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The Staphylococci
The staphylococci are gram-positive spherical cells, usually arranged in
grapelike irregular clusters.
They grow readily on many types of media and are active metabolically,
fermenting carbohydrates and producing pigments that vary from white to
deep yellow.
Some are members of the normal microbiota of the skin and mucous
membranes of humans; others cause suppuration, abscess formation, a
variety of pyogenic infections, and even fatal septicemia.
The genus Staphylococcus has at least 45 species. The four most frequently
encountered species of clinical importance are Staphylococcus aureus, S.
epidermidis, S. lugdunensis, and S. saprophyticus.
S aureus is coagulase positive, which differentiates it from the other species.
S aureus is a major pathogen for humans. The coagulase-negative
staphylococci (CoNS) are normal human microbiota and sometimes cause
infection, often associated with implanted devices, such as shunts and
intravascular
catheters,
especially
in
very
young,
old,
and
immunocompromised patients.
Approximately 75% of these infections caused by coagulase-negative
staphylococci are caused by S epidermidis; S saprophyticus is a relatively
common cause of urinary tract infections in young women, although it rarely
causes infections in hospitalized patients.
Morphology and Identification
A. Typical Organisms
Staphylococci are spherical cells about 1μm in diameter arranged in irregular
clusters. Single cocci, pairs, tetrads, and chains are also seen in liquid cultures.
Microbiology
Medical bacteriology
Dr. Zainab D. Degaim
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Young cocci stain strongly gram positive; on aging, many cells become gram
negative. Staphylococci are nonmotile and do not form spores.
B. Culture
Staphylococci grow readily on most bacteriologic media under aerobic or
microaerophilic conditions. They grow most rapidly at 37°C but form
pigment best at room temperature (20–25°C). Colonies on solid media are
round, smooth, raised, and glistening. S aureus usually forms gray to deep
golden yellow colonies.
S epidermidis colonies usually are gray to white on primary isolation; many
colonies develop pigment only upon prolonged incubation. Various degrees
of hemolysis are produced by S aureus and occasionally by other species.
C. Growth Characteristics
1- The staphylococci produce catalase, which differentiates them from the
streptococci.
2- Staphylococci slowly ferment many carbohydrates, producing lactic acid but not
gas.
3- Staphylococci are relatively resistant to drying, heat (withstand 50°C for 30
minutes), and 10% NaCl, but are inhibited by certain chemicals (3%
hexachlorophene).
4- Staphylococci are variably susceptible to many antimicrobial drugs. Resistance
is caused by several mechanisms:
1. β-Lactamase production is common, is under plasmid control, and makes the
organisms resistant to many penicillins (penicillin G, ampicillin, ticarcillin,
piperacillin).
2. Resistance to nafcillin (and to methicillin and oxacillin) is independent of β-
lactamase production. Resistance to nafcillin is encoded and regulated by a
sequence of genes found in a region of the chromosome called the staphylococcal
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cassette chromosome mec (SCCmec). The mecA and newly described mecC genes
on this locus encode a low-affinity penicillin-binding protein (PBP2a) that is
responsible for the resistance.
3. In the United States, S aureus and S lugdunensis are considered to be susceptible
to vancomycin if (MIC) is 2 μg/mL or less; of intermediate susceptibility if the
MIC is 4–8 μg/mL; and resistant if the MIC is 16 μg/mL or greater. Strains of S
aureus with intermediate susceptibility to vancomycin have been isolated in Japan,
the United States and several other countries. These are often known as
vancomycin-intermediate S aureus (VISA). They generally have been isolated from
patients with complex infections who have received prolonged vancomycin
therapy. The mechanism of resistance is associated with increased cell wall
synthesis and alterations in the cell wall and is not caused by the van genes found in
enterococci. S aureus strains of intermediate susceptibility to vancomycin usually
are nafcillin resistant but generally are susceptible to oxazolidinones.
4. Since 2002, several isolates of vancomycin-resistant S aureus (VRSA) strains
(MICs ≥ 16 μg/mL) were isolated from patients in the United States. The isolates
contained the vancomycin resistance gene vanA likely derived from enterococci
and the nafcillin resistance gene mecA. Both of the initial VRSA strains were
susceptible to other antibiotics. Vancomycin resistance in S aureus is of major
concern worldwide.
5. Plasmid-mediated resistance to tetracyclines, erythromycins, aminoglycosides,
and other drugs is frequent in staphylococci.
6. ―Tolerance‖ implies that staphylococci are inhibited by a drug but not killed by
it. Patients with endocarditis caused by a tolerant S aureus may have a
prolonged
clinical course compared with patients who have endocarditis caused by a fully
susceptible S aureus. Tolerance can at times be attributed to lack of activation of
autolytic enzymes in the cell wall.
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Antigenic Structure
S aureus has amazing adaptive capacity. S aureus has acquired many mobile
genetic elements (insertion sequences, transposons) that determine both
pathogenicity and antimicrobial resistance. Staphylococci contain antigenic
polysaccharides and proteins as well as other substances important in cell wall
structure.
Cell wall virulence factors:
Protein A is a major component of the S. aureus cell wall. It binds to the Fc
moiety of IgG, exerting an antiopsonin (and therefore strongly antiphagocytic)
effect.
Fibronectin-binding protein (FnBP) and other staphylococcal surface proteins
promote binding to mucosal cells and tissue matrices.
*Peptidoglycan,
* Teichoic acids,
*Most S aureus strains of clinical importance have polysaccharide capsules.
Enzymes and Toxins
Staphylococci can produce disease both through their ability to multiply and spread
widely in tissues and through their production of many extracellular substances.
A. Catalase
Staphylococci produce catalase, which converts H
2
O
2
into water and oxygen. The
catalase test differentiates the staphylococci, which are positive, from the
streptococci, which are negative.
B. Coagulase and Clumping Factor
S aureus produces an extracellular coagulase, an enzyme like protein that clots
oxalated or citrated plasma. Coagulase binds to prothrombin; together they become
enzymatically active and initiate fibrin polymerization. Coagulase may deposit
fibrin on the surface of staphylococci, perhaps altering their ingestion by
phagocytic cells or their destruction within such cells.
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Clumping factor is cell wall bound and that is responsible for adherence of the
organisms to fibrinogen and fibrin. When mixed with plasma, S aureus forms
clumps. Clumping factor is distinct from coagulase.
C. Other Enzymes
Other enzymes produced by staphylococci include a hyaluronidase, or spreading
factor—a staphylokinase resulting in fibrinolysis but acting much more slowly than
streptokinase, proteinases, lipases, and β-lactamase.
D. Hemolysins
S aureus possesses four hemolysins. α-Hemolysin is a heterogeneous protein that
acts on a broad spectrum of eukaryotic cell membranes. The β-toxin degrades
sphingomyelin and therefore is toxic for many kinds of cells, including human
RBC. The δ-toxin is heterogeneous, it disrupts biologic membranes and may have a
role in S aureus diarrheal diseases. The γ-hemolysin is a leukocidin that lyses white
blood cells.
E. Panton–Valentine Leukocidin
PVL is encoded on a mobile phage. It can kill white blood cells of humans and
rabbits.
F. Exfoliative Toxins
These epidermolytic toxins of S aureus are two distinct proteins of the same
molecular weight. Exfoliative toxin A is encoded by eta located on a phage and is
heat stable. Exfoliative toxin B is plasmid mediated and heat labile. These
epidermolytic toxins yield the generalized desquamation of the staphylococcal
scalded skin syndrome (SSSS) by dissolving the mucopolysaccharide matrix of the
epidermis. The toxins are superantigens.
G. Toxic Shock Syndrome Toxin
Most S aureus strains isolated from patients with toxic shock syndrome produce a
toxin called toxic shock syndrome toxin-1 (TSST-1). TSST-1 is the prototypical
superantigen. The gene for TSST-1 is found in about 20% of S aureus isolates,
including MRSA.
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*Superantigen exotoxins: These toxins have an affinity for the T cell receptor MHC
Class II antigen complex. They stimulate enhanced T lymphocyte response (as
many as twenty percent of T cells respond, compared with 0.01 percent responding
to the usual processed antigens). This difference is a result of their ability to
recognize a relatively conserved region of the T cell receptor. This major T cell
activation can cause toxic shock syndrome, primarily by release into the circulation
of inordinately large amounts of T cell cytokines, such as interleukin-2 (IL-2),
interferon, and tumor necrosis factor.
H. Enterotoxins
There are 15 enterotoxins (A–E, G–P) that, similar to TSST-1, are superantigens.
Approximately 50% of S aureus strains can produce one or more of them. The
enterotoxins are heat stable and resistant to the action of gut enzymes. Important
causes of food poisoning, enterotoxins are produced when S aureus grows in
carbohydrate and protein foods.
Pathogenesis
Staphylococci, particularly S epidermidis, are members of the normal
microbiota of the human skin and respiratory and gastrointestinal tracts.
Nasal carriage of S aureus occurs in 20–50% of humans. Staphylococci are
also found regularly on clothing, bed linens, and other fomites in human
environments.
The pathogenic capacity of a given strain of S aureus is the combined effect
of extracellular factors and toxins together with the invasive properties of the
strain.
At one end of the disease spectrum is staphylococcal food poisoning,
attributable solely to the ingestion of preformed enterotoxin
.
Pathogenic, invasive S aureus produces coagulase and tends to produce a
yellow pigment and to be hemolytic.
Nonpathogenic, noninvasive staphylococci such as S epidermidis are
coagulase negative and tend to be non-hemolytic.
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S saprophyticus is typically non-pigmented, novobiocin resistant, and non-
hemolytic; it causes urinary tract infections in young women.
Pathology
The prototype of a staphylococcal lesion is the furuncle or other localized
abscess. Groups of S aureus established in a hair follicle lead to tissue
necrosis (dermonecrotic factor). Coagulase is produced and coagulates fibrin
around the lesion and within the lymphatics, resulting in formation of a wall
that limits the process and is reinforced by the accumulation of inflammatory
cells and, later, fibrous tissue.
Focal suppuration (abscess) is typical of staphylococcal infection. From any
one focus, organisms may spread via the lymphatics and bloodstream to
other parts of the body. Suppuration within veins, associated with
thrombosis, is a common feature of such dissemination.
In osteomyelitis, the primary focus of S aureus growth is typically in a
terminal blood vessel of the metaphysis of a long bone, leading to necrosis of
bone and chronic suppuration.
S aureus may cause pneumonia, meningitis, empyema, endocarditis, or
sepsis with suppuration in any organ. Staphylococci of low invasiveness are
involved in many skin infections (eg, acne, pyoderma, or impetigo).
Staphylococci also cause disease through the elaboration of toxins without
apparent invasive infection. Bullous exfoliation, the scalded skin syndrome,
is caused by the production of exfoliative toxins. Toxic shock syndrome is
associated with TSST-1.
Clinical Findings
1. Localized skin infections: The most common S. aureus infections are small,
superficial abscesses involving hair follicles (folliculitis) or sweat or sebaceous
glands. Subcutaneous abscesses called furuncles (boils) often form around foreign
bodies, such as splinters. These generally respond to local therapy, that is, removal
of the foreign body, soaking, and drainage as indicated. Carbuncles are larger,
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deeper, multiloculated skin infections that can lead to bacteremia and require
antibiotic therapy and debridement. Impetigo is usually a localized, superficial,
spreading crusty skin lesion generally seen in children. It can be caused by S.
aureus, although more commonly by Streptococcus pyogenes (see p. 80), or both
organisms together.
*S aureus infection can also result from direct contamination of a wound, such as a
postoperative staphylococcal wound infection or infection after trauma (chronic
osteomyelitis subsequent to an open fracture, meningitis after skull fracture).
2.Deep, localized infections: These may be metastatic from superficial infections or
skin carriage, or may result from trauma. S. aureus is the most common cause of
acute and chronic infection of bone marrow. S. aureus is also the most common
cause of acute infection of joint space in children (septic joint).
3. Acute endocarditis, generally associated with intravenous drug abuse, is caused
by injection of contaminated preparations or by needles contaminated with S.
aureus. S. aureus also colonizes the skin around the injection site, and if the skin is
not sterilized before injection, the bacteria can be introduced into soft tissues and
the bloodstream, even when a sterilized needle is used.
4- Septicemia is a generalized infection with sepsis or bacteremia.
5- Pneumonia: S. aureus is a cause of severe, necrotizing pneumonia
6-Nosocomial infections: S. aureus is one of the most common causes of hospital-
acquired infections, often of wounds (surgical) or bacteremia associated with
catheters. Progression to septicemia is often a terminal event.
7-Toxinoses are diseases caused by the action of a toxin, frequently when the
organism that secreted the toxin is undetectable. Toxinoses caused by S. aureus
include:
*Toxic shock syndrome, which results in high fever, rash (resembling a sunburn,
with diffuse erythema followed by desquamation), vomiting, diarrhea, hypotension,
and multi-organ involvement (especially renal, and/or hepatic damage).
*Staphylococcal gastroenteritis is caused by ingestion of food contaminated with
enterotoxin-producing S. aureus. Often contaminated by a food-handler, these
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foods tend to be protein-rich (for example, egg salad, cream pastry) and improperly
refrigerated. Symptoms, such as nausea, vomiting, and diarrhea, are acute
following a short incubation period (1–8 hours). There is no fever.
*Scalded skin syndrome involves the appearance of 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.
Diagnostic Laboratory Tests
A. Specimens
Surface swab pus or aspirate from an abscess, blood, endonasotracheal aspirate,
sputum, or spinal fluid for culture, depending on the localization of the process.
The anterior nares are frequently swabbed to determine nasal colonization.
B. Smears
Typical staphylococci appear as gram + cocci in clusters in Gram-stained smears of
pus or sputum. It is not possible to distinguish non-pathogenic (S epidermidis) from
the pathogenic S aureus organisms on smears.
C. Culture
Specimens planted on blood agar plates give rise to typical colonies in 18 hours at
37°C, but hemolysis and pigment production may not occur until several days later
and are optimal at room temperature. S aureus but no other staphylococci ferment
mannitol. Specimens contaminated with a mixed microbiota can be cultured on
media containing 7.5% NaCl; the salt inhibits most other normal microbiota, but
not S aureus.
D. Catalase Test
This test is used to detect the presence of cytochrome oxidase enzymes. A drop of
3% H
2
O
2
solution is placed on a slide, and a small amount of the bacterial growth is
placed in the solution. The formation of bubbles (the release of oxygen) indicates a
positive test result.
E. Coagulase Test
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Citrated rabbit (or human) plasma diluted 1:5 is mixed with an equal volume of
broth culture or growth from colonies on agar and incubated at 37°C. A tube of
plasma mixed with sterile broth is included as a control. If clots form in 1–4 hours,
the test result is positive.
G. Serologic and Typing Tests
Serologic tests for diagnosis of S aureus infections have little practical value.
Molecular typing techniques have been used to document the spread of epidemic
disease-producing clones of S aureus.
Treatment
The multiple skin infections (acne, furunculosis) occur most often in
adolescents. Tetracyclines are used for long-term treatment.
Abscesses and other closed suppurating lesions are treated by drainage,
which is essential, and antimicrobial therapy.
Bacteremia, endocarditis, pneumonia, and other severe infections caused by
S aureus require prolonged intravenous therapy with a β-lactamase-resistant
penicillin. Vancomycin is often reserved for use with nafcillin-resistant
staphylococci.
Alternative agents for the treatment of MRSA bacteremia and endocarditis
include newer antimicrobials such as daptomycin, linezolid, and
quinupristin–dalfopristin.
S epidermidis is more often resistant to antimicrobial drugs than is S aureus;
approximately 75% of S epidermidis strains are nafcillin resistant.
dalbavancin, a longacting intravenous lipoglycopeptide; tedizolid phosphate,
an intravenous and oral oxazolidinone, similar to linezolid; and oritavancin, a
semisynthetic glycopeptide.