MODULE 3: BACTERIAL STRUCTURE, Cytoplasmic/ Plasma/Cell Membrane

GROWTH, AND METABOLISM • Phospholipid w/c form 2 parallel surfaces

(phospholipid bilayer)
• Polar phosphate groups - outside
2 Basic Types of Microorganisms • Nonpolar lipid chains are – inside
• Acts as permeability barrier, restricting
Prokaryotic (Before – nucleus)
molecules that enter and leave the cell
Eukaryotic (True – nucleus)
Peptidoglycan

• Determines the shape of the cell

Prokaryotes • Composed of a cross-linked polymeric
mesh
➢ include the bacteria and cyanobacteria
• Glycan – linear polymer of alternating
which were formerly classified as blue-
monosaccharide subunits (backbone)
green algae.
• Peptido – short string of AA forming a
➢ They possess a simple make-up that does
network w/ high tensile strength
not contain sub-cellular organelles. The
typical size of a prokaryote is about 1um
diameter.
➢ Archaebacteria and Eubacteria (MONERA) Difference b/w Gram (+) & Gram (-) Cell Walls:

Eukaryotes Gram (+) Bacteria Cell Wall

➢ possess a complex cellular structure. • Thick, multilayered, peptidoglycan cell

➢ contain membrane-bound organelles walls that are exterior to the cytoplasmic
such as mitochondria, lysosomes, (plasma) membrane
endoplasmic reticulum and golgi bodies. • The peptidoglycan in most gram (+)
species is covalently linked to teichoic acid,
which is essentially a polymer of
substituted glycerol units linked by
2 Kinds of Cells phosphodiester bonds
Eukaryotic – cells with a well-defined nucleus • Teichoic acids are major cell surface
antigens & are integrated into the
Prokaryotic – cells without a nucleus peptidoglycan layers but not tethered to the
All bacteria are prokaryotes cytoplasmic membrane
• Lipoteichoic acids are lipid modified and
Unorganized DNA (single double-stranded integrated by this moiety into the outer
molecule) leaflet of the cytoplasmic membrane
Gram (-) Bacteria Cell Wall
Bacterial Structure • More complex cell wall structure composed
of 2 membranes (an outer membrane and
Cell Envelope
a periplasmic membrane/space) (inner
• Term applied to all material external to and membrane = cytoplasmic / plasma
enclosing the cytoplasm membrane)
• Layers: Cell Wall, Plasma / Cell / • The 2 membranes are separated by
Cytoplasmic Membrane, * Capsule (some / periplasmic space, w/c contains the
not all) peptidoglycan layer
• It consists of several chemically and • The periplasmic space also contains
functionally distinct layers, the most degradative enzymes and transport
prominent of which are the cell wall and proteins
the cytoplasmic membrane • The peptidoglycan layer of gram (-) cells is
• Also includes the capsule or glycocalyx, if thin, and cells are consequently more
present susceptible to physical damage
 • The outer membrane is distinguished by
the presence of embedded
lipopolysaccharide (LPS) that is the
major constituent of the outer leaflet of the
outer membrane
• The polysaccharide portion of LPS (O-
polysaccharide) is antigenic and can,
therefore, be used to identify different
strains and species
• The lipid portion (lipid A) is imbedded in
the membrane and is toxic to humans and
animals
• It is called endotoxin
External Capsule and Glycocalyx
Sporulation
• sticky, viscous, slime material that forms an
extracellular coating around the cell –
glycocalyx
• allows cells to adhere to surfaces, protect
bacteria from antibodies & phagocytosis
• Capsules can also protect bacteria against
desiccation, or drying, which facilitates
transmission
Appendages
Flagella
• Long, semirigid, helical, hollow tubular
structures composed of several thousand
molecules of the protein flagellin
• Enable bacteria to move in a directed
fashion
• Anchored in the cell membranes by a basal
body, which is a complex molecular
machine that rotates the flagellum like the
screw propeller of a ship
• Cells may have one or many flagella
• Highly antigenic
Pili (Fimbriae)
• Shorter and thinner than flagella and
function as attachment structures that
Medical Significance of Sporulation
promote specific cell-to-cell contact
• The attachment can be between the
bacterial cell and the host eukaryotic cell or
between one bacterial cell and another
Spores and Sporulation
• To enhance survival during periods of
environmental hostility (such as
nutritional deprivation), some
gram-positive rods undergo profound
structural and metabolic changes
• These result in the formation of a dormant
cell called an endospore inside the original
cell
• Endospores can be released from the
original cell as free spores
• Spores are the most resistant life forms
known
• They are remarkably resistant to heat
(they survive boiling), desiccation,
ultraviolet light, and bactericidal chemical
agents
• In fact, sterilization procedures are
assessed by their ability to inactivate
Spores
• Repackaging a copy of bacterial DNA into
a new form that
- contains very little water
- no metabolic activity
- does not divide
- has a restructured, highly impermeable,
multilayered envelope
• Begins with the invagination of the parent
cell membrane, producing a double
membrane that encapsulates and isolates
a copy of the bacterial DNA in what will
become the core of the spore
• Mature spore retains the complete
machinery for protein synthesis, and new
spore-specific enzymes are synthesized in
the core of the spore
• The core also has high levels of a unique
compound called calcium dipicolinate,
which is thought to be important for
protection of the spore DNA from
environmental damage
• Many enzymes of the original vegetative
(non-dividing) cells are degraded
• When the endospore is completed, the
parent cell lyses, releasing the spore
• Some of the most notorious pathogens are
spore-formers, including:
- B. anthracis
- Bacillus cereus (gastroenteritis)
- Clostridium tetani
- Clostridium botulinum
- Clostridium perfringens (gas gangrene)
- Clostridium difficile
• Spores of these organisms can remain
viable for many years and are generally not
killed by boiling, but they can be killed by
 autoclaving (temps > 120oC at elevated
pressure)
• In the absence of an autoclave, spores can
be largely eliminated by a primary boiling to
activate germination and, after a short
period of vegetative growth, a second
boiling
Bacterial Growth and Metabolism
➢ All cells must accomplish certain
metabolic tasks to grow and divide
➢ All cells, whether bacterial or human,
accomplish these metabolic tasks by
similar pathways
➢ There are, however, some important
differences that set bacteria apart
metabolically from eukaryotic cells
Characteristics of Bacterial Growth
• If bacterial cells are suspended in a liquid
nutrient medium, the increase in cell
number or mass can be measured in
several ways
• Techniques include:
- microscopically counting the cells in a
given volume using a ruled slide
- counting the number of appropriately
diluted cells that are able to form colonies
following transfer to a solid nutrient (agar)
surface
- quantitating the turbidity—which is
proportional to the cell mass—of a culture
in liquid medium
Stages of Bacterial Growth Cycle
1. Lag Phase
2. Log (exponential) Phase
3. Stationary Phase
4. Death (decline) Phase
• Because bacteria reproduce by binary
fission (1 becomes 2, 2 become 4, 4
become 8, etc.), the number of cells
increases exponentially with time (the
exponential, or log, phase of growth)
• Depending on the species, the minimum
doubling time can be as short as 10 mins
or as long as several days
• Ex: for a rapidly growing species such as E.
coli in a nutritionally complete medium, a
single cell can give rise to some 10 million
cells in just 8 hours
• Eventually, growth slows and ceases
entirely (stationary phase) as nutrients
are depleted, and toxic waste products
accumulate
• Most cells in a stationary phase are not
dead, however
• If they are diluted into fresh growth
medium, exponential growth will resume
after a lag phase
Energy Production
• A distinctive feature of bacterial
metabolism is the variety of mechanisms
used to generate energy from carbon
sources
• Depending on the biochemical mechanism
used, bacterial metabolism can be
categorized into 3 types:
- aerobic respiration
- anaerobic respiration
- fermentation
Aerobic Respiration
• Metabolic process in which molecular
oxygen serves as the terminal electron
acceptor of the electron transport chain
• In this process, oxygen is reduced to
water
• Respiration is the energy-generating mode
used by all aerobic bacteria
Anaerobic Respiration
• Metabolic process in which inorganic
compounds other than molecular oxygen
serve as the terminal electron acceptors
• Depending on the species, acceptors can
be molecules such as nitrate or sulfate
• Anaerobic respiration can be used as an
alternative to aerobic respiration in some
species (facultative organisms), but is
obligatory in other species (some obligate
anaerobes)
Fermentation
• Anaerobic process utilized by some
bacterial species
• It is the metabolic process by which an
organic metabolic intermediate derived
from a “fermentable” substrate serves as
the final electron acceptor
MODULE 4: STAPHYLOCOCCI
Gram Staining
• Staining method using crystal violet
(primary stain) & a counterstain – safranin
or fuchsine
• To distinguish & classify bacteria into Gram
(+) or Gram (-)
• Based on its peptidoglycan cell wall
• Developed by Hans Christian Gram (1884)
➢ Gram (+) bacteria stains (DARK) VIOLET
(retains the Crystal Violet)
➢ Gram (-) bacteria stains PINK or RED
(retains the counterstain Safranin or
Fuchsine)
➢ Gram-variable / Gram-indeterminate 🡪
unclear or no stain
- Ex: Typhus bacillus
Staphylococci General Features
• Stains dark violet 🡪 Gram (+)
• Round and tend to occur in bunches like
grapes
• Because growth of staphylococci requires
supplementation with various amino acids
and other growth factors, they are routinely
cultured on enriched media containing
nutrient broth and/or blood
• Facultatively anaerobic organisms
• Produce catalase 🡪 Catalase (+)
Catalase Test
(+) Bubbles 🡪 Catalase (+) 🡪 Staphylococci
(-) Bubbles 🡪 Catalase (-) 🡪 Streptococci
🡪 A small drop of HYDROGEN PEROXIDE is
placed on a microscope slide
🡪 Applicator stick is touched to a bacterial colony
and the tip is smeared onto the hydrogen peroxide
drop
• S. aureus - most virulent specie secrete
coagulase, an enzyme that causes
citrated plasma to clot
• S. aureus 🡪 COAGULASE (+)
Coagulase Test
🡪 Used to differentiate S. aureus from coagulase
(-) species (S. epidermidis, S. saprophyticus)
🡪 2 drops saline 🡪 slide (T) and slide (C)
🡪 Emulsified with the organism using a wire loop
slide (T)
🡪 A drop of plasma anticoagulated w/ EDTA
(EthyleneDiamineTetraacetic Acid) is placed on
the slide (T) & (C)
🡪 Macroscopic clumping in the plasma within 10
secs in slide (T) w/ no clumping in the slide (C) 🡪
Coagulase (+)
🡪 No clumping in both slides 🡪 Coagulase (-)
* followed by a tube test
• Other species that occasionally cause
disease and lack coagulase are often
referred to as coagulase-negative
staphylococci
• Resistant to heat and drying; can persist
for long periods on fomites (inanimate
objects), which can then serve as sources
of infection
• Frequent hand-washing before and after
contact with food or potentially infected
individuals decreases the transmission
Staphylococcus Aureus
• Generally, significant host compromise
is required like:
- break in the skin or insertion of a foreign
body (ex: wounds, surgical infections)
- obstructed hair follicle (folliculitis)
- compromised immune system
Disease may be:
1) largely or wholly the result of actual
invasive infection, overcoming host
defense mechanisms, and the production
of extracellular substances which facilitate
invasion
2) a result of toxins in the absence of
invasive infection (“pure” toxinoses)
3) a combination of invasive infection and
Intoxication

Epidemiology
• Frequently carried by healthy individuals on
the skin and mucous membranes
• Carriers serve as a source of infection to
themselves and others (ex: direct contact,
by contamination of fomites (objects such
as a doorknob, which in turn can be a
source of infection) or contamination of
food, which can then result in food
poisoning
Pathogenesis
Cell wall virulence factors:
- Capsule: thin (microcapsule) & resistant to
phagocytosis
- Protein A: major component of cell wall
- Fibronectin-binding protein (FnBP) 🡪 adhesin 🡪
promote binding to mucosal cells & tissue
- Clumping factor: Coagulase
🡪 Cytolytic exotoxins: attack RBC (hemolysin)
🡪 Panton-Valentine leukocidin: pore-forming
toxin making the strain more virulent (community-
acquired methicillin-resistant S. aureus – MRSA)
🡪 Superantigen exotoxins
- Enterotoxins: cause food poisoning
- Toxic shock syndrome toxin (TSST-1)
- Exfoliatin toxin (ET): scalded skin syndrome in
children
Clinical Significance
🡪 S. aureus causes disease by infecting tissues,
typically creating abscesses and/or by producing
toxins
🡪 A common entry point into the body is a break
in the skin, which may be a minute needlestick or
a surgical wound
🡪 Another portal of entry is the respiratory tract
(Ex: staphylococcal pneumonia)
🡪 Localized host response to staphylococcal
infection:
- inflammation (swelling)
- accumulation of pus (pyogenic)
- necrosis of tissue
🡪 Fibroblasts and their products may form a wall
around the inflamed area, which contains bacteria
and leukocytes
🡪 This creates a characteristic pus-filled boil or
abscess
🡪 Serious consequences occur when the bacteria
invade the bloodstream 🡪 septicemia (presence
and persistence of pathogenic microorganisms
or their toxins in the blood) 🡪 rapidly fatal
🡪 Bacteremia (presence of viable bacteria
circulating in the bloodstream) may result in
- seeding internal abscesses
- skin lesions
- infections in the lung, kidney, heart, skeletal
muscle, or meninges
1. Localized skin infections
- small, superficial abscesses involving hair
follicles (folliculitis) or sweat or sebaceous glands
- Ex: sty (external hordeolum) – eyelash
- Furuncle (boil) – subcutaneous abscess
(neck, face, axilla, buttocks)
- Carbuncle – larger, deeper skin infections
- Impetigo – localized, superficial, spreading
crusty skin lesion in children
2. Deep, localized infections
- metastatic from superficial infections
- most common cause of acute and chronic
bone marrow infection
- most common cause of acute infection of
joint space in children (septic joint) – emergency
because pus can rapidly cause irreparable
cartilage damage
3. Acute endocarditis
- generally associated with IV drug abuse by
injection of contaminated preparations or needles
- also colonizes skin around the injection site,
and if the skin is not sterilized before injection,
bacteria can be introduced into soft tissues and the
bloodstream, even when a sterilized needle is
used
4. Septicemia
- generalized infection with sepsis or bacteremia
that may be associated with a known focus (for
example, a septic joint) or not (an occult focus)
5. Pneumonia
6. Nosocomial infections
- hospital-associated infections, often of
wounds (surgical, decubital) or bacteremia
associated with catheters
7. Toxinoses
a. Toxic shock syndrome:
- high fever
- rash ('sunburn', w/ diffuse erythema followed
by desquamation)
- vomiting, diarrhea 🡪 hypotension
- multiorgan involvement (GI, renal, and/or
hepatic damage)
b. Staphylococcal gastroenteritis:
- ingestion of food contaminated with
enterotoxin
- protein rich (egg salad or cream pastry) or
salty, like ham, and improperly refrigerated
- nausea, vomiting, and diarrhea, are acute
following a short incubation period (< 6 hours)
because toxin in the food has already been formed
before food is ingested
c. Scalded skin syndrome:
- superficial bullae resulting from the action of
an exfoliative toxin that attacks the intercellular
adhesive of the stratum granulosum, causing
marked epithelial desquamation
- the bullae may be infected or may result from
toxin produced by organisms infecting a different
site
Lab Identification
🡪 Stain strongly gram (+), and are frequently seen
in grapelike clusters
🡪 Relies largely on microscopic and colony
morphology and catalase positive
🡪 Distinguished from the coagulase-negative
staphylococci primarily by coagulase positive
🡪 Tend to be yellow (“aureus,”- golden) and
hemolytic, rather than gray and nonhemolytic like
the coagulase-negative staphylococci
🡪 S. aureus is also distinguished from most
coagulase-negative staphylococci by being
mannitol-positive
Immunity
🡪 Do not elicit strong or long-lasting immunity, as
demonstrated by the continuing susceptibility of
individuals to S. aureus infections throughout life
Treatment
🡪 Require aggressive treatment, including incision
and drainage (I&D) of localized lesions, as well as
systemic antibiotics
- Oxacillins
- Cephalosporins
🡪 Vancomycin for methicillin-resistant S. aureus
Coagulase-negative Staphylococci
🡪 Of 12 species recovered as normal commensals
of human skin and anterior nares, the most
abundant and important is S. epidermidis
🡪 The second most important coagulase-negative
staphylococcus is S. saprophyticus
🡪 Important agents of hospital-acquired infections
associated with the use of implanted prosthetic
devices and catheters
Staphylococcus Epidermidis
• Present in large numbers as part of the
normal flora of the skin
• As such, it is frequently recovered from
blood cultures, generally as a contaminant
from skin
• Despite its low virulence, it is a common
cause of infection of implants such as heart
valves and catheters
• Acquired drug resistance by S. epidermidis
is even more frequent than by S. aureus
• Produces an extracellular polysaccharide
material called polysaccharide intercellular
adhesin (“slime”), that facilitates adherence
to bioprosthetic material surfaces, such as
intravenous catheters, and acts as a barrier
to antimicrobial agents
Staphylococcus Saprophyticus
• Frequent cause of cystitis in women,
probably related to its occurrence as part of
normal vaginal flora
• It tends to be sensitive to most antibiotics,
even penicillin G
• Distinguished from S. epidermidis and
most other coagulase-negative
staphylococci by its natural resistance to
novobiocin
MODULE 5: STREPTOCOCCI
Streptococci General Features
• Gram (+)
• Non-motile
• Catalase (-)
• Ovoid to spherical
• Pairs or chains
• Aerotolerant / Facultative anaerobes
• Blood enriched medium
2 Ways of Classification
1.) Hemolytic properties on blood agar
2.) Serologic (Lancefield) groupings
Hemolytic Properties on Blood Agar
➢ α-Hemolytic: chemical change in the
hemoglobin in blood agar, resulting in a
green pigment that forms a ring around
the colony
➢ β-Hemolytic: gross lysis of RBC, resulting
in a clear ring around the colony
➢ γ-Hemolytic: is a term applied to
streptococci that cause no color change
or lysis of RBC
Serologic (Lancefield) Groupings
🡪 Many species of streptococci have a
polysaccharide in their cell walls known as C-
substance, which is antigenic and easily
extractable with dilute acid
🡪 Classifies primarily β-hemolytic streptococci
into groups A through U on the basis of their C-
substance
🡪 The clinically most important groups of β-
hemolytic streptococci are:
- Groups A and B
Group A β-Hemolytic Streptococci (GABHS)
🡪 S. pyogenes (Streptococcus Pyogenes)
• most clinically important member of this
group of gram (+) cocci
• one of the most frequently encountered
bacterial pathogens of humans worldwide
• can invade apparently intact skin or
mucous membranes, causing some of the
most rapidly progressive infections known
• cause rheumatic fever and acute
glomerulonephritis
• Nasopharyngeal carriage is common
especially in colder months and particularly
among children
• does not survive well in the
environment (unlike staph)
• instead, its habitat is infected patients and
also normal human carriers in whom the
organism resides on skin and mucous
membranes
• usually spread person to person by skin
contact and via the respiratory tract
• usually form long chains when recovered
from liquid culture but may appear as
individual cocci, pairs, or clusters of cells in
Gram stains of samples from infected
tissue
Capsule:
🡪 Hyaluronic acid (Hyaluronan), identical to that
found in human connective tissue, forms the
outermost layer of the cell
🡪 Not recognized as foreign by the body and,
therefore, is nonimmunogenic
🡪 Antiphagocytic
Cell Wall:
🡪 contains a number of clinically important
components:
➢ M Protein (evasive protein)
- not infectious if absent
- highly variable, w/ 80 different antigenic
types
- antiphagocytic
➢ Protein F (fibronectin-binding protein)
- mediates adhesion / attachment in the
pharyngeal epithelium
Extracellular Products:
🡪 secretes a wide range of exotoxins that often
vary from one strain to another
Epidemiology:
🡪 the only known reservoir is the skin and
mucous membranes of the human host
🡪 respiratory droplets or skin contact spreads from
person to person, especially in crowded
environments (classrooms & play areas)
Clinical Significance
🡪 Major cause of cellulitis
🡪 Acute tonsillopharyngitis (ATP) /
pharyngotonsillitis
- most common type of infection
- “strep throat” is associated with severe,
purulent inflammation of the posterior oropharynx
and tonsillar areas
🡪 Impetigo
- classic cause
- begins on any exposed surface (legs)
- extensive lesions on face & limbs
- treated with a topical agent such as mupirocin, or
systemically with penicillin or a first-generation
cephalosporin (cephalexin)
🡪 Erysipelas
- affecting all age groups
- fiery red, advancing erythema, especially on
the face or lower limbs
🡪 Puerperal sepsis
- initiated during, or following soon after, delivery
caused by exogenous transmission (nasal droplets
from an infected carrier or from contaminated
instruments) or endogenously, from the mother’s
vaginal flora
🡪 Streptococcal toxic shock syndrome
- mediated by production of pyrogenic exotoxins
- initially flulike symptoms, followed shortly by
necrotizing soft tissue infection, shock, acute
respiratory distress syndrome, and renal failure
- Tx: penicillin G + clindamycin
🡪 Post-streptococcal sequelae
- Acute rheumatic fever after 2-3 weeks of
pharyngitis characterized by fever, rash, carditis, &
arthritis
- Acute glomerulonephritis after a week of
impetigo or pharyngitis causing renal failure
Lab Identification
🡪 Rapid latex antigen kits
• (+) test: latex particles clump together
• (-) test, they stay separate, giving the
suspension a milky appearance
Treatment
🡪 Penicillin
🡪 Clarithromycin or Azithromycin
🡪 Plus Clindamycin to inhibit protein (toxin)
synthesis released from rapidly dying bacteria
Group B β-Hemolytic Streptococci (GBBHS)
🡪 S. Agalactiae (Streptococcus Agalactiae)
• gram (+), catalase (-)
• found in vaginocervical tract of female
carriers, and the urethral mucous
membranes of male carriers as well as in
the gastrointestinal (GI) tract
 • can be transmitted sexually among adults
and from an infected mother to her infant at
birth
• leading cause of meningitis and septicemia
in neonates, with a high mortality rate
• occasional cause of infections in
postpartum women (endometritis) and
individuals with impaired immune systems,
in whom the organism may cause
septicemia or pneumonia
• Latex agglutination tests can also
demonstrate the presence of group B
antigen in these samples
• Group B streptococci are β hemolytic, with
larger colonies and less hemolysis than
group A
• Most remain sensitive to penicillin G and
ampicillin, which are still the antibiotics of
choice
🡪 S. Pneumoniae (Streptococcus
Pneumoniae) (Pneumococcus)
• capsular swelling observed when reacted
with type-specific antisera (Quelling
reaction)
• most common cause of community-
acquired pneumonia and adult bacterial
meningitis and is an important cause of
otitis media, sinusitis and mastoiditis
• Fastidious (complex nutritional
requirements) and routinely cultured on
blood agar
• Releases an α hemolysin that damages
red cell membranes, causing colonies to
be α hemolytic

• gram-positive, nonmotile, encapsulated

cocci
• lancet shaped, and their tendency to
occur in pairs accounts for their earlier
designation as Diplococcus pneumoniae

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