Microbiology viriology and immunology

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Gram Positive Cocci (I+II)

● Staphylococcus
● Streptococcus
● Enterococcus

Gram Negative Cocci (II)

● Neisseria (gonorrhea, meningitis)

Both staphylococci and streptococci are gram-positive cocci, but they are differentiated by
two main criteria:

1. Microscopically, staphylococci appear in grapelike clusters, whereas streptococci are

in chains.
2. Biochemically, staphylococci produce catalase (i.e., they degrade hydrogen
peroxide), whereas streptococci do not.

Staphylococci

Staphylococci are gram-positive aerobic nonmotile cocci about 1m in diameter. They form
clusters of cells as they divide. Common inhabitants of the skin and mucous membranes. As
a group, Staphylococci do not have spores or flagella, but may form capsules. They grow
well on routine laboratory media and are facultative anaerobes.

Important human pathogens are: S. aureus, S. capitis, S. epidermidis, and S. hominis Of

these, S. aureus in the most serious.

Virulence Factors of Staphylococcus aureus

Different strains of Staphylococcus aureus produce a variety virulence factors (enzymes and
toxins) harmful to their human hosts

Exoenzymes:

1. Coagulases are enzymes that clot plasma and blood. The presence of this enzyme
indicates the pathogenic potential of the
strain (coagulase test - fig. 18.6).
2. -lactamases break down penicillin and cephalosporins, making these antibiotics
useless.
3. Hyaluronidase breaks down hyaluronic acid, that holds cells together in connective
tissue; thus facilitates the spread of
bacteria.
4. Lipases hydrolyze host cellular membrane fats and lipids, causing lysis of host cells.
Found in Staphylococcus strains that cause
pimples and boils.

Exotoxins found in Staphylococcus aureus are often responsible for the symptoms of the
disease.

1. Leukocidin is a cytolytic toxin that disrupts the plasma membrane of

polymorphonuclear leukocytes (e.g. neutrophils) and
macrophages. It attacks the phospholipids in the cell membrane.
2. Hemolysins are toxins that destroy RBCs.
3. Exfoliative toxin – causes epidermis to separate from dermis; skin peels away in
scalded skin syndrome.
4. Enterotoxins – exotoxins that act on the gastrointestinal tract inducing nausea,
vomiting, and diarrhea; one of the top causes of
food borne illness.
5. Toxic Shock Syndrome Toxin (TSST) – exotoxin present in some strains that leads to
a characteristic and potentially fatal
condition known as toxic shock syndrome.

The Scope of Staphylococcal Disease

Staphylococcal infections

Like many invading organisms, Staphylococci can begin as a local infection of the skin and
then spread systemically, leading to much more severe conditions. A local staph infection
can cause an abscess.

Localized Cutaneous Infections.

1. folliculitis – a mild, superficial inflammation of hair follicles

2. impetigo – characterized by bubble-like epidermal swellings that can break and peel
away (fig. 18.5a).
3. Boils (furuncles) result when the inflammation of a single hair follicle or sebaceous
gland progresses into a large, red, and
tender abscess or pustule. A cluster of furuncles is referred to as a carbuncle.
4. scalded skin syndrome (SSSS) – exfoliative toxin in local infections causes blistering
and peeling away of outer skin layers.

Treatment: Fluid and electrolyte replacement to reverse hypotension and shock. Drain
abscesses and use antibiotics (-lactamase- resistant penicillins and cephalosporins).
Host Defenses Against S. aureus

Humans have a well-developed defense against staph infections. Unbroken skin is a good
defense against staph. Specific antibodies are produced against staph, but they are not very
effective. Neutrophils and macrophages serve as the better defense. Abscess formation
helps to prevent the spread of staph in the body.

Other Important Staphylococci

Coagulase-negative staphylococci.

1. Staphylococcus epidermidis – normal skin flora and mucous membranes; also S.

hominis lives near apocrine glands, S. capitis lives on the face, scalp and external
ear. Each bacterium is able to enter breaks in the skin at their location and cause
infection.
2. S. saprophyticus – a common urinary tract infection in sexually active young women.

Identification of Staphylococcus Isolates in Clinical Samples

Staphylococci can be isolated from body fluids and infected tissues. Can use sheep’s blood
agar and/or mannitol salt agar. It is Gram positive with irregular clusters of cocci cells.
Differentiate from streptococci by a catalase test , and other cocci by biochemical tests.

S. aureus is differentiated from other staph species by the production of coagulase where S.
aureus produces coagulase PCR can also be used to identify S. aureus. See table 18.2 for
the different characteristics of the clinically relevant staphylococci.

Clinical Concerns in Staphylococcal Infections

MRSA carries multiple antibiotic drug resistance genes. HA-MRSA are hospital acquired;
CA-MRSA are community acquired.

Streptococcus

They are all Gram-positive, catalase negative, non-spore forming, non- motile, facultative
aerobic cocci arranged in chains and pairs. The most important pathogens are
Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, and
Streptococcus mutans and Enterococcus faecalis.

They are classified into Lancefield groups, based on their surface antigens; in this case,
surface polysaccharides (A-H, J & K). Group A & B contain the most important human
pathogens. An alternative classification is based on their hemolytic activity.
Alpha hemolytic Streptococci break down red blood cells in blood agar and cause a zone of green
discoloration around the colonies. Beta hemolytic Streptococci produces a clear zone of hemolysis
around the colony. “N” hemolytic Streptococci produces no hemolysis.

Strep throat is caused by infection with a bacterium known as Streptococcus

pyogenes, also called group A streptococcus. Streptococcal bacteria are
contagious. They can spread through droplets when someone with the
infection coughs or sneezes, or through shared food or drinks.

Beta-Hemolytic Streptococci: Streptococcus pyogenes

The most serious streptococcal pathogen is Strep. pyogenes, a Group A strep. It is a strict
pathogen that inhabits the throat,nasopharynx, and occasionally the skin of humans.

Streptococcal pathogenicity resides in virulence factors including: M-protein, hemolysins and

erythrogenic toxins:

1. The M-protein of Group A is a fimbria protein that inhibits phagocytosis and helps the
bacterial cell bind to respiratory epithelial cells .
2. C-carbohydrates, specialized polysaccharides found on the cell wall.
3. Lipoteichoic acid contributes to the adherence of epithelial cells in the skin and
pharynx.
4. Capsule made of hyaluronic acid (HA).
5. C5a protease catalyzes a protein in the complement system.

Major Extracellular Toxins:

● Erythrogenic toxins are toxins that damage blood vessels beneath the skin and result
in the characteristic rash of scarlet fever.
● Streptolysins are hemolysins (destroy blood cells). Two types:
a. Streptolysin O (SLO) is an oxygen-sensitive enzyme produced by Group A
streptococci. SLO affects leukocytes and myocardial (heart) cells.
b. Streptolysin S is an oxygen-tolerant enzyme that contributes to beta4
hemolysis.
● Superantigens cause an over stimulation of T cells which leads to tumor necrosis
factor.
● Like the Staphylococci, Streptococcal species also cause a broad range of diseases.
These conditions can start as local infections and spread systemically.
● Erythrogenic/pyogenic toxin: scarlet fever
●

Major Extracellular Enzymes

1. Streptokinase will digest fibrin clots.

2. Hyaluronidase breaksdown connective tissue.
3. Streptodornase is a DNAse that hydrolyzes DNA

Epidemiology and Pathogenesis of Streptococcus pyogenes

Humans are the only significant reservoir. It gains access to the human host when immune
resistance is low or there is a break in the skin. Children tend to be the most susceptible
group in the population.

Skin Infections - Streptococcal Cutaneous Infections

Streptococcal infections also cause pyogenic lesions. They result from invasion of skin
through scratches, insect bites, cuts or sores. Occasionally, superficial streptococcal
infections can result in complications.

1. Streptococcal impetigo – a crusty, flaking of the epidermis, is caused by -hemolytic

Group A streptococci.

2. Erysipelas is an acute febrile (fever-related) disease with inflammation, redness of the

skin, head and face lesions accompanied by headache, nausea and vomiting. Untreated
infections can result in septicemia, abscesses, nephritis or rheumatic fever as the result of
toxins.

3. Necrotizing fasciitis – extensive necrosis of skin, and underlying connective tissue

associated with S. pyogenes infections5. The so- called “flesh eating disease”. Starts as a
normal infection. Enzymes digest connective tissue and toxins poison epidermal and dermal
tissue. As the flesh is killed (necrosis), it separates and sloughs off, forming a pathway for
deeper microbial invasion.

Throat Infections

Pharyngotonsillitis (“strep throat”) is inflammation of the pharynx accompanied by fever,

malaise, throat pain and post nasal secretions . The throat is scarlet red with pus-containing
material. Primary cause6: Group A, -hemolytic Streptococcus pyogenes.

"Strep throat" is common in school-age children (5-15 years) during the winter months. It is
transmitted by aerosols and occasionally food.

Systemic Infections

1. Scarlet fever – a fine, red "sandpaper" rash resulting from the action of erythrogenic toxins
on blood vessels. A complication of streptococcal pharyngotonsillitis. Begins with a rash on
the chest which spreads to other parts of the body. Fever, vomiting, and prostration
accompany the rash.

Long-Term Complications of Group A Infections

1. Rheumatic fever or rheumatic heart disease causes 10-20,000 deaths each year
(about 3% of human streptococcal infections develop into rheumatic fever). It is
characterized by arthritis and carditis (including permanent scarring and distortion of
heart valves), fever and inflammation of small blood vessels .

2. Acute glomerulonephritis is an acute inflammation of the glomeruli in the kidney. It

is characterized by blood in the urine (hematuria) and hypertension.
 3. Acute epiglottitis is grave inflammation of the epiglottis. The disease progresses
quickly causing fever, sore throat, extreme difficulty in swallowing and a continuing
enlargement of the epiglottis. If the airway becomes blocked, apnea and death will
follow. Other etiologic agents include Haemophilus influenzae, Streptococcus
pneumoniae, streptococci, staphylococci, and viruses.

Group B: Streptococcus agalactiae

Streptococcus agalactiae represents the group B streptococci (GBS), a potential normal flora
of the human vagina, pharynx, and large intestine. GBS is transferable to infants during
delivery and is the most prevalent cause of neonatal pneumonia, sepsis, and meningitis in
the U.S. and Europe. Approximately 19,000 babies a year acquire infection (5% mortality).

Group D Enterococci and Groups C and G Streptococci

1) Group D Enterococcus faecalis, E. faecium and E. durans are the “enterococci”,

normally inhabiting the human large intestine. Enterococci are emerging as a serious
nosocomial infection that is becoming highly drug resistant to vancomycin (VRE).
2) Groups C and G found in domestic animals, seen in severely compromised patients.

Formerly known as Streptococcus faecalis, Enterococcus faecalis was assigned its own
genus based on DNA hybridization tests. It shares many of the cellular characteristics of
Streptococcus (i.e. Gram-positive, non-spore forming, non-motile, etc.). Lancefield Group D,
hemolysis type α, β, and N. Habitat = human and animal intestine. Causative agent of
endocarditis and UTI (urinary tract infection).

Infections common in elderly patients having undergone surgery. These are emerging
nosocomial opportunists whose multi-drug resistance is becoming more prevalent.
Vancomycin- resistant strains (VRE) are of particular concern.

Laboratory Identification Techniques

It is important to recognize group A streptococcal infections. A rapid identification test is

used to identify Group A strep from throat samples. The test is based on monoclonal
antibodies that react with the C carbohydrate. Other tests include the CAMP test.

Treatment and Prevention of Group A, B, and D Streptococcal Infections

Treatment:
Penicillin remains the drug of choice since streptococci have not developed extensive
resistance to the drug. However, there are now strains of S. pneumoniae that are penicillin
resistant (PRSP). Lincomycin, erythromycin, clindamycin and third-generation
cephalosporins are also effective.

Alpha-Hemolytic Streptococci: The Viridans Group

Viridans strep are of human origin but do not fall into a Lancefield serology. They are mostly
in the oral cavity. They produce an alpha hemolysis. Usually enter via dental or surgical
work. The most important complication is subacute endocarditis

Streptococcus pneumoniae: The Pneumococcus

Bacteria in the mouth and pharynx can sometimes gain access (via the eustachian tube) to
the middle ear chamber and cause infections called otitis media

In young children Streptococcus pneumoniae is the most common cause of otitis media.
Inflammation of this sensitive area of the ear results in very painful earaches and even
temporary deafness.

Pneumonia is an inflammation of the lungs accompanied by fluid buildup in the alveolar

sacs. It can result from infectious and non-infectious processes. Bacterial pneumonia is often
caused by pneumococci, pyogenic cocci, and bacilli. It is characterized by high fever, chest
pains, chills, and a purulent cough (purulent = pus-containing). Worldwide, pneumonia is the
highest ranking of infectious disease and among top ten causes of death.

Pneumococcal pneumonia is caused by Streptococcus pneumoniae. Characterized by

acute onset of fever, chills, dyspnea (difficulty in breathing), pleurisy (inflammation of the
pleura), and productive cough. Sputum has purulent discharge tinged with blood.
Streptococcus pneumoniae is a Gram-positive coccus arranged in lancet-shaped pairs.

Various strains can be differentiated by the Quellung test, a serological test which
differentiates the strains based on surface antigens. Virulent strains are encapsulated
(antiphagocytosis), demonstrate alpha-hemolysis on blood agar, and form small mucoid
colonies with a central depression. The organism can be found as part of the normal flora in
up to 50% of adults.

The Family Neisseriaceae: Gram-Negative Cocci

Two species within the Neisseria genus are of most relevance to human health: N.
gonorrhoeae and N. meningitidis. Both are Gram negative, so caution must be taken if the
infection spreads systemically as septicemia. Their morphology is diplococcus. Most in the
genus Neisseria are strict parasites, so they do not survive long outside the human body.
Endotoxic shock can result from improper antibiotic administration (especially for N.
meningitidis).

Neisseria gonorrhoeae and Neisseria meningitidis are both Gram Negative Bacteria.
They are diplococci, non-sporing, non-motile and oxidase positive. But they have some
differences which are as follows:

Neisseriae occur in pairs that are non-motile. Facultative aerobes that are catalase and
oxidase positive

Neisseriae gonorrhoeae (gonococci) and Neisseria meningitidis (Meningococci) are

pathogenic for humans. Some neisseriae are normal inhabitants of respiratory tract, rarely
causing disease

Neisseriae meningtidis commonly cause of meningitis and septicemia

Neisseriae gonorrhoeae causes gonorrhea, conjunctivitis, pharyngitis, proctitis, urethritis

and prostatitis.

● They are strict aerobes, no growth occurs anaerobically.

● The optimum temperature for growth is 35-36 oC.
● No growth takes place below 30oC.
● Optimum pH is 7.4- 7.6.
● Growth is facilitated by 5-10 percent CO2 and high humidity.

Resistance

Meningococci are very delicate organisms being highly susceptible to heat,

dessication, alterations in pH and to disinfectants. They are sensitive to penicillin and
other antibiotics, but resistance strains have emerged and become common in many
areas.

Pathogenicity

Cerebrospinal meningitis and meningococcal septicemia are the two main types of
meningococcal disease. Meningococci are strict human parasites inhabiting the
nasopharynx. Infection is usually asymptomatic. In some, local inflammation ensues,
with rhinitis and pharyngitis. Dissemination occurs only in a small proportion. Most
common complication include Waterhouse-Friderichsen syndrome, a massive,
usually bilateral hemorrhage into the adrenal glands caused by fulminant
meningococcemia, adrenal insufficiency and disseminated intravascular coagulation.

Laboratory diagnosis

In meningococcal meningitis, the cocci are present in large numbers in the spinal
fluid and, in the early stage in the blood as well. Demonstration of meningococci in
the nasopharynx helps in the detection of carriers.

(a) Examination of CSF

The fluid will be under pressure and turbid, with a large number of pus cells.
For bacteriological examination, if a sufficient quantity is available, the CSF is
 divided into three portions. One portion is centrifuged and Gram- stained
smears are prepared from the deposit. Meningococci will be seen mainly
inside polymorphs but often extracellularly also. The second portion of the
CSF is inoculated iin blood agar or chocolate agar plates and incubated at 35-
36oC under 5-10% CO2. Colonies appear after 18-24 hrs which may be
identified by morphological and biochemical reactions. The third portion of the
CSF is incubated overnight either as it is or after adding an equal volume of
glucose broth and then subcultured on chocolate agar.
(b) Blood culture
Meningococcemia and in early cases of meningitis, blood culture is often
positive. Cultures should be incubated for 4-7 days, with daily subcultures.
(c) Nasopharyngeal swab
This is useful for the detection of carriers. The swab should be held in a
suitable transport medium like stuart’s medium
(d) Petechial lesions
Meningococci may sometimes be demonstrated in petechial lesions by
microscopy and culture.
(e) Molecular diagnosis
Group-specific diagnosis of infection can be made by detection of
meningococcal DNA sequence in CSF or blood by PCR amplifications.

Treatment

Prompt treatment is essential to ensure recovery without sequelae. Intravenous

penicillin G is the treatment of choice. Chloramphenicol is equally effective.

Neisseria gonorrhoeae: The Gonococcus

Neisseria gonorrhoeae is a Gram negative, nonmotile “bean-shaped” coccus. Strict

parasites, they do not survive long outside the body, i.e. you cannot catch it from inanimate
objects. Etiologic agent of gonorrhea. Also, see notes below on Neisseria meningitidis.

Morphology

The organism appears as a diplococcus with the adjacent sides concave, being
typically kidney shaped. It is predominantly within the polymorphs. Gonococci
possess pilli on their surface. Pili facilitate adhesion of the cocci to the mucosal
surfaces and promote virulence by inhibiting phagocytosis.

Cultural characteristics

Gonococci are more difficult to grow than meningococci. They are aerobic but may
grow anaerobically also. Growth occurs best at pH 7.2-7.6 and at a temperature of
35-360c with 5-10% CO2. They grow well on chocolate agar and Mueller-Hinton
agar. A popular selective medium is the Thayer-Martin medium which inhibits most
contaminants including nonpathogenic neisseria. Colonies are small, round,
translucent, convex and slightly umbonate, with a finely granular surface and lobate
margins.
Biochemical reactions

Gonococci resemble meningococci except in the effect of maltose. Gonococci acidify

only glucose and not maltose.

Antigenic properties

Gonococci are antigenically heterogeneous. They are capable of changing their

surface structures in vitro. Pili, which are hair like structures act as virulence factors
by attaching to host cells and inhibiting phagocytosis. The trilaminar outer membrane
of gonococci contains protein I and II which acts as ligands attaching the coccus to
the host cells. The outer membrane also contain lipopolysaccharide which may be
responsible for the toxicity in gonococcal infections.

Resistance

The gonococcus is a very delicate organism, readily killed by heat, drying and
antiseptics. In cultures, the coccus dies in 3-4 days but survives in slant cultures at
35oC.

Pathogenicity

The name gonorrhea, meaning flow of seed. The disease is acquired by sexual
contact. Infection of the lower genital tract can result in a purulent or pus-like
discharge from the genitals which may be foul smelling. N.gonorrhoeae can also
cause conjunctivitis, pharyngitis, proctitis or urethritis, prostatitis and orchitis.
Conjunctivitis is common in neonates and silver nitrate or antibiotics are often
applied to their eyes as a preventive measure against gonorrhea. Infection of the
genitals in females with N.gonorrhoeae can result in pelvic inflammatory disease if
left untreated, which can result in infertility.

Laboratory diagnosis Specimens

Pus and secretions are taken from the urethra, cervix, rectum, conjunctiva, throat, or
synovial fluid for culture and smear.

Smears

Gram-stained smears of urethral or endocervical exudates reveal many diplococci

within pus. These give a presumptive diagnosis.

Cultures

Immediately after collection, pus or mucus is streaked on enriched selective medium

like modified Thayer-Martin medium and incubated in an atmosphere containing 5%
CO2 (candle jar) at 36oc. to avoid overgrowth by contaminants, the selective medium
contains antimicrobial drugs like vancomycin, colistin, amphotericin. Forty-eight
hours after culture, the organisms can be quickly

identified by their apperancce on Gram-stained smear, by oxidase positivity, and by

coagglutination, immunofluorescence staining, or other laboratory test. The species
of bacteria may be determined by rapid carbohydrate utilization tests.

Nucleic Acid Amplification Tests

Several food and drug administration cleared nucleic acid amplification assays are
available for detection of N gonorrhoeae in genitourinary specimens.

Treatment

Penicillin G for inhibition (MIC - 2μg/ml). Pencillinase producing N gonorrhoeae

(PPNG) also have increased in prevalence. Uncomplicated genital or rectal
infections are treated with ceftriaxone 250mg given intramuscularly as a single dose.
Additional therapy with azithromycin 1 gm / doxycycline, orally twice a day for 7
days, is recommended for the possible concomitant chlamydial infection.

Factors Contributing to Gonococcal Pathogenicity

Gonorrhea is characterized by acute inflammation of the genital mucosal epithelium and a

purulent discharge. Only piliated strains are pathogenic, and attach to mucosal epithelium by
pili. Apparently, pili alone do not make N. gonorrhoeae virulent, they also contain a cell wall
component that destroys anti-gonococcal IgA antibodies (secretory antibodies)

Epidemiology and Pathology of Gonorrhea

Females: Approximately 50% of cases are asymptomatic. Signs and symptoms include
purulent exudates from the vagina and painful urination. Salpingitis, also known as PID
(pelvic inflammatory disease) results when the infection spreads to the fallopian tubes where
inflammation and scarring can result in the blockage of the oviducts which can result in
infertility or ectopic pregnancy

Males: anterior urethritis (inflammation of the urethra) together with painful urination and
characteristic purulent exudates (containing leukocytes – often with gonococci located
intracellularly, cellular debris, and gonococci) . With antibiotics, epididymitis (inflammation of
epididymis) and prostatitis (inflammation of prostate glands) have all but disappeared as
complications.

Treatment:
Penicillin is the preferred drug of choice, but some strains have begun to produce
penicillinase and new drug resistant strains have appeared over the past two decades.
Alternative drugs: Tetracycline, spectinomycin, and the quinolones. There is no vaccine and
no permanent immunity after an active case.

Neisseria meningitidis: The Meningococcus

Neisseria meningitidis, which causes meningococcal meningitis, is the only one to

cause small epidemics (e.g. crowded army barracks, crowded slum areas, daycare centers,
refugee camps). It is transmitted by droplets. Meningococcemia is when the organism
spreads to the bloodstream, where toxins can overwhelm the body in as little as 2 hours ;
this is associated with the appearance of subcutaneous hemorrhages called petechiae .
Drug of choice: Penicillin G.

Epidemiology and Pathogenesis of Meningococcal Disease

Sporadic or epidemic incidence in late winter or early spring. The reservoir is inhabited by
humans who harbor it in the nasopharynx. Easily transmitted to people who live in close
quarters, like the military or college dorms. It can cross the blood-brain barrier and grow in
the CSF.

Clinical Diagnosis of Meningococcal Disease

Bacterial meningitis is an emergency. CSF and blood samples are stained. Cultures may be
used to help in the identification. Specific rapid tests are also available.

Immunity, Treatment, and Prevention of Meningococcal Infection

Most people have a natural immunity. Drug treatment for those infected are given a third
generation cephalosporin called ceftriaxone. Vaccination is recommended for those 11-18
who may come into contact with the infectious agent. Two vaccines are available – Menactra
and Menomune.

Differentiating Pathogenic from Nonpathogenic

Lab tests include gram stain, growth on enriched media, growth at high CO2 levels, and
oxidase testing. Also PCR is used.

Other Genera of Gram-Negative Cocci and Coccobacilli

Branhamella catarrhalis is normal flora of the human nasopharynx. It can cause purulent
disease, associated with meningitis, endocarditis, and other infections. It is important to
differentiate it from the meningococcus.

Acinetobacter baumannii lives in water and soil and can survive harsh environments. It can
survive on fomites. It can cause nosocomial infections.

Moraxella species are found on mucous membranes of domestic animals and humans.
Rarely causes infections in humans.
S.N. Characteristics Neisseria gonorrhoeae Neisseria meningitidis

1 Referred as Referred to as gonococcus. Referred to as meningococcus.

2 Agents N. gonorrhoeae is the agent of N. meningitidis is a major

gonorrhoea. cause of cerebrospinal
meningitis.

3 Vaccine No Serogroup A, B, C, Y and W-

Development 135 meningococcal infections
can be prevented by vaccines.

4 Colony Morphology N. gonorrhoeae form smooth, N. meningitides would form

round, moist, uniform smooth, round, moist, uniform

grey/brown colonies with a large grey/brown colonies with

greenish colour underneath on a glistening surface and entire
primary isolation medium. edges.

5 Morphology N. gonorrhoea is kidney N. meningitidis is semicircular

shaped with apposing ends diplococcus with flat apposing
concave. ends.

6 Autolyse May autolyse Autolyse

7 Maltose No Yes
Fermentation
8 Nitrite Reduction N. gonorrhoeae doesn’t N. meningitidis can reduce
reduce nitrites. nitrites in low concentrations.

9 Growth on Blood N. gonorrhoeae grow less N. meningitidis grow well on

Agar blood agar than N.
gonorrhoeae.
well on blood agar than N.
meningitidis.

10 Capsule No Yes. The capsule is anti-

phagocytic and is an important
virulence factor.

11 Site of Infection Primarily causing infection of Colonizes the upper respiratory

the anogenital tract. tract as a commensal and
occasionally invades to

cause systemic disease.

12 Pathogens It is always considered a It is not always considered as

pathogen. pathogens.

13 Enzyme Production It doesn’t produce gamma- It produces gamma-

glutamylaminotransferase. glutamylaminotransferase.

14 Specimen Collection Transport swab of endocervix, Collect cerebrospinal fluid

urethra, rectum, pharynx, (CSF) and blood, swab skin
conjunctiva, blood, joint fluid, lesions and nasopharynx.
aspirates from skin lesions.
15 Β-Lactamase Common Rare
Production

16 Movement Movement of N. Movement of N.

gonorrhoeaeoccurs at lower meningitidisoccurred at higher
speed. speed and with a larger
number of retracting pili.

17 Pili N. gonorrhoeae most often N. meningitidis most often used

moved using one retracting four pili.
pilus.

18 Prevalence and N. gonorrhoeae infections N. meningitidis infections have

Mortality have a high prevalence and a low prevalence and high
low mortality mortality.

19 Superbug Considered as “superbug” Not considered as “superbug”

20 Pathogenesis N. gonorrhoeae can also Cause meningitis and other

cause conjunctivitis, forms of meningococcal
pharyngitis, proctitis or disease such as
urethritis, prostatitis, and meningococcemia, a life-
orchitis. threatening sepsis.
1. No spores, motility
2. Capsules
3. Slime layers
4. Facultative
5. Fermenters
6. Enriched media
7. Sensitive to drying, heat, disinfectants
8. Many drug-resistant
9. Enterococcus
10. Shared between humans and pets
Escherichia, shigella, Salmonella and vibrio cholerae food intoxication and poisioning

Escherichia

● Features:
○ Stain: gram-negative
○ Morphology: bacillus (rod)
○ Facultative anaerobic
○ Either nonmotile or motile (flagellated)
○ Catalase-positive
○ Ferments lactose
● Special media and biochemical test:
○ MacConkey agar: grows as pink colonies
○ Eosin–methylene blue (EMB) agar: grows as metallic, green colonies
○ Indole test–positive

Pathogenesis and Virulence Factors

Virulence
● Antigenic structures:
○ O antigen: component of the lipopolysaccharide (LPS) in the cell wall
 ○ H antigen: flagellar protein
○ K antigen: polysaccharide capsule
● Adherence factors and toxins are specific to the E. coli strain.

Avirulent variants of Escherichia coli

● Reservoir:
○ Humans are the primary reservoir.
○ Strains are part of the normal gut flora.
● Transmission:
○ In urinary tract infections (UTIs):
■ Bacterium ascends urethra to cause infection.
■ More common in women or with catheter use
■ Type 1 fimbriae (pili): virulence factor that allows bacterial
attachment to uroepithelial cells.
○ In neonatal meningitis:
■ Infant infected with maternal E. coli through rupture of
membranes or during childbirth
■ K1 capsular polysaccharide: virulence factor in most cases

Pathogenic variants of Escherichia coli

● Pathogenic strains are found exogenously and are introduced through
unsanitary food production or preparation.
● Transmission:
○ Fecal–oral route
○ Contaminated meat or produce
● Employ various virulence factors in producing illness, depending on the
pathogen

Pathogenic strains of E. coli

The following strains of E. coli are diarrheagenic:
1. Enterotoxigenic E. coli (ETEC); Also known as travelerts diarrhea with 2

types of toxins which are heat labile toxins (LT) and heat stable toxins (ST).

Fimbrial adhesins allow binding to the intestinal mucosa. Heat-labile (LT)

enterotoxin increases cAMP → altered electrolyte transport (↑ chloride

secretion) and diarrhea. Heat-stabile (ST) enterotoxin increases cGMP →

diarrhea
○ No inflammation or invasion
○ Short incubation period, lasting ≤ 5 days
○ Diagnosis: detection of heat-labile or heat-stable enterotoxins (by
polymerase chain reaction (PCR))

1. Enteropathogenic E. coli (EPEC) : Destruction of surface microvilli can lead

to fever,diarrhea,vomiting and non-bloody stools.For colonization, requires
bundle-forming pilus (BFP) (encoded by a plasmid EPEC adherence factor or
EAF)Carries chromosomal locus of enterocyte effacement (LEE), which
encodes intimin, a specific adhesin that binds intestinal epithelium .No toxin
production
○ Watery diarrhea without blood nor pus
○ Diagnosis: stool PCR
2. Enteroaggregative E. coli (EAEC): Aggregative adherence fimbriae (AAF) to
adhere to intestinal mucosa; forms a biofilm.“Stacked-brick” adherence pattern.
Some possess a heat-stable (ST) enterotoxin, similar to ETEC.
○ Treatment: fluoroquinolones to prevent chronic infection

3. Enteroinvasive E. coli (EIEC): Direct invasion of the intestinal epithelium

and formation of enterotoxins → necrosis and inflammation

○ EIEC directly invades the intestinal epithelium, causing bloody diarrhea.
○ Treatment is supportive.
 4. Enterohemorrhagic E. coli (EHEC): Binds to intestinal epithelium via
bacterial fimbriae.

Employs phage-encoded Shiga toxin:

○ Cytotoxic to both intestinal villi and colon epithelial cells

○ Primarily a food-borne infection
○ Painful, bloody diarrhea
○ Diagnosis: detection of Shiga toxin by enzyme immunoassay or PCR
○ Treatment:
■ Supportive
■ Avoid antibiotics, as they have been associated with the
development of HUS.
○ Inhibits protein synthesis → cell death

○ O157:H7 strain may lead to hemolytic uremic syndrome.

Shigella

● General characteristics:
○ Structure: bacilli
○ Gram stain: gram negative
○ Facultative intracellular
○ Motility: immotile, non-flagellated
○ Lactose fermentation: non-lactose fermenting
○ Oxidase negative
○ Acid stable
○ Culture:
■ No hydrogen sulfide (H₂S) production
■ On Hektoen enteric (HE) agar: green transparent colonies
● Associated disease: shigellosis (dysentery or Shigella diarrhea)

Clinically relevant species

Serogroups defined by specific O antigens:
 ● S. dysenteriae (serogroup A)
● S. flexneri (serogroup B)
● S. boydii (serogroup C)
● S. sonnei (serogroup D)

Pathogenesis

Reservoir and transmission

● Reservoir: human intestinal tract
● Transmission:
○ Contaminated food or water, usually fecal-oral transmission
○ Person-to-person contact (e.g., lack of hand washing, sexual
transmission in men who have sex with men)

Mnemonic

To help remember the modes of transmission of Shigella, remember the “4 Fs:”

● Fingers
● Flies
● Food
● Feces

Virulence factors
● Endotoxin:
 ○ Toxic lipopolysaccharide
○ Causes bowel-wall irritation
● Shiga toxin:
○ Produced by S. dysenteriae type 1
○ Inhibits binding of aminoacyl-tRNA to the 60S ribosome, leading to
cessation of protein synthesis
○ Causes colonic mucosal damage, leading to sloughing and dysentery
○ Other changes (noted in hemolytic uremic syndrome (HUS)):
■ The toxin is translocated into circulation and induces endothelial
damage, particularly in the glomeruli.
■ Damaged endothelium causes platelet aggregation.
■ RBCs are lysed → schistocytes/schizocytes (fragmented RBCs)
● Shigella enterotoxin 1 (ShET1; S. flexneri) and Shigella enterotoxin 2 (ShET2;
4 species) cause fluid secretion and subsequent watery diarrhea.
● Type III secretion system:
○ Directly delivers virulence effectors to the host cell
○ Facilitates bacterial invasion of epithelial cells
● Resistance to gastric acids:
○ Shigella can survive the acidic environment of the stomach during
transit.
○ Thus, only a small inoculum is required to produce disease.

Disease process
1. Pathogen invades and is engulfed by the microfold (M) M cell

(transcytosis).
2. . Pathogen reaches subepithelial macrophages and dendritic cells,

then induces macrophage apoptosis. Shigella sp. is released along

with interleukin-1 (IL-1) and other cytokines. The pathogen is
then engulfed into the adjacent epithelial cell in a membrane-
bound compartment (epithelial-cell entry).
 3. . Released interleukins also recruit polymorphonuclear leukocytes

(PMNs), which destabilize the cellular junctions. This is another

entryway for the pathogen (PMN transmigration).
4. Shigella sp. passes through the disrupted tight junctions and then
the pathogen enters the epithelial cell. Once inside, the host cell
actin nucleators are hijacked. Actin-based motility (ABM; actin
tail) propels the pathogen to reach the plasma membrane, where
the infected cell contacts another cell.
5. Multiplication and intercellular spread are facilitated by cellular

protrusion (with Shigella) and elongation into the adjacent cell.

The protrusion resolves as a double-membrane vacuole. The
pathogen lyses the membrane, escapes, and enters the adjacent
cell. The adjacent cell is infected and this process is repeated in
other cells.
6. As each invaded epithelial cell dies, fluids are lost.

Clinical Presentation

● Shigellosis:
○ Incubation period: 1–4 days
○ Watery diarrhea initially, then dysentery (diarrhea with blood and
mucus)
○ Accompanied by abdominal pain, tenesmus, and fever
○ In the majority of cases, resolution is noted within 5 days.
○ In high-risk populations (immunocompromised, elderly, and children <
5 years of age), fluid and electrolyte losses can lead to dehydration and
possible death.

Diagnosis
● Gram stain and culture: fresh stool or rectal swab
 ○ Gram negative
○ Differential media: eosin methylene blue (EMB) or MacConkey’s agar,
showing non-lactose-fermenting colonies
○ Selective media: HE agar (green transparent colonies) or xylose-lysine-
deoxycholate agar
○ Triple sugar iron (TSI): alkaline slant, acid at the bottom, and no H₂S
● Polymerase chain reaction (PCR): Shigella-specific deoxyribonucleic acid
(DNA) in stool detected
● Additional work-up especially in severe infections:
○ Complete blood count (anemia and thrombocytopenia in HUS)
○ Metabolic panel (renal failure and electrolyte abnormalities in
dehydration or HUS)

Table: Shigella and Salmonella

Shigella Salmonella

Gram stain/structure Gram-negative bacilli Gram-negative bacilli

Lactose fermentation Non-lactose-fermenting Non-lactose-fermenting

Oxidase Negative Negative

H2S production No Yes

Motility No Yes (with flagella)

Virulence factors Endotoxin, Shiga toxin Endotoxin, Vi capsular

antigen

Reservoir Humans Humans (S. typhi), animals

Dose to produce disease Small inoculum (acid Large dose (inactivated by

stable) acids)

Infection spread Cell-to-cell (no Can spread

hematogenous spread) hematogenously
 Salmonella

● Features:
○ Gram stain and structure: gram-negative bacilli
○ Motility: motile and flagellated
○ Non-lactose-fermenting microbes; ferment glucose and mannose
○ Oxidase-negative bacilli
○ Inactivated by acids
○ Ability to produce hydrogen sulfide (H2S)
○ Growth medium:
■ Eosin methylene blue (EMB), MacConkey, or deoxycholate
medium used to detect non-lactose-fermenting microbes
■ Selective for Salmonella-Shigella (SS): Hektoen enteric (HE)
agar, SS agar
● Associated diseases: typhoid fever, gastroenteritis/enterocolitis, bacteremia

Clinically relevant species

Salmonella enterica is the primary species and has the following serotypes:

● Typhoidal (serotypes that cause typhoid or enteric fever):

○ S. enterica serotype typhi (formerly S. typhi)
○ S. paratyphi
● Nontyphoidal (all other serotypes associated with foodborne gastroenteritis,
most common in the United States):
○ S. enteritidis
○ S. typhimurium

Pathogenesis
Transmission and virulence
● Reservoirs:
○ Typhoidal: human GI tract
○ Nontyphoidal:
■ Farm animals: poultry, cattle, and pigs
■ Pets: turtles, parrots, rodents, cats, and dogs
● Transmission:
○ Fecal-oral route
○ Ingestion of food products (commonly contaminated or undercooked
foods, including poultry and eggs)
○ Infecting dose of S. enterica infection:
■ Varies with serotype
■ Considerably higher than Shigella (human-to-human transmission
by direct contact unlikely)
○ S. enterica is inactivated by acids. Smaller inocula can infect individuals
on antacids or patients with achlorhydria.
● Virulence:
○ Vi capsular polysaccharide antigen:
■ Surface polysaccharide in Salmonella typhi
■ Interferes with phagocytosis
○ H (flagellar) antigen: required for motility
○ O (lipopolysaccharide) antigen: produces smooth colonies on agar
○ Pili:
■ Bind to D-mannose receptors on eukaryotic cell types
■ Similar to Escherichia coli type 1 pili in function and
morphology

Disease process
● Entry in humans via ingestion:
 ○ Some organisms survive gastric-acid exposure → small bowel
○ From the lumen, Salmonella spp., mediated by their pili, can:
■ Penetrate M (microfold) cells of Peyer’s patches (gut-associated
lymphoid system)
■ Penetrate epithelial cells of the bowel
● In the bowel:
○ Organisms proliferate → Peyer’s patch hyperplasia and inflammation

(fever, abdominal symptoms)

○ Salmonella spp. go further into the lamina propria.
○ Proliferation leads to:
■ Further invasion and recruitment of mononuclear cells and

lymphocytes → may cause ulcerations

■ Disruption of sodium and chloride transport between cells and

intestinal lumen → diarrhea

Diseases Caused by Salmonella

Typhoidal species (Salmonella typhi and paratyphi)

Typhoid fever or enteric fever:

● Description:
○ Severe systemic illness in which bacteria enter the bloodstream and
disseminate in organs and lymphoid tissue
○ More common in:
■ Children, young adults
■ Impoverished regions
■ Travelers (to endemic areas)
Nontyphoidal species (Salmonella enteritidis and
typhimurium)
Enterocolitis/gastroenteritis:

● Description:
○ Most common presentation of Salmonella infection
○ Most common occurrence:
■ Consumption of poultry, eggs, and egg products (Salmonellae
passed from chicken to eggs transovarially)
■ Consumption of contaminated water, meat, and other food
products
■ Contact with animal reservoirs (especially pets) also increases the
risk.

vibrio cholerae

Epidemiology
● Primarily occurs in areas with limited access to clean water
● Endemic in some countries in Africa and Asia
● Cholera affects only humans.

Transmission
● Through contaminated food or water
● Fecal-oral route (person-to-person)

Basic features of Vibrio

● Curved gram-negative bacilli
● Facultative anaerobes
● Highly motile: 1–3 polar flagella
● Non-spore forming
● Oxidase positive

Biochemistry and growth characteristics

● Halophilic: require sodium chloride (NaCl) for growth
● Acid labile: grows well in alkaline media
● Thiosulfate-citrate-bile-sucrose (TCBS) agar:
○ V. cholerae ferments sucrose → forms yellow colonies

Pathogenesis
● Not all strains are pathogenic.
● V. cholerae pathogenicity genes code for proteins directly or indirectly
involved in the virulence of the bacteria. To adapt the host intestinal
environment and to avoid being attacked by bile acids and antimicrobial
peptides, V. cholera uses its outer membrane vesicles (OMVs). Upon entry, the
bacteria sheds its OMVs, containing all the membrane modifications that make
it vulnerable for the host attack
● Pathogenesis is determined by production of cholera toxin (CT):
○ Carried by a lysogenic bacteriophage (CTXΦ)
○ Heat-labile enterotoxin: composed of 1 A subunit (toxic domain) and 5
B subunits (receptor-binding domain)
○ B-subunit binds to the mucosal receptor ganglioside
monosialotetrahexosylganglioside (GM1).
○ CT is internalized by endocytosis: The A1 subunit of the toxin activates
adenylyl cyclase, which converts adenosine triphosphate (ATP) to cyclic
adenosine monophosphate (cAMP).
○ cAMP causes chloride secretion into lumen and inhibition of sodium
absorption.
 ○ Water follows the osmotic gradient and moves into the lumen, resulting
in watery diarrhea with electrolyte concentrations isotonic to those of
plasma.
○ Stool contains large amounts of sodium, chloride, bicarbonate, and
potassium with few cells.
● O lipopolysaccharide antigens:
○ Confer serologic specificity; > 200 serotypes
○ Only strains of O1 (classic and El Tor biotypes) and O139 serogroups
cause epidemic and pandemic cholera (they are the most virulent).
● Fimbriae (pili):
○ Aid in attachment to the intestinal mucosa
○ V. cholerae does NOT invade the intestinal mucosa.
○ Co-expressed (co-regulated) with cholera toxin and needed for
adherence, biofilm formation, colonization, and as receptors for the
bacteriophage that carries the genes for cholera toxin
● Because V. cholerae are acid labile, a high inoculum is required to overcome
the acidity of the gastric mucosa. The infectious dose is reduced:
○ In hypochlorhydric persons
○ In those using antacids
○ When gastric acidity is buffered by a meal
● The higher the bacteria number, the more severe the symptoms.
● Incubation period: 1–2 days
● Fluid loss originates in the duodenum and upper jejunum; the ileum is less
affected.
● The colon is relatively insensitive to the toxin, but the large volume of fluid
overwhelms its absorptive capacity.

food intoxication and poisioning

Food infection the presence of bacteria or other microbes which infect the body after
consumption
Food intoxication ingestion of toxins contained within the food, including bacterially produced
exotoxins.
Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases and
results from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by
enterotoxigenic strains of Staphylococcus aureus.
Staphylococcus aureus

S.aureus is gram positive anaerobic cocci that occurs in singles, pairs, short chains,and irregular

grape-like clusters. Only those strains that produce enterotoxin can cause food poisoning .

Causes: Food containing protein.

Properties of Staphylococcal Enterotoxins

• Resistant to heat treatment
• Stable over a wide pH range
• Resistant to gastrointestinal and other proteases

Tuberculosis,whooping cough,Diphtheria and Lepreae (Myconacterium Leprae)

TB is an airborne disease caused by the bacterium Mycobacterium tuberculosis (M.

tuberculosis)

Mycobacterium tuberculosis, is a non-motile, slow-growing, rod-shaped bacillus.

They are acid fast however they appear bright- red colored rods when a Ziehl–
Neelsen stain is used.

Other causative organisms

Mycobacterium africanum
Mycobacterium microti
Non-Mycobacterium Genus
Mycobacterium leprae
Mycobacterium avium
Mycobacterium asiaticum
M.tuberculosis complex
M. africanum
 M. Bovis
M. Canetti
M. microti

Severe Symptoms
● Persistent cough
● Chest pain
● Coughing with bloody sputum
● Shortness of breath
● Urine discoloration
● Cloudy & reddish urine Fever with chills.
● Fatigue

Characteristics:
● Acid fast:
○ Property conferred by mycolic acid
○ Do not destain by acid alcohol after being stained with aniline
dyes
● Gram stain:
○ Usually cannot penetrate Mycobacterium tuberculosis complex
waxy cell wall
○ Most commonly produce no stain or variable results
● Slow growing
● Obligate aerobes ( an aerobe that requires oxygen for aerobic
respiration.
Virulence factors:
● Cell envelope:
○ Major constituent: mycolic acid
○ Mycolic acid is attached to glycolipids.
○ Glycolipids are responsible for “cord formation” on microscopy
(grossly corresponds to granuloma formation).
● Catalase-peroxidase: resists host cell oxidative response
● Sulfatides and trehalose dimycolate: triggers toxicity
● Lipoarabinomannan (LAM): induces cytokines

Transmission:
● Exclusively airborne
● From patients with active TB

Pathogenesis
● 1st step is inhalation of aerosol droplets.
● Droplets are deposited in the lungs.
● 3 possible outcomes:
○ Clearance of bacteria
○ Primary active disease
○ Latent infection (clinical disease may occur many years later)

Primary active disease:

● Proliferation of bacteria within alveolar macrophages
● Cytokines produced by macrophages attract other phagocytic cells.
● A tubercle (granulomatous structure) forms.
● Tubercle expands into lung parenchyma → Ghon’s complex
● Bacteria then can spread to draining lymph nodes → lymphadenopathy
● Ghon’s complex + lymphadenopathy/calcification → Ranke complex
● If spread is not controlled by the immune cells, bacteremia with seeding of
other organs may occur → miliary TB
 ● When bacteria erode into airways (caseating granulomas), the patient
becomes contagious.
● Infection may progress to a chronic stage with episodes of healing and
subsequent scarring of the lesions.
● Spontaneous eradication is rare.

Latent infection:
● Lifetime risk of reactivation is 5%–10%.
● Immunosuppression is a definite factor in reactivation.
● Risk factors:
○ HIV
○ Kidney disease
○ Diabetes
○ Steroids
○ Lymphoma
○ Advanced age
○ Smoking

Primary TB
● Symptomatic primary disease develops in only about 10% of infected
people.
● Fever:
○ Most common symptom
○ Mostly low grade, but may be up to 39°C (102.2°F)
○ Lasts up to 10 weeks, but on average 14–21 days
● Pleuritic chest pain (may or may not be associated with effusion)
● Retrosternal/interscapular pain (due to bronchial lymphadenopathy)
● Cough
● Fatigue
● Arthralgia
● Pharyngitis
Reactivation TB
● Apical segments of upper lobes and superior segments of lower lobes
are most commonly involved, likely because of:
○ Increased oxygen tension
○ Poor lymphatic drainage
● Onset of symptoms is gradual; may go undiagnosed for 2–3 years
○ Fever:
■ Low grade first, worsening with more advanced disease
■ Classically diurnal: peaks in the afternoon, afebrile at night
and in the early morning
○ Night sweats
○ Cough:
■ Mild first, gradually worsening
■ Initially only in the mornings
■ Becomes more productive (greenish-yellow sputum) as
disease progresses
■ Nocturnal cough and hemoptysis: advanced disease
○ Pneumothorax or effusions may present with dyspnea (rare).
○ Anorexia, wasting, malaise
○ Ulcers of mouth, tongue, larynx, and esophagus: due to infected
expectorated secretions

Extrapulmonary and miliary TB

● Can develop in the course of primary or reactivation disease
● In up to 15%–20% of active cases
● Most common in children and immunocompromised individuals
● Extrapulmonary manifestations (can affect any organ system):
○ Tuberculous pleurisy
○ Adrenal gland insufficiency
○ Meningitis
 ○ Spondylitis tuberculosa (Pott’s disease, TB infection of > 1
vertebra)
○ Constrictive pericarditis
○ Lupus vulgaris (reddish-brown nodules that usually appear on the
face around the nose, eyelids, lips, cheeks, ears, and neck)
○ Scrofula (cervical lymphadenitis)
○ Sterile pyuria
● Miliary TB:
○ Massive spread of infection through blood and lymphatics
○ Small granulomatous lesions through multiple organs

Diagnosis

History
● Travel to endemic areas
● Exposure to individuals with known or suspected active infection
● HIV or other immune deficiencies
● Working in healthcare
● Living in a homeless shelter or correctional institution

Physical exam
● Findings are often non-specific.
● Pulmonary:
○ Dullness to percussion (effusions)
○ Crackles on auscultation
○ Distant hollow breath sounds
● Extrapulmonary (depends on organ involvement):
○ Cervical lymphadenopathy
○ Hepatomegaly/splenomegaly
○ Ascites, jaundice
 ○ Meningismus, altered mental status
○ Skin changes (lupus vulgaris)

Imaging
● Chest X-ray:
○ Can be normal in primary TB
○ Hilar lymphadenopathy
○ Ghon’s complex: enlarged hilar lymph nodes + local shadowing
○ Assmann’s focus: infraclavicular infiltration
○ Pleural effusions
○ Reactivation TB:
■ Simon foci (calcified small scar from primary infection)
■ Infiltrates in apical segments and upper segments of lower
lobes
■ Cavities with air-fluid levels
● Computed tomography (CT) scan:
○ More sensitive than plain X-ray
○ Used if chest X-ray is non-specific or alternative diagnosis is
considered

Laboratory identification
○ Sputum:
■ 3 specimens, at least 1 in the early morning
■ Acid-fast bacillus (AFB) smear
■ Mycobacterial culture
■ Nuclear acid amplification (NAA) test
○ Blood or urine mycobacterial culture: in HIV or
immunocompromised patients
 ○ Tuberculin skin test (TST; purified protein derivative (PPD) or
Mantoux test):
■ Intradermal injection of tuberculin antigen
■ Can detect active or latent infection
■ Measure induration area in 48–72 hours; positive if:
■ > 5 mm in patients with HIV,
immunosuppression, or recent contact with TB
■ > 10 mm in patients from high-risk countries, IV
drug users, medical and lab workers
■ > 15 mm in patients with no known risk factors
for TB
○ lnterferon-y release assay (IGRA): no distinction between
active and inactive TB

Pertussis (Whooping Cough)

Bordetella pertussis is a small (approximately 0.8 μm by 0.4 μm), rod-shaped,
coccoid, or ovoid, non motile with no flagella Gram-negative bacterium that is
encapsulated and does not produce spores. It is a strict aerobe. It is arranged singly
or in small groups and is not easily distinguished from Haemophilus
species.mesophilic coccobacillus .
Its virulence factors include
● pertussis toxin: It is a major virulent factor playing a central role in pathogenesis of
whooping cough. It is made up of two units, A and B. A unit is enzymatically active. The
B-unit binds the toxin to the target cells and helps A-unit to cross the membrane.The toxin
causes increased concentration of cAMP within the target cell. This toxin increases
sensitivity to histamine and serotonin resulting in the increased susceptibility to
anaphylaxis.It is a potent T cell mitogen that causes accumulation of lymphocytes in the
intravascular compartment.
Adhesins

● Adherence of B.pertussis to ciliated epithelial cells is essential for the pathogenesis

of the organisms.
● This is mediated by capsular fimbriae or agglutinogens.
● The most important is filamentous haemagglutinin which also causes agglutination
of red blood cells.
● adenylate cyclase toxin: It is produced by all Bordetellae except B. avium.ACT enters non-
infected cells and causes intracellular increase in cAMP leading to intoxication of
mammalian cells.It also inhibits chemotaxis and superoxide burst in polymorphonuclear
leukocytes, which helps in intracellular survival of B. pertussis.
●
● filamentous hæmagglutinin:
● Pertactin:
● Fimbria:
● tracheal cytotoxin: It is a Bordetella toxin derived from the peptidoglycan of the cell wall
and present in all species of Bordetellae.TCT induces ciliostasis ( loss of movement ) in
the epithelial cells, followed by extrusion of these cells from the epithelium of the
respiratory tract.This makes a person more prone to secondary infection.
●
 ■

■
■
■ PT can then enter the bloodstream.
■ Primary isolation of B pertussis requires enriched media. Bordet-Gengou medium (potato-
blood-glycerol agar) that contains penicillin G, 0.5 μg/mL, can be used; however, a charcoal-
containing medium supplemented with horse blood (Regan Lowe) is preferable because of
the longer shelf life. The plates are incubated at 35–37°C for 3–7 days aerobically in a moist
environment (eg, a sealed plastic bag). The small, faintly staining gram-negative rods are
identified by immu- nofluorescence staining. B pertussis is nonmotile.

Presentation
Pertussis typically starts with mild coryzal symptoms, a low grade fever and possibly a mild dry

cough.

More severe coughing fits start after a week or more. These involve sudden and recurring

attacks of coughing with cough free periods in between. This is described as a paroxysmal

cough. Coughing fits are severe and keep building until the patient is completely out of breath.
Patient typically produces a large, loud inspiratory whoop when the coughing ends. Patients

can cough so hard they faint, vomit or even develop a pneumothorax. Bear in the mind that not

all patients will “whoop” and infants with pertussis may present with apnoeas rather than a

cough.

Diagnosis

● Diagnosis is strongly suspected with clinical history but requires

laboratory confirmation.
● History: possible contact with other “whooping cough” cases and
vaccination (as the vaccine does not provide full protection, pertussis
needs to be considered even in vaccinated children!)
● Laboratory tests:
○ Nasopharyngeal swab → culture (gold standard) or polymerase chain
reaction
■ Only reliable during the first 2–3 weeks of the infection
○ Complete blood count shows nonspecific lymphocytosis.
○ Serology testing can be used up to several weeks after the onset
of symptoms.
■ A 2-fold rise in the antibody titer against pertussis is
diagnostic.

Diphtheria
Corynebacterium diphtheriae is a nonmotile, noncapsulated, club-shaped, Gram-positive bacillus
C. diphtheriae is transmitted between humans via droplets, secretions or direct contact
Corynebacterium diphtheriae is classified into biotypes (mitis, intermedius, and gravis) .
Metachromatic granules when stained with albert stain.
Mitis - black colonies with gray periphery
Gravis : Large grray colonies
Intermedius : small,dull,to black
Virulence factor :
Pili
The pili found on the surface of C. diphtheriae are beneficial in the adherence to host cells. There are three
distinct types of pili expressed including SpaA-, SpaD-, and SpaH- (Spa for sortase-mediated pilus
assembly)
diphtheria toxin (DT), an exotoxin, released by the bacteria after entering the human body. DT is classified
as an AB toxin because it has two components, one for activation and one for binding

Identification
To accurately identify C. diphtheriae, a Gram stain is performed to show Gram-positive, highly pleomorphic
organisms with no particular arrangement. Special stains like Albert's stain and Ponder's stain are used to
demonstrate the metachromatic granules formed in the polar regions. The granules are called polar
granules.An enrichment medium, such as Löffler's medium, is used to preferentially grow C.
diphtheriae.
● Blood tellurite agar
Virulence factor:
The main virulence factor of C. diphtheriae is diphtheria toxin (DT), an exotoxin,
released by the bacteria after entering the human body. DT is classified as an AB
toxin because it has two components, one for activation and one for binding.The
ADP-ribosylation activity of diphtheria toxin is determined completely by the A
fragment, and no portion of the B fragment is required for catalytic activity.
Diphtheria toxin kills cells by inhibiting eukaryotic protein synthesis,
This potent toxin inactivates elongation factor (EF-2) required for protein synthesis
Treatment :
Patient is isolated
- Penicliin G is given or erthythromycin
- There are vaccines available
-

Lepreae (Myconacterium Leprae)

M. leprae is a strongly acid-fast, rod-shaped bacterium.

It is an intracellular, pleomorphic ( having variation in the size and shape of

cells or their nuclei.), acid-fast, pathogenic bacterium. M. leprae is an
aerobicbacillus (rod-shaped bacterium) with parallel sides and round ends,
surrounded by the characteristic waxy coating unique to mycobacteria. In size
and shape, it closely resembles Mycobacterium tuberculosis. This bacterium
often occurs in large numbers within the lesions of lepromatous leprosy that
are usually grouped together like bundles of cigars or arranged in a palisade.
- Though they are Gram-positive, they cannot be easily stained
by Gram stain as they contain wax like substance (mycolic
 acid) in their cell wall; but they can be stained by Z-N
method
-
Due to its thick waxy coating, M. leprae stains with a carbol fuchsin rather
than with the traditional Gram stain.

Mycobacterium leprae is a bacterium that causes leprosy, also known as

"Hansen’s disease", which is a chronic infectious disease that damages the
peripheral nerves and targets the skin, eyes, nose, and muscles
Virulence factors:
The virulence factors exhibited by M. leprae includes:

1. Fibronectin:
● Enhances the attachment and ingestion of M. leprae by epithelial and Schwann
cell lines.

3. Lipoarabinomannan (LAM):

● LAM is a lipoglycan in the cell wall.

● It is thought to hinder macrophage activity as well as provide resistance to
oxidative radicals.
● It inhibits antigen presentation and prevents release of IFN-8, a macrophage
activating cytokine.

4. Phenolic glycolipid -1 (PGL-1):

● PGL-1 is a prominent surface lipid found on the outermost layer of

the cell wall.
● It binds to C3 component of the complement which leads to
phagocytosis mediated by CR1, CR3 and CR4 receptors found on
the cell surfaces.
● PGL-1 protects the lepra bacillus once inside the phagocytic cells,
from oxidative killing by macrophages by removing hydroxyl
radicals and superoxide anions.

5. Intracellular bacilli:

● The intracellular location of the bacteria makes them resistant to killing by

phagocytes.

6. Mycolic acid:
 ● The lipid rich waxy cell wall containing mycolic acids also acts as an important
virulence factor.

Reservoir
Besides humans, the only known resevoir is the armadillo. It is
thought that they are a good host for Mycobacterium leprae
because of their low body temperature.

Microbiology
It is an intracellular, pleomorphic, acid-fast, pathogenic bacterium. M. leprae is
an aerobicbacillus (rod-shaped bacterium) with parallel sides and round ends,
surrounded by the characteristic waxy coating unique to mycobacteria. In size
and shape, it closely resembles Mycobacterium tuberculosis. This bacterium
often occurs in large numbers within the lesions of lepromatous leprosy that
are usually grouped together like bundles of cigars or arranged in a
palisade.Due to its thick waxy coating, M. leprae stains with a carbol fuchsin
rather than with the traditional Gram stain.