Unit 2: Bacteriology
78
Lecture 1 - Staphylococcus
Staphylococci are Gram-positive cocci occurring in
clusters. They can be cultured on normal nutrient
mediums both aerobically and anaerobically. The most
important species from the viewpoint of human medicine
is S. aureus. A number of extracellular enzymes and
exotoxins such as coagulase, alphatoxin, leukocidin,
exfoliatins, enterotoxins, and toxic shock toxin are
responsible for the clinical symptoms of infections by this
pathogen, which are observed in the three types invasive
infections, pure toxicoses, and mixed forms. The
antibiotics of choice for therapy of these infections are
penicillinase-resistant penicillins. Laboratory diagnosis
involves identification of the pathogen by means of
microscopy and culturing. S. aureus is a frequent
pathogen in nosocomial infections and limited outbreaks
in hospitals. Hand washing by medical staff is the most
important prophylactic measure in hospitals.
Coagulase-negative staphylococci are classic
opportunists. S. epidermidis and other species are frequent
agents in foreign body infections due to their ability to
form biofilms on the surfaces of inert objects. S.
saprophyticus is responsible for between 10 and 20% of
acute urinary tract infections in young women.
Staphylococci are small spherical cells (1 µm) found in
grapelike clusters.Staphylococci are nonmotile, catalase-
producing bacteria. The genus Staphylococcus includes
over 30 species and subspecies. S. aureus (and E. coli) are
among the most frequent causal organisms in human
bacterial infections.
Staphylococcus aureus
Morphology and culturing
. This is a facultative
anaerobe that is readily cultured on normal nutrient
mediums at 37 ˚C. Hemolytic zones are frequently
observed around the colonies.
Fine structure
. The cellwall consists of a thick layer of
murein. Linear teichoic acids and polysaccharides are
covalently coupled to the murein polysaccharide. The
lipoteichoic acids permeating the entire murein layer are
anchored in the cell membrane. Teichoic and lipoteichoic
acids can trigger activation of complement by the
alternative pathway and stimulate macrophages to secrete
cytokines. Cell wall-associated proteins are bound to the
peptide components of the murein. Clumping factor,
fibronectin-binding protein, and collagen-binding protein
bind specifically to fibrinogen, fibronectin, and collagen,
respectively, and are instrumental in adhesion to tissues
and foreign bodies covered with the appropriate matrix
protein. Protein A binds to the Fc portion of
immunoglobulins (IgG). It is assumed that “false” binding
of immunoglobulins by protein A prevents “correct”
binding of opsonizing antibodies, thus hindering
phagocytosis.
Extracellular toxins and enzymes
.
S. aureus secretes numerous enzymes and toxins that
determine, together with the fine structures described
above, the pathogenesis of the attendant infections. The
most important are:
1) Plasma coagulase is an enzyme that functions like
thrombin to convert fibrinogen into fibrin. Tissue
microcolonies surrounded by fibrin walls are difficult to
phagocytose.
2) a-toxin can have lethal CNS effects, damages membranes
(resulting in, among other things, hemolysis), and is
responsible for a form of dermonecrosis.
3) Leukocidin damages microphages and macrophages by
degranulation.
4) Exfoliatins are responsible for a form of epidermolysis.
5) Food poisoning symptoms can be caused by eight
serologically differentiated enterotoxins (A-E, H, G, and
I). These proteins (MW: 35 kDa) are not inactivated by
heating to 100 ˚C for 15–30 minutes. Staphylococcus
enterotoxins are superantigens.
6) Toxic shock syndrome toxin-1 (TSST-1) is produced by
about 1% of Staphylococcus strains. TSST-1 is a
superantigen that induces clonal expansion of many T
lymphocyte types (about 10%), leading to massive
production of cytokines, which then give rise to the
clinical symptoms of toxic shock.
Pathogenesis and clinical pictures
.
The pathogenesis and symptoms of S. aureus infections
take one of three distinct courses:
1) Invasive infections. In this type of infection, the
pathogens tend to remain in situ after penetrating through
the derma or mucosa and to cause local infections
characterized by purulence. Examples include furuncles
,carbuncles,wound infections, sinusitis, otitis media, and
mastitis puerperalis. Other kinds of invasive infection
include postoperative or posttraumatic
ostitis/osteomyelitis, endocarditis following heart surgery
(especially valve replacement), postinfluenza pneumonia,
and sepsis in immunocompromised patients. S. aureus and
E. coli are responsible for approximately equal shares of
nearly half of all cases of inpatient sepsis.
Inert foreign bodies can be colonized by S. aureus.
Colonization begins with specific binding of the
Unit 2: Bacteriology
79
staphylococci, by means of cell wall-associated adhesion
proteins, to fibrinogen or fibronectin covering the foreign
body, resulting in a biofilm that may function as a focus
of infection.
2) Toxicoses. Food poisoning results from ingestion of food
contaminated with enterotoxins. The onset a few hours
after ingestion takes the form of nausea, vomiting, and
massive diarrhea.
3) Mixed forms. Dermatitis exfoliativa (staphylococcal
scalded skin syndrome, Ritter disease), pemphigus
neonatorum, and bullous impetigo are caused by
exfoliatin-producing strains that infect the skin surface.
Toxic shock syndrome (TSS) is caused by strains that
produce TSST-1. These strains can cause invasive
infections, but may also only colonize mucosa. The main
symptoms are hypotension, fever & a scarlatiniform rash.
Diagnosis.
This requires microscopic and culture-based pathogen
identification. Differentiating S. aureus from the
coagulase-negative species is achieved by detection of the
plasma coagulase and/or the clumping factor. The
enterotoxins and TSST-1 can be detected by means of
immunological and molecular biological methods (special
laboratories).
Plasma Coagulase and Clumping Factor Test
To detect plasma coagulase, suspend several colonies in
0.5 ml of rabbit plasma, incubate the inoculated plasma
for one, four, and 24 hours and record the levels of
coagulation.
For the clumping factor test, suspend colony material in a
drop of rabbit plasma on a slide. Macroscopically visible
clumping confirms the presence of the factor.
Therapy
.
Aside from surgical measures, therapy is based on
dministration of antibiotics. The agents of choice for severe
infections are penicillinase resistant penicillins, since 70–
80% of all strains produce penicillinase. These penicillins
are, however, ineffective against methicillin-resistant
strains, & this resistance applies to all betalactams.
Epidemiology and prevention.
S. aureus is a frequent colonizer of skin and mucosa. High
carrier rates (up to 80%) are the rules among hospital
patients and staff. The principle localization of
colonization in these persons is the anterior nasal mucosa
area, from where the bacteria can spread to hands or with
dust into the air and be transmitted to susceptible
persons.S. aureus is frequently the causal pathogen in
nosocomial infections. Certain strains are known to cause
hospital epidemics. Identification of the epidemic strain
requires differentiation of relevant infection isolates from
other ubiquitous strains. Lysotyping can be used for this
purpose, although use of molecular methods to identify
genomic DNA “fingerprints”is now becoming more
common.
The most important preventive measure in hospitals is
washing the hands thoroughly before medical and nursing
procedures. Intranasal application of antibiotics
(mupirocin) is a method of reducing bacterial counts in
carriers.
Coagulase-Negative Staphylococci (CNS)
CNS is an element in the normal flora of human skin and
mucosa. They are classic opportunists that only cause
infections given a certain host disposition.
S. epidermidis. This is the pathogen most frequently
encountered in CNS infections (70–80% of cases). CNS
cause mainly foreign body infections. Examples of the
foreign bodies involved are intravasal catheters,
continuous ambulant peritoneal dialysis (CAPD)
catheters, endopro- stheses, metal plates and screws in
osteosynthesis, cardiac pacemakers, artificial heart valves,
and shunt valves. These infections frequently develop
when foreign bodies in the macroorganism are covered by
matrix proteins (e.g., fibrinogen, fibronectin) to which the
staphylococci can bind using specific cell wall proteins.
They then proliferate on the surface and produce a
polymeric substance—the basis of the developing biofilm.
The staphylococci within the biofilm are protected from
antibiotics and the immune system to a great extent. Such
biofilms can become infection foci from which the CNS
enter the bloodstream and cause sepsis like illnesses.
Removal of the foreign body is often necessary.
S. saprophyticus is responsible for 10–20% of acute
urinary tract infections, in particular dysuria in young
women, and for a small proportion of cases of nonspecific
urethritis in sexually active men.
Antibiotic treatment of CNS infections is often
problematic due to the multiple resistances often
encountered in these staphylococci, especially S.
hemolyticus.