STRUCTURE AND CLASSIFICATION
OF PROKARYOTES-PART 2
DR NAZIA KHAN
ASSISTANT PROFESSOR
OBJECTIVES
• EXPLAIN THE DIFFERENCE IN THE CELL WALL STRUCTURES OF GRAM POSITIVE
AND GRAM NEGATIVE ORGANISMS .
• CORRELATE GRAM REACTION TO DIFFERENTIATE CELL WALL STRUCTURE OF
GRAM POSTIVE AND GRAM NEGATIVE ORGANISMS
• DESCRIBE CLINICAL IMPORTANCE OF GRAM POSITIVE AND GRAM NEGATIVE
ORGANISMS HOSPITAL SETTINGS
CELL WALL
• The bacterial cell wall owes its
strength to a layer composed of
murein, mucopeptide, or
peptidoglycan (all are synonyms).
• Comprised of alternating Nacetylglutamic acid and Nacetylmuramic acid molecules
• Attached to each NAM is four
amino acid peptide: tetrapeptide
• provides strong, flexible support to
keep bacteria from bursting or
collapsing because of changes in
osmotic pressure
GRAM STAINING
• Basis of bacterial classification and identification, named after the histologist Hans
Christian Gram
• Most bacteria are classified as
1. gram-positive or 2. gram-negative according to their response to the Gram staining
procedure.
• STEPS OF GRAM STAINING
• The Gram stain depends on the ability of certain
bacteria (the gram-positive bacteria) to retain a
complex of crystal violet (a purple dye) and iodine after
a brief wash with alcohol or acetone.
• Gram-negative bacteria do not retain the dye-iodine
complex and become translucent, but they can then
be counterstained with safranin (a red dye).
• Thus, gram-positive bacteria look purple under the
microscope, and gram-negative bacteria look red.
• The distinction between these two groups turns out to
reflect fundamental differences in their cell envelopes
DIFFERENCE BETWEEN GRAM NEGATIVE AND GRAM POSITIVE CELL WALL
• Functions of cell wall:
1. Gives osmotic protection,
2. plays an essential role in cell division Serves as a primer for its own
biosynthesis.
• Various layers of the wall are the sites of major antigenic determinants of the
cell surface, and one component—the lipopolysaccharide of gramnegative cell walls—is responsible for the nonspecific endotoxin activity of
gram-negative bacteria.
• The cell wall is, in general, nonselectively permeable; one layer of the gramnegative wall, however—the outer membrane—hinders the passage of
relatively large molecules
SPECIAL COMPONENTS OF GRAM-POSITIVE CELL WALLS
1. 20-30 layers of peptidoglycan
2. Teichoic and teichuronic acids,
provides functions relating to the elasticity, porosity,
tensile strength, and electrostatic properties of the
envelope
The teichoic acids constitute major surface antigens :
chains of ribitol-phosphate or glycerol-phosphate to
which sugars or alanine attached
• Techoic Acid sticks out above the peptidoglycan layer
3. some gram-positive walls may contain polysaccharide
molecules
SPECIAL COMPONENTS OF GRAM-NEGATIVE CELL WALLS
• Gram-negative cell walls contain
1.
2.
3.
4.
an outer membrane containing lipopolysaccharide (LPS)
thin shell of peptidoglycan
periplasmic space
inner membrane
• The outer membrane is chemically distinct from all other
biological membranes.
• the outer membrane has special channels, consisting of
protein molecules called porins
(exemplified by OmpC, D, and F and PhoE of E coli and
Salmonella Typhimurium)
• The OmpA protein is an abundant protein in the outer membrane. The
OmpA protein participates in the anchoring of the outer membrane to the
peptidoglycan layer; it is also the sex pilus receptor in F-mediated bacterial
conjugation .
• involved in the transport of specific molecules such as vitamin B12 and ironsiderophore complexes.
Lipopolysaccharide (LPS)
• The LPS of gram-negative cell walls
consists of a complex glycolipid,
called lipid A, to which is attached
a polysaccharide made up of a
core and a terminal series of repeat
unit
• It is an endotoxin that may become
toxic when released during infections
and hence is Important in sepsis
• Bacteria may have different shapes( cocci, bacilli, spiral,filamentous,
comma shaped) , arrangemets (pairs,clusters,chains,tetrads) and sizes
Four major groups based on gram staining
BACTERIA WITH ATYPICAL CELL
 Not all bacteria are classified based on gram
WALLS
staining
 Some bacterial groups lack typical cell wall
structure and may stain poorly with gram stain
• Mycobacterium
• cell wall has lipid called mycolic acid
• basis for acid-fast stain
 Some have no cell wall
• Mycoplasma
 Flexible thin walled bacteria(spirochetes)
• Treponema sp
• Leptospira sp
• Borrelia sp
 Obligate intracellular bacteria
• Chlamydia sp
• Rickettsia sp
CLINICAL IMPORTANCE OF GRAM POSITIVE &
GRAM NEGATIVE ORGANISMS IN HOSPITAL
SETTINGS
GRAM NEGATIVE SEPTICEMIA
• Gram negative Septicemia is a medical
term referring to the presence of gram
negative organisms in the bloodstream,
leading to sepsis
• Sepsis is a deadly condition
characterized by a severe whole body
inflammatory response caused by
severe infection
• The endotoxin (LPS layer of gram
negative cell wall) of rapidly dividing
bacteria in the blood stream lead to
widespread cytokine release from
theinflammatory cells resulting in
• hypotension and shock,
• high fever and
• multiorgan failure
HOSPITAL ACQUIRED INFECTIONS
• Among the gram positive
bacteria staphylocoous aureus
and enterococcus sp are
commonly isolated from
patients specimens in the
hospital and cause hospital
acquired infections
• Wound infections
• Hospital acquired pneumonia
• urinary tract infections esp
catheter associated
• MRSA and VRE are multi drug
resistant forms of these
bacteria
HOSPITAL ACQUIRED INFECTIONS
• Many of these gram negative
bacteria(e.g. Escherichia coli,
Klebsiella pneumoniae, Proteus sp.,
Enterobacter sp., Serratia spp,
Citrobacter spp ,pseudomonas sp,
acinetobacter sp etc.) are
frequently multidrug resistant and a
common cause of hospital
acquired infections e.g
• Wound infections
• Hospital acquired pneumonia
• urinary tract infections esp catheter
associated
• Bacteremia and septicemia
GRAM STAIN GUIDES INITIAL
ANTIBIOTIC THERAPY
• Clinicians suspecting infections send
relevant clinical specimens from the
patients
• Sputum, CSF, pus, urine stool,blood
etc.
• Gram stain is a quick laboratory
procedure that guides initial
antibiotic therapy pending culture
results
• Quick and same day result
PROVISIONAL DIAGNOSIS OF INFECTION
• Bacterial arrangement on gram stain
may give an early clue to diagnosis so a
targeted antibiotic can be started
RIGHT CHOICE OF ANTIBIOTIC THERAPY
Although majority of the antibiotic work with
both gram positive and gram negative
bacteria some of the antibiotics only work with
either of them
• e.g colistin, a life saving end resort antibiotic
only acts on gram negative bacteria
• Vancomycin another life end resort
treatment usually has its action on gram
positive bacteria
 Full Culture & sensitivity result takes three days
Some serious patients may not survive that
long
• e.g septicemia, meningitis( a timely start of
antibiotic therapy can save life)
CASE SCENARIO 1
• A 50 year old male alcoholic presents with
right sided chest pain and high fever and
chills. His CXR reveals a dense infiltrate in his
right base. The presumed diagnosis is
pneumonia and he is treated with a
cephtriaxone and a macrolide. 18 hours
after admission the patient becomes
lethargic and hypotensive 80/35 mmhg.
• He is shifted to the ICU and his blood and
sputum sent for culture & sensitivity
Sputum gram stain
• Numerous pus cells
• Gram negative rods
• After about 6 hours the blood culture
machine gives a positive signal
• Gram stain from the blood culture
bottle shows gram negative rods
• The laboratory technician alerts the ICU
physcian and antibiotic therapy of the
patient is changed to inj imipenem( a
broad spectrum antibiotic against
gram negative rods)
• Patient gets well in few days and is
discharged
• His culture report show klebsiella
pneumoniae resistant to ceftriaxone
but sensitive to imipenem
SCENARIO 2
• A 30 yrs old male presents to the STD
clinic with purulent uretheral
discharge from his penis and dysuria
of three days duration. On history
taking he admits visit to prostitutes
His treating physician advises a lab
investigation
• Uretheral smear
• In the laboratory his
uretheral smear was
prepared and gram
stained
• Gram stain of the uretheral smear,
• Pus cells
• Gram negative diplococci both
inside and outside of pus cells
• A diagnosis of gonorrhea( a sexually
transmitted disease caused by
Neisseria gonorrhea) was made
• He was given a single dose of
125mg I/m ceftriaxone injection
• Patient was relieved of his symptoms
and moves on with his life
CASE SCENARIO 3
• A 35 yrs-old man was admitted to
the University Hospital of majmaah
with a 2-day history; of pain and
swelling of the left knee associated
with fever and rigors.
• On examination He had a tender,
warm, left knee joint and restricted
movements
• His synovial fluid was aspirated and
sent for culture sensitivity
• Gram stain of the synovial fluid
showed
• Gram positive cocci in clusters
• Numerous pus cells
• Patient was started on inj
vancomycin 15mg/kg q12 hrly
• Culture /sensitivity result showed
methicillin resistant staphylococcus
aureus sensitive to vancomycin
THANK YOU
SELF ASSESMENT
1. Gram staining is which type of staining
a. simple
b. differential
c.negative
d. fluorescent
2. In gram staining if bacteria are retaining the dye iodine complex, then the bacteria is
a. gram positive
b.gram negative
c.gram variable
d. improper staining
3. Counter stain used in gram staining is
a. crystal violet
b. malachite green
c. safranin
d. carbol fuschin
• How is gram staining useful to us in hospital?