Gram Positive Bacteria
Karen F Ross
2007
• Spore forming Gram+ve Bacilli
– Bacilli
– Clostridium
• Non-pore forming Gram+ve Bacilli
– Corynebacterium
– Propionibacterium
– Listeria
• Staphylococci
• Streptococci
Bacillus anthracis
• Capsule, endospores viable up to 40 yrs
• Anthrax protein exotoxins- protective antigen, edema
factor and a toxic factor
• Skin- a papule first develops within 12-36 hours, then
pustule, then necrotic ulcer from which dissemination
may lead to scepticemia
• Inhalation- mild respiratory symptoms, sepsis,
meningitis, or hemorrhagic pulmonary edema.
Hemorrhagic pneumonia with shock is a terminal event.
Few patients survive.
• Lab tests- stained smears, culture, serological tests
• Treatment- Penicillin, but treatment must be started
early.
• Prevention and control- soil contimination, carcasses of
dead animals, spores viable for decades. Burning and
immunization
Sheep blood agar plate culture
Bacillus cereus
Bacillus anthracis
CDC/Dr. James Feeley
Bacillus cereus
• Lives in soil, water, and the gastrointestinal tract of
humans and animals
• Food poisoning- vomiting and diarrhea, usually mild and
brief
• Endotoxin is formed in food or in gut by growth of
bacteria
• Heat stable enterotoxin causes vomiting; rice dishes,
onset 1-6 hs
• Heat labile enterotoxin-diarrheal type; meat dishes and
vegetables, onset 1-24 hrs
• Diagnosis- 105 or more bacteria/g of food
Corynebacterium
diphtheriae
•
•
•
•
•
Gram+ve rod, pleomorphic, club shaped ends
Aerobic, or facultative anaerobe
Nasopharynx, skin, normal flora, respiratory droplets
Toxin inhibits protein synthesis in eukaryote cells
Formation of pseudomembrane that can block the throat and
cause suffocation
• Cutaneous diphtheria (extra-respiratory disease)
• Vaccine DTP-Diphtheria, Tetanus and Pertussis
Listeria monocytogenes
• Gram-positive rod-shaped, direct smears they may be
coccoid, so they can be mistaken for streptococci
• Listeriosis, a serious infection caused by eating food
contaminated with the bacteria
• Disease affects primarily pregnant women, newborns, and
adults with weakened immune systems
• Virulence factors
– Growth at low temperatures, grow and accumulate in
contaminated food stored in the refrigerator
– Motility
– Adherence and invasion
– listeriolysin O
– Act A
– phospholipase
Staphylococci
• Spherical 1µm in diameter arranged in
irregular clusters, cocci, pairs, tetrads
• Gram+ve, aerobic, microaerophilic
• Catalase +ve
• Coagulase +ve
• Hyaluronidase
• TSST-1
Gram positive Staphylococcus aureus
Staphylococcus aureus
• facultative anaerobic, gram+ bacteria, cocci, irregular clusters
• normal flora of humans found on nasal passages, skin and
mucous membranes
• causes a wide range of suppurative (pus forming) infections,
as well as food poisoning and toxic shock syndrome
• Virulence factors: toxin, superantigens,TSST-1, enterotoxin,
dermonecrotic toxin, coagulase, hemolysin, hyaluronidase,
leukocidin, lipase, fibronectin binding protein, protein A, FC
binding protein, FC receptor, antibiotic resistance
Virulence
factors
•
•
•
•
•
•
•
•
surface proteins that promote colonization of host tissues;
invasins that promote bacterial spread in tissues (leukocidin, kinases,
hyaluronidase);
Surface factors that inhibit phagocytic engulfment (capsule, Protein A);
Biochemical properties that enhance their survival in phagocytes (carotenoids,
catalase production);
Immunological disguises (Protein A, coagulase, clotting factor); and
Membrane-damaging toxins that lyse eukaryotic cell membranes (hemolysins,
leukotoxin, leukocidin;
Exotoxins that damage host tissues or otherwise provoke symptoms of disease
(SEA-G, TSST, ET)
Inherent and acquired resistance to antimicrobial agents
HA-MRSA and CA-MRSA
Hospital or Community Acquired-Methicillin
Resistant Staphylococcus aureus
• MRSA infection is an infection with
a strain of Staphylococcus aureus
bacteria that is resistant to
antibiotics known as beta-lactams.
These antibiotics include
methicillin, amoxicillin, and
penicillin.
Rebecca Lancefield (1895-1981)
Streptococci
Facultative anaerobe
Gram-positive coccichains or pairs
Catalase negative
(staphylococci are catalase positive)
Lancefield groups
A, B and D -most important
C, G, F -rare
Non-groupable -S. pneumoniae
Viridans streptococci eg. S. mutans
Lancefield classification is based on the antigenicity of a
carbohydrate which is soluble in dilute acid and called the C
carbohydrate
Streptococcus in chains
Streptococcus pneumoniae (diplococcus). Fluorescent stain
Sheep blood agar
Alpha hemolysis
Streptococcus pneumoniae
(and viridans stpreptococci)
Beta hemolysis
Gamma hemolysis
Streptococcus pyogenes (Group A)
(also Group B)
Enterococcus faecalis
Alpha hemolysis occurs when the RBCs are intact, but hemoglobin
is converted to biliverdin This causes a greening of the plate.
Beta hemolysis is true hemolysis due to the actions of a hemolysin,
an erythrocyte lysing enzyme. The plate becomes clear where
the blood cells have been lysed.
Mixed hemolysis on blood agar
Columbia CNA agar has the antibiotics colistin and
nalidixic acid added which inhibit the growth of
gram-negative bacteria but not the growth of
gram-positives. Staphylococcus (growth, yellow
colonies) vs. Streptococcus (growth,white colonies)
Dental abcess culture
Streptococcus pyogenes
• Group A, affect all ages, peak age 5-15 y
• Non-invasive
– Phayngitis
– Skin infection, impetigo
• Invasive bacteremia
– Toxic shock-like syndrome
– “flesh eating” bacteria
– Pyrogenic toxin
• Superantigen
• T cell mitogen
• Activates immune system
• Scarlet fever
– Rash
– Erythrogenic toxin
Pathogenesis of S pyogenes infections
Epidemiology
• Person to person via droplets or direct contact with
infected skin, fomites
• Impetigo-like skin infections are often due to secondary
infections of insect bites
Pathogenesis
• attachment to epithelial cells of the pharynx or skin via
hairlike fimbriae
• Systemic disease spread initiated by breaks in skin or
mucosal barriers or by direct cellular invasion by GAS
• Scarlet fever is a result of pyrogenic exotoxin
production by the organism
• Pyrogenic exotoxins are also felt to play a principle role
in triggering the deleterious immune activation of STSS
Surface virulence factors
M protein
hyaluronic acid capsule
F protein
Lipotechoic acid (LTA)
immunoglobulin binding proteins
C5a peptidase
The group A carbohydrate
Secreted virulence Factors
Streptococcal pyrogenic exotoxins
Streptolysin S
Streptolysin O (SLO)
Streptokinase
Hyaluronidase
NADase and DNAases
Rapid “Strep” Test
Antibody
Latex beads
Streptococcal antigenic extract
(eg. throat swab)
Latex Agglutination
Gram Positive Cocci Flow Chart
Gram Positive
Cocci
Staphylococcus
species
Positive
Catalase Test
Coagulase Test
Negative
Negative
A CoagulaseNegative
Stapylococcus
species
Positive
Staphylococcus aureus
Streptococcus
species
Some bacteria and macrophages can reduce diatomic oxygen to hydrogen peroxide or
superoxide. Both of these molecules are toxic to bacteria. Some bacteria, however, possess a
defense mechanism which can minimize the harm done by the two compounds. These
resistant bacteria use two enzymes to catalyze the conversion of hydrogen peroxide and
superoxide back into diatomic oxygen and water. One of these enzymes is catalase and its
presence can be detected by a simple test. The catalase test involves adding hydrogen
peroxide to a culture sample or agar slant. If the bacteria in question produce catalase,
they will convert the hydrogen peroxide and oxygen gas will be evolved. The evolution of gas
causes bubbles to form and is indicative of a positive test.
Gram Positive Cocci Flow Chart
Gram Positive
Cocci
Staphylococcus
species
Positive
Catalase Test
Coagulase Test
Negative
Negative
A CoagulaseNegative
Stapylococcus
species
Positive
Staphylococcus aureus
Streptococcus
species
Like the mannitol salts agar, the coagulase test is
another method for differentiating between pathogenic
and non-pathogenic strains of Staphylococcus. Bacteria
that produce coagulase use it as a defense mechanism
by clotting the areas of plasma around them, thereby
enabling themselves to resist phagocytosis by the host's
immune system. The sample in question is usually
inoculated onto 0.5 ml of rabbit plasma and incubated
at 37 degrees celsius for one to four hours. A positive
test is denoted by a clot formation in the test tube
after the allotted time.
Gram Positive Cocci Flow Chart
Gram Positive
Cocci
Staphylococcus
species
Positive
Catalase Test
Coagulase Test
Negative
Negative
A CoagulaseNegative
Stapylococcus
species
Positive
Staphylococcus aureus
Streptococcus
species
Growth on
Sheep’s
Blood Agar
Alpha Hemolytic
Growth in
Presence of
Bacitracin
Growth in
Presence of
Optochin
No
Sensitive
Streptococcus
pneumoniae
Beta Hemolytic
Yes
Resistant
No
Sensitive
α-hemolytic
streptococci
Streptococcus
pyogenes
(GAS)
Yes
Resistant
Another
β-hemolytic
streptococci
The optochin test is a presumptive test that is used to
identify strains of Streptococcus pneumoniae. Optochin
(ethyl hydrocupreine) disks are placed on inoculated
blood agar plates. Because S. pneumoniae is not
optochin resistant, a zone of inhibition will develop
around the disk where the bacteria have been lysed.
This zone is typically 14mm from the disk or greater.
Growth on
Sheep’s
Blood Agar
Alpha Hemolytic
Growth in
Presence of
Bacitracin
Growth in
Presence of
Optochin
No
Sensitive
Streptococcus
pneumoniae
Beta Hemolytic
Yes
Resistant
No
Sensitive
α-hemolytic
streptococci
Streptococcus
pyogenes
(GAS)
Yes
Resistant
Another
β-hemolytic
streptococci
Sensitivity to bacitracin
Streptococcus agalactiae
• Group B strep
• Facultative anaerobe, Gram+ve, cocci, beta hemolytic,
fastidious
• Diseases
– Neonatal sepsis, pneumonia (1-5 days of age)
– Neonatal meningitis (10 -60 days of age)
– Endometritis
– Osteomyelitis
– Septic arthritis
• Contracted vertically, as the baby passes through the birth
canal. Group B Strep colonizes the vagina of about 25% of
young women, and approximately 1% of infants born via a
vaginal birth to colonized mothers will become infected.
Mortality is high, between 50 and 70%
Enterococcus faecalis (previously
streptococcus)
• group D strep
• facultative anaerobic, gram+ bacteria, cocci
• opportunistic urinary tract infections and wound
infections.
• Infection is often associated with bacteremia
which can lead to endocarditis or colonization of
previously damaged heart valves. Little is
known about its pathogenesis.
Streptococcus pneumoniae
• Pneumococcus, Diplococcus pneumoniae
• facultative anaerobic, gram+ bacteria, cocci in pairs, lancetshaped, fastidious
• Pneumonia, meningitis, sometimes occult bacteremia
• Major virulence factor is the capsule
– Antiphagocytic
– non-encapsulated organisms are avirulent
• Protection -normal mucociliary barrier and intact phagocytic
and T-independent immune responses. Type-specific anticapsule antibody is protective.
• Common in elderly, debilitated, or immunosuppressed
individuals. The disease often sets in after a preceding viral
infection damages the respiratory ciliated epithelium;
incidence therefore peaks in the winter.
• Treatment is usually with penicillin.