2. Acute circulatory failure, which may be peripheral or cardiac

Laboratory
diagnosis
1. Specimens
Swabs from the nose, throat, or other suspected
lesions must be obtained before antimicrobial
drugs are administered.
2. Microscopy
Direct microscopy of a smear is unreliable since C.
diphtheriae is morphologically similar to other
coryneforms. Smears stained with alkaline
methylene blue or Gram’s stain show beaded rods
in typical arrangement.
3. Culture The swab should be inoculated on
Loffler’s serum slope, tellurite blood agar, and
blood agar. The cultures should be incubated
aerobically at 37°C.

• Media
• Cystine-tellurite: Corynebacterium spp.
form black colonies
• from hydrolysis of tellurite.
• Tinsdale's agar: Corynebacterium
spp. form brown to black colonies
with halos from hydrolysis of tellurite.
• Loeffler agar is a nonselective medium
that supports growth and enhances
pleomorphism and the formation of
metachromatic granules.
• Most Corynebacterium spp. produce small,
white to gray colonies,
• C. diphtheriae will grow on SBA as
small, white, dry colonies. Most strains
are nonhemolytic.

• b. Corynebacterium
jeikeium
• C.jeikeium is an important
cause of nosocomial
infections and produces
infections after prosthetic
device implants and in
immunocompromised
patients.
• Pyrazidamidase
positive
• Resistant to most
antimicrobial agents
• c. Corynebacterium
urealyticum
• Cause UTIs
• Is rapid urease positive
and grows very slowly

GRAM NEGATIVE RODS
6. Enterobacteriaceae

Enterobacteriaceae
Characteristics
• Named, as well coliforms or
enterobacilli:
• Found as normal flora in intestinal
tract of humans and animals.
• Major cause nosocomial infection
• Gram-negative, non-spore forming,
aerobic and facultative anaerobic
bacteria.
• Most are motile.
• Grow over a wide range of
temperature in ordinary media.
• All ferment glucose with acid
production.
• Oxidase negative.
• Release endotoxin from their cell
wall.
• Some release exotoxin.
• They reduce nitrate to nitrites

Enteric culture media
Characteristics
• MacConkey agar: Lactose fermenters are pink/red, and
lactose nonfermenters are white.
• Eosin methylene blue agar: Colonies of lactose fermenters
have a dark center, and non lactose fermenters are colorless
• Hectoen enteric agar: Lactose and/or sucrose fermenters
from yellow/ orange colonies

Classification
It comprises the following
bacterial groups
1. Oxidase negative
a. Lactose-fermenters
• Escherichia spp.
• Klebsiella spp.
• Enterobacter
spp.
• Citrobacter spp.
b. Non-lactose
fermenters
• Salmonella spp.
• Shigella spp.
• Proteus spp.
• 2. Oxidase Positive
• Pseudomonas
spp.
• Vibrio
• Campylobacter
• Helicobacter

Escherichia
coli
Introduction
• Escherichia coli is a Gram-negative, non-
sporulating
• Facultative anaerobic and rod-shaped
Bacterium
• Commonly found in the lower intestine of
warm-blooded organisms (endotherms).
• Strains that possess flagella are motile
• Most E. coli strains are harmless,
• Some serotypes can cause serious food
poisoning in humans,
• The harmless strains are part of the
normal flora of the gut, and can benefit
their hosts by producing vitamin K, and by
preventing the establishment of
pathogenic bacteria within the intestine.

Pathogenicity
Urinary tract infection- cystitis, pyelonephritis
Wound infection- appendicitis, peritonitis
Neonatal septicemia and meningitis
E.coli-associated diarrheal disease:
• 1. Enteropathogenic E.coli (EPEC)
• Causes outbreaks of self-limiting infantile diarrhea
• They also cause severe diarrhea in adults
• Antibiotic treatment shorten the duration of illness
and cure diarrhea
• 2. Enteroinvasive E.coli (EIEC)
• Non-motile, non-lactose fermenting E.coli invade the
mucosa of the ileum and colon, and causes
shigellosis-like dysentery in children in developing
countries and travellers to these countries

3. Enterotoxigenic E.coli(ETEC)
• Colonization factor of the organism
promote adherence to epithelial cells of
small intestine followed by release of
enterotoxin which causes toxin-mediated
watery diarrhea in infants and young adults.
• Antibiotic prophylaxis can be effective but
may increase drug resistance
4. Entero haemorrhagic E.coli( EHEC)
• Cytotoxic verotoxin producing E.coli
serotype O157:H7 causes haemorrhagic
colitis (severe form of diarrhea), and
hemolytic uremic syndrome characterized
by acute renal failure, hemolytic anemia
and low platelet count

5. Enteroaggressive E.coli ( EAEC)
• Adhere to human intestinal mucosal cells
and produce ST-like toxin and hemolysin,
and causes acute and chronic diarrhea as
well as food-borne illness.
6. Diffusely adherent E coli
7. Uropathogenic E. coli

Virulence
factors
Complex surface antigens contribute to
pathogenicity and trigger immune
response:
• H – flagellar Ag
• K – capsule and/or fimbrial Ag
• O – somatic or cell wall Ag – all have
• Endotoxin
• Exotoxins

Pathogenicity
The commonest infection caused by E. coli is
infection of the urinary tract,
They cause of cystitis (infection of the bladder), the
infection may spread up the urinary tract to the
kidneys, causing pyelonephritis.
E. coli bacteria may also cause infections in the
intestine. Diarrhoeal infections(intestinal)
Infection to the bloodstream may cause blood
poisoning (E. coli bacteraemia).
In rare instances, E. coli may cause meningitis in very
young children.

Samples: stool, urine, blood or other
relevant sample
Culture:
• Isolation media
a) nutrient agar,
b)MacConkey’s agar.
b. Susceptibility

Culture characteristic and
biochemical test
Temperature 37°C for 24 hrs MacConkey Agar
Size in mm
Shape
Color
Margin
Elevation
Opacity
Consistency
1
Circular
Rose Pink colonies
Complete
Slightly
Opaque
Soft
Biochemical Test Results
Oxidase
Urease
TSI
MR
VP
Nitrate
Citrate
Indole (TW)
Gelatin
Negative
Negative
Acid butt, with gas, acid slant
Positive
Negative
Positive
Negative
Positive
Negative

Escherichia coli
Gram staining of a urine
sediment preparation: rounded
gram-negative rods, some
coccoid.
Can cause acute cystitis.
Culture on endo agar, a
combined selective/indicator
medium. The red color of the
colony and agar indicates the
lactose breakdown process.

11. klebsiella

Introduction
• Klebsiella pneumoniae can be found as a commensal in
the mouth and upper respiratory tract, it can be found
intestinal tract of humans and animals.
• These are also found in plants, water and soil.
Characteristics
• Non-motile,
• lactose-fermenting,
• Capsulated ,
• Gram-negative rods.
• Main species of medical importance:
• K. pneumoniae
• K. rhinoscleromatis
• K. ozenae

K. pneumoniae
• It is found as a commensal in the intestinal tract, and
also found in moist environment in hospitals.
• It is an important nosocomial pathogen.
• It causes:
• Pneumonia
• Urinary tract infection
• Septicaemia and meningitis (especially in neonates)
• Wound infection and peritonitis
• The virulence factor is the Capsular polysaccharide.
• The treatment is based on the result of antimicrobial
sensitivity test.

K. rhinoscleromatis
• It causes rhinoscleroma of nose and pharynx to
extensive destruction of nasopharynx.
K.ozaenae
• It causes ozena manifesting with foul smelling
nasal discharge leading to chronic atrophic rhinitis.

1.Specimen: Sputum, urine, pus, CSF, body fluid.
2.Culture on
(a) nutrient agar,
(b) MacConkey’s agar
3.Gram stain
4.Biochemical test
Serology: Capsular polysaccharide serotyping

Culture characteristic and biochemical results
Temperature 37°C for 24 hours Mac Conkey Agar
Size in mm
Shape round
Color Pink
Margin Complete
Elevation Slightly Raised
Opacity Translucent
Consistency Mucoid
3-4
Round
Pink
Complete
Slightly raised
Translucent
Mucoid
Test K. aerogenes Organism K. pneumoniae
Oxidase
Urease
TSI,
MR
VP
Citrate
Indole
Negative
Positive
acid slant, with gas, acid butt
negative
Positive
positive
Negative
Negative
Positive slow
acid slant, with gas, acid butt
positive
negative
positive
Negative

12.Enterobacter
• It is gram-negative lactose fermenting motile rods,
and found as a commensal in the intestinal tract of
humans and animals and moist environments.
• Medical important species is Enterobacter aerogens.
• It produces mucoid colony resembling Klebsiella on
Mac Conkey agar.
• Enterobacter aerogens is associated with urinary
tract infection, wound infection and septicaemia in
immunocompromised patients.

• Sample: wounds,
urine, blood and CSF
• Major characteristics
Colonies resemble
Klebsiella
• Motile
• Indole: Negative
• MR: negative
• VP: positive
• Citrate: Positive

13.Citrobacter
It is gram-negative lactose
fermenting motile rods, and
opportunistic pathogen.
Medical important species is
Citrobacter freundii.
Citrobacter freundii is associated
with urinary tract infection,
wound infection and septicaemia
in immunocompromised.

Diagnosis
Sample can be
urine, pus,
blood
Smear: Gram
stain
Culture
Biochemical
test:
Indole:- Methyl Red:+
VP:- Citrate:+

14.Salmonella
• Coliform bacilli (enteric rods)
• Motile gram-negative facultative anaerobes
• Non-lactose fermenting
• Resistant to bile salts
•
• H2S producing
• Species of medical importance are:
• S. typhi
• S. paratyphi
• S. enteritidis

Causes
1. Caused by the bacterium Salmonella Typhi .
2. Ingestion of contaminated food or water.
3. Contact with an acute case of typhoid
fever.
4. Water is contaminated where inadequate
sewerage systems and poor sanitation.
5. Contact with a chronic asymptomatic carrier.
6. Eating food or drinking beverages that
handled by a person carrying the bacteria.
7. Salmonella enteriditis and Salmonella
typhimurium are other salmonella bacteria,
cause food poisoning and diarrhoea.

How the bacteria cause disease
Ingestion of contaminated
food or water
Salmonella bacteria
Invade small intestine and
enter the bloodstream
Carried by white blood
cells in the liver, spleen,
and bone marrow
Multiply and reenter the
bloodstream
Bacteria invade the
gallbladder, biliary system,
and the lymphatic tissue
of the bowel and multiply
in high numbers
Then pass into the
intestinal tract and can be
identified for diagnosis in
cultures from the stool
tested in the laboratory

Clinical features
1. Enteric fever
• It is caused by S. typhi and S. paratyphi, and
• transmitted by fecal-oral route via contaminated
food and drinks
• Incubation period: 10-14 days
Predisposing factors:
• Reduced gastric acidity
• Disrupted intestinal microbial flora
• Compromised local intestinal immunity

Both manifest with persistent fever, headache, malaise,
chills, enlargement of liver and spleen, and skin rashes.
Paratyphoid fever is milder than typhoid fever
Complications:
• Intestinal perforation
• Lower gastrointestinal bleeding
• Dissemination to different body organs
• including meninges and brain
Mortality rate
• Untreated cases: 10-15%
• Treated cases: < 1%

2. Bacteremia with focal lesions
• Causative agent: S. choleraesuis
• Manifests with blood stream invasion with focal
lesions in lungs, bones and meninges
3. Gastroenteritis
• It is caused by S. enteritidis
• S. typhimurium
• IP= 8-48 hrs
• It manifests with initial watery diarrhea, and later
bloody mucoid diarrhea associated with crampy
abdominal pain.

Specimen:
1. Blood, Bone marrow, stool,
urine and serum for enteric fever.
• Blood – 80% positive in the first week.
• Stool- 70-80% positive in the second and
third week.
• Urine- 20% positive in the third and
fourth week.
• Serum for widal test- positive after the
second week of illness.

2. Stool for gastroenteritis.
• Gram reaction: Gram-negative rods
• Culture: Bacteriologic methods for
salmonella isolation
1. Differential medium
• For rapid isolation of lactose non-
fermenters Egs. Mac Conkey agar (Non-
lactose fermenting , H2S producing
colonies)
• Deoxycholate agar
2. Selective medium
• SS agar
• Deoxycholate-Citrate agar
Serology: (widal test)

Susceptibility and
prevention
Susceptibility
1. For cases
• Chloramphenicol
• Fluoroquinolones
• 3rd generation cephalosporins
Prevention and control
• Sanity measures like hygienic food and
drink handling, and avoid carriers from food
handling until properly treated
• Provision of vaccine

15.SHIGELLA
• Species of medical importance
are:
• S. dysenteriae A
• S. flexneri B
• S. boydii C
• S. sonnei D
• In developing countries,
shigellosis (bacillary dysentery) is
caused by S. flexneri and S.
dysenteriae.
It is found in human intestinal tract
as pathogen.

15. SHIGELLA
Coliform bacilli (enteric rods)
 Nonmotile gram-negative
facultative anaerobes
 Non-lactose fermenting
 Resistant to bile salts
• Shigellosis = Generic term for
disease
Low infectious dose (102-104)
Humans are only reservoir
Transmission by fecal-oral
route
Watery diarrhea with fever; changing to dysentery
Major cause of bacillary dysentery (severe 2nd stage)
in pediatric age group (1-10 yrs) via fecal-oral route
Outbreaks in daycare centers, nurseries, institutions
Estimated 15% of pediatric diarrhea in U.S.
Leading cause of infant diarrhea and mortality
(death) in developing countries

Virulence Factors
Virulence attributable to:
 Invasiveness
 Attachment (adherence) and internalization
with complex genetic control
 Large multi-gene virulence plasmid regulated
by multiple chromosomal genes
 Exotoxin (Shiga toxin)
 Intracellular survival & multiplication

Pathogenesis
and Clinical
features
• Pathogenicity determinant:
• Toxins:
• Endotoxin: irritate the bowel wall
• Exotoxin: Enterotoxin and neurotoxin
• IP: 1-2 days
• It causes shigellosis (bacillary dysentery)
characterized by bloody mucoid diarrhea, abdominal
cramp, fever, generalized muscle ache and
weakness.
Complication: Dehydration
• Electrolyte and acid-base disturbance
High prevalence:
• Poor sanitation, personal hygiene
• Polluted water supply
• Young children are frequently affected.

Laboratory
diagnosis
Specimen: Stool, serum
Gram reaction: Gram-negative non-
motile rods.
Culture: Non-lactose fermenting colonies
on Macconkey agar and SS agar.
It produces acid but not gas from
carbohydrate.

Susceptibility and
prevention
Susceptibility Ciprofloxacin Cotrimaxazole
Suppress acute
clinical attacks of
dysentry
Shorten the
duration of
symptoms
Prevention and
control:
Sanitary control of
water, food and
milk, sewage
disposal and fly
Control
Antibiotic
treatment of
infected individuals

16.Proteus
Proteus species are found in the intestinal tract of humans
and animals, soil, sewage and water.
They are gram-negative, motile, non-capsulated ,oxidase
negative and non lactose fermenting, pleomorphic rods.
Species of medical importance:
P. mirabilis
P. vulgaris

Clinical features
P. mirabilis
• Urinary tract infection
• Septicemia
• Abdominal and wound infection
• Secondary invader of ulcer, burn, pressure
sores and chronic discharging ear.
• P. vulgaris
• Important nosocomial pathogen.
• Isolated in wound infection and urinary
tract infection

Specimen: Urine, pus,
blood, ear discharge
Smear: Gram-negative
rods
Culture: Produce
characteristic swarming
growth over the surface
of blood agar.
Non-lactose fermenting
colonies in Macconkey
agar.
Proteus species have a
characteristic smell.
Biochemical reaction:
MR:+, VP:- AND
Citrate:-
Proteus spp………..
Urease positive
P. vulgaris………... Indole
positive
P. mirabilis……….. Indole
negative
Susceptibility: Based
on sensitivity testing.

17.Pseudomonas
• Gram-negative motile aerobic rods.
• Can be found in water, soil, sewage, vegetation, human and
animal intestine.
Species of medical importance:
• P. aeruginosa
• P. pseudomallei

Pseudomonas
aeruginosa
Found in human and animal intestine, water,
soil and moist environment in hospitals.
Primarily a nosocomial pathogen.
Invasive and toxigenic
Produce green pigment on culture medium

Antigenic
characteristic
• Pili: Adhere to epithelial cells
• Exopolysaccharide: Anti-phagocytic
property/ inhibit pulmonary clearance
• Lipopolysaccharide: Endotoxic effect
Enzymes
• Elastases: Digests protein (elastin,
collagen, IgG)
• Proteases
• Hemolysins
• Phospholipases C (heat labile): Degrade
cytoplasmic membrane components
• Exotoxin A: Cytotoxic by blocking protein
synthesis

Clinical
features
• Pathogenic only when introduced into areas devoid
of normal defenses eg. Breached mucus membrane
or skin, use of IV line or urinary catheterization,
neutropenia of any cause
• Urinary tract infection- chronic, complicated Urinary
tract infection and associated with indwelling
catheter.
• Wound infection of burn sites, pressure sores and
ulcers.
• Septicaemia
• Pneumonia- Infection of the lung in patients with
cystic fibrosis.
• Eye infection- Secondary to trauma or surgery.

• Specimen: pus, urine, sputum,
blood, eye swabs, surface swabs
• Smear: Gram-negative rods
• Culture:
• Obligate aerobe, grows readily on all
routine media over
• Wide range of temperature (5-42
OC).
• Bluish-green pigmented large
colonies
• Catalase positive
• Citrate positive
• Indole negative

Susceptibility and prevention

18.Vibrios
• Actively motile, gram-negative
curved rods.
• Species of medical importance:
Vibrio cholerae-01

Vibrio
cholerae
Man is the major reservoir of V. cholerae-
01, which causes epidemic cholera.
Found in fresh water, shellfish and other
sea food.
Characteristics:

Antigenic structure
O antigen
• Six major subgroups.
• All strains possess a distinctive O
antigen and belong to subgroup I with
subdivision into three serotypes;
• Ogawa, Inaba, Hikojima.
H antigen
• Little value in identification

Clinical features
Causing excessive fluid secretion resulting in diarrhea (rice water stool) characterized by passage of
voluminous watery diarrhea containing vibrios, epithelial cells and mucus; and result in severe
dehydration.
The bacteria adheres to the intestinal wall with out invasion then
produces an exotoxin
After ingestion of the V. cholerae-01,
Route of infection is fecal-oral route.

Laboratory
diagnosis
• Specimen: Stool flecks
• Smear: Gram-negative motile curved rods
• Motility of vibrios is best seen using dark-field
microscopy.
• Culture:
• 1. TCBS (thiosulphate citrate bile salt sucrose
agar)
• 2. Alkaline peptone water: Enrichment media
for
Biochemical Reaction:
• Oxidase-positive.
• Ferment sucrose and maltose(acid; no gas).
• Do not ferment L-arabinose.

Susceptibility
Susceptibility to tetracycline
and chloramphenicol.
Fluid and electrolyte
replacement are the first line
of management for cholera.

19. Brucella
The genus Brucella consists of very small,
nonmotile, aerobic, gram-negative coccobacilli.
Brucellae are essentially pathogens of goats,
sheep, cattle and pigs. Man acquires infection by
direct or indirect contact with infected animals.
Human infection is a zoonosis that is acquired
from animals or animal products.
Species: Six species are currently recognized: B.
melitensis, B. abortus, B. suis, B. ovis, B.
neotomae and B. canis.

Classification of Brucellae
Brucellae may be categorized into species and biovars
The three major species are: B. melitensis, B. abortus, B suis infecting
primarily goats or sheep, cattle and swine, respectively. Many biotypes
have been recognized in these species
.i. B. melitensis: three biotypes.
ii. B. abortus: 7 biotypes (1-6 and 9, biotypes 7 and 8 have been
discarded as invalid).
iii. B. suis: 5 biotypes. B. suis strains that produce H2 S are known as
‘American’ strains and those that do not as ‘Danish’ strains.

Epidemiology
Infection is transmitted among animals directly or
through blood-sucking arthropods, particularly ticks.
The animals that commonly act as sources of human
infection are goat, sheep, cattle, buffaloes, and swine.
Human brucellosis is acquired from animals, directly or
indirectly.
Brucellosis is a zoonosis of worldwide importance,
particularly in developing countries.

Pathogenesis
The three major species of brucellae are
pathogenic to human beings. B. melitensis is the
most pathogenic, B. abortus and B. suis of
intermediate pathogenicity.
Mode of infection: The modes of infection are by
ingestion, contact, inhalation or accidental
inoculation.
Person-to-person spread does not ordinarily occur,
but very rarely transmission has been reported
through the placenta, breastfeeding and sex

Pathogenesis
Ingestion: The most important
vehicle of infection is raw milk.
ii. Contact: Contact infection is especially important as an
occupational hazard in agricultural workers, veterinarians,
butchers, animal handlers, and others in occupations that
involve handling of animals or uncooked animal tissues are at
higher risk for direct inoculation.
iii. Inhalation: Infection is transmitted by inhalation of dried
material of animal origin such as dust from wool

Pathogenesis
The incubation period is usually about 10–30 days. Human infection may
be of three types:
1. Latent or subclinical infection: There is no clinical evidence of disease
but is detectable only by serological tests.
2. Acute brucellosis: Acute brucellosis is mostly due to B. melitensis. It is
associated with prolonged bacteremia and irregular fever. It is also
known as undulant fever or Malta fever because of the periodic noctural
fever that may occur over weeks, months or years particularly in
untreated cases.
3. Chronic brucellosis: Chronic brucellosis is a low grade infection with
periodic exacerbations. It is usually nonbacteremic. The illness lasts for
years.

Laboratory
Diagnosis
Laboratory methods include culture of
brucellae, serology, polymerse chain reaction
and hypersensitivity type skin tests.
1. Specimens
Blood culture is most important. Material from
bone marrow or liver biopsy, is also cultured,
lymph nodes, cerebrospinal fluid, urine and
abscesses, and on occasion, also from sputum,
breast milk, vaginal discharges and seminal
fluid.
2. Culture
a. Blood culture Blood culture is the most
definitive method for the diagnosis of
brucellosis

Prophylaxis
Prevention consists of checking brucellosis
in dairy animals.
2. Control of this disease in cattle has been achieved by
serologic surveillance, vaccination (B. abortus strain 19), and
elimination of reactor cattle.
3. Pasteurization of infected milk or milk
products.
4. Vaccines have been developed for use in
animals.
The live-attenuated B. abortus strain S19 vaccine has been is now
being replaced by the rough strain B. abortus RB51, which gives
comparable protection, but does not induce interfering antibodies
and is less hazardous to man

20.Helicobacter
pylori
• Spiral-shaped gram negative,
microaerophilic, motile rods
with polar flagella
• Pili
• Protease
• Urease

Pathogenesis
and clinical
features
Route of entry:
Ingestion of
contaminated food
and drinks
Type B chronic
gastritis
Peptic ulcer
disease (gastric and
duodenal ulcer)
Gastric carcinoma Gastric lymphoma

Lab. Diagnosis
• Specimen: Gastric biopsy, serum
• Smear: Giemsa or silver stain
• Culture:
• Urease positive
Serology:
• Detection of antibodies in the serum specific for H. pylori
• Detection of H. pylori antigen in stool specimen

Susceptibility
and prevention
Susceptibility
• Triple or quadruple therapy:
• . Amoxicillin + clarithromycin/
metronidazole +
• Proton pump inhibitors (PPI
(Omeprazole or
• lansoprazole))
• or
• Metronidazole + Bismuth
subsalicylate/ Bismuth
• subcitrate + Amoxicillin / Tetracycline
+ PPI
• Improving sanitary hygiene

21.Mycobacterium
Introduction:
The genus Mycobacterium belongs to the family
Mycobacteriaceae.
There are over 80 named species of mycobacteria.
The most familiar of the species are Mycobacterium
tuberculosis (MTB) and Mycobacterium leprae, the
causative agents of tuberculosis (TB) and Hansen’s
disease (leprosy), respectively.

Classification of
Mycobacterium

Mycobacteria
Characteristics:
• Non-spore forming, non-motile, aerobic,
Acid-fast bacilli.
• Acid-fastness depends on the waxy
envelope-mycolic acid of cell wall.
• More resistant to chemical agents than other
bacteria.
• Once stained with primary stain, they resist
decolonization by acid-alcohol.
• All bacteria are decolorized by acid-alcohol
except Mycobacteria.
• Mycobacteria of medical importance:
• M. tuberculosis
• M. leprae

Mycobacterium
tuberculosis
Strictly aerobic acid-fast bacilli.
The main reservoir is an infected human.
1. Lipids: Mycolic acid, waxes, phosphatides responsible for acid-
fastness, granuloma formation and caseation necrosis.
2. Proteins
Elicits the tuberculin reaction and antibody production.
3. Polysaccharides
• Induce the immediate type of hypersensitivity.

Clinical
manifestation
Incubation period: 4-6 weeks.
Source of infection: Tuberculous patients
Route of infection: Respiratory- Inhalation of droplet nuclei
Ingestion of infected milk
Disease: Pulmonary and extrapulmonary tuberculosis
The disease generally manifests with low-grade persistent
fever, night sweating, significant weight loss, fatigue and
generalized weakness.

Diagnosis: Bacteriological
diagnosis of tuberculosis can
be established by direct
microscopy, culture
examination
Specimen: Sputum; pleural,
peritoneal and
cerebrospinal fluid
Microscopy: Z–N staining
and auramine rhodamine
staining for demonstration
of AFB in stained smears is
most method used
Smear: Acid fast bacilli from
primary specimen.
Culture:Lowenstein-Jensen
medium
It is the ordinary selective
media for tubercle bacilli
Raised, dry, cream colored
colonies of tubercle bacilli
after 3-6 wks of incubation
The recent rapid and
automated methods include
automated radiometric
culture methods (e.g.
BACTEC) , SEPTICHEK,
MGITs, etc

Susceptibility
and
prevention
Susceptibility
• Anti-tuberculosis drugs
1. First-line drugs
• Isoniazid
• Ethambutol
• Rifampin
• Pyrazinamide
• Streptomycin

Susceptibility
2. Second-line drugs
•Kanamicin.
•Cycloserine
•Capreomycin
•Ofloxacin
• Ethionamide
•Para-aminosalicylic acid (PAS)

Prevention
and control
Prompt and effective treatment of
patients with active tuberculosis.
Immunization with BCG (Bacillus-
Calmette-Guerin) vaccine
Pasteurization of milk and milk
products

Mycobacterium
leprae
Characteristics:
Typical acid-fast bacilli, arranged in singly, parallel
bundles or in globular masses.
Not grown in non-living bacteriologic media.
Characteristic lesions are grown in laboratory
animals.
Eg. mice

Clinical
features
Incubation period is
months to years.
Route of infection is
through nasal
mucus secretion,
skin contact.
Disease: Hansen’s
disease or leprosy.
The lesion involves
the cooler parts of
the body, Eg. Ear
lobes.
Peripheral neuritis
Presence of acid-fast
bacilli from skin
lesion
Two major types of
leprosy
• 1. Lepromatous leprosy
• 2. Tuberculoid leprosy

Laboratory
diagnosis
Specimen: Skin
scrapings from
the ear lobe.
Smear: Acid fast
bacilli from the
primary specimen.
Bacterial index (BI)
indicates number
of organisms
present in a smear.

Susceptibility
•Dapsone
•Rifampicin
•Clofazimine
Anti-leprosy drugs

22.Nontuberculous
Mycobacteria
• Mycobacteria other than human or
bovine tubercle bacilli, may
occasionally cause human disease
resembling tuberculosis.
• This large group of mycobacteria
have been known by several names;
atypical, anonymous, unclassified,
paratubercle, tuberculoid,
environmental or nontuberculous
mycobacteria (NTM),
• ‘nontuberculous mycobacteria
(NTM)’ has gained wide

Epidemiology
Environmetal mycobacteria are widely distributed
in nature. Infection with them is quite common,
from soil, water and air. Infection is mainly
asymptomatic.
In countries in which tuberculosis is ‘uncommon,
opportunist mycobacterial infections are relatively
common.
In addition, the absolute incidence is increasing as
a result of the growing number of
immunocompromised individuals, notably
patients with AIDS.

Differentiating characterstics of M. tuberculosis and ‘nontuberculous
mycobacteria (NTM)

Classification
The N TM are classified
into four groups based on
phenotypic characteristics
of the various species,
most notably pigment
(yellow or orange)
production and rate of
growth.

Pathogenesis
Four main types of
opportunist
mycobacterial disease
have been described in
man:

Laboratory
diagnosis
• Specimen: Sputum, pus or exudates. B.
Microscopy: Ziehl–Neelsen staining of
smear showsacid-fastt bacilli.
• Reapeted smear examination is necessary.
C. Culture: They grow well on LJ medium.
Several LJ media should be inoculated
with the specimen.
• These are incubated in the dark and in the
light at different temperatures for
distinguishing the species.

Differentiation between tubercle bacilli and some species of
atypical mycobacteria

23.The anaerobic bacteria
The anaerobic bacteria are widespread in nature. A
range of anaerobic bacteria are found in the mouth
and oropharynx, gastrointestinal tract and female
genital tract of healthy individuals as part of the
commensal flora.
Previously considered to be harmless commensals
of our indigenous flora, are now recognized as
opportunistic pathogens that may produce disease
when the host’s resistance is reduced.

Anaerobic infections
Most of the anaerobic bacteria that cause infection are members of our normal indigenous
flora.
Anaerobic infections are usually endogenous and are caused by tissue invasion by bacteria
normally resident on the respective body surfaces. Anaerobic bacteria are normally present on
the skin, mouth, nasopharynx and upper respiratory tract, intestines and vagina.
Anaerobic infections generally follow some precipitating factor, such as trauma, tissue
necrosis, impaired circulation, hematoma formation or the presence of foreign bodies.
Diabetes, malnutrition, malignancy or prolonged treatment with aminoglycoside antibiotics,
corticosteroids and cytotoxic agents may act as predisposing factors. Anaerobic infections are
typically polymicrobial

Classification

Classification ( Con’t)

Anaerobic cocci
• Strictly anaerobic gram-positive
cocci have been assigned to the
genera Peptococcus,
Peptostreptococcus, Coprococcus,
Ruminococcus and Sarcina.
• Strictly anaerobic gram-negative
cocci are included in Veillonella,
Megasphera and
Acidaminococcus.

Anaerobic cocci
• Strictly anaerobic gram-positive cocci
have been assigned to the genera
Peptococcus, Peptostreptococcus,
Coprococcus, Ruminococcus and
Sarcina.
• Strictly anaerobic gram-negative
cocci are included in Veillonella,
Megasphera and Acidaminococcus.

Anaerobic cocci
• Peptococcus
• Peptococcus niger is the only surviving member of the genus Peptococcus. They are
gram-positive, nonsporing, anaerobic cocci, that occur singly or in pairs or in
clusters.
• They produce black colonies on blood agar on prolonged incubation due to the
production of H2 S. They occur as normal flora of skin, intestine and genitourinary
tract.
• They may cause pyogenic infections of wounds, puerperal sepsis and urinary tract
infections.
• Peptostreptococcus
• They are cocci of small size (0.2–2.5 µm). Many of them are aerotolerant and grow
well under 10% CO2 in an aerobic atmosphere. Peptostreptococcus anaerobic is
most often responsible for puerperal sepsis and Pst. magnus for abscesses.

Anaerobic Gram (+)cocci
• Peptococcus
• Peptococcus niger is the only surviving member of the genus Peptococcus. They are gram-
positive, nonsporing, anaerobic cocci, that occur singly or in pairs or in clusters.
• They produce black colonies on blood agar on prolonged incubation due to the production
of H2 S.
• They occur as normal flora of skin, intestine and genitourinary tract. They may cause
pyogenic infections of wounds, puerperal sepsis and urinary tract infections.
• Peptostreptococcus
• They are cocci of small size (0.2–2.5 µm). Many of them are aerotolerant and grow well
under 10% CO2 in an aerobic atmosphere. Peptostreptococcus anaerobius is most often
responsible for puerperal sepsis and Pst. magnus for abscesses.

Anaerobic Gram
(ve)cocci
Veillonella Veillonellae
are gram-negative cocci
of varying sizes, and
measure 0.3–2.5 μm in
diameter.
Occurring as diplococci,
short chains or groups.
They are normal
inhabitants of the
mouth, intestinal and
genital tracts.
Veillonella parvula has
been reported from
clinical specimens but
its pathogenic role is
uncertain

Anaerobic,
nonspore-
forming,
gram-positive
bacilli
This group contains many genera, of which
medically relevant are Eubacterium,
Propionibacterium, Lactobacillus, Mobiluncus,
Bifidobacterium and Actinomyces.
1. Eubacterium
Members of the genus Eubacterium are strictly
anaerobic and grow very slowly. They are
members of the normal mouth and intestinal
flora. Some species (E. brachy, E. timidum and
E. nodatum) are commonly seen in
periodontitis.
2. Bifidobacterium
Bifidobacterium is gram-positive bacilli
nonmotile, nonsporing and pleomorphic,
showing true and false branching. They occur
as normal flora of mouth, gastrointestinal tract
(GI) tract and genitourinary tract.
They are usually nonpathogenic.

Anaerobic,
nonspore-
forming,
gram-positive
bacilli
3. Lactobacillus.
• Lactobacilli are gram-positive bacilli, straight
or slightly curved which frequently show
bipolar and barred staining.
• They are nonsporing and most strains are
nonmotile.
• They form considerable amount of lactic acid
from carbohydrates and grow best at pH of 5
or less.
• Lactobacilli are normally present in the
mouth and have been incriminated in the
pathogenesis of dental caries.

It is believed that lactobacilli ferment sucrose to
produce lactic acid, which dissolves the mineral
components of enamel and dentine causing dental
caries.
Lactobacillus species are major members of the
normal flora of the vagina and, typically, in the adult
vagina and these are collectively known as
Doderlein’s bacilli.
They ferment the glycogen deposited in the vaginal
epithelial cell and form lactic acid, which accounts
for the highly acidic pH of vaginal mucus epithelia.
They protect adult vagina from infections.

Anaerobic,
nonspore-
forming, gram-
positive bacilli
4.Mobiluncus
• Members of the genus Mobiluncus are
obligate anaerobic, gram-variable or gram-
negative, curved bacilli with tapered ends.
Two species, Mobiluncus curtisii and
Mobiluncus mulieris, have been identified in
humans.
• Mobiluncus curtisii and Mobiluncus mulieris
have been isolated from the vagina in
bacterial vaginosis, along with Gardnerella
vaginalis.
• Their microscopic appearance is a useful
marker for this disease, but the precise role
of these organisms in the pathogenesis of
bacterial vaginosis is unclear.

Anaerobic,
nonspore-
forming,
gram-positive
bacilli
5. Propionibacterium
• Propionibacterium species are members
of the normal flora of the skin and cause
disease when they infect plastic shunts
and appliances.
• Their metabolic products include
propionic acid, from which the genus
name derives.
• Species: The two most commonly isolated
species are Propionibacterium acnes and
Propionibacterium propionicus.
• P. acnes are responsible for oportunistic
infections in patients with prosthetic
devices or intravascular lines.

Anaerobic
gram-
negative
bacilli
• Gram-negative, anaerobic,nonsporeforming, non
motile rods were previously classified in the the
family Bacteroidaceae within three genera,
Bacteroides, Fusobacterium and Leptotrichia
• Bacteroides
• The genus Bacteroides can be divided into:
• Bile-tolerant
• i. Members of the B. fragilis group—B. fragilis, B.
ovatus, B. distasonis, B. vulgatus, B.
thetaiotaomicron, and others.
• ii. Two other species—Bacteroides eggerthii and
Bacteroides splanchnicus.

Anaerobic
gram-
negative
bacilli
• b. Bile Sensitive species
• i. Pigmented: Have been reclassified into—
the genera Porphyromonas, Prevotella and
porphyromonas.
• ii. Nonpigmented species: Many non-
pigmented species of Bacteroides also have
been transferred to the genus Prevotella.
• 2. Fusobacterium (Bacilli with pointed ends)
Fusobacterium necrophorum, Fusobacterium
nucleatum, Fusobacterium necrogenes.
• 3. Leptotrichia (Large bacilli) Leptotrichia
buccalis—the only species

1. Bacteroides
• Members of the B. fragilis group, which contains
about 10 related species, are especially
pathogenic.
• B. fragilis is the most common species of
anaerobic bacteria isolated from infectious
processes of soft tissue and anaerobic
bacteremia.
• B. fragilis is the most frequent of the nonsporing
anaerobes isolated from clinical specimens.
• It is often recovered from blood, pleural and
peritoneal fluids, CSF, brain abscesses, wounds
and urogenital infections.
• Their polysaccharide capsule is an important
virulence factor, conveying resistance to
phagocytosis

b. Bile-sensitive Species
• Pigmented species
• Prevotella
• Prevotella spp. appears as gram-negative coccobacilli or
bacilli, very similar to Bacteroides spp.
• Prevotella melanogenic is part of the normal flora of the
mouth and upper alimentary and respiratory tracts.
• Its cell wall contains a strong endotoxin.
• Prevotella infections are usually associated with the upper
respiratory tract, causing, for example, dental and sinus
infections, pulmonary infections and abscesses, brain
abscesses, and infections caused by a human bite

Laboratory
diagnosis
• P. melaninogenica forms black colonies on
blood agar—a characteristic from which its
name was derived.
• The color is not due to the melanin pigment
• Cultures of P. melaninogenica and even
dressings from wounds infected with the
bacillus give a characteristic red fluorescence
when exposed to ultraviolet light.
• P. melaninogenica exhibits less drug resistance
than do the bacteroides, and are susceptible to
penicillin.
• Porphyromonas: Porphyromonas species can be
cultured from gingival and periapical tooth
infections and, more commonly, breast, axillary,
perianal, and male genital infections

Nonpigmented
Species
Many non pigmented
species of bacteraies have
been transferred to the
genus prevotella.
Nonpigmented
Prevotella spp. includes
Prevotella bivia,
Prevotella buccae,

Fusobacterium
Fusobacteria are spindle-shaped gram-
negative bacilli with pointed ends a
morphology characteristically referred to as
fusiform. They are found as part of the normal
flora of the mouth, female genital tract, and
colon. They grow slowly in vivo, and are
therefore of limited virulence.
Species:
i. F. nucleatum: It is frequently recovered
from mixed infections of the head and
neck region, including dental abscesses
and the central nervous system, from
transtracheal aspirates and pleural fluid.
ii. F. necrophorum: Is an important animal
pathogen.

Leptotrichia
• They are long, straight or slightly
curved rods, often with pointed
ends.
• This species was originally classified
in the genus Fusobacterium, and
was formerly known as Vincent’s
fusiform bacillus or Fusobacterium
fusiforme.
• The genus Leptotrichia contains the
single species, L. buccalis.

The association of L. buccalis with disease is not clearcut, although it
has been reported in acute necrotizing ulcerative gingivitis (Vincent’s
gingivitis) or Vincent’s angina, together with Treponema,
Porphyromonas and Fusobacterium species.
It is seen in patients with malnutrition, debility and poor oral
hygiene.
It is characterized by pain, hemorrhage, foul odor,
destruction of interdental.

Selected infections

Specimen Collection and Transport.
Aspirated or tissue specimens are preferable to swabs
whenever feasible. Swabs are unsatisfactory should be sent in
Stuart’s transport medium.
specimens should be placed in an anaerobic transport device
that consists of a tube or vial containing an anaerobic gas
mixture substituted for air.
Specimens should be delivered within 20 minutes) for culture.
Recapping a syringe and transporting the needle and syringe
to the laboratory is no longer acceptable because of safety
concerns involving needle stick injuries.

• Specimen Collection and Transport.
• Even aspirates must be injected into oxygen-free
transport tube or vial.
• Gas-liquid chromatography may be done on pus
in order to detect metabolic products, such as
butyric and propionic acids, that are defines
certain anaerobes.
• B. Direct Microscopy Examination of a gram
stained smear is very useful. Pus in anaerobic
infection usually shows a large variety of different
organisms and numerous pus cells. Examination
of the specimen under ultraviolet light may show
the bright red fluorescence of P. melaninogenica.

C. Culture
Several special media have been described for anaerobes but for
routine diagnostic work, freshly prepared blood agar with neomycin,
yeast extract, hemin and vitamin K is adequate. Plates are incubated at
37°C in an anaerobic jar, with 10% CO2 .
The Gas-Pak system provides a convenient method of routine anaerobic
cultures. Plates are examined after 24 or 48 hours. Anaerobic
fusobacteria, require longer periods of incubation.
Since many anaerobes are relatively slow-growing, it is essential that
cultures are incubated for several days before being discarded. In mixed
infections, fast-growing aerobic or facultatively anaerobic organisms are
often detected within 24 hours, whereas some anaerobes may require
incubation for 7–10 days before their colonies can be recognized

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