Staphylococci: Classification, Structure, Virulence factor, Pathogenesis and Laboratory Diagnosis

The History of Discovery

Robert Koch discovered staphylococci in 1878. L. Pasteur obtained the pure culture of these bacteria in 1880. Later they were thoroughly studied by F. Rosenbach (1884).

Classification of Staphylococci

The genus Staphylococcus belongs to the family Staphylococcaceae. More than 40 species comprise the genus Staphylococcus. Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus saprophyticus are the three most common species.

One of the major human pathogens is S. aureus. In various tissues, wound infections, food poisoning, septicemia and many other serious disorders, it causes suppurative lesions. Representatives of normal human microflora of the skin and mucosal tissues are usually other staphylococci. S. epidermidis, however, affects immunocompromised patients and implanted appliance patients (intravenous catheters, drains, etc.)

Prosthetic device infections can also be caused by S. S. and Hominis. Haemolyticus. Hemolytic. In rare instances, S. Saprophyticus can predominantly cause urinary tract infections in young women. The remaining staphylococcal species (S. schleifeiri, S. warneri, S. capitis and many others are not considered to cause human infection.

Structure and Properties of Staphylococci

Morphology of Staphylococci

Staphylococci are small spherical microbes, 0.5-1 μm in diameter. Microbial cells are usually grouped into grape-like, irregular clusters, but it is easy to observe single cells, diplococci, and short microbial chains. Under adverse circumstances, they may turn into L-forms.

Staphylococci are non-motile bacteria without flagella and spores that are gram-positive. During infection, they can form capsules, particularly in the host tissues.

Cultivation of Staphylococci

At 37 ° C and 7.2-7.4 pH, staphylococci easily grow on basic nutrient media. They are able to produce water-insoluble pigments during cultivation: golden (mostly S. aureus), grey (S. epidermidis), and white or yellow (S. saprophyticus). In milk-supplemented media, pigment synthesis is facilitated. As a selective medium for the culture of staphylococci, egg yolk salt agar containing up to 10% NaCl is applied. The growth of concomitant bacteria is inhibited by elevated NaCl concentrations.

All staphylococci produce medium-sized pigmented, smooth, convex, glistening colonies. S. aureus generates positive lecithinase activity when growing on egg yolk agar, degrading egg yolk lecithin. Other staphylococci species do not express lecithinase in most cases. As a selective medium for staphylococci with additional detection of mannitol fermentation, mannitol salt agar with egg yolk is also used.

Selective S. aureus Baird-Parker isolation agar contains an enrichment of lithium chloride and egg yolk tellurite that prevents the growth of other bacteria. S. aureus reveals shiny convex black colonies resulting from the reduction of tellurite following overnight incubation. In blood-containing media, staphylococci can cause hemolysis of rabbits, sheep and human erythrocytes. Staphylococcal cultures in liquid nutrient media develop diffuse opacity.

Biochemical properties of Staphylococci

Facultative anaerobes are staphylococci. They ferment carbohydrates that without gas, yield acid metabolites (e.g. lactic acid). Proteins with hydrogen sulphide production are used by these bacteria. Staphylococci (predominantly S. aureus) liquefy gelatin, milk is coagulated and nitrates are reduced to nitrites. They also generate catalase that distinguishes them from streptococci, as well as urease, phosphatase, and certain other enzymes. The production of coagulase sets S. aureus apart from other members of the same genus (with rare exceptions). Therefore, S. aureus refers to bacteria that are coagulase-positive, while other staphylococci are coagulase-negative. Similarly, S. aureus refers to thermostable nucleases.

Antigenic structure of Staphylococci

Staphylococci possess antigenic polysaccharides and proteins in peptidoglycan of microbial cell wall and microcapsule. Cell wall teichoic acids carry additional antigenic determinants of staphylococci.

Virulence factors of Staphylococci

A great variety of virulence factors, including exo- and endotoxins, are expressed by S. aureus. Most of them are controlled by plasmids; some of them may be under chromosomal control. S. aureus manufactures α-, β-, γ- and δ-hemolysins.

The deadly, necrotic, and hemolytic activity is alpha-hemolysin or alpha-toxin. It is a pore-forming toxin capable of being incorporated into the target cell membrane with subsequent impairment of the membrane. This toxin readily lyses rabbit erythrocytes, damages smooth muscle cells and platelets, etc. For rabbits, it is lethal when injected. β-Toxin restores the activity of sphingomyelinase. The membranes of human erythrocytes and many other cells are damaged by it. Erythrocytes of many mammalian species, as well as white blood cells (neutrophils and macrophages), can be affected by γ-Hemolysin. δ-Hemolysin damages different mammalian cells’ cytoplasmic membranes. Within the membrane lipid bilayer, it is able to aggregate, thus forming membrane channels that mediate cell lysis. During infection, leukocytes and bone marrow precursors of blood cells are destroyed by Poreforming leukocidin (or Panton-Valentine toxin).

More than 10 variations of heat-stable enterotoxins are synthesized by S. aureus, causing food poisoning. They are resistant to proteolytic intestinal enzymes. Enterotoxins exhibit high biological ability and activate a large subset of T-lymphocytes. The latter is followed by the production of redundant proinflammatory cytokines by T cells and macrophages (IL-1, IL-2, IL-6, IL-12, alpha-TNF, etc The release of cytokines causes systemic inflammation with severe tissue damage. The gastrointestinal tract that results in vomiting and diarrhea is mostly affected by enterotoxins.

The toxin toxic shock syndrome (TSST) resembles enterotoxins B and C in structure. In particular, it can induce toxic shock syndrome in menstruating women who have used absorbent tampons. Tampons can be contaminated by staphylococci that produce TSST. TSST has strong super antigenic activity that with fever, collapse, desquamative skin rashes and multi-organ dysfunction, can ultimately result in systemic shock.

Staphylococcal exfoliative toxins (ETA and ETB) promote similar actions. Exfoliatins are taken from the primary site of the skin infection and carried to large areas of the skin through the bloodstream. Deep cellular layers of the epidermis are destroyed, resulting in staphylococcal skin scalding syndrome. This disease primarily affects and may be fatal in newborn infants. More than 50 percent of the area of the skin may be damaged. The skin becomes red, wrinkled, and large clear fluid-filled blisters appear. General symptoms are also essential for the disease, such as malaise and fever. Specific antitoxic antibodies can prevent the development of syndromes.

In addition, Staphylococcal peptidoglycan has super antigenic activity. It stimulates inflammation and encourages host leukocyte (endotoxin-like activity) chemotaxis. In most S. aureus strains, Protein A is anchored within the cell wall. It binds IgG molecules of various mammalian species including humans, to the Fc portion. Protein A is considered to hinder the activation of the complement and the binding of IgG to immune cells. The S. aureus capsule promotes microbial survival within the phagocytes.

Staphylococci can develop a large number of destructive enzymes in addition to exo- and endotoxin production. S.aureus coagulase is capable of converting serum prothrombin into thrombin that with fibrin clotting, activates blood coagulation. On the microbial surface, fibrin threads enable staphylococci to avoid the attachment of phagocytes. Staphylokinase activates the plasminogen, promoting blood clot fibrinolysis within 24-48 hours.

Staphylococci generate hyaluronidase, or spreading factor, which breaks down connective tissue hyaluronic acid, facilitating microbial invasion. S. aureus lecithinase hydrolyzes the phospholipid component of cellular membranes, lecithin. Staphylococcal β-lactamases break down the bonds within the beta-lactam ring, causing beta-lactam antibiotics to be microbially insusceptible. Beta-lactamase action is overcome only by specially designed beta-lactam drugs (e.g. methicillin, oxacillin, several cephalosporins and carbapenems).

The production of β-Lactamase is usually under plasmid control. Nonetheless, methicillin resistant Staphylococcus aureus (or MRSA) strains have emerged from penicillin-binding protein (PBP) chromosome-dependent alteration. Bacteria produce modified low-affinity PBP2a protein with beta-lactam antibiotics. The chromosomal gene mecA is encoded. It was later found that staphylococcal methicillin resistance confers microbial insusceptibility to nearly all beta-lactams. MRSA has now become a huge public health problem because it causes numerous life-threatening infections that are not responsive to antibiotic therapy.

Resistance of Staphylococci

Staphylococci are bacteria which are relatively resistant. In a 10% medium of sodium chloride, they can propagate. These microbes develop drying, freezing, heating resistance (maintaining their viability at 70° for more than 1 hour) and certain chemical substances. Boiling inactivates microbial cells rapidly. Staphylococci are also sensitive to disinfectants containing chlorine and certain aniline dyes.

Pathogenesis and Clinical Findings of Staphylococcal Infections

Staphylococcal infections infect a large number of mammalian species including humans. Staphylococci, in particular S. epidermidis and S. saprophyticus, are nevertheless representatives of the normal human skin and respiratory tract flora. In 40-50% of humans, nasal S. aureus carriage is revealed. But staphylococcal virulence is ensured by a large number of pathogenic factors, including toxins and destructive enzymes, and a considerable invasive capacity.

Staphylococci, predominantly S. aureus, cause local and generalized (i.e, invasive) infections. Through the skin and mucous tissues that are followed by local microbial propagation, staphylococci enter the host. They can, finally overcome tissue barriers and infect the blood. In all body tissues, furuncles (boils), carbuncles, paronychia, hidradenitis, chronic pyoderma, abscesses and phlegmons, periostitis, osteomyelitis, otitis, appendicitis, cholecystitis, pyelonephritis and many other diseases, Staphylococcus aureus can cause or participate in suppurative local lesions.

Pneumonia, peritonitis and meningitis are also caused, as are post-operative wound infections. Almost all of these situations can lead to staphylococcal septicemia, leading to disseminative infection. S. aureus participates actively in mixed infections.

Clinical manifestations of specific staphylococcal infections are produced by the actions of various toxins. They should be considered to be toxic infections. After ingestion of foods (daily products, cakes, pastry, ice cream, etc.) contaminated with pathogenic bacteria, staphylococcal food poisoning appears. Enterotoxins are thermostable and withstand 30 minutes of heating at 100oC.

Scalded skin syndrome and toxic shock syndrome result from infections caused by specific strains of staphylococci producing toxins. Anti-toxic antibodies appearing in toxic staphylococcal infections may neutralize the action of the toxin. Nevertheless most staphylococcal infections are shown to only trigger short-term low-grade immune responses. The substantial mechanism for staphylococci elimination is considered to be phagocytosis.

In general, S. epidermidis is less pathogenic than S. aureus, but highly deleterious complications are emphasized in immunocompromised patients and in implanted prosthetic devices (e.g., bacterial endocarditis and septicemia). Overall, the most common causative agents for hospital-acquired infections are S. aureus and S. epidermidis. S. saprophyticus may affect young women’s urogenital tract and may be a rare cause of wound infections.

Laboratory Diagnosis of Staphylococcal Infections

Pus, wound discharge, tracheal aspirate, spinal fluid, sputum, urine, blood, contaminated foods, lavage fluids, feces, etc are obtained from specimens.

For staphylococcal infection validation, microscopy is used as a preliminary test. Gram-stained smear examination typically reveals gram-positive cocci arranged or separately settled into grape-like clusters. Molecular genetic tests, e.g., can be used to develop rapid differential diagnosis of different staphylococcal species directly in clinical samples, e.g. Uh. PCR.

Specimens are planted on blood agar and egg yolk salt agar for microbial culture isolation. For staphylococci, the latter medium is selective. Blood is inoculated into the glucose broth in cases of septicemia. Hemolysis on blood agar is rendered by S. aureus culture. Golden pigment and positive lecithinase activity in yolk salt agar are also produced. The catalase test enables staphylococci and streptococci to be discerned (the latter are devoid of catalase activity).

A positive coagulase test for S. aureus is essential. By inoculation of microbial culture into citrated rabbit plasma, the identification is carried out. If the clot forms within hours, it is determined that the test is positive. S. aureus ferments mannitol and produces thermostable nucleases, but not other staphylococci. These tests for discrimination against S. aureus may be valuable. Serological tests are of limited value for staphylococcal infection verification. Susceptibility testing finalizes the staphylococci investigation. Methods of Disk Diffusion and broth microdilution are used. S. aureus (i.e., MRSA strains) resistant to methicillin are determined by PCR.

Treatment and Prophylaxis of Staphylococcal Infections

For staphylococcal infection therapy, drugs that block cell wall synthesis are most appropriate.  Unfortunately, beta-lactamases are produced by most S. aureus isolates, thus conferring resistance to penicillin G or amoxycillin. Therefore for antibacterial treatment, beta-lactamase-resistant penicillin (e.g., oxacillin and methicillin) and cephalosporins, as well as carbapenems, are used here. Specific inhibitors of β-lactamases (e.g., clavulanic acid) in combination with antibiotics give an additional beneficial effect on the outcome of treatment.
In about 20 percent of S. aureus (i.e., MRSA strains) and about 75 percent of S. epidermidis strains, resistance to oxacillin and methicillin appears. Other cell wall synthesis inhibitors, vancomycin glycopeptides or teicoplanin, are used for the treatment of these bacteria. Aminoglycosides, macrolides, lincosamides, tetracyclines (e.g., tigecycline), and linezolid’s should be administered in combination with antibiotics, blocking the synthesis of microbial proteins.

Specific passive immune therapy (e.g., anti-staphylococcal γ-globulin) may be given in cases of chronic staphylococcal infections, particularly in immunocompromised patients and infants. It is also possible to administer a toxoid derived from S. aureus alpha-toxin to activate anti-staphylococcal immunity. Specific staphylococcal toxoid prophylaxis is recommended for patients who are susceptible to staphylococcal infections. The spread of staphylococcal infections can be limited by adequate hospital disinfection and prevention of staphylococcal transport among medical personnel.


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