Clinical Manifestations

Staphylococci can cause many forms of infection. (1) S aureus causes superficial skin lesions (boils, styes) and localized abscesses in other sites. (2) S aureus causes deep-seated infections, such as osteomyelitis and endocarditis and more serious skin infections (furunculosis). (3) S aureus is a major cause of hospital acquired (nosocomial) infection of surgical wounds and, with S epidermidis, causes infections associated with indwelling medical devices. (4) S aureus causes food poisoning by releasing enterotoxins into food. (5) S aureus causes toxic shock syndrome by release of superantigens into the blood stream. (6) S saprophiticus causes urinary tract infections, especially in girls. (7) Other species of staphylococci (S lugdunensis, S haemolyticus, S warneri, S schleiferi, S intermedius) are infrequent pathogens.

Structure

Staphylococci are Gram-positive cocci 1μm in diameter. They form clumps.

Classification

S aureus and S intermedius are coagulase positive. All other staphylococci are coagulase negative. They are salt tolerant and often hemolytic. Identification requires biotype analysis.

Natural Habitat

S aureus colonizes the nasal passage and axillae. S epidermidis is a common human skin commensal. Other species of staphylococci are infrequent human commensals. Some are commensals of other animals.

Pathogenesis

S aureus expresses many potential virulence factors. (1) Surface proteins that promote colonization of host tissues. (2) Factors that probably inhibit phagocytosis (capsule, immunoglobulin binding protein A). (3) Toxins that damage host tissues and cause disease symptoms. Coagulase-negative staphylococci are normally less virulent and express fewer virulence factors. S epidermidis readily colonizes implanted devices.

Host Defenses

Phagocytosis is the major mechanism for combatting staphylococcal infection. Antibodies are produced which neutralize toxins and promote opsonization. The capsule and protein A may interfere with phagocytosis. Biofilm growth on implants is impervious to phagocytosis.

Treatment

Infections acquired outside hospitals can usually be treated with penicillinase-resistant β-lactams. Hospital acquired infection is often caused by antibiotic resistant strains and can only be treated with vancomycin.

Antibiotic Resistance

Multiple antibiotic resistance is increasingly common in S aureus and S epidermidis. Methicillin resistance is indicative of multiple resistance. Methicillin-resistant S aureus (MRSA) causes outbreaks in hospitals and can be epidemic.

Epidemiology

Epidemiological tracing of S aureus is traditionally performed by phage typing, but has limitations. Molecular typing methods are being tested experimentally.

Diagnosis

Diagnosis is based on performing tests with colonies. Tests for clumping factor, coagulase, hemolysins and thermostable deoxyribonuclease are routinely used to identify S aureus. Commercial latex agglutination tests are available. Identification of S epidermidis is confirmed by commercial biotyping kits.

Control

Patients and staff carrying epidemic strains, particularly MRSA, should be isolated. Patients may be given disinfectant baths or treated with a topical antibiotic to eradicate carriage of MRSA. Infection control programs are used in most hospitals.

Structure

Staphylococci are Gram-positive cocci about 0.5 – 1.0 μm in diameter. They grow in clusters, pairs and occasionally in short chains. The clusters arise because staphylococci divide in two planes. The configuration of the cocci helps to distinguish micrococci and staphylococci from streptococci, which usually grow in chains. Observations must be made on cultures grown in broth, because streptococci grown on solid medium may appear as clumps. Several fields should be examined before deciding whether clumps or chains are present.

Catalase Test

The catalase test is important in distinguishing streptococci (catalase-negative) staphylococci which are catalase positive. The test is performed by flooding an agar slant or broth culture with several drops of 3% hydrogen peroxide. Catalase-positive cultures bubble at once. The test should not be done on blood agar because blood itself will produce bubbles.

Isolation and Identification

The presence of staphylococci in a lesion might first be suspected after examination of a direct Gram stain. However, small numbers of bacteria in blood preclude microscopic examination and require culturing first.

The organism is isolated by streaking material from the clinical specimen (or from a blood culture) onto solid media such as blood agar, tryptic soy agar or heart infusion agar. Specimens likely to be contaminated with other microorganisms can be plated on mannitol salt agar containing 7.5% sodium chloride, which allows the halo-tolerant staphylococci to grow. Ideally a Gram stain of the colony should be performed and tests made for catalase and coagulase production, allowing the coagulase-positive S aureus to be identified quickly. Another very useful test for S aureus is the production of thermostable deoxyribonuclease. S aureus can be confirmed by testing colonies for agglutination with latex particles coated with immunoglobulin G and fibrinogen which bind protein A and the clumping factor, respectively, on the bacterial cell surface. These are available from commercial suppliers (e.g., Staphaurex). The most recent latex test (Pastaurex) incorporates monoclonal antibodies to serotype 5 and 8 capsular polysaccharide in order to reduce the number of false negatives. (Some recent clinical isolates of S aureus lack production of coagulase and/or clumping factor, which can make identification difficult.)

The association of S epidermidis (and to a lesser extent of other coagulase-negative staphylococci) with nosocomial infections associated with indwelling devices means that isolation of these bacteria from blood is likely to be important and not due to chance contamination, particularly if successive blood cultures are positive. Nowadays, identification of S epidermidis and other species of Staphylococcus is performed using commercial biotype identification kits, such as API Staph Ident, API Staph-Trac, Vitek GPI Card and Microscan Pos Combo. These comprise preformed strips containing test substrates.

S aureus Adheres to Host Proteins

S aureus cells express on their surface proteins that promote attachment to host proteins such as laminin and fibronectin that form part of the extracellular matrix (Figure 12-3). Fibronectin is present on epithelial and endothelial surfaces as well as being a component of blood clots. In addition, most strains express a fibrinogen/fibrin binding protein (the clumping factor) which promotes attachment to blood clots and traumatized tissue. Most strains of S aureus express fibronectin and fibrinogen-binding proteins.

Figure 12-3

Summary of virulence factors of Staphylococcus aureus.

The receptor which promotes attachment to collagen is particularly associated with 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 these staphylococcal matrix-binding proteins are virulence factors has come from studying defective mutants in vitro adherence assays and in experimental infections. Mutants defective in binding to fibronectin and to fibrinogen have reduced virulence in a rat model for endocarditis, suggesting that bacterial attachment to the sterile vegetations caused by damaging the endothelial surface of the heart valve is promoted by fibronectin and fibrinogen. Similarly, mutants lacking the collagen-binding protein have reduced virulence in a mouse model for septic arthritis. Furthermore, the soluble ligand-binding domain of the fibrinogen, fibronectin and collagen-binding proteins expressed by recombinant methods strongly blocks interactions of bacterial cells with the corresponding host protein.

Role of Adherence in Infections Associated with Medical Devices

Infections associated with indwelling medical devices ranging from simple intravenous catheters to prosthetic joints and replacement heart valves can be caused by S aureus and S epidermidis (Figure 12-2). Very shortly after biomaterial is implanted in the human body it becomes coated with a complex mixture of host proteins and platelets. In one model system involving short-term contact between biomaterial and blood, fibrinogen was shown to be the dominant component and was primarily responsible for adherence of S aureus in subsequent in vitro assays. In contrast, with material that has been in the body for longer periods (e.g., human intravenous catheters) the fibrinogen is degraded and no longer promotes bacterial attachment. Instead, fibronectin, which remains intact, becomes the predominant ligand promoting attachment.

Adherence to Endothelial Cells

S aureus can adhere to the surface of cultured human endothelial cells and become internalized by a phagocytosis-like process. It is not clear if attachment involves a novel receptor or a known surface protein of S aureus. Some researchers think that S aureus can initiate endocarditis by attaching to the undamaged endothelium. Others feel that trauma of even a very minor nature is required to promote attachment of bacteria.

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 after antibody labeling, unlike the copious capsules of other bacteria which are visualized by light microscopy. S aureus isolated from infections expresses high levels of polysaccharide but rapidly loses it upon laboratory subculture. The function of the capsule is not clear. It may impede phagocytosis, but in in vitro tests this was only demonstrated in the absence of complement. Conversely, comparing wild-type and a capsule defective mutant strain in an endocarditis model suggested that polysaccharide expression actually impeded colonization of damaged heart valves, perhaps by masking adhesins.

Protein A

Protein A is a surface protein of S aureus which binds immunoglobulin G molecules by the Fc region (Fig. 12-3). In serum, bacteria will bind IgG molecules the wrong way round by this non-immune mechanism. In principle this will disrupt opsonization and phagocytosis. Indeed mutants of S aureus lacking protein A are more efficiently phagocytozed in vitro, and studies with mutants in infection models suggest that protein A enhances 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. This toxin is discussed in more detail in the next section.

Membrane Damaging Toxins

Superantigens: enterotoxins and toxic shock syndrome toxin

S aureus can express two different types of toxin with superantigen activity, enterotoxins, of which there are six serotypes (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. When expressed systemically, enterotoxins can cause toxic shock syndrome (TSS) - indeed enterotoxins B and C cause 50% of non-menstrual TSS. TSST-1 is very weakly related to enterotoxins and 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. Tampon-associated TSS is not a true infection, being caused by growth of S aureus in a tampon and absorption of the toxin into the blood stream. TSS came to prominence with the introduction of super-absorbent tampons; and although the number of such cases has decreased dramatically, they still occur despite withdrawal of certain types of tampons from the market.

Superantigens stimulate T cells non-specifically without normal antigenic recognition (Figure 12-4). Up to one in five T cells may be activated, whereas only 1 in 10,000 are stimulated during 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 Vβ element of the T cell receptor. Thus any T cell with the appropriate Vβ element can be stimulated, whereas normally antigen specificity is also required in binding.

Figure 12-4

Superantigens and the non-specific stimulation of T cells.

Coagulase

Coagulase is not an enzyme. It 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. This is the basis of the tube coagulase test, in which a clot is formed in plasma after incubation with the S aureus broth-culture supernatant. Coagulase is a traditional marker for identifying S aureus in the clinical microbiology laboratory. However, there is no evidence that it is a virulence factor, although it is reasonable to speculate that the bacteria could protect themselves from host defenses by causing localized clotting. Notably, coagulase deficient mutants have been tested in several infection models but no differences from the parent strain were observed.

There is some confusion in the literature concerning coagulase and clumping factor, the fibrinogen-binding determinant on the S aureus cell surface. This is partly due to loose terminology, with the clumping factor sometimes being referred to as bound coagulase. Also, although coagulase is regarded as an extracellular protein, a small fraction is tightly bound on the bacterial cell surface where it can react with prothrombin. Finally, it has recently been shown that the coagulase can bind fibrinogen as well as thrombin, at least when it is extracellular. 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. 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 evidence that staphylokinase is a virulence factor, although it seems reasonable to imagine that localized fibrinolysis might aid in bacterial spreading.

Antimicrobial Drugs

Ever since the first use of penicillin, S aureus has shown a remarkable ability to adapt. Resistance has developed to new drugs within a short time of their introduction. Some strains are now resistant to most conventional antibiotics. It is worrisome that there do not seem to be any new antibiotics on the horizon. Any recent developments have been modifications to existing drugs.

The original strategy used by the pharmaceutical industry to find antimicrobial drugs was to screen natural products and synthetic chemicals for antimicrobial activity. The mechanism of action was then investigated.

New approaches are being adopted to find the next generation of antimicrobials. Potential targets such as enzymes involved in an essential function (e.g., in cell division) are identified based on knowledge of bacterial physiology and metabolism. Screening methods are then developed to identify inhibitors of a specific target molecule. In addition, with detailed molecular knowledge of the target molecule, specific inhibitors can be designed.

Vaccines and New Approaches to Combatting Nosocomial Infections

No vaccine is currently available to combat staphylococcal infections. There may now be a case for considering methods to prevent disease, particularly in hospitalized patients.

Hyperimmune serum from human volunteer donors or humanized monoclonal antibodies directed towards surface components (e.g., capsular polysaccharide or surface protein adhesions) could both prevent bacterial adherence and also promote phagocytosis of bacterial cells. Indeed a prototype vaccine based on capsular polysaccharide from S aureus has been administered to volunteers to raise hyperimmune serum, which could be given to patients in hospital before surgery. A vaccine based on fibronectin binding protein induces protective immunity against mastitis in cattle and might also be used as a vaccine in humans.

When the molecular basis of the interactions between the bacterial surface proteins and the host matrix protein ligands are known it might be possible to design compounds that block the interactions and thus prevent bacterial colonization. These could be administered systemically or topically.