The Staphylococci
Staphylococci are Gram-positive spherical bacteria that occur in microscopic clusters resembling grapes. Bacteriological culture of the nose and skin of normal humans invariably yields staphylococci. In 1884, Rosenbach described the two pigmented colony types of staphylococci and proposed the appropriate nomenclature:
Staphylococcus aureus (yellow) and
Staphylococcus albus (white). The latter species is now named
Staphylococcus epidermidis. Although more than 20 species of
Staphylococcus are described in Bergey's Manual (2001), only
Staphylococcus aureus and
Staphylococcus epidermidis are significant in their interactions with humans.
S. aureus colonizes mainly the nasal passages, but it may be found regularly in most other anatomical locales.
S epidermidis is an inhabitant of the skin.
Taxonomically, the genus Staphylococcus is in the Bacterial family Staphylococcaceae, which includes three lesser known genera, Gamella, Macrococcus and Salinicoccus. The best-known of its nearby phylogenetic relatives are the members of the genus Bacillus in the family Bacillaceae, which is on the same level as the family Staphylococcaceae. The Listeriaceae are also a nearby family.
Staphylococcus aureus forms a fairly large yellow colony on rich medium, S. epidermidis has a relatively small white colony. S. aureus is often hemolytic on blood agar; S. epidermidis is non hemolytic. Staphylococci are facultative anaerobes that grow by aerobic respiration or by fermentation that yields principally lactic acid. The bacteria are catalase-positive and oxidase-negative. S. aureus can grow at a temperature range of 15 to 45 degrees and at NaCl concentrations as high as 15 percent. Nearly all strains of S. aureus produce the enzyme coagulase: nearly all strains of S. epidermidis lack this enzyme. S. aureus should always be considered a potential pathogen; most strains of S. epidermidis are nonpathogenic and may even play a protective role in their host as normal flora. Staphylococcus epidermidis may be a pathogen in the hospital environment.
Staphylococci are perfectly spherical cells about 1 micrometer in diameter. They grow in clusters because staphylococci divide in two planes. The configuration of the cocci helps to distinguish staphylococci from streptococci, which are slightly oblong cells that usually grow in chains (because they divide in one plane only). The catalase test is important in distinguishing streptococci (catalase-negative) from staphylococci, which are vigorous catalase-producers. The test is performed by adding 3% hydrogen peroxide to a colony on an agar plate or slant. Catalase-positive cultures produce O2 and bubble at once. The test should not be done on blood agar because blood itself contains catalase.
FIGURE 1. Gram stain of Staphylococcus aureus in pustular exudate
Table 1. Important phenotypic characteristics of Staphylococcus aureus
Gram-positive, cluster-forming coccus nonmotile, nonsporeforming facultative anaerobe fermentation of glucose produces mainly lactic acid ferments mannitol (distinguishes from S. epidermidis) catalase positive coagulase positive golden yellow colony on agar normal flora of humans found on nasal passages, skin and mucous membranes pathogen of humans, causes a wide range of suppurative infections, as well as food poisoning and toxic shock syndrome
Pathogenesis of S. aureus infections
Staphylococcus aureus causes a variety of suppurative (pus-forming) infections and toxinoses in humans. It causes superficial skin lesions such as
boils,
styes and
furunculosis; more serious infections such as
pneumonia,
mastitis,
phlebitis,
meningitis, and
urinary tract infections; and deep-seated infections, such as
osteomyelitis and
endocarditis.
S. aureus is a major cause of
hospital acquired (nosocomial) infection of surgical wounds and infections associated with indwelling medical devices.
S. aureus causes
food poisoning by releasing enterotoxins into food, and
toxic shock syndrome by release of superantigens into the blood stream.
S. aureus expresses many potential virulence factors: (1) surface proteins that promote colonization of host tissues; (2) invasins that promote bacterial spread in tissues (leukocidin, kinases, hyaluronidase); (3) surface factors that inhibit phagocytic engulfment (capsule, Protein A); (4) biochemical properties that enhance their survival in phagocytes (carotenoids, catalase production); (5) immunological disguises (Protein A, coagulase, clotting factor); and (6) membrane-damaging toxins that lyse eukaryotic cell membranes (hemolysins, leukotoxin, leukocidin; (7) exotoxins that damage host tissues or otherwise provoke symptoms of disease (SEA-G, TSST, ET (8) inherent and acquired resistance to antimicrobial agents.
FIGURE 2. Virulence determinants of Staphylococcus aureus
For the majority of diseases caused by S. aureus, pathogenesis is multifactorial, so it is difficult to determine precisely the role of any given factor. However, there are correlations between strains isolated from particular diseases and expression of particular virulence determinants, which suggests their role in a particular diseases. The application of molecular biology has led to advances in unraveling the pathogenesis of staphylococcal diseases. Genes encoding potential virulence factors have been cloned and sequenced, and many protein toxins have been purified. With some staphylococcal toxins, symptoms of a human disease can be reproduced in animals with the purified protein toxins, lending an understanding of their mechanism of action.
Human staphylococcal infections are frequent, but usually remain localized at the portal of entry by the normal host defenses. The portal may be a hair follicle, but usually it is a break in the skin which may be a minute needle-stick or a surgical wound. Foreign bodies, including sutures, are readily colonized by staphylococci, which may makes infections difficult to control. Another portal of entry is the respiratory tract. Staphylococcal pneumonia is a frequent complication of influenza. The localized host response to staphylococcal infection is inflammation, characterized by an elevated temperature at the site, swelling, the accumulation of pus, and necrosis of tissue. Around the inflamed area, a fibrin clot may form, walling off the bacteria and leukocytes as a characteristic pus-filled boil or abscess. More serious infections of the skin may occur, such as furuncles or impetigo. Localized infection of the bone is called osteomyelitis. Serious consequences of staphylococcal infections occur when the bacteria invade the blood stream. A resulting septicemia may be rapidly fatal; a bacteremia may result in seeding other internal abscesses, other skin lesions, or infections in the lung, kidney, heart, skeletal muscle or meninges.
FIGURE 3. Sites of infection and diseases caused by Staphylococcus aureus
Adherence to Host Cell Proteins
S. aureus cells express on their
surface proteins that promote attachment to host proteins such as laminin and fibronectin that form the extracellular matrix of epithelial and endothelial surfaces. In addition, most strains express a fibrin/fibrinogen binding protein (clumping factor) which promotes attachment to blood clots and traumatized tissue. Most strains of
S. aureus express both fibronectin and fibrinogen-binding proteins. In addition, an adhesin that promotes attachment to collagen has been found in strains that cause osteomyelitis and septic arthritis. Interaction with collagen may also be important in promoting bacterial attachment to damaged tissue where the underlying layers have been exposed.
Evidence that staphylococcal matrix-binding proteins are virulence factors has come from studying defective mutants in adherence assays. Mutants defective in binding to fibronectin and to fibrinogen have reduced virulence in a rat model for endocarditis, and mutants lacking the collagen-binding protein have reduced virulence in a mouse model for septic arthritis, suggesting that bacterial colonization is ineffective. Furthermore, the isolated ligand-binding domain of the fibrinogen, fibronectin and collagen receptors strongly blocks attachment of bacterial cells to the corresponding host proteins.
Invasion
The invasion of host tissues by staphylococci apparently involves the production of a huge array of extracellular proteins, some of which may occur also as cell-associated proteins. These proteins are described below with some possible explanations for their role in invasive process.
Membrane-damaging toxins
a-toxin (a-hemolysin) The best characterized and most potent membrane-damaging toxin of S. aureus is a-toxin. It is expressed as a monomer that binds to the membrane of susceptible cells. Subunits then oligomerize to form heptameric rings with a central pore through which cellular contents leak.
In humans, platelets and monocytes are particularly sensitive to a-toxin. Susceptible cells have a specific receptor for a-toxin which allows the toxin to bind causing small pores through which monovalent cations can pass. The mode of action of alpha hemolysin is likely by osmotic lysis.
ß-toxin is a sphingomyelinase which damages membranes rich in this lipid. The classical test for ß-toxin is lysis of sheep erythrocytes. The majority of human isolates of S. aureus do not express ß-toxin. A lysogenic bacteriophage is known to encode the toxin.
d-toxin is a very small peptide toxin produced by most strains of S. aureus. It is also produced by S. epidermidis. The role of d-toxin in disease is unknown.
Leukocidin is a multicomponent protein toxin produced as separate components which act together to damage membranes. Leukocidin forms a hetero-oliogmeric transmembrane pore composed of four LukF and four LukS subunits, thereby forming an octameric pore in the affected mwembrane. Leukocidin is hemolytic, but less so than alpha hemolysin.
Only 2% of all of S. aureus isolates express leukocidin, but nearly 90% of the strains isolated from severe dermonecrotic lesions express this toxin, which suggests that it is an important factor in necrotizing skin infections.
Coagulase and clumping factor
Coagulase is an extracellular protein which binds to prothrombin in the host to form a complex called staphylothrombin. The protease activity characteristic of thrombin is activated in the complex, resulting in the conversion of fibrinogen to fibrin. Coagulase is a traditional marker for identifying S aureus in the clinical microbiology laboratory. However, there is no overwhelming evidence that it is a virulence factor, although it is reasonable to speculate that the bacteria could protect themselves from phagocytic and immune defenses by causing localized clotting.
There is some confusion in the literature concerning coagulase and clumping factor, the fibrinogen-binding determinant on the S. aureus cell surface. Partly the confusion results from the fact that a small amount of coagulase is tightly bound on the bacterial cell surface where it can react with prothrombin leading to fibrin clotting. However, genetic studies have shown unequivocally that coagulase and clumping factor are distinct entities. Specific mutants lacking coagulase retain clumping factor activity, while clumping factor mutants express coagulase normally.
Staphylokinase
Many strains of
S aureus express a plasminogen activator called staphylokinase. This factor lyses fibrin.The genetic determinant is associated with lysogenic bacteriophages. A complex formed between staphylokinase and plasminogen activates plasmin-like proteolytic activity which causes dissolution of fibrin clots. The mechanism is identical to streptokinase, which is used in medicine to treat patients suffering from coronary thrombosis. As with coagulase, there is no strong evidence that staphylokinase is a virulence factor, although it seems reasonable to imagine that localized fibrinolysis might aid in bacterial spreading.
Other extracellular enzymes
S. aureus can express proteases, a lipase, a deoxyribonuclease (DNase) and a fatty acid modifying enzyme (FAME). The first three probably provide nutrients for the bacteria, and it is unlikely that they have anything but a minor role in pathogenesis. However, the FAME enzyme may be important in abscesses, where it could modify anti-bacterial lipids and prolong bacterial survival.
Avoidance of Host Defenses
S. aureus expresses a number of factors that have the potential to interfere with host defense mechanisms. This includes both structural and soluble elements of the bacterium.
Capsular Polysaccharide
The majority of clinical isolates of
S aureus express a surface polysaccharide of either serotype 5 or 8. This has been called a microcapsule because it can be visualized only by electron microscopy unlike the true capsules of some bacteria which are readily visualized by light microscopy.
S. aureus strains isolated from infections express high levels of the polysaccharide but rapidly lose the ability when cultured in the laboratory. The function of the capsule in virulence is not entirely clear. Although it does impede phagocytosis in the absence of complement, it also impedes colonization of damaged heart valves, perhaps by masking adhesins.
Protein A
Protein A is a surface protein of
S. aureus which binds IgG molecules by their Fc region. In serum, the bacteria will bind IgG molecules in the wrong orientation on their surface which disrupts opsonization and phagocytosis. Mutants of
S. aureus lacking protein A are more efficiently phagocytosed in vitro, and mutants in infection models have diminished virulence.
Leukocidin
S. aureus can express a toxin that specifically acts on polymorphonuclear leukocytes. Phagocytosis is an important defense against staphylococcal infection so leukocidin should be a virulence factor.
Exotoxins
S. aureus can express several different types of protein toxins which are probably responsible for symptoms during infections. Those which damage the membranes of cells were discussed above under
Invasion. Some will lyse erythrocytes, causing hemolysis, but it is unlikely that hemolysis is a relevant determinant of virulence in vivo. Leukocidin causes membrane damage to leukocytes, but is not hemolytic.
Systemic release of a-toxin causes septic shock, while enterotoxins and TSST-1 are superantigens that may cause toxic shock. Staphylococcal enterotoxins cause emesis (vomiting) when ingested and the bacterium is a leading cause of food poisoning.
The exfoliatin toxin causes the scalded skin syndrome in neonates, which results in widespread blistering and loss of the epidermis. There are two antigenically distinct forms of the toxin, ETA and ETB. The toxins have esterase and protease activity and apparently target a protein which is involved in maintaining the integrity of the epidermis.
Superantigens: enterotoxins and toxic shock syndrome toxin
S. aureus secretes two types of toxin with superantigen activity, enterotoxins, of which there are six antigenic types (named SE-A, B, C, D, E and G), and toxic shock syndrome toxin (TSST-1). Enterotoxins cause diarrhea and vomiting when ingested and are responsible for staphylococcal food poisoning. TSST-1 is expressed systemically and is the cause of toxic shock syndrome (TSS). When expressed systemically, enterotoxins can also cause toxic shock syndrome. In fact, enterotoxins B and C cause 50% of non-menstrual cases of TSS. TSST-1 is weakly related to enterotoxins, but it does not have emetic activity. TSST-1 is responsible for 75% of TSS, including all menstrual cases. TSS can occur as a sequel to any staphylococcal infection if an enterotoxin or TSST-1 is released systemically and the host lacks appropriate neutralizing antibodies.
Superantigens stimulate T cells non-specifically without normal antigenic recognition (Figure 4). Up to one in five T cells may be activated, whereas only 1 in 10,000 are stimulated during a usual antigen presentation. Cytokines are released in large amounts, causing the symptoms of TSS. Superantigens bind directly to class II major histocompatibility complexes of antigen-presenting cells outside the conventional antigen-binding grove. This complex recognizes only the Vb element of the T cell receptor. Thus any T cell with the appropriate Vb element can be stimulated, whereas normally, antigen specificity is also required in binding.
FIGURE 4. Superantigens and the non-specific stimulation of T cells. Superantigens bind directly to class II major histocompatibility complexes (MHC II) of antigen-presenting cells outside the normal antigen-binding groove. Up to one in five T cells may be activated. Cytokines are released in large amounts, causing the symptoms of toxic shock.
Exfoliatin toxin (ET)
The exfoliatin toxin, associated with scalded skin syndrome, causes separation within the epidermis, between the living layers and the superficial dead layers. The separation is through the stratum granulosum of the epidermis. This is probably why healing occurs with little scarring although the risks of fluid loss and secondary infections are increased. Staphylococcal exfoliative toxin B has been shown to specifically cleave desmoglein 1, a
cadherin that is found in desmosomes in the epidermis.
Pathogenic Staphylococcus epidermidis
In contrast to
S. aureus, little is known about mechanisms of pathogenesis of
S. epidermidis infections. Adherence is obviously a crucial step in the initiation of foreign body infections. Bacteria-plastic interactions are probably important in colonization of catheters, and a polysaccharide adhesion (PS/A) has been identified. In addition, when host proteins deposit on the implanted device
S. epidermidis will bind to fibronectin.
A characteristic of many pathogenic strains ofS. epidermidis is the production of a slime resulting in biofilm formation. The slime is predominantly a secreted teichoic acid, normally found in the cell wall of the staphylococci. This ability to form a biofilm on the surface of a prosthetic device is probably a significant determinant of virulence for these bacteria.
Resistance of Staphylococci to Antimicrobial Drugs
Hospital strains of
S. aureus are usually resistant to a variety of different antibiotics. A few strains are resistant to all clinically useful antibiotics except vancomycin, and vancomycin-resistant strains are increasingly-reported. The term
MRSA refers to
Methicillin resistant Staphylococcus aureus. Methicillin resistance is widespread and most methicillin-resistant strains are also multiply resistant. A plasmid associated with vancomycin resistance has been detected in
Enterococcusfaecalis which can be transferred to
S. aureus in the laboratory, and it is speculated that this transfer may occur naturally (e.g. in the gastrointestinal tract). In addition,
S. aureus exhibits resistance to antiseptics and disinfectants, such as quaternary ammonium compounds, which may aid its survival in the hospital environment.
Staphylococcal disease has been a perennial problem in the hospital environment since the beginning of the antibiotic era. During the 1950's and early1960's, staphylococcal infection was synonymous with nosocomial infection. Gram-negative bacilli (e.g. E. coli and Pseudomonas aeruginosa) have replaced the staphylococci as the most frequent causes of nosocomial infections, although the staphylococci have remained a problem, especially in surgical wounds.. S aureus responded to the introduction of antibiotics by the usual bacterial means to develop drug resistance: (1) mutation in chromosomal genes followed by selection of resistant strains and (2) acquisition of resistance genes as extrachromosomal plasmids, transducing particles, transposons, or other types of DNA inserts. S. aureus expresses its resistance to drugs and antibiotics through a variety of mechanisms.
Beginning with the use of the penicillin in the 1940's, drug resistance has developed in the staphylococci within a very short time after introduction of an antibiotic into clinical use. Some strains are now resistant to most conventional antibiotics, and there is concern that new antibiotics have not been forthcoming. New strategies in the pharmaceutical industry to find antimicrobial drugs involve identifying potential molecular targets in cells (such the active sites of enzymes involved in cell division), then developing inhibitors of the specific target molecule. Hopefully, this approach will turn up new antimicrobial agents for the battle against staphylococcal infections. In fact, in the past two years alternatives to vancomycin have been approved with the increase in VRSA (vancomycin resistant S. aureus) isolates.
Host Defense against Staphylococcal Infections
Phagocytosis is the major mechanism for combatting staphylococcal infection. Antibodies are produced which neutralize toxins and promote opsonization. However, the bacterial capsule and protein A may interfere with phagocytosis. Biofilm growth on implants is also impervious to phagocytosis. Staphylococci may be difficult to kill after phagocytic engulfment because they produce carotenoids and catalase which neutralize singlet oxygen and superoxide which are primary phagocytic killing mechanisms within the phagolysosome.
Treatment
Hospital acquired infection is often caused by antibiotic resistant strains (MRSA) and can only be treated with vancomycin or an alternative. Until recently, infections acquired outside hospitals have been treated with penicillinase-resistant ß-lactams. However, many of the community acquired (CA) Staphylococcal infections are now methicillin resistant. Particularly in Georgia, Texas, and California, the prevalence of CA-MRSA is widespread. Over 60% of abscess isolates from the emergency department of an Austin, Texas hospital yielded MRSA. These organisms are uniformly resistant to penicillins and cephalosporins. The infections have been treated with combination therapy using sulfa drugs and minocycline or rifampin.
Vaccines
No vaccine is yet available that stimulates active immunity against staphylococcal infections in humans. A vaccine based on fibronectin binding protein induces protective immunity against mastitis in cattle and might also be used as a vaccine in humans.
Hyperimmune serum or monoclonal antibodies directed towards surface components (e.g., capsular polysaccharide or surface protein adhesions) could theoretically prevent bacterial adherence and promote phagocytosis by opsonization of bacterial cells. Also, human hyperimmune serum could be given to hospital patients before surgery as a form of passive immunization.
When the precise molecular basis of the interactions between staphylococcal adhesins and host tissue receptors is known it might be possible to design compounds that block the interactions and thus prevent bacterial colonization. These could be administered systemically or topically.
In February, 2002, an experimental bivalent vaccine against Staphylococcus aureus was reported to be safe and immunogenic for approximately 40 weeks in patients with end-stage renal disease undergoing hemodialysis. The vaccine called StaphVAX is composed of S. aureus type 5 and 8 capsular polysaccharides conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin A. In randomized trials, one injection of the vaccine was administered to 892 hemodialysis patients. Between weeks 3 and 40, 11 cases of S. aureus bacteremia were diagnosed in the vaccinated group compared with 26 cases in a control group. Nearly 90% of patients receiving the vaccine generated antibodies to the two capsular polysaccharides. A decrease in vaccine efficacy after week 40 correlated with a decrease in S. aureus antibodies. The investigators did not believe that use of StaphVAX would be limited to hemodialysis patients. For example, the vaccine might be used in cases where healthy individuals come into the hospital for elective surgery, such as a joint replacement. Such patients do not require protection for the rest of their lives; what they need is protection for a short period while they are in the hospital. The vaccine manufacturer will experiment with booster shots to maintain immunity for longer periods of time, and with passive immunization for such at-risk populations as premature infants. They hope to gain FDA approval for the vaccine in 2006.
Table 2. Possible virulence determinants expressed in the pathogenesis of Staphylococcus aureus infections
boils and pimples (folliculitis) Colonization: cell-bound (protein) adhesins
Invasion:
Invasins: staphylokinase
Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.)
Resistance to phagocytosis: coagulase, leukocidin
Resistance to immune responses: coagulase
Toxigenesis: cytotoxic toxins (hemolysins and leukocidin)
pneumonia
Colonization: cell-bound (protein) adhesins
Invasion:
Invasins: staphylokinase, hyaluronidase
Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.)
Resistance to phagocytosis: coagulase, leukocidin, hemolysins, carotenoids, superoxide dismutase, catalase, growth at low pH
Resistance to immune responses: coagulase, antigenic variation
Toxigenesis: Cytotoxic toxins (hemolysins and leukocidin)
food poisoning (emesis or vomiting)
Toxigenesis: Enterotoxins A-G
septicemia (invasion of the bloodstream)
Invasion:
Invasins: staphylokinase, hyaluronidase
Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.)
Resistance to phagocytosis: coagulase, protein A, leukocidin, hemolysins, carotenoids, superoxide dismutase, catalase, growth at low pH
Resistance to immune responses: coagulase, protein A, antigenic variation
Toxigenesis: cytotoxic toxins (hemolysins and leukocidin)
osteomyelitis (invasion of bone)
Colonization: cell-bound (protein) adhesins
Invasion:
Invasins: staphylokinase, hyaluronidase
Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.)
Resistance to phagocytosis: coagulase, protein A, leukocidin, hemolysins, carotenoids, superoxide dismutase, catalase, growth at low pH
Resistance to immune responses: coagulase, protein A, antigenic variation
Toxigenesis: cytotoxic toxins (hemolysins and leukocidin)
toxic shock syndrome
Colonization: cell-bound (protein) adhesins
Resistance to immune responses: coagulase, antigenic variation
Toxigenesis: TSST toxin, Enterotoxins A-G
surgical wound infections
Colonization: cell-bound (protein) adhesins
Invasion:
Invasins: staphylokinase, hyaluronidase
Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.)
Resistance to phagocytosis: coagulase, protein A, leukocidin, hemolysins, carotenoids, superoxide dismutase, catalase, growth at low pH
Resistance to immune responses: coagulase, protein A, antigenic variation
Toxigenesis: cytotoxic toxins (hemolysins and leukocidin)
scalded skin syndrome (analogous to scarlet fever)
Colonization: cell-bound (protein) adhesins
Invasion:
Invasins: staphylokinase, hyaluronidase
Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.)
Resistance to phagocytosis: coagulase, leukocidin, hemolysins
Resistance to immune responses: coagulase, antigenic variation
Toxigenesis: Exfoliatin toxin
