Dr.

Samer Al-Hilali Infectious Diseases

Post-Graduate Lecture ( ): GRAM-POSITIVE COCCI
GRAM-POSITIVE COCCI
Streptococci
Streptococci and staphylococci are both gram-positive spheres (cocci) and are responsible for a wide
variety of clinical diseases. It is often necessary to differentiate between these two organisms to prescribe
the appropriate antibiotic.
The first way to differentiate them is to examine their appearance on a Gram stain. Streptococci line
up one after the other like a strip of button candy, while staphylococci appear as a cluster that can be
visualized as a cluster of hospital staff members posing for a group shot.

A second method to differentiate streptococci from staphylococci involves the enzyme catalase.
staphylococci possess the enzyme catalase, whereas streptococci do not.

Streptococcal Classification
Certain species of streptococci can either completely or partially hemolyze red blood cells (RBCs).
The streptococci are divided into three groups based on their specific hemolytic ability. The streptococci are
incubated overnight on a blood agar plate. Beta-hemolytic streptococci completely lyse the RBCs, leaving a
clear zone of hemolysis around the colony. Alpha-hemolytic streptococci only partially lyse the RBCs, leaving
a greenish discoloration of the culture medium surrounding the colony. This discolored area contains unlysed
RBCs and a green-colored metabolite of hemoglobin. Gamma-hemolytic streptococci are unable to
hemolyze the RBCs, and therefore we should really not use the word "hemolytic" in this situation (the term
non-hemolytic streptococci is often used to avoid confusion).
The streptococci can also be classified based on the antigenic characteristics of the C carbohydrate
(a carbohydrate found on the cell wall). These antigens are called Lancefield antigens and are given letter
names (from A, B, C, D, E, through S). Historically, the Lancefield antigens have been used as a major way of
differentiating the many streptococci. However, there are so many different types of streptococci that we
now rely less on the Lancefield antigens and more on a combination of tests such as the above-mentioned
patterns of hemolysis, antigenic composition (including Lancefield), biochemical reactions, growth
characteristics, and genetic studies. Although there are more than 30 species of streptococci, only 5 are
significant human pathogens. Three of these pathogens have Lancefield antigens: Lancefield group A, B, and
D. The other two pathogenic species of the streptococcal genus do not have Lancefield antigens and are
therefore just called by their species names: One is Streptococcus pneumoniae and the other is actually a
big group of streptococci collectively called the Viridans group streptococci.

GROUP A BETA-HEMOLYTIC STREPTOCOCCI (also called Streptococcus pyogenes)

These organisms are so-named because they possess the Lancefield group A antigen and are beta-
hemolytic on blood agar. They are also called Streptococcus pyogenes (which means pus-producing) and
cause the diseases "strep throat," scarlet fever, rheumatic fever, and poststreptococcal glomerulonephritis.
The components of the streptococcal cell wall that are antigenic include:
1. C carbohydrate: The C carbohydrate was used by Rebecca Lancefield to divide streptococci into
groups. Streptococcus pyogenes has the "Lancefield Group A" type of C carbohydrate.
2. M protein (80 types): This is a major virulence factor for the group A streptococcus. It inhibits the
activation of complement and protects the organism from phagocytosis. However, it is also the
weakest point in the organism's defense, because plasma (B) cells generate antibodies against the M
Dr. Samer Al-Hilali Infectious Diseases
Post-Graduate Lecture ( ): GRAM-POSITIVE COCCI
protein. These antibodies bind to the M protein (opsonization), aiding in the destruction of the
organism by macrophages and neutrophils.
Beta-hemolytic group A streptococci also have many enzymes that contribute to their pathogenicity:
1. Streptolysin 0: The 0 stands for oxygen labile as it is inactivated by oxygen. This enzyme destroys red
and white blood cells and is the reason for the beta-hemolytic group A streptococci's beta-hemolytic
ability. This enzyme is also antigenic. Following pharyngeal or systemic beta-hemolytic group A
streptococcal infection, anti-streptolysin 0 CASO) antibodies develop. On the wards, you may order
ASO titers on a patient's blood to confirm a recent infection.
2. Streptolysin S: The S stands for oxygen Stabile. This is also responsible for beta-hemolysis but is not
antigenic.
3. Pyrogenic exotoxin (also called erythrogenic toxin): This is found in only a few strains of
beta-hemolytic group A streptococci, but when these strains invade, they can cause scarlet fever.
Some strains produce pyrogenic exotoxins that are superantigens. The exotoxins directly super
stimulate T cells to pour out inflammatory cytokines. This causes a streptococcal toxic shock
syndrome. More on scarlet fever and toxic shock syndrome later . . .
4. Other enzymes include streptokinase (activates the proteolytic enzyme plasmin, which breaks up
fibrin blood clots), hyaluronidase, DNAases, anti-C5a peptidase, and others. Staphylococcus aureus
has many enzymes that are similar to those of streptococci.
Beta-hemolytic group A streptococci cause 4 types of disease by local invasion and/or exotoxin release.
These include:
1. Streptococcal pharyngitis
2. Streptococcal skin infections
3. Scarlet fever
4. Streptococcal toxic shock syndrome
Beta-hemolytic group A streptococci can also cause 2 delayed antibody mediated diseases:
1. Rheumatic fever
2. Glomerulonephritis

GROUP B STREPTOCOCCI (Streptococcus agalactiae)

Group B streptococci (GBS) produce short chains and diplococcal pairs of spherical or ovoid Gram-
positive cells. Colonies are larger and β-hemolysis due to a pore forming cytolysin (β-hemolysin) is less
distinct than with GAS and may even be absent. In addition to the Lancefield B antigen, GBS produce
polysaccharide capsules of nine antigenic types (Ia, Ib, II–VIII), all of which contain sialic acid in the form of
terminal side chain residues. Pili and surface proteins are also present.
S. agalactiae is found in the vaginocervical tract of female carriers, and the urethral mucous
membranes of male carriers as well as in the gastrointestinal (GI) tract. S. agalactiae can be transmitted
sexually among adults and from an infected mother to her infant at birth. Group B streptococci are a leading
cause of meningitis and septicemia in neonates, with a high mortality rate. They are also an occasional cause
of infections in postpartum women (endometritis) and individuals with impaired immune systems, in whom
the organism may cause septicemia or pneumonia.
Dr. Samer Al-Hilali Infectious Diseases
Post-Graduate Lecture ( ): GRAM-POSITIVE COCCI
Viridans Group Streptococci
The members of this huge group include the Mitis group (S. mitis, S. sanguis, S. parasanguis, S.
gordonii, S. crista, S. infantis, S. oralis, S. peroris), Salivarius group (S. saliuarius, S. uestibularis, S.
thermophilus), the Mutans group (S. mutans, S. sobrinus, S. criceti, S. rattus, S. downei, S. macacae), and
the Anginosus group (S. anginosus, S. constellatus, and S. intermedius). Note that many of these names
refer to the mouth and saliva since the viridans group streptococci are indigenous to the GI tract. These
critters represent more than 30% of the culturable bacteria from dental plaque, gingival crevices, the tongue,
and saliva! This is a big, heterogeneous group of streptococci that are not identified based on one Lancefield
group. Viridis is the Latin word for green, and most of the viridans streptococci are alpha-hemolytic,
producing greenish discoloration on blood agar. They are normal human gastrointestinal (G.I.) tract flora
that is frequently found in the nasopharynx and gingival cervices.
The viridans streptococci cause 3 main types of infection: dental infections, endocarditis, and abscesses.

1. Dental infections:
2. Endocarditis:
3. Abscesses:

GROUP D STREPTOCOCCI
(Enterococci and Non-enterococci) These bacteria, which can be alpha or gamma-hemolytic,
traditionally have been divided into two sub-groups: the enterococci (comprised of Enterococcus faecalis
and Enterococcus faecium) and the non-enterococci (comprised of many organisms including Streptococcus
bovis and Streptococcus equinus). Recently the enterococci have been shown to be sufficiently different
from the streptococci to be given their own genus enterococcus. S. bovis and S. equinus are still classified as
streptococci.

Enterococcus (faecalis and faecium)

The enterococci take up residence in the human intestines and are considered normal bowel flora.
They are variably hemolytic and unique in that they all grow well in 40% bile or 6.5% NaCl. Clinically, the
enterococci are commonly the infecting agents in urinary tract infections, biliary tract infections (as they
grow well in bile), bacteremia, and subacute bacterial endocarditis (SBE). While these bugs are not as virulent
as Streptococcus pyogenes, they are always around in the G.I. tract and prey on weak hospitalized patients.
In fact, the enterococci are currently the second to third most common cause of hospital-acquired
(nosocomial) infection! In hospitalized patients, the enterococci frequently cause urinary tract infections,
wound infections, native and prosthetic valve endocarditis (like Viridans group streptococci), and bacteremia
and sepsis after infecting intravenous catheters.

Non-Enterococci (Streptococcus bovis and equinus)

Like the enterococci, Streptococcus bovis is hardy, growing in 40% bile (but not in 6.5% NaCl). It lives
in the G.I. tract, and it causes similar diseases. An important unique property is that there is a remarkable
association between S. bovis infection and colon cancer!!! In some series, 50% of people with S. bovis
bacteremia have a colonic malignancy. We do not know if S. bovis is a cause of colon cancer or just a marker
of the disease.
Dr. Samer Al-Hilali Infectious Diseases
Post-Graduate Lecture ( ): GRAM-POSITIVE COCCI

Streptococcus pneumoniae (Alias the pneumococcus; No Lancefield antigen)

The pneumococcus is a very important organism because it is a major cause of bacterial pneumonia
and meningitis in adults and otitis media in children. Pneumococcus is to Parents what group B streptococcus
is to Babies.
The pneumococcus does not have Lancefield antigens! Under the microscope, they appear as lancet-
shaped gram-positive cocci arranged in pairs (diplococci).
The major virulence factor of the pneumococcus is its polysaccharide capsule, which protects the
organism from phagocytosis. Fortunately, the capsule is antigenic, and antibodies specific for the capsule
can neutralize the pneumococcus. The only problem is that there are 84 different capsule serotypes, so
surviving an infection with this organism only provides immunity to 1 out of the 83 possible capsule types.
There are 2 important lab tests to identify the pneumococcus:

1. Quellung reaction: When pneumococci on a slide smear are mixed with a small amount of anti-serum
(serum with antibodies to the capsular antigens) and methylene blue, the capsule will appear to
swell. This technique allows for rapid identification of this organism.
2. Optochin sensitivity. Streptococcus pneumoniae is alpha-hemolytic (partial hemolysis-greenish
color) but Streptococcus viridans is also alpha-hemolytic! To differentiate the two, a disc impregnated
with optochin (you don't want to know the real name) is placed on the agar dish. The growth of
Streptococcus pneumoniae will be inhibited, while Streptococcus viridans will continue to grow.
Streptococcus pneumoniae is the most common cause of pneumonia in adults, and also the most
common cause of otitis media (middle ear infection) in children, and the most common cause of
bacterial meningitis in adults.

Staphylococci
Staphylococci are forever underfoot, crawling all over hospitals and living in the nasopharynx and
skin of up to 50% of people. While at times they cause no symptoms, they can become mean and nasty. They
will be one of your future enemies, so know them well.
The 3 major pathogenic species are Staphylococcus aureus, Staphylococcus epidermidis, and
Staphylococcus saprophyticus.
It is extremely important to know how to differentiate staphylococci from streptococci because most
staphylococci are penicillin G resistant! You can do 3 things to differentiate them-Gram stain, catalase test,
and culture.
1. Gram stain: Staphylococci lie in grape-like clusters as seen on Gram stain. Visualize this cluster of
hospital staff posing for a group photo. Staphylococcus aureus is catalase-positive, thus explaining
the cats in the group photo. Staphylococcus aureus (aureus means "gold") can be differentiated from
the other beta-hemolytic cocci by their elaboration of a golden pigment when cultured on sheep
blood agar. Notice that our hospital Staff (Staph) all proudly wear gold medals around their necks.
2. Catalase test: All staphylococci have the enzyme catalase (streptococci do not!). Catalase testing,
showing a cluster of staphylococci (catalase-positive) blowing oxygen bubbles.
Dr. Samer Al-Hilali Infectious Diseases
Post-Graduate Lecture ( ): GRAM-POSITIVE COCCI
To test, rub a wire loop across a colony of gram-positive cocci and mix on a slide with H202.
If bubbles appear, this indicates that H202 is being broken down into oxygen bubbles and water;
catalase-positive staphylococci are present.
3. Culture: Staphylococcus aureus and certain streptococci are beta-hemolytic (completely hemolyze
red blood cells on an agar plate), but Staphylococcus aureus can be differentiated from the other
beta-hemolytic cocci by their elaboration of a golden pigment on sheep blood agar.
Now that we can differentiate staphylococci from streptococci, it is important to know which species of
staphylococcus is the actual pathogen. The key point: Of the 3 pathogenic staphylococcal species, only
Staphylococcus aureus is coagulase positive!!! It elaborates the enzyme, coagulase, which activates
prothrombin, causing blood to clot.

Staphylococcus aureus
This critter has a microcapsule surrounding its huge peptidoglycan cell wall, which in turn surrounds
a cell membrane containing penicillin-binding protein. Numerous powerful defensive and offensive protein
weapons stick out of the microcapsule or can be excreted from the cytoplasm to wreak havoc on our bodies:

Proteins That Disable Our Immune Defenses

1. Protein A: This protein has sites that bind the Fe portion of IgG. This may protect the organism from
opsonization and phagocytosis.
2. Coagulase: This enzyme can lead to fibrin formation around the bacteria, protecting it from
phagocytosis.
3. Hemolysins (4 types): Alpha, beta, gamma, and delta. They destroy red blood cells, neutrophils,
macrophages, and platelets.
4. Leukocidins: They destroy leukocytes (white blood cells). Community-acquired methicillin-resistant
Staphylococcus aureus (CA-MRSA) produces a particular leukocidin called Panton-Valentine
Leukocidin (PVL), which is associated with a propensity to form abscesses.
5. Penicillinase: This is a secreted form of beta-lactamase. It disrupts the beta-lactam portion of the
penicillin molecule, thereby inactivating the antibiotic.
6. Novel penicillin-binding protein: This protein, also called transpeptidase, is necessary for cell wall
peptidoglycan formation and is inhibited by penicillin. Some strains of Staphylococcus aureus have
new penicillin-binding proteins that are resistant to penicillinase-resistant penicillins and
cephalosporins.

Proteins to lysis Tissue (Tunnel Through)

1. Hyaluronidase ("Spreading Factor"): This protein breaks down proteoglycans in connective tissue.
2. Staphylokinase: This protein lyses formed fibrin clots (like streptokinase).
3. Lipase: This enzyme degrades fats and oils, which often accumulate on the surface of our body. This
degradation facilitates Staphylococcus aureus' colonization of sebaceous glands.
4. Protease: destroys tissue proteins.

Exotoxin (Assault Weaponry)

1. Exfoliatin: A diffusible exotoxin that causes the skin to slough off (scalded skin syndrome).
2. Enterotoxins (heat stable): Exotoxins that cause food poisoning, resulting in vomiting and diarrhea.
Dr. Samer Al-Hilali Infectious Diseases
Post-Graduate Lecture ( ): GRAM-POSITIVE COCCI
3. Toxic Shock Syndrome toxin (TSST- 1): This exotoxin is analogous to the pyrogenic toxin produced
by Lancefield group A beta-hemolytic streptococci, but is far more deadly. This exotoxin causes toxic
shock syndrome and is found in 20% of Staphylococcus aureus isolates. These pyrogenic toxins are
called superantigens and bind to the MHC class II molecules on antigen-presenting cells (such as
macrophages). The toxin-MHC II complex causes a massive T-cell response and outpouring of
cytokines, resulting in toxic shock syndrome.
Staphylococcus aureus causes a broad range of human disease and can infect almost any organ system. The
diseases can be separated into 2 groups:

Disease caused by exotoxin release:

1. Gastroenteritis (food poisoning).
2. Toxic shock syndrome.
3. Scalded skin syndrome.

Disease resulting from direct organ invasion by the bacteria:

1. Pneumonia
2. Meningitis
3. Osteomyelitis
4. Acute bacterial endocarditis
5. Septic arthritis
6. Skin infections
7. Bacteremia/sepsis
8. Urinary tract infection

Methicillin-resistant Staphylococcus aureus (MRSA)

Most staphylococci are penicillin-resistant because they secrete penicillinase. Methicillin, Nafcillin,
and other penicillinase-resistant penicillins are not broken down by penicillinase, thus enabling them to kill
most strains of Staphylococcus aureus. MRSA is a strain of Staphylococcus aureus that has acquired multi-
drug resistance, even against methicillin and nafcillin. This resistance is mediated by an acquired
chromosomal DNA segment (mecA) encoding a new penicillin-binding protein 2A that can take over the job
of peptidoglycan cell wall assembly when the normal penicillin-binding protein (transpeptidase) is inhibited.
Until recently, most strains of MRSA developed in the hospital under the influence of heavy antibiotic
pressure. These strains had been classified as health care or hospital-acquired MRSA or HA-MRSA (this
distinction is starting to go away). Generally, these hospital-acquired MRSA infections have exhibited
extensive antibiotic resistance. In these cases, vancomycin remains one of the most useful antibiotics.
Unfortunately, strains of Staphylococcus aureus resistant to vancomycin are now being reported. These
VRSA isolates have acquired a chromosomal transposon DNA element called vanA from vancomycin-
resistant enterococcus (VRE) that encodes a series of proteins that modify the D-alanine-D-alanine terminus
of the peptidoglycan cell wall, changing it to D-alanine-D-lactate, which has a low affinity for vancomycin.

Community-Acquired Methicillin-Resistant Staphylococcus aureus (CA-MRSA)

In recent years we have seen the emergence of multiple clones of MRSA that have been dubbed
"community-acquired" MRSA due to its emergence outside the hospital. There have been numerous highly
publicized outbreaks of CA-MRSA infections among sports teams. Humans are frequently colonized in their
nasopharynx and skin folds with Staphylococcus aureus, and this leads to a propensity to develop skin and
Dr. Samer Al-Hilali Infectious Diseases
Post-Graduate Lecture ( ): GRAM-POSITIVE COCCI
soft tissue infections with the same organism. Exposure to CA-MRSA in close contact settings may lead to
the contacts becoming carriers or developing skin and soft tissue infections. CA-MRSA carries a gene that
encodes for the Panton-Valentine Leukocidin (PVL) toxin which is associated with a propensity to form skin
abscesses. Both HA-MRSA and CA-MRSA pass their resistance genes via mobile genetic elements that can
be passed between bacteria. The genes encoding methicillin resistance are carried on a genomic strand
called SCCmec (staphylococcal cassette chromosome conferring resistance to methicillin). It just so happens
that the old HA-MRSA strains have an SCCmec element that is large and bulky because it also carries multiple
resistance elements to antibiotics in addition to methicillin. As a result, transfer of this mobile element is a
bulky time-consuming process. In contrast, the new kid on the block CA-MR.SA has a much smaller SCCmec
transferable element that is easily transferred among staph bacteria but carries few resistant mutations with
it. As a result CA-MRSA is much more efficient at spreading its seed (or SCCmec in this case) and it is now the
predominant methicillin-resistant staphylococcus bacterium acquired both in and outside the hospital.
Fortunately, CA-MRSA still tends to be susceptible to several oral antibiotics such as clindamycin and
trimethoprim-sulfamethoxazole.

Staphylococcus epidermidis
This organism is part of our normal bacterial flora and is widely found in the body. Unlike
Staphylococcus aureus, it is coagulase-negative.
This organism normally lives peacefully on our skin without causing disease. However, compromised
hospital patients with Foley urine catheters or intravenous lines can become infected when this organism
migrates from the skin along the tubing.
Staphylococcus epidermidis is a frequent skin contaminant of blood cultures. Contamination occurs
when the needle used to draw the blood passes through skin covered with Staphylococcus epidermidis.
Drawing blood from 2 sites will help determine if the growth of Staphylococcus epidermidis represents a real
bacteremic infection or is merely a contamination. If only one of the samples grows Staphylococcus
epidermidis, you can suspect that this is merely a skin contaminant. However, if 2 cultures are positive, the
likelihood of bacteremia with Staphylococcus epidermidis is high.
Staphylococcus epidermidis also causes infections of prosthetic devices in the body, such as
prosthetic joints, prosthetic heart valves, and peritoneal dialysis catheters. In fact, Staphylococcus
epidermidis is the most frequent organism isolated from infected indwelling prosthetic devices. The
organisms have a polysaccharide capsule that allows adherence to these prosthetic materials.
Staphylococcus epidermidis often forms biofilms on intravascular catheters and leaches out to cause
bacteremia and catheter-related sepsis. A biofilm is an extracellular polysaccharide network, similar to the
capsule polysaccharides, that forms a mechanical scaffold around bacteria. The biofilm allows bacteria to
bind to prosthetic devices, like intravenous catheters and protects them from attack by antibiotics and the
immune system. Imagine bacteria secreting their polysaccharide concrete around themselves to form a
biological bunker.

Staphylococcus saprophyticus
This organism is a leading cause (second only to E. coli) of urinary tract infections in sexually active
young women. It is most commonly acquired by females (95%) in the community (NOT in the hospital). This
organism is coagulase-negative.