Staphylococcus

The family Micrococcaceae is composed of three genera : Micrococcus, Planococcus and Staphylococcus. They are catalase positive, gram positive, spherical cocci which divide incompletely in three perpendicular planes to form pairs, tetrads, short chains. Micrococci are coagulase negative and usually oxidase positive, are rarely associated with infections. They have a tendency to produce a yellow pigmented colony. Planococci are capable of withstanding saline concentrations of upto12%, arranged in tetrads, produce a yellow – brown pigment on nutrient agar. Staphylococci are associated with colonisation and/or infection of man. Some are coagulase negative while some are positive. They are ubiquitous, cause localized lesions. Snce they develop resistance to penicillin and other antibiotics they are important as human pathogen, especially in the hospital environment.

Staphylococci were first seen in pus by Koch in 1878, first cultivated in the medium by Pasteur in 1880 and named by Alexander Ogston in 1881.

Important species are:

                                    S. aureus

                                    S. epidermidis

Staphylococcus aureus :

Morphology- Spherical cocci, 1 mm in diameter, grape-like clusters. Cluster formation is due to cell division occurring in 3 planes, with daughter cells tending to remain in close proximity. Also found singly, in pairs, short chains, never long chains. They are non-motile and non-sporing. Many non-capsulated, few capsulated. Under the influence of Penicillin and certain chemicals, they may change to L-forms (irregularly shaped naturally occurring with defective cell walls).

Cultural characteristics - They grow on ordinary media at 10-40°C, pH 7.4-7.6. They are aerobes and facultative anaerobes.

On nutrient agar, after 24 hours incubation the colonies are large, circular, convex, smooth, shiny, opaque and easily emulsifiable. Most strains produce golden yellow pigment, some may be white, orange or yellow. Pigment production occurs optimally at 22°C and only in aerobic cultures. The pigment is a lipoprotein allied to carotene. On nutrient agar slope, the confluent growth presents a characteristic “oil-paint appearance”. In liquid media, uniform turbidty is produced.

On blood agar most strains are hemolytic, ie., b-type.

They grow on Mac Conkey’s medium, producing small colonies that are pink due to lactose fermentation.

Phenolphthalein phosphate agar is an indicator medium, aids in the identification of S. aureus in mixed cultures. They produce phosphatase which liberates phenolphthalein from sodium phenolphthalein diphosphate. To detect it, 0.1ml of ammonia solution is placed in the lid and the culture plate, with the culture is inverted over it. Colonies become bright pink in a minute as phenolphthalein is pink in alkaline pH.

Mannitol Salt Agar is both selective and indicator medium. Most strains ferment mannitol and produce acids and their colonies are surrounded by yellow zones. The medium contains 7.5% NaCl, which favors the growth of staphylococci.

Biochemical reactions - They ferments glucose, maltose, lactose, sucrose and mannitol producing acid but no gas. They are catalase positive, oxidase negative, hydrolyse urea, reduce nitrates to nitrites, liquefy gelatin, MR,VP positive, indole negative. Most strains are lipolyitc, produce phosphatase, coagulase positive, produce thermostable nucleases.

Susceptibility - Highly resistant to dryness, killed at 60°C for 30 minutes. Some require 80°C for 1 hr to be killed. Heat resistant strains grow at higher temperature, 45°C. Most strains grow in the presence of 10% NaCl. They resist 1% phenol for 15 minutes, sensitive to aniline dyes, crystal violet, fatty acids and resistant to lysozyme. Sensitive to Penicillin but now resistant strains has emerged. The resistance is because of the production of penicillinase, changes in bacterial surface receptors, development of tolerance.

Antigenic structure

·         Capsular polysaccharide : Some virulent strains are encapsulated. The capsule is composed of antigenic polysaccharide. It prevents ingestion of the organism by polymorphonuclear leucocytes. The capsular material may promote the adherence of the organisms to the host cells.

·         Teichoic acid : is the group-specific ribitol teichoic acid of the cell wall, is associated with peptidoglycan. S. epidermidis contains glycerol teichoic acid. It functions in the adherence of gram positive bacteria to mucosal surfaces.

·         Peptidoglycan : is a polysaccharide polymer which provides rigidity to the cell wall. It stimulates both cellular and humoral responses of the host.

·         Protein A : is a group specific antigen found in the cell wall of 90% strains. During growth of the bacteria, it is released into the culture medium. It has a molecular weight of 13, 000 daltons. It is chemotactic, anticomplementary, antiphagocytic and elicits platelet injury and hypersensitivity reaction. It binds IgG molecules, non specifically, through Fc region leaving Fab sites free to combine with specific antigen. When suspension of such sensitized cells is treated with test antigen, the antigen combines with free Fab sites of IgG attached to staphylococcal cells, this is known as coagglutination.

·         Clumping factor (bound coagulase) : is a surface component that causes the organisms to clump when mixed with plasma. This factor reacts directly with fibrinogen in plasma, causing rapid cell agglutination.

Virulence factors – S. aureus possesses a large number of cell wall associated and extracellular toxins and enzymes :

·         Extracellular toxins

1.      Haemolysins : Almost every strain of S. aureus produces one or more haemolytic, membrane damaging exotoxins known as α, β, γ and δ lysins. They differ from each other in their activity against RBC of different animal species, lethal activity dermonecrosis and leucocidal activity.

·         Αlpha lysin – In cultures it is produced under aerobic conditions and its production is enhanced by high concentration of CO₂. it is a protein consisting of a single polypeptide chain, with a molecular weight of 28,000-30,000 daltons. It is inactivated at 60⁰C, but the activity can be retained on further heating to 80⁰ – 100⁰C, because the toxin combines with a heat labile inhibitor at 60⁰C, but at higher temperature the inhibitor is inactivated and thus toxin regains activity. Above 100⁰C the toxin itself gets inactivated. The toxin lyses sheep and rabbit erythrocytes. It has haemolytic, dermonecrotic, lethal and leucocidal properties, also damages smooth muscles and toxic to human macrophages and platelets and causes degranulation of PMNLs through disruption of their lysosomes.

·         Βeta lysin – It is stongly active on sheep and weakly active on rabbit and human RBCs. It is a phospholipase C, acts specifically on sphingomyelin and lysolecithin. It exhibits ‘hot – cold hemolysis’, the cells that have been exposed to the lysin at 37⁰C lyse only when cooled at 4⁰C. It lyses platelets.

·         Gamma lysin – acts on sheep, rabbit and human RBCs.

·         Delta lysin – is a polypeptide with a molecular weight of 1600 daltons. It lyses RBCs of sheep, rabbit, horse and man. They are lethal,dermonecrotic and leucocidal.

2.      Leucocidins : consists of two protein components, based on the migration in    carboxymethylcellulose columns they are designated as F (fast) and S (slow)  components, they are dermonecrotic and damages PMNLs and macrophages.

3.      Epidermolytic toxins : Type A and Type B. They are proteins with molecular weight of 30,000 and 29,500 daltons respectively. A is heat stable and its production is determined by a chromosomal gene, while B is heat labile and plasmid controlled.

4.      Enterotoxins : are exotoxins that cause food poisoning in man. They are proteins with molecular weight of 26,000 and 30,000 daltons, are heat stable, therefore once formed enterotoxins may not be destroyed even if food is heated sufficiently. These are also not destroyed by gut enzymes. Nine antigenic types, enterotoxin F is toxic shock syndrome toxin-1. patient develops nausea, vomiting and diarrhoea. They are also pyrogenic, mitogenic, produces thrombocytopenia and hypotension.

5.      Toxic shock syndrome toxin – 1(TSST-1) : is a protein with molecular weight of 22,000 daltons. They are superantigens, ie., they are potent activators of T lymphocytes resulting in the liberation of cytokines and bind to mononuclear cells.

·         Extracellular enzymes

1.      Coagulase : It activates a coagulase– reacting factor(CRF)present in plasma, and causes the plasma to clot by the conversion of fibrinogen to fibrin. The enzyme is produced during the logarithmic phase of growth in a variety of media.

2.      Staphylokinase : is a protein with a molecular weight of 13,000 – 15,000 daltons. It has fibrinolytic activity and breakdown fibrin clots and allow spread of infection to tissues.

3.      Hyaluronidase : it hydrolyzes hyaluronic acid present in the intercellular ground substance of connective tissue, thus facilitating the spread of the organisms to adjacent areas.

4.      Deoxyribonuclease : Degrades DNA.

5.      Lipase : degrades lipid.

6.      Phospholipases : degrades phospholipids.

7.      Proteases : cause proteolysis.

Pathogenicity - S. aureus possess cytolytic toxins, which are membrane active substances. The organism produces mainly 2 types of diseases - infections and intoxications. In infections, cocci gain access to damaged skin, mucosal or tissue sites, colonize by adhering to cells, evade host defensive mechanisms, multiply and cause tissue damage. In intoxications, the disease is caused by the bacterial toxins produced either in the infected host or preformed in vitro. It is an opportunistic pathogen, causes infections at sites of lowered host resistance.

Staphylococcal diseases may be classified as cutaneous infections, exfoliative diseases, food poisoning and toxic shock syndrome.

Staphylococcal Skin Infections : Pimples caused by S. aureus. Staphylococcal skin infections are common because organisms are nearly always present on the skin. Strains of staphylococci colonize the skin and upper respiratory tract of infants within 24 hrs of birth. Infection occurs when these organinsms invade the skin through a hair follicle, producing folliculitis or pimples or pustules. An infection at the base of eyelash is sty. A larger, deeper, pus-filled infection is an abscess, an exterior abscess is known as a furuncle or boil. Further spread of infection, particularly on the neck and upper back creates a massive lesion called a carbuncle. Encapsulation of abscesses prevents them from shedding organisms into the blood, but it also prevents circulating antibodies from reaching the abscesses in effective quantities. So it is necessary to drain abscesses surgically.

            This infections transmitted through asymptomatic carriers, hospital personnel, visitors, by nasal droplets, fomites.

Exfoliative diseases : Epidermolytic toxins separate the outer layer of epidermis from the underlying tissues leading to blistering disease. The effect of these toxins is scalded skin syndrome or Ritter’s disease in which toxin spreads systemically. Patient develops painful rash which sloughs off and skin surface resembles scalding. Staphylococcal scalded skin syndrome (SSSS) occurs primarily in new borns.

Staphylococcal Enterotoxicosis (Food poisoning): Some strains causes food poisoning or enterotoxicosis by releasing enterotoxins. These toxins inflame the intestinal lining and inhibit water adsorption from the intestine. These enterotoxins also cause neural stimulation of the vomiting center of the brain. Because of their resistance to heat and drying, foods easily contaminated from food handlers or environment. The organisms multiply and release toxin in uncooked or inadequated cooked foods or refrigerated. The toxin is stable and withstands boiling for 30 minutes. Hence cooking foods kill organisms, not toxins. When toxin comes in contact with mucosa, it causes tissue damage only after it has entered the blood and has circulated back to intestine. Symptoms are abdominal pain, nausea, vomiting, diarrhoea, appear 1-6 hrs of ingestion. The best way of preventing food poisoning is to use sanitary food handling procedures.

Toxic shock syndrome(TSS): Most cases of infections develop TSS. Organisms enter the blood and produce exotoxin which enhances the effect of an endotoxin. Symptoms include fever, low blood pressure (toxic shock), red rash on the trunk later peels. Treatment with nafcillin. Death, if occurs, by shocks.

Laboratory diagnosis - Diagnosis by collecting specimens such as pus, blood, urine, faeces, sputum suspected food. Examine a Gram stained smear of pus or wound exudate which may show pus cells and gram positive cocci in clusters. The specimens are cultured on blood agar plate and the colonies are confirmed by coagulase test, slide or tube. 0.1 ml of an overnight broth culture with 0.5 ml of plasma. The mixture is incubated in a water bath at 37⁰C for 3-6 hrs. If positive, the plasma clots and does not flow when the tube is inverted. Other tests are mannitol fermentation, DNAse and phosphatase production and gelatin liquefaction.

Treatnent - Benzyl penicillin is most effective.

S. epidermidis  is the most common cause of hospital acquired urinary tract infections.. They can adhere to plastic catheters and prosthetic devices. Slime producing strains utilize their adherence factor to inhibit the action of lymphocytes and neutrophils.