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STAPHYLOCOCCUS 1 STAPHYLOCOCCI Staphylococci are Gram-positive cocci that occur in grape-like clusters. They are ubiquitous and are the most common cause of localised suppurative lesions in human beings. Their ability to develop resistance to penicillin and other antibiotics enhances their importance as a human pathogen, especially in the hospital environment. The name Staphylococcus (staphyle, in Greek, meaning 'bunch of grapes'; kokkos, meaning a berry) is due to the typical occurrence of the cocci in grape-like clusters in pus and in cultures. The genus Staphylococcus is now classified into 32 species and 15 subspecies based on the chemical composition of their cell wall components and other properties. Besides Staph aureus, three coagulase- negative speciesStaph epidermidis, Staph haemolyticus and Staph saprophyticus-can also cause human disease. Some other coagulase-negative species such as Staph hominis and Staph capitus are part of the commensal flora of the human skin. Other species are parasitic on animals. Morphology: They are spherical cocci, approximately 1 urn in diameter, arranged characteristically in grape- like clusters Cluster formation is due to cell division occurring in three planes, with daughter cells tending to remain in close proximity. They may also be found singly, in pairs and in short chains of three or four cells, especially when examined from liquid culture. Long chains never occur. They are nonmotile and nonsporing. A few strains possess microscopically visible capsules, particularly in young cultures. Many apparently noncapsulated strains have small amounts of capsular material on the surface. They stain readily with aniline dyes and are uniformly Gram positive. Under the influence of penicillin and certain chemicals, they may change to L forms. Cultural characteristics: They grow readily on ordinary media within a temperature range of 10 to 42°C, the optimum being 37 DC, and a pH of 7.4-7.6. They are aerobes and facultative anaerobes. On nutrient agar, after incubation for 24 hours, the coloniesare large (2-4 mm diameter), circular, convex, smooth, shiny, opaque and easily emulsifiable. Most strains produce golden yellow pigment, though some may be white, orange or yellow. The pigment does not diffuse into the medium. Pigment production occurs optimally at 22°C and only in aerobic cultures. Pigment production is enhanced when 1% glycerol monoacetate or milk is incorporated in the medium. The pigment is believed to be a lipoprotein allied to carotene. The colonies on blood agar are similar to those on nutrient agar.Most strains are hemolytic, especially when incubated.under 20-25% carbon dioxide. Hemolysis is marked on rabbit or sheep blood and weak on horse blood agar. In liquid media, uniform turbidity is produced. Several selective media have been devised for isolating Staph aureus from specimens such as feces containing other bacteria. These include media containing 8~ 10 per cent NaCI (salt-milk agar, salt broth), lithium chloride and tellurite (Ludlam's medium) , and polymyxin. Biochemical reactions: They ferment a number of sugars, producing acid but no gas. Sugar fermentation is of no diagnostic value except for mannitol, which is usually fermented by Staph aureus but not by other species. They are catalase positive (unlike streptococci) and usually hydrolyse urea, reduce nitrates to nitrites, liquefy gelatin and are MR and VP positive but indole negative. Most strains are lipolytic and when grown on media containing egg yolk, produce a dense opacity. Production of phosphatase can be demonstrated by culturing on nutrient agar containing phenolphthalein diphosphate. When such a STAPHYLOCOCCUS 2 culture is exposed to ammonia vapour, colonies assume a bright pink colour due to the presence of free phenolphthalein. Staph aureus strains usually exhibit the following characteristics: 1. coagulase positive; 2. greater biochemical activity, ferment mannite; 3. produce clear hemolysis on blood agar; 4. produce a golden yellow pigment; 5. liquefy gelatin; 6. produce phosphatase; 7. in a medium containing potassium tellurite, reduce tellurite to form black colonies and 8. produce thermostable nucleases which can be demonstrated by the ability of boiled cultures to degrade DNA in an agar diffusion test Resistance: Staphylococci are among the more resistant of non sporing bacteria. Dried on threads, they retain their viability for 3-6 months. Pathogenicity and virulence: Staphylococci produce two types of diseases-infections arid intoxications. In the former the cocci gain access to damaged skin, mucosal or tissue sites, colonize by adhering to cells or extracellular matrix, evade host defence 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. The virulence factors described include the following: Cell Associated Polymers The cell wall polysaccharide peptidoglycan confers rigidity and structural integrity to the bacterial cell. It activates the complement and induces release of inflammatory cytokines. Teichoic acid, an antigenic component of the cell wall, facilitates adhesion of the cocci to the host cell surface and protects them from complement- mediated opsonisation. Capsular polysaccharide surrounding the cell wall inhibits opsonisation. Cell Surface Proteins: Protein A, present on most Staph aureus strains, has many biological properties, including chemotactic, antiphagocytic and anti- complementary effects. It also induces platelet damage and hypersensitivity. Clumping factor, another surface protein, is the 'bound coagulase' which is responsible for the 'slide coagulase' test. When a saline suspension of Staph aureus is mixed on a slide with a drop of human plasma the cocci are clumped. The slide coagulase test is routinely used for the identification of Staph aureus isolates. Capsulated strains may sometimes show a negative test because the clumping factor may be enveloped by the capsular polysaccharide. Extracellular enzymes: Coagulase is an enzyme which brings about clotting of human or rabbit plasma. It acts with a 'coagulase reacting factor' (CRF) present in plasma, binding to prothrombin and converting fibrinogen to fibrin.Coagulase is an enzyme secreted into the medium. Lipases: Staphylococci produce a number of lipid hydrolases which help them infect the skin and subcutaneous tissues. Hyaluronidase breaks down the connective tissue. Staphylokinase (fibrinolysin), fatty acid modifying enzymes and proteases help in initiation and spread of infection. STAPHYLOCOCCUS 3 Nuclease: A heat stable nuclease is a characteristic feature of Staph aureus. Protein receptors: Staphylococci possess receptors for many mammalian proteins such as fibronectin, fibrinogen. Toxins Cytolytic toxins: Cytolytic toxins are membrane- active substances, consisting of four hemolysins and a leucocidin. Alpha hemolysin (alpha toxin, lysin) is the more important among them. It is a protein inactivated at 70°C, but reactivated paradoxically at 100°C. Beta hemolysin is a sphingomyelinase, hemolytic sheep celis, but not for human or rabbit cells. It exhibits a hot-cold phenomenon, the hemolysis being initiated at 37°C, but becoming evident only after chilling. Gamma hemolysin is composed of two separate proteins, both of which are necessary for hemolyti activity. Delta hemolysin has a detergent-like effect on the eel membranes of erythrocytes, leucocytes, macrophag and platelets. Leucocidin is also a two-component toxin, like the gamma lysin, being composed of two componen (S and F). Such bicomponent, membrane-active toxins as the staphylococcal leucocidin and gamma lysin has been grouped as synergohymenotropic toxins. Enterotoxin: This toxin is responsible for the manifestations of staphylococcal food poisoning- nausea, vomiting and diarrhea 2-6 hours after consuming contaminated food containing preform toxin. The toxin is relatively heat stable, resisting 100 for 10 to 40 minutes Toxic shock syndrome toxin (TSST): Toxic shock syndrome (TSS) is a potentially fatal multisystem disease presenting with fever, hypotension, myalgia, vomiting, diarrhea, mucosal hyperemia and an erythematous rash which desquamates subsequently. This is associated with infection of mucosal or sequestered sites by TSSTproducing Staph aureus strains usually belonging to bacteriophage group I. TSST type-l (formerly also known as enterotoxin type F or pyrogenic exotoxin C depending on the concentration ~ the toxin and nature of the medium. Exfoliative (epidermolytic) toxin: This toxin, also known as ET or 'exfoliatin', is responsible for the staphylococcal scalded skin syndrome' (SSSS), an exfoliative skin disease in which the outer layer of the epidermis gets separated from the underlying tissues. The severe form of SSSS is known as Ritter's disease in the newborn and toxic epidermal necrolysis in older patients. Staphylococcal Diseases Staphylococcal infections are among the most common of bacterial infections and range from the trivial to the fatal. Staphylococcal infections are characteristically localised pyogenic lesions, in contrast to the spreading nature of streptococcal infections. Cornmon staphylococcal infections are as follows: Skin and soft tissue: Folliculitis, furuncle (boil), abscess (particularly breast abscess), wound infection, carbuncle, impetigo, paronychia, less often cellulitis. Musculoskeletal: Osteomyelitis, arthritis, bursitis, pyomyositis Respiratory: Tonsillitis, pharyngitis, sinusitis, otitis, bronchopneumonia, lung abscess, empyema, rarely pneumonia Central nervous system: Abscess, meningitis, intracranial thrombophlebitis , STAPHYLOCOCCUS 4 Endovascular: Bacteremia, septicemia, pyemia, endocarditis. Urinary: Staphylococci are uncommon in routine urinary tract infections, though they do cause infection in association with local instrumentation, implants or diabetes. Urinary isolates of staphylococci are to be considered significant even with low colony counts, as they may be related to bacteremia. Bacteriophage Typing Staphylococci may be typed, based on their susceptibility to bacteriophages. An internationally accepted set of phages is used for typing. Staphylococcal phage typing is done by a pattern method. The strain to be typed is inoculated on a plate of nutrient agar to form a lawn culture. After drying, the phages are applied over marked squares in a fixed dose (routine test dose). After overnight incubation, the culture will be observed to be lysed by some phages but not by others. The phage type of the strain is expressed by the designations of all the phages that lyse it. Thus, if a strain is lysed only by phages 52, 79 and 80, it is called phage type 52/79/80. Phage typing is of great importance in epidemiological studies of staphylococcal infections. International basic set of phages for typing Staph aureus of human origin Epidemiology: Staphylococci are primary parasites of human beings and animals, colonising the skin, skin glands and mucous membranes. The most common sources of infection are human patients and carriers, animals and inanimate objects being less important. Hospital infections by staphylococci deserve special attention because of their frequency and because they are caused by strains resistant to various antibiotics. Staphylococci are a common cause of postoperative wound infection and other hospital cross-infections. Measures for the control of staphylococcal infection in hospitals include: isolation of patients with open staphylococcal lesions; Group I- 29, 52, 52A, 79, 80 detection of staphylococcal lesions among surgeons, nurses Group II -3A, 3C, 55, 71 and other hospital staff and keeping them away from work till the Group III- 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85 lesions are healed; Group IV strict aseptic techniques in theatres; the oldest, simplest and Group V -94,96 the most effective method of checking hospital cross-infection is Not allocated-81,95 hand washing, which unfortunately is often neglected. Laboratory diagnosis: The specimens to be collected depend on the type of lesion (for example, pus from suppurative lesions, sputum from respiratory infections). In cases of food poisoning, feces and the remains of suspected food should be collected. For the detection of carriers, the usual specimen is the nasal swab. Swabs from the perineum, pieces of hair and umbilical stump may be necessary in special situations. Direct microscopy with Gram-stained smears is useful in the case of pus, where cocci in clusters ma_ be seen. This is of no value for specimens like sputum where mixed bacterial flora are normally present. Diagnosis may readily be made by culture. The coagulase test can be done using two methods: tube and slide. The tube coagulase test detects free coagulase. About 0.1 ml of a young broth culture or agar culture suspension of the isolate is added to about 0.5 ml of human or rabbit plasma in a narrow test tube. EDTA, oxalate or heparin may be used as the anticoagulant for preparing the plasma.Positive and negative controls are also set up. The tubes are incubated in a water bath at 37 DC for 3-6 hours. If positive, the plasma clots and does not flow when the tube is tilted. Continued incubation is not recommended as the clot may get lysed by the STAPHYLOCOCCUS 5 fibrinogen formed by some strains. For the slide test, the isolate is emulsified in a drop of saline on a slide. After checking for absence of autoagglutination, a drop of human or rabbit plama is added to the emulsion and mixed. Prompt clumping of the cocci indicates a positive test. Positive and negative controls also are set up. Antibiotic sensitivity tests should be performed as a guide to treatment. Bacteriophage typing may be done if the information is desired for epidemiological purposes. Other typing methods include antibiogram pattern, plasmid profile, DNA fingerprinting, ribotyping and PCR-based analysis for genetic pleomorphism. Treatment As drug resistance is so common among staphylococci, the appropriate antibiotic should be chosen based on antibiotic sensitivity tests. Benzyl penicillin is the most effective antibiotic, if the strain is sensitive. For life-threatening staphylococcal infections, vancomycin is the drug of choice. Coagulase-negative Staphylococci Coagulase-negative staphylococci constitute a major component of the normal flora of the human body. Some species of coagulase-negative staphylococci can produce human infections-Staph epidermidis, Staph haemolyticus and Staph saprophyticus. MICROCOCCI These are Gram - positive cocci which occur mostly in pairs, tetrads or irregular clusters. They are catalase and oxidase positive. They are aerobic with strictly respiratory metabolism. They are parasitic 0 mammalian skin and are ordinarily nonpathogenic, They resemble staphylococci but in stained smears the cells are generally larger and more Gram variable than staphylococci. In cultures they form smaller colonies. *********** pass word:staph