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Staphylococci Virulence

Introduction

Staphylococcus is a gram positive, cocci shaped, genus of bacteria. Observed under a microscope will reveal they exist in microscopic ‘grape-like’ clusters. One species of staphylococci, Staphylococcus aureus, can grow at temperature ranges of 15-45ºC and at  a relatively high NaCl concentration of 15%.

Catalase Test

One of the main tests used to differentiate between bacterial species is the catalase test. This test determines whether or not the enzyme catalase is present. Catalase, is responsible for the breakdown of hydrogen peroxide. The test consists of adding a bacterial colony to a drop of hydrogen peroxide, if the bacteria is catalase-positive (i.e. has the catalase enzyme) bubbles of oxygen will be produced. This will not occur in catalase-negative bacteria. Staphylococci species are catalase-positive.

Oxidase Test

The oxidase test, is another major test used to differentiate between bacteria. It tests to see if certain cytochrome c oxidase enzymes are present. These enzymes are involved with the electron transport chain. There are a number of ways to perform this procedure, but they all typically involve adding a reagent to the sample bacteria and observing for a colour change. Typically the development of a blue colour indicates a positive result (oxidase enzymes are present), with no colour change indicating a negative result. Staphylococci species are oxidase-negative.

Coagulase Test

The final major test used for differentiation is the coagulase test. This test determines whether the enzyme coagulase is present, an enzyme responsible for the formation of blood clots and primarily associated with staphylococci species. However, within the Staphylococcus genus, there are both coagulase-positive and coagulase-negative species. S. aureus is an example of a coagulase-positive species, and S. epidermidis is an   example of a coagulase-negative species. The procedure involves adding blood plasma to the test sample, the development of agglutination after a short period of time indicates a positive result, with no agglutination indicating a negative result.

Staphylococci & Animals

Currently, around 34 species of staphylococci have been identified, with many of these being found in multiple species of animals. Different species of Staphylococcus have different preferred hosts, Staphylococcus tend to co-evolve with their hosts, but they are still able to cross species barriers. The major Staphylococcus found in dogs and cats are:

  • S. intermedius
  • S. felis

S. aureus is not commonly found in dogs and cats, it is found more often in other domesticated species however.

Staphylococci can be found in certain locations all over the body where they result in no disease. These are known as resident populations and include; mucosal surfaces, mucocutaneous junctions (such as the lips, nostrils, vagina, etc.) and the ear canal.

Staphylococci of Veterinary Importance

Coagulase positive:

  • S. aureus
  • S. intermedius
  • S. hyicus

Coagulase negative:

  • S. sciuri
  • S. equorum
  • S. epidermidis

Staphylococcus pseudintermedius

S. pseudintermedius is most commonly found on the skin of domestic dogs and cats. This species is zoonotic and thus has the potential to infect humans, although human cases of infection with this species are rare. Of increasing concern is the rise in human S. pseudintermedius infectious, particularly because of the resistance to antibiotics shown by this species.

Some clinical signs associated with infection of this bacteria are; otitis externa (inflammation of the outer ear), mastitis, infective endocarditis (inflammation of the inner layer of the heart), abscess formation, infection of wounds and primarily, chronic pyoderma (an inflammatory skin disease).

Alterations of the skin’s micro-environment can promote the growth of bacteria such as S. pseudintermedius. For example, inflammation provides humidity and warmth, both of which promote bacterial multiplication. Any trauma caused to the skin will further reduce epidermal defenses. Continuing with the inflammation example, irritation may cause the the animal to scratch intensely which can lead to damage of the upper layers of the epidermis and make infection for S. pseudintermedius easier. Any allergic skin diseases, or underlying immunological disorders will also contribute to the degree of infection.

Pyoderma

Pyoderma is a bacterial skin disease, caused by bacteria normally found on healthy skin (commensal) such as Staphylococcus. Because these bacteria are opportunistic, if skin becomes diseased or damaged, they may proliferate which can cause problems. Whilst pyoderma is primarily caused by underlying skin problems, it is also possible for pyoderma to occur on healthy skin, however this often indicates underlying immune system problems.

Pyoderma becomes a problem when commensal bacteria breach the epidermis and begin to proliferate and adhere to keratinocytes – the predominant cell type of the epidermis. Areas prone to infection are those where the skin creates folds which reduces air circulation and provides a warm, humid environment, perfect for bacterial growth.

Treatment of pyoderma typically involves the use of antimicrobials either topically or systemically for around 8 weeks. Whilst this may remove the initial pyoderma, it is also important to treat the underlying conditions responsible for the outbreak as reinfection may occur. Reinfection will require more treatment, which is of concern as this can lead to antimicrobial resistance. Methicillin-resistant Staphylococcus pseudintermedius (MRSP, i.e. similar to MRSA) has recently been identified.

Staphylococcus hyicus

S. hyicus is responsible for causing exudative epidermitis, an oozing inflammation of the skin, also known as ‘Greasy Pig Disease’. It occurs when abraded skin is invaded by S. hyicus bacteria which then cause infection. Lesions begin to develop after infection, which then spread to the hair follicles (known as folliculitis). Inflammation soon follows this causing erosion and ulceration and the lesions continue to growth, engulfing large amounts of the skin surface. Alongside this, the sebaceous glands will produce a black, greasy, exudate.

Whilst this disease can be treated with antibiotics, death often occurs as a result of starvation of dehydration, so it is important to offer the infected pigs electrolytes by mouth to ensure a steady recovery.

Virulent strains of S. hyicus produce an ‘exfoliative toxin’. A toxin responsible for the epidermal necrolysis. Isolation of this toxin and reintroduction into a healthy pig will interestingly, reproduce the observed disease.

Exfoliative Toxin

The histopathology of S. hyicus induced exudative epidermitis, is very similar to that of S. aureus scalded skin syndrome. A disease which causes fluid filled blisters to appear on the skin of humans. Their similarities are owed to the exfoliative toxins, A & B which they secrete. These toxins cause a detachment of the epidermal layer and result in the observed lesions and deformations of the skin. This is beneficial to the bacteria as it allows them to further penetrate and proliferate beneath the skin.

Staphylococcus aureus Diseases

Staphylococcus aureus is responsible for a wide array of diseases, these include:

  • Superficial lesions
    • Skin and soft tissue infections
    • Mastitis
  • Invasive infections
    • Osteomyelitis – inflammation of the bone marrow
    • Endocarditis – inflammation of the inner layer of the heart
    • Pneumonia – inflammatory condition of the lungs
    • Septicaemia – The presence of pathogenic organisms in the blood which can lead to a body-wide inflammatory state
  • Toxinoses
    • Food poisoning
    • Scalded skin syndrome – Formation of fluid filled blisters on the skin
    • Toxic shock syndrome – A potentially fatal disease caused by a S. aureus toxin

Virulence & Virulence Factors

Staphylococcus species can express virulence in a number of ways, these include:

  • Adherence and surface associated proteins
  • Bacterial capsules which prevent phagocytosis
  • Exoenzymes, extracellular enzymes secreted by bacteria
  • Proteases, enzymes which break down proteins e.g. nuclease
  • Exotoxins, toxins secreted by the bacteria

Adhesion

Bacterial adhesion to host cells is the first step in colonization. Adhesion typically requires the presence of bacterial adhesins. These include pili, fimbrae, the flagella or the cell surface itself. These surface proteins allow for the attachment to host cells, in particular the host proteins laminin and fibronectin. Adhesins which promote attachment to collagen fibres can lead to osteomyelitis (infection of bone marrow) and arthritis.

Clumping Factor

Clumping factor is a protein which binds to fibrinogen, it is responsible for the formation of blood clots and it what causes the agglutination in the coagulase test. Clumping factor also helps to evade the immune system, by coating the itself in fibrinogen the bacterium avoid opsonisation and thus phagocytosis.

Protein A

A surface associated protein found in S. aureus, it binds to the Fc regions (tail regions of antibodies, which bind to Fc receptors on cell surfaces) of immunoglobulins, particularly IgG. This causes incorrect orientation of the bacteria in relation to the IgG, this is beneficial  because it disrupts opsonisation and thus phagocytosis. Protein A can also bind to IgM associated with B-cells (the lymphocytes associated with the humoral immune response), this induces apoptosis in these cells and thus causes depletion.

The gene which encodes for this protein is the spa gene, this gene is often used for molecular typing of Staphylococcus.

Capsular Polysaccharides

Capsules are bacterial structures which encase the entire bacterium, they are useful for protecting the bacterium against phagocytosis but they can also be a virulence factor. The presence of certain capsules can act as a potent abscess potentiator, isolation and injection of such a capsule can produce sterile intra-abdominal abscesses.

Super Antigens

Normal antigens are able to generate an immune response which induces a reaction from only the appropriate cells of the immune system. Typically much less than 1% of body T-cells (the lymphocytes associated with cell-mediated immunity) are activated. A super antigen on the other hand, generates a much more potent immune response. It is believed super antigens can activate up to 20% of the T-cells in the body.

Such a large immune response actually works in favour of Staphylococcus species. It is thought that these bacteria can produce more than 20 types of super antigens, in the form of enterotoxins and exotoxins. Enterotoxins specifically target the intestines and can cause diarrhoea and vomiting. In severe responses to super antigens (such as the exotoxins) the host can develop Toxic Shock Syndrome (TSS). TSS is where the body cannot cope with the large amounts of inflammatory cytokines released by the T-cells due to the binding of the super antigen, this can lead to shock and multiple organ failure.

The vast majority of TSS cases are caused by S. aureus and S. pyogenes. The most potent inducer of TSS however is Toxic shock syndrome toxin 1, a toxin produced by S. aureus which is responsible for around 75% of all TSS cases. Again, this leads to over stimulation of T-cells and a drastic systemic release of inflammatory cytokines which leads to lowered blood pressure, fever and in severe cases shock and organ failure.

Exfoliative Toxins

Another virulence factor of staphylococci is their ability to produce exfoliative toxins, exfoliative toxin A and B (ETA & ETB). These toxins result in blister formation at the epidermis surface which allows the bacteria to further penetrate and spread beneath the skin.

Membrane Damaging Toxins

There are three main forms of membrane damaging toxins (also known as type-II exotoxins) excreted by Staphylococcus, these are:

  • α-toxin – A potent membrane damaging toxin which is important for tissue invasion. In humans, platelets and monocytes are particularly sensitive to it. Cells susceptible to the α-toxin have specific receptors which it binds to, allowing it to cause damage.
  • β-toxin – This membrane damaging toxin targets membranes rich in lipids. A classic test to determine whether β-toxin is present, is to plate a sample on sheep erythrocyte-enriched growth medium. If β-toxin is present, it will lyse the erythrocytes which will be apparent on the growth medium as a clear area (as opposed to red).
  • Panton-Valentine Leukocidin (PVL) – This membrane damaging toxin, targets the cell membrane of leukocytes for which it has a high affinity. It can cause skin and soft tissue infections as well as pneumonia. Its structure allows for the formation of a pore through membranes, causing leukocyte cell contents to leak out.

MRSA – Methicillin resistant Staphylococcus aureus

Methicillin resistant S. aureus are bacteria which have developed resistance to beta-lactam antibiotics – those which target cell wall synthesis of bacteria. Such antibiotics includes the penicillin family of drugs. Their resistance is conferred by the gene meAc, which encodes for the protein penicillin-binding protein 2a (PBP2)

To determine whether a species of S. aureus is resistant:

  • The sample is first cultured
  • It must then be identified as S. aureus
  • The next step is to determine wether or not the strain is methicillin resistant
  • Once resistance is confirmed, the strain is then typed

Samples should be obtained from typical sites of carriage which include; the nasal passageway, skin, perineum and faeces. To culture MRSA, the growth medium must be selective i.e. contains a beta-lactam antibiotic, so only MRSA will grow.

At-risk patients include:

  • Dogs/Cats – Recent surgery, presence of non-healing wounds, recent or current treatment with multiple courses of broad spectrum antibiotics.
  • Horses – As above, also any length in-patient hospitalization