Microbiology of Infectious

Diseases

C. Gram Negative Cocci

• Gram Negative Cocci and Rods
•
• Pathogenesis of Gram negative bacteria:
• 1-Toxins:
• -Endotoxins
• -Exotoxins (enterotoxin)
• 2-Antigens:
• O-antigen
• H-antigen
• K-antigen
Toxins

•Gram-negative bacteria contain endotoxin in their cell

walls. This is the bacterial lipopolysaccharide (LPS).

•In addition, several exotoxins are produced; eg, E. coli

and Vibrio cholerae secrete exotoxins, called

enterotoxins that activate adenylate cyclase within the

cells of the small intestine, causing diarrhea.

Antigens

•They are used for identification purposes both in the clinical laboratory and

in epidemiologic investigations. The 3 surface antigens are as follows:

•The cell wall antigen (also known as the somatic or O antigen) is the outer

polysaccharide portion of the lipopolysaccharide. The O antigen, which is

composed of repeating oligosaccharides consisting of 3 of 4 sugars

repeated 15 or 20 times, is the basis for the serologic typing of many

enteric rods

•The H antigen is on the flagellar protein.

The capsular of K polysaccharide antigen is particularly prominent in

heavily encapsulated organisms such as Klebsiella.

 Gram-Negative Cocci
NEISSERIA
•The genus Neisseria contains 2 important human pathogens: Neisseria
meningitidis and Neisseria gonorrhoeae.
•Neisseria are gram-negative cocci that resemble paired kidney beans. They
possess endotoxin in their outer membrane.
•The endotoxin of N. meningitidis is a lipo-oligosaccharide (LOS) that contain
lipid A, but lacks the long repeating sugar side chains of LPS.
•The growth of Neisseria is inhibited by toxic trace metals and fatty acids, found

in certain culture media, e.g. blood agar plates. They are therefore cultured on

“chocolate” agar containing blood heated to 80oC, which inactivates the

inhibitors.

•Neisseriae are oxidase-positive; i.e. they possess the enzyme Cytochrome c.

This is an important laboratory diagnostic test in which colonies exposed to

phenylenediamine turn black as a result of oxidation of the reagent by the

enzyme.

•The genus Neisseria is one of several in the family Neisseriaceae. A separate

genus contains the organism Moraxella catarrhalis, which is part of the normal

throat flora and an occasionally opportunistic cause of pneumonia.

 Diseases

•N. meningitidis mainly causes meningitis and

meningococcemia.

•N gonorrhoeae causes gonorrhea, the most common

notifiable bacterial disease in the USA. It also causes

neonatal conjunctivitis (ophthalmia neonatorum) and

pelvic inflammatory disease (PID).

 (Table-1). Neisseriae of medical importance.

Portal of Poly- Maltose - Available

Species Entry saccharid Fermentatio Lactamase Vaccine
e Capsule n Production

N. meningitidis Respiratory
(meningococcus tract present yes None yes
)

N gonorrhoeae Genital tract absent no Some no

(gonococcus)
Neisseria meningitidis

Important Properties

•N. meningitidis (meningococcus) has a prominent

polysaccharide capsule that enhances virulence by its

antiphagocytic action and induces protective antibodies.

•Meningococci are divided into at least 13 serologic

groups based on the antigenicity of their capsular

polysaccharides.
 Pathogenesis & epidemiology

•Humans are the only natural hosts for meningococci.

•The organisms are transmitted by airborne droplets; they

colonize the membranes of the nasopharynx and become part of

the transient flora of the upper respiratory tract.

•Carriers are usually asymptotic.

•From the nasopharynx, the organism can enter the bloodstream

and spread to specific sites, such as the meninges or joints, or be

disseminated throughout the body (meningococcemia).

•About 5% of people become chronic carriers and

serve as a source of infection for others. The carriage

rate can be as high as 35% in people who live in close

quarters, e.g. military recruits; this explains the high

frequency of outbreaks of meningitis in the armed

forces prior to the use of the vaccine. The carriage rate

is also high in close (family) contacts of patients

Table-2 Properties of the polysaccharide capsule of the Meningococcus

(1) Enhances virulence by its antiphagocytic action

(2) Is the antigen that defines the serologic groups

(3) Is the antigen detected in the spinal fluid of patients

with meningitis

(4) Is the antigen in the vaccine

The same 4 features apply to the capsule of the

pneumococcus and Haemophilus influenzae.

•Three organisms cause more than 80% of cases of bacterial

meningitis in persons over 2 months of age: Haemophilus

influenzae, Streptococcus pneumoniae, and N meningitidis.

•Virulence:

•Meningococci have 3 important virulence factors:

•Polysaccharide capsule that enables the organism to resist

phagocytosis by polymorph nuclear leukocytes (PMNs).

•Endotoxin (LPS), which causes fever, shock, and other patho-

physiologic changes (in purified from, endotoxin can reproduce

many of the clinical manifestations of meningococcemia); and

•Immunoglobulin A (IgA) protease, which, by cleaving secretory

IgA, helps the bacteria to attach to the membranes of the upper

respiratory tract.

•Resistance to disease correlates with the presence of antibody

to the capsular polysaccharide. Most carriers develop protective

antibody titers within 2 weeks of colonization.

•Immunity is group specific, and so it is possible to

have protective antibodies to one group of organisms

yet be susceptible to infection by organisms or the

other groups. Complement is an important feature of

the host defense, because people with complement

deficiencies, particularly in the late-acting complement

components (C6-C9), have an increased incidence of

meningococcal bacteremia.
 Clinical Findings

• Meningococcemia: The intravascular multiplication of

Neisseria meningitidis results in an abrupt on-set of spiking fevers,

chills, arthralgia (joint pains), and muscle pains, as well as the

petechial rash.

• These pa-tients usually look acutely ill.

• Once in the bloodstream, the meningococci rapidly

disseminate throughout the body.

• This can lead to meningitis and/or fulminant meningococcemia.

• 2) Fulminant meningococcemia

(Waterhouse-Friderichsen syndrome):

•This is septic shock. Bilateral hemorrhage into the adrenal

glands occurs, which causes adrenal insuffi-ciency. Abrupt

onset of hypotension and tachycardia occurs, along with

rapidly enlarging petechial skin lesions. Disseminated

intravascular coagulation (DIC) and coma may develop.

Death can occur rapidly (6-8 hours).

•3) Meningitis: This is the most common form of meningococcal

disease, usually striking infants < 1 year of age. Infants usually display

nonspecific findings of an infection, including fever, vomiting, irritability,

and/or lethargy.

•A bulging open anterior fontanelle may be a sign of meningitis in neonates,

while slightly older infants may display a stiff neck. The classic petechial skin

rash may occur when meningococcemia occurs in conjunction with meningitis.

• This allows the physician to make a presumptive diagnosis of meningococcal

meningitis even before per-forming a diagnostic spinal tap

 Laboratory Diagnosis
•The principal laboratory procedures are smear and culture of

blood and spinal fluid samples.

• A presumptive diagnosis of meningococcal meningitis can be

made if gram-negative cocci are seen in a smear of spinal fluid.

•The organism grows best on chocolate agar incubated at 37oC in

a 5% CO2 atmosphere.

• A presumptive diagnosis of Neisseria can be made if oxidase

positive colonies of gram-negative diplococci are found

•The differentiation between N. meningitidis and N. gonorrhoeae is

made on the basis of sugar fermentation: meningococci ferment

maltose, whereas gonococci do not (both organisms ferment

glucose).

•Immunofluorescence can also be used to identify these species.

Tests for serum antibodies are not useful for clinical diagnosis.

• A test that can assist in the rapid diagnosis of meningococcal

meningitis is the latex agglutination test, which detects capsular

polysaccharide in the spinal fluid

 Treatment
•Prompt treatment with penicillin G or ceftriaxone is required at the first
indication of disseminated meningococcemia..
Prevention
•Chemoprophylaxis and immunization are both used to prevent
meningococcal disease. Rifampin is use for prophylaxis in household
and other close contacts. It is preferred because it is efficiently secreted
into the saliva, in contrast to penicillin G.
The meningococcal vaccine, which contains the capsular
polysaccharides of group A, C, Y, and W-135 strains, is effective in
preventing epidemics of meningitis and in reducing the carrier rate,
especially in military personnel. The vaccine does not contain the group
B polysaccharide, which is poorly immunogenic in humans
Neisseria gonorrhoeae
Important Properties
•N. gonorrhoeae (gonococcus) has no polysaccharide
capsule but has multiple serotypes based on the
antigenicity of its pilus protein.
• There is a marked antigenic variation in the
gonococcus pili as a result of chromosomal
rearrangement; more than 100 serotypes are known.
Gonococci have 3 outer membrane proteins (proteins I,
II, and III). Protein II plays a role in attachment of the
organism to cells and varies antigenically as well.
 Pathogenesis and Epidemiology
•Gonococci, like meningococci, cause disease
only in humans.
•The organism is usually transmitted sexually;
newborns can be infected during birth.
•Because the gonococcus is quite sensitive to
dehydration and cool conditions, sexual
transmission favors its survival.
•Gonorrhea is usually symptomatic in men but often asymptotic in

women.

•Infections of the anorectal area and pharynx, as well as those of

the genital tract, can act as the source of the organisms.

Virulence factors

Pili constitute one of the most important virulence factors,

because they mediate attachment to mucosal cell surfaces and

are antiphagocytic. Piliated gonococci are usually virulent,

whereas non-piliated strains are non virulent.

The virulence factors in the cell wall are
endotoxin
IgA, which could otherwise block
attachment to the mucosa.
Repeated gonococcus infections are common
primarily as a result of antigenic changes of
pili and the outer membrane proteins
NB. Gonococci have no capsules.
•The main host defenses against gonococci are antibodies
(IgA and IgG), complement, and neutrophils. Antibody-
mediated opsonization and killing within phagocytes
occur.
•Gonococci infect primarily the mucosal surfaces, e.g. the
urethra and vagina, but dissemination occurs.
•Certain strains of gonococci cause disseminated
infections more frequently than others because they
possess the following features:
 (1) Resistance to the bactericidal action of serum
(2) Marked sensitivity to penicillin
(3) Auxotroph for arginine, uracil, and hypoxanthine; i.e., for growth,
they require these substances in the medium.
The occurrence of a disseminated infection is a function not only of
the strain of gonococcus but also of the effectiveness of the host
defenses. Persons with a deficiency of the late-acting complement
components (C6-C9) are at risk for disseminated infections, as are
women during menses and pregnancy. Disseminated infections usually
arise from asymptomatic infections, indicating that local inflammation

may deter dissemination

 Clinical Findings :
1-Gonococcal Disease in Women
Like men, women can also develop a gonococcal urethritis,
with painful burning on urination and purulent discharge from
the urethra. However, urethritis in women is more likely to be
asymptomatic with minimal urethral discharge.
Neisseria gonorrhoeae also infects the columnar epithelium
of the cervix, which becomes reddened and friable, with a
purulent exudate. A large percentage of women are
asymptomatic.
 If symptoms do develop, the Woman may
complain of lower abdominal discomfort, pain with
sexual intercourse (dyspareunia), and a purulent
vaginal discharge. Both asymptomatic and
symptomatic women can transmit this infection.
A gonococcal infection of the cervix can progress to
pelvic inflammatory disease (PID). PID is an
infection of the uterus (endometritis), fallopian
tubes (salpingitis), and/or ovaries (oophoritis).
Clinically, patients can present with fever, lower abdominal
pain, abnormal menstrual bleeding, and cervical motion
tenderness (pain when the cervix is moved by the doctor's
examining finger). Menstruation allows the bacteria to
spread from the cervix to the upper genital tract. It is
therefore not surprising that over 50% of cases of PID
occur within one week of the onset of menstruation. The
presence of an intrauterine device (IUD) increases the risk
of a cervical gonococcal infection progressing to PID.
Chlamydia trachomatis is the other major cause of PID.
 Complications of PID include:
1) Sterility: The risk of sterility appears to increase with each
gonorrhea infection. Sterility is most commonly caused by scarring of
the fallopian tubes, which occludes the lumen and prevents sperm from
reaching the ovulated egg.
2) Ectopic pregnancy: The risk of a fetus developing at a site other
than the uterus is significantly increased with previous fallopian tube
inflammation (salpingitis). The fallopian tubes are the most common site
for an ectopic pregnancy. Again, with scarring down of the fallopian
tubes, there is resistance to normal egg transit down the tubes.
3) Abscesses may develop in the fallopian tubes, ovaries, or
peritoneum
 Gonococcal Disease in Both Men and Women
1) Gonococcal bacteremia: Rarely, Neisseria
gonorrhoeae can invade the bloodstream. Manifestations
include fever, joint pains, and skin lesions (which usually
erupt on the extremities). Pericarditis, endocarditis, and
meningitis are rare but serious complications of a
disseminated infection.
2) Septic arthritis: Acute onset of fever occurs along
with pain and swelling of 1 or 2 joints. Without prompt
antibiotic therapy, progressive destruction of the joint will
occur.
 Examination of synovial fluid usu-ally reveals
increased white blood cells.
Gram stain and culture of the synovial fluid
confirms the diagnosis, revealing gram-negative
diplococci within the white blood cells.
Gonococcal arthritis is the most common kind of
septic arthritis in young, sexually active
individuals.
 Gonococcal Disease in Infants:

Neisseria gonorrhoeae can be transmitted from

a pregnant woman to her child during delivery,

resulting in ophthalmia neonatorum.

This eye infection usually occurs on the first or

second day of life and can damage the cornea,

causing blindness.
Laboratory Diagnosis
1-The diagnosis of localized infections depends on Gram staining
and culture of the discharge.
In men, the finding of gram-negative diplococci within PMNs in a
sample of urethral discharge is sufficient for diagnosis. In women,
the use of the Gram stain alone can be difficult to interpret, so
cultures should be done. Gram stains on cervical specimens can
be falsely positive because of the presence of Gram-negative
diplococci in the normal flora and can be falsely negative because
of the inability to see small numbers of gonococci when using oil
immersion lens. Cultures must also be used in diagnosing
suspected pharyngitis or anorectal infections.
2-Specimens are cultured on Thayer-Martin medium, which is a
chocolate agar containing antibiotics (vancomycin, colistin,
Trimethoprim, and nystatin) to suppress the normal flora, and are
incubated at 37oC in a 5% CO2 atmosphere. The finding of an oxidase-
positive colony composed of gram-negative diplococci is sufficient to
diagnose Neisseria.
3-Specific identification of the gonococcus can be made either by its
fermentation of glucose (but not maltose) or by fluorescent-antibody
staining.
4-Two rapid, sensitive, and specific test are being increasingly used for
diagnosis, namely, the ELISA, which detects gonococcus antigens, and
the DNA probe assay, which detects gonococcus ribosomal genes.
5-Serologic tests to determine the presence of antibody to
gonococci are not useful for diagnosis.
Treatment
•Ceftriaxone is the treatment of choice in uncomplicated
gonococcus infections.
•Spectinomycin or Ciprofloxacin should be used if the patient is
allergic to penicillin.
•Because mixed infections with C. trachomatis are common,
tetracycline or erythromycin is recommended .
•A follow-up culture should be done 1 week after completion of
treatment to determine whether gonococci are still present.
Prevention
•The key prevention of gonorrhea involves complete chastity
(abstention from unlawful sexual intercourse) and avoidance of
infidelity (marital unfaithfulness).
•Prompt treatment of symptomatic patients and their contacts.
•Cases of gonorrhea must be reported to the public health
department to ensure proper follow up.
•A major problem is the detection of asymptomatic carriers.
• Gonococcus conjunctivitis in newborns is prevented most often
by the use of erythromycin ointment.
•No vaccine is available.
The End