Staphylococcus aureus (Student Notes)

Staphylococcus aureus is one of the most frequent agents of human bacterial infections.

This catalase-positive bacteria which can be found on the skin has a variety of virulence factors, enabling it to cause various human infections ranging from mild skin infection to severe, life-threatening systemic infections.

While the average staph aureus is responsive to several types of antibiotics, strains of methicillin resistant staphylococcus aureus (MRSA) are more resistant and can be challenging to treat.

The prevalence of MRSA is increasing worldwide and is becoming a significant problem in medical practice.

Laboratory features

Being one of the most frequent human pathogens, staphylococcus aureus is often cultivated in the lab. Here it expresses certain laboratory characteristics that enable us to distinguish it from other general and species.

Microscopic appearance

Like all the staphylococci, Staphylococcus Aureus (SA) are seen as small, gram + cocci that are stained blue/purple when observed under the microscope with gram staining.

Another microscopical feature is that the bacteria usually aggregate in grape-like clusters on the slide.

Cultivation

SA is a facultative anaerobic bacteria that are easily cultivated on a bouillon medium, as well as on normal agar and blood agar medium.

On agar medium, SA forms 2-3 mm wide circular, yellow colonies. The yellow color comes from their synthesis of a yellow/golden non-diffusible pigment, which stains the colonies. Hence its name aureus, which means golden in Latin.

On blood agar, the colonies also express their characteristic yellow/golden pigment as well as extensive beta-hemolysis, which results in the otherwise red medium becoming more or less transparent around the colonies.

Finally, SA is able to survive and grow in high salt concentrations, therefore,  cultivation with ~7.5 NaCl can be used as an effective selective cultivation method.

Biochemical properties

Staphylococcus Aureus is both catalase and coagulase-positive, which represents 2 of its biochemical features. However, SA expresses 2 types of coagulases, the exo-and endocoagulase.

The exocoagulase is an enzyme that is released by the bacteria, and if present in the blood, it is able to convert fibrinogen to fibrin, clotting the blood.

The endocoagulase (also called clumping factor) is a coagulase enzyme present on its surface which can also convert fibrinogen to fibrin, causing blood clotting.

Both coagulases help the bacteria form a fibrin coat around it which can help it evade detection by the immune system. 

In the lab it can be used to determine coagulase positivity, as the exocoagulase will cause agglutination in a tube test, while the endocoagulase can cause agglutination on slides, using latex beads (latex agglutination). Both are serological tests.

Virulence factors

The ability of SA to cause disease in the human body comes from its virulence factors. These include membrane bound proteins, such as protein A, enzymes, such as invasins and adhesins, as well as the release of various exotoxins.

Membrane bound proteins

There are various molecules, proteins, as well as structural complexes that make up the membrane-bound virulence factors of Staphylococcus Aureus.

These include its polysaccharide capsule, an outer slime layer, the teichoic and lipoteichoic acids in the cell wall, adhesion proteins, endonucleases, and the already mentioned protein A.

The polysaccharide capsule is found outside its peptidoglycan cell wall and is made up of sugars (polysaccharides). This layer covers the surface AG’s of the bacteria making it difficult for the immune cells to detect and react to them, protecting the bacteria against macrophages and other cells of the human immune system.

Staphylococcus Aureus also has an outermost slime layer which enables it to adhere/stick to plastic and metal medical equipment, such as IV catheters, urinary catheters and metal prosthesis.

When doing so, they form so-called biofilms. This presents a challenge when trying to prevent nosocomial infections (hospital-acquired infections), as Staphylococcus Aureus can gain access to the body through these devices.

Staphylococcus Aureus also expresses various non-specific and specific adhesion proteins on their cell surface, which allows them to attach to various surfaces and tissues/cells.

One example is the laminin-binding protein, which allows Staphylococcus Aureus to adhere to collagen in the extracellular matrix.

As mentioned, Staphylococcus Aureus express coagulase enzymes on their cell surface. These enzymes can convert fibrinogen to fibrin, enabling the bacteria to obtain an outer fibrin coat.

This protects them from immune cells such as macrophages as the fibrin coat buries the bacterias’ surface antigens, while the fibrin coat is recognized as self-tissue.

Finally, Staphylococcus Aureus also expresses another protein called protein A. This protein is able to bind the immune system Immunoglobulins, which  can prevent antibody mediated phagocytosis of the bacterium, another way in which Staphylococcus Aureus evades the human immune system.

Enzymes

In addition to the membrane-bound virulence factors, Staphylococcus Aureus also releases various enzymes (exoenzymes) that can help the bacteria to infiltrate bodily tissues (invasins), as well as disturb the normal homeostasis of blood coagulation (exocoagulase).

These enzymes includes, exocoagulase, fibrinolysin, DNase, hyaluronidase, phosphatase and lipase

The release of exogoaculase and fibrinolysin can cause blood clotting, as exocoagulase can convert fibrinogen to fibrin, as well as fibrinolysis, as fibrinolysin will degrade fibrin.

The release of DNAse enables Staphylococcus Aureus to break down host DNA.

Other exoenzymes include hyaluronidase which breaks down hyaluronic acid found in the connective tissue, lipase, which breaks down fatty acids, and phosphatase, which can break down human bone (contain lots of phosphate). 

This enables the bacteria to break down various types of human tissue,  enabling it to spread. This includes bone, which can result in osteomyelitis which is an infection causing inflammation of  the bone or bone marrow.

Toxins

Staphylococcus Aureus also produce and release toxins, which are called exotoxins. The most frequent includes the Toxic Shock Syndrome Toxin (TSST-1), enterotoxin and exfoliative toxin

TSST is a very dangerous bacterial toxin that belongs to a group of Antigens called superantigens. These superantigens are able to interfere with the antigen presentation to T-cells via MHC molecules.

This can lead to an overactivation and subsequent overproduction of cytokines by the T-cells in an event that is known as a cytokine storm.

This leads to a septic state in the patient who develops a high fever, a drop in blood pressure, and altered consciousness. 

This is known as septic shock which can cause multi-organ failure, which has a mortality rate of up to 25-50%. Luckily, not all strains of Staphylococcus Aureus are able to produce TSST-1.

Staphylococcus Aureus can also produce and release staphylococcal enterotoxin, which can cause staphylococcal enteritis.

This is inflammation of the small intestines caused by the toxin. This toxin is not as dangerous as TSST-1, and the symptoms are usually milder, including fever, nausea, vomiting, and diarrhea. 

The condition is usually not fatal in adults, but can be dangerous to infants and the elderly as lots of fluid and electrolytes can be lost through vomit and diarrhea, causing dehydration.

The release of exfoliative toxin can cause skin damage as it is able to destroy the intercellular connections (cell adhesion molecules) between epithelial cells of the skin.

This results in the development of a dermatological condition known as staphylococcal scaled skin syndrome also called Ritter’s disease, which is usually not a severe condition.

Finally, Staphylococcus Aureus also releases some cytotoxins which essentially are enzymes causing the lysis of human cells.

Staphylococcus Aureus can release a group of hemolysins (α, β, δ, and γ), which results in lysis of erythrocytes. these enzymes are responsible for the b-hemolysis observed when cultivating Staphylococcus Aureus on blood agar.

Staphylococcus Aureus also releases an enzyme called leukocidin, which can cause the lysis of leukocytes.

Diseases/infections

Around 5-10% of the population are asymptomatic carriers of Staphylococcus Aureus in the nose and nasopharynx, from which it can spread through direct contact or through respiratory droplets.

In addition, it can survive for days on dry environmental surfaces such as towels and clothing, from which they can be contracted.

Staphylococcus Aureus can also be spread from infected individuals through contact with pus from infected wounds or by direct skin-skin contact. 

When contracting the bacterium, severe infections may develop, however, the infections are grouped into purulent, invasive, and toxin-mediated infections, depending on the site of infection and the symptoms presented by the infection.

Purulent infections

First of all, we can have purulent infections which are infections at the site of contraction on the skin or through skin lesions.

These infections can through the virulence factors of SA lead to the development of superficial and deep dermatological infections.

Impetigo

The least invasive of these infections is impetigo. This is a superficial skin infection caused by staphylococcus aureus or streptococcus pyogenes which appear as yellow-ish crusting on the skin. 

The bacteria typically enter through breaks in the skin such as mosquito bites, eczema, or herpes blisters. It is most typical among children and can be effectively treated with topical antibiotic creams.

Folliculitis, furuncles, carbuncles and abscesses

If the Staphylococcus Aureus invades into hair follicles of the skin it may cause folliculitis. This is a localized skin infection in the hair follicle accompanied by pus formation. 

Pus develops as the virulence factors of SA degrade the CT under the skin and attracts neutrophils which try to fight the bacteria and clear the infected area of cellular debris.

If left untreated, or if the patient suffers from any condition disabling the body’s ability to fight the infection (e.g. diabetes or AIDS), the infection can go deeper and grow bigger creating what is known as furuncles.

If several furuncles appear in the same area (e.g. on the neck), they are known as carbuncles.

Staphylococcus aureus can also enter the skin through small lesions such as superficial wounds, or following needle injections. Once under the skin, they can cause abscesses, which appear as painful and tender collections of pus in the subcutaneous tissue.

Furuncles, carbuncles, and abscesses are more common among diabetics than the general population. Another population prone to abscesses is IV drug users who inoculate the bacteria through injections. 

Deep purulent infections

If the bacterium or infection is able to penetrate deep into the tissues, it can cause deep purulent infections. Also, the bacteria may enter the bloodstream, known as bacteremia from which they can spread to the entire body creating abscesses and infect various tissues.

Endocarditis

Staphylococcus aureus is known to be a cause of endocarditis. This is an infection of the endocardium on the heart valves (typically the aortic valve) that occurs following the dissemination of the bacterium into the bloodstream.

Septic arthritis

In some cases, the bacterium may enter joints following bacteremia, causing septic arthritis (purulent infection of the joint).

Other deep purulent infections have different names depending on their location and include conditions such as:

Other deep purulent infections

• Osteomyelitis (infection of the bone and/or bone marrow) 
• Peritonitis (Infection of the peritoneum (inner lining of the abdominal cavity)
• Meningitis (infection of the meningeal membrane surrounding the brain)
• Mastoiditis (infection of the mastoid air cells in the skull)
• Mastitis (infection in the breast tissue)

Diabetic patients are also prone to develop infections in wounds in their feet. Staphylococcus aureus is a common agent in these wound infections.

If they penetrate deep, they can cause severe infections and gangrene which might necessitate amputation of the affected limb.

Toxin mediated diseases

When infected by Staphylococcus Aureus, either in the blood or in organs/areas with high blood supply, their exotoxins can be absorbed/released into the circulation where they can cause toxic shock syndrome and staphylococcal enteritis (discussed earlier).

Treatment

Antibiotics are usually required to treat infections caused by Staphylococcus aureus.

In order to treat it most effectively, one should do laboratory diagnostics to confirm that the infection is caused by Staphylococcus Aureus and antibiogram in order to determine which antibiotic agent the strain is sensitive to.

This can be done by selective cultivation of the bacteria from blood, pus, or by swabs from the infected area, after testing various antibiotic agents on the culture.

This typically takes 3-4 days. Therefore, you have to start treatment based on suspicion that the infection can be caused by Staphylococcus Aureus.

This is called empiric treatment and for Staphylococcus Aureus, β-lactams are usually given together with β-lactamase inhibitors (e.g. amoxicillin + clavulanic acid).

β-lactams inhibit the synthesis of the peptidoglycan cell wall of bacterias by targeting the penicillin-binding protein (PBP). This protein is a transpeptidase responsible for cross-linking the peptidoglycan chains.

Because 95% of the cell wall in gram + bacteria is made up of peptidoglycans, it is very efficient against Staphylococcus Aureus which is a G+ bacteria.

Unfortunately, most strains (about 99%) produce an enzyme called β-lactamase, which is able to cleave the β-lactam ring (the functional group of the antibiotic), thereby inactivating the antibiotic.

Because of this, we also administer β-lactamase inhibitors (like clavulanic acid) which are able to inhibit the β-lactamase enzyme.

Although the problem with β-lactamase enzymes was overcome, from the 1970s, alterations in the structure of the PBP appeared, which rendered all β-lactam antibiotics ineffective against these strains.

These strains are resistant against all β-lactam antibiotics and are termed methicillin-resistant Staphylococcus aureus (MRSA)

Fortunately, other antibiotics with other target molecules are available, such as vancomycin, linezolid, clindamycin, and daptomycin, which enables us to treat strains of MRSA.

Nevertheless, MRSA infections can be difficult to treat and represent a growing health problem.

Vancomycin is considered the most effective antibiotic against MRSA strains. Vancomycin also inhibits cell wall synthesis in G+ bacterias but targets D-alanine AA’s in the peptide cross-bridges between the peptidoglycan chains, instead of the PBP.

Unfortunately, vancomycin-resistant strains of Staphylococcus Aureus (VRSA) have been detected, which can present an even greater challenge when treating Staphylococcus Aureus infections in the future.

In VRSA, the D-alanine amino acids are linked to lactate, which makes vancomycin unable to target them and inhibits cell wall synthesis.