GRAM NEGATIVE BACTERIA

Gram staining
- Christian Gram (1884) developed gram staining process.
- Stains interact differently with a particular type of cell
wall.
- Cells will be either Gram positive / Gram negative.

Gram Negative Bacteria

a group of bacteria that do not retain the crystal
violet stain used in the Gram staining method of
bacterial differentiation, making positive
identification possible.

Gram negative: A group of bacteria that do not

retain the crystal violet dye after the differential
staining procedure known as Gram staining. They
appear pink due to the counterstain.
Gram positive appears purple. The difference
between Gram negative and Gram positive bacteria
is the cell wall structure, which accounts for the
different staining characteristics

GRAM-STAINING
Gram Negative PINK
Gram Positive PURPLE
 The Gram-negative cell wall is composed of a thin, inner layer
of peptidoglycan and an outer membrane consisting of
molecules of phospholipids, lipopolysaccharides (LPS),
lipoproteins and surface proteins. The lipopolysaccharide
consists of lipid A and O polysaccharide.

Characteristics:
1. Cell membrane (cytoplasmic).
2. Thin peptidoglycan layer (which is much thicker in
gram-positive bacteria)
3. Outer membrane containing lipopolysaccharide
(LPS, which consist of lipid A, core polysaccharide, Thin peptidoglycan layer (which is much thicker in gram-
and O antigen) in its outer leaflet and phospholipids positive bacteria)
in the inner leaflet
4. Porins exist in the outer membrane, which act like
pores for particular molecules
5. There is a space between the peptidoglycan layer
and the secondary cell membrane called
the periplasmic space
6. The S-layer is directly attached to the outer
membrane rather than the peptidoglycan
7. If present, flagella have four supporting rings instead
of two
8. No teichoic acids or lipoteichoic acids are present
9. Lipoproteins are attached to the polysaccharide
backbone. Outer membrane containing lipopolysaccharide (LPS, which
consists of lipid A, core polysaccharide, and O antigen) in its
Lipopolysaccharides - large molecules consisting of a outer leaflet and phospholipids in the inner leaflet
lipid and a polysaccharide.

Porins are involved in the exchange of nutrients over the

outer membrane of Gram-negative bacteria but are also
involved in pathogenesis

S-layer (surface layer) is a part of the cell envelope

commonly found in bacteria, as well as
among archaea. It consists of a monomolecular layer
composed of identical proteins or glycoproteins.

The main function of teichoic acids is to provide rigidity

(stiffness) to the cell-wall by attracting cations such as
magnesium and sodium
Porins exist in the outer membrane, which act like pores for
particular molecules
There is a space between the peptidoglycan layer and
the secondary cell membrane called the periplasmic
space

The S-layer is directly attached to the outer membrane

rather than the peptidoglycan

S Layer Crystalline layer on top of either gram-positive

or gram-negative bugs. The S Layer may increase
virulence, and it may also help in adhesion.

If present, flagella have four supporting rings instead of

two

Enterobacteriaceae
most common group of gram-negative rods cultured in
clinical laboratories
They are gram-negative rods, either motile with
peritrichous flagella or nonmotile; grow on peptone or
meat extract media without the addition of sodium
chloride or other supplements; grow well on MacConkey
agar; grow aerobically and anaerobically.
Morphology and Identification
- short gram-negative rods,
- Capsules are large and regular in Klebsiella species,
less so in Enterobacter species, and uncommon in
the other species.
Culture
- E coli and most of the other enteric bacteria form
circular, convex, smooth colonies with distinct
edges. Enterobacter colonies are similar but
somewhat more mucoid.
- Klebsiella colonies are large and very mucoid and
tend to coalesce with prolonged incubation.
- The salmonellae and shigellae produce colonies
similar to E coli but do not ferment lactose.
- Some strains of E coli produce hemolysis on blood
agar.
Pathogenesis and Clinical Findings:
E.coli
1. Urinary tract infectionE coli is the most common
cause of urinary tract infection and accounts for
approximately 90% of first urinary tract infections in
young women
Most of the urinary tract infections that involve
the bladder or kidney in an otherwise healthy
host are caused by a small number of O antigen
types that have specifically elaborated virulence
factors that facilitate colonization and
subsequent clinical infections
2. E coliassociated diarrheal diseases
E coli that causes diarrhea are extremely
common worldwide.
This E coli are classified by the characteristics of
their virulence properties
The small or large bowel epithelial cell
adherence properties are encoded by genes on
plasmids.
Similarly, the toxins often are plasmid or phage
mediated.
Enteropathogenic E coli (EPEC)
EPEC is important cause of diarrhea in infants,
especially in developing countries.
EPEC adhere to the mucosal cells of the small
bowel.
After attachment, there is loss of microvilli
(effacement); formation of filamentous actin
pedestals or cuplike structures;
Characteristic lesions can be
seen on electron micrographs of small bowel
biopsy lesions.
Shiga toxin-producing E coli (STEC)
named for the cytotoxic toxins they produce.
There are at least two antigenic forms of the
toxin referred to as Shiga-like toxin 1 and Shiga-
like toxin 2.
SepsisWhen normal host defenses are inadequate, E
coli may reach the bloodstream and cause sepsis.
MeningitisE coli and group B streptococci are the
leading causes of meningitis in infants
- Approximately 75% of E coli from meningitis
cases have the K1 antigen.
- This antigen cross-reacts with the group B
capsular polysaccharide of N meningitidis.
- The mechanism of virulence associated with the
K1 antigen is not understood.
KlebsiellaEnterobacterSerratia;
ProteusMorganellaProvidencia; and Citrobacter
1. Klebsiella
pneumoniae is present in the respiratory tract
and feces of about 5% of normal individuals
K pneumoniae can produce extensive
hemorrhagic necrotizing consolidation of the
lung.
2. Enterobacter
Three species of Enterobacter, Enterobacter
cloacae, Enterobacter aerogenes, and
Enterobacter sakazakii (now in the genus
Cronobacter), cause the majority of Enterobacter
infections.
These bacteria ferment lactose, may contain
capsules that produce mucoid colonies, and are
motile
resistance to third generation cephalosporin.
3. Serratia
Serratia marcescens is a common opportunistic
pathogen in hospitalized patients. Serratia
(usually nonpigmented) causes pneumonia,
bacteremia, and endocarditis, especially in
narcotics addicts and hospitalized patients.
Infections can be treated with third-generation
cephalosporins.
4. Proteus
Proteus species produce infections in humans
only when the bacteria leave the intestinal tract.
They are found in urinary tract infections and
produce bacteremia, pneumonia, and focal
lesions in debilitated patients or those receiving
contaminated intravenous infusions.
Proteus species produce urease, resulting in
rapid hydrolysis of urea with liberation of
ammonia.
Thus, in urinary tract infections with Proteus
species, the urine becomes alkaline, promoting
stone formation and making acidification
virtually impossible.
5. Providencia
Providencia species (Providencia rettgeri,
Providencia alcalifaciens, and Providencia
stuartii) are members of the normal intestinal
flora.
All cause urinary tract infections and occasionally
other infections and are often resistant to
antimicrobial therapy.
6. Citrobacter
Citrobacter species can cause urinary tract
infections and sepsis.
SHIGELLAE
The natural habitat of shigellae is limited to the
intestinal tracts of humans and other primates,
where they produce bacillary dysentery.
Morphology and Identification
Shigellae are slender gram-negative rods;
coccobacillary forms occur in young cultures.
Shigellae are facultative anaerobes but grow
best aerobically.
Convex, circular, transparent colonies with intact
edges reach a diameter of about 2 mm in 24
hours.
All shigellae ferment glucose. With the exception
of Shigella sonnei, they do not ferment lactose.
Shigella infections are almost always limited to the
gastrointestinal tract; bloodstream invasion is quite
rare. Shigellae are highly communicable;
The essential pathologic process is invasion of the
mucosal epithelial cells (eg, M cells) by induced
phagocytosis, escape from the phagocytic vacuole,
multiplication and spread within the epithelial cell
cytoplasm, and passage to adjacent cells.
THE SALMONELLA-ARIZONA GROUP
Salmonellae vary in length. Most isolates are motile
with peritrichous flagella. Salmonellae grow readily
on simple media, but they almost never ferment
lactose or sucrose
The Enteric Fevers (Typhoid Fever)
Bacteremia with Focal Lesions
C. Enterocolitis This is the most common
manifestation of salmonella infection.
Treatment and Prevention
Gram-negative bacteria cause infections including
pneumonia, bloodstream infections, wound or
surgical site infections, and meningitis in healthcare
settings.
Gram-negative bacteria are resistant to multiple
drugs and are increasingly resistant to most available
antibiotics.
Combination antibiotic therapy for invasive
infections with Gram-negative bacteria is employed
in many health care facilities
Sanitary measures must be taken to prevent
contamination of food and water by rodents or other
animals that excrete salmonellae. Infected poultry,
meats, and eggs must be thoroughly
cooked. Carriers must not be allowed to work as food
handlers and should observe strict hygienic
precautions.
Vaccination is recommended for travelers to
endemic regions,
List of Gram Negative Bacteria
Acetic acid bacteria
derive their energy from the oxidation
of ethanol to acetic acid during fermentation.
aerobic, rod-shaped bacteria.
Acetic acid bacteria are airborne and are ubiquitous
in nature.
Acinetobacter baumannii
It can be an opportunistic pathogen in humans,
affecting people with compromised immune
systems
Colloquially, A. baumannii is referred to as
'Iraqibacter' due to its seemingly sudden emergence
in military treatment facilities during the Iraq War.
Agrobacterium tumefaciens
the causal agent of crown gall disease (the
formation of tumours) in over 140 species
of eudicots.
Economically, A. tumefaciens is a serious pathogen
of walnuts, grape vines, stone
fruits, nut trees, sugar beets, horse radish,
andrhubarb.
Arcobacter
It shows an unusually wide range of habitats, and
some species can be human and animal pathogens
Symptoms of infections include diarrhea associated
with abdominal pain, nausea, and vomiting or fever
Bacteroides
obligate anaerobic bacteria
Some species (B. fragilis, for example)
are opportunistic human pathogens, causing
infections of the peritoneal cavity, gastrointestinal
surgery, and appendicitis via abscess formation
Bdellovibrio
obligate anaerobic bacteria
The Bdellovibrio cell uses hydrolytic enzymes to
break down the host cell molecules, which it
uses to elongate and form a filament and gets
the host cell nutrients
Diseases that are caused by gram negative bacteria
Gram Negative Spiral Bacteria
Slender and flexible, come in a lot of different
shapes
More rigid than spirochetes
Ex. Campylobacter jejuni
Symptom tenesmus: the sensation of desire
to defecate, which is common and occurs
frequently , with out the production of
significant amounts of feces (often small
amounts of mucous or blood are alone passed).
Gram Negative Spirochetes
pathogenic
very flexible
tightly coiled, helically coiled
Example
o syphilis
o Treponema pallidum
 Gram Negative Facultative Rods
Vibrio

V. cholera
Most of pathogenic - Most well known of group
Very flexible - Very severe dysentery.
Tightly coiled, helically coiled - Can lose 10-15 liters of water/day. Leads to
Example hypovolemia low water, hardly any water
o Lyme disease in body
Borrelia burgdorferi V. vulnificus
(organism gets lodged in tissues) o Very pathogenic
o Can cause flesh eating disease, if it gets
in a wound
Gram Negative Aerobic Rods
V. parahaemolyticus
o Found in shellfish oysters
o Halophile loves salt (will find in
oceans, estuaries)
o Self limiting

Enteric
Salmonella
Legionella pneumophila Shigella
Lower respiratory tract infection E. coli (0157H7)
Needs oxygen
Gram Negative Anaerobic Rods
Fusobacterium
o Live in between teeth and gums
o Cause tooth abscesses and periodontal
disease
o Teeth have nothing to anchor bone is
destroyed

Bordetella pertussis Gram Negative Cocci or Coccobaccilli (plump rods)

whooping cough Neisseria gonorrhoeae
Needs oxygen - Usually a diplococcus in PMN
- Sexually Transmitted Disease
- very antibiotic resistant

Neisseria meningitidis
- very infectious and communicable.
Pseudomonas aeruginosa (pigmented)
Needs moisture Acinetobacter baumanni
Common in hospitals - opportunistic, UTI, skin, and upper respiratory
Opportunistic pathogen causes UTI, skin, and
lung infection
Chlamydia Gram Negative Rods (Transitional)
- Very short little rods
- Transitional doesnt hold stain well
- Do not have the ability to synthesize own ATP, therefore and obligate intracellular parasite of other animals
(humans)
- Can go asymptomatic for a long time
- Ex.
o C. trachomatis STD, causes eye infection
o C. psittaci parrot (associated with birds)

Rickettsia Gram Negative Rod (Transitional)

- Small gram negative rods
- Transitional doesnt hold stain well
- Cant synthesize its own NAD, coenzyme A, therefore an obligate intracellular parasite
- Causative agent of Rocky Mountain Spotted Fever
- Example
o R. Prowazekii