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Part III - Chapter 21 - Staphylococci_F.ppt
- 1. Universities Press © Universities Press (India) Private Limited Universities Press 3-6-747/1/A & 3-6-754/1, Himayatnagar Hyderabad 500 029 (A.P.), India Part III Bacteriology Email: info@universitiespress.com marketing@universitiespress.com Phone: 040-2766 5446/5447
- 2. Universities Press © Universities Press (India) Private Limited STAPHYLOCOCCI
- 3. Universities Press © Universities Press (India) Private Limited STAPHYLOCOCCI • Gram-positive cocci – grape-like clusters • Localised suppurative lesions • Resistance to penicillin • Human pyogenic lesions – von Recklinghausen • Sir Alexander Ogston – Staphylococcus • Rosenbach – S. aureus and S. albus • Passet – S. citreus
- 4. Universities Press © Universities Press (India) Private Limited STAPHYLOCOCCI MORPHOLOGY Staphylococcus aureus in Gram stain
- 5. Universities Press © Universities Press (India) Private Limited MORPHOLOGY • Spherical cocci, approximately 1ɥm in diameter • Arranged in grape-like clusters • Cluster formation – cell division in three planes, daughter cells close proximity • Under influence of penicillin – change to L forms STAPHYLOCOCCI
- 6. Universities Press © Universities Press (India) Private Limited CULTURAL CHARACTERISTICS • Nutrient agar – colonies are large, circular, opaque • Golden yellow pigment • Pigment production enhanced by incorporation of milk in the medium • Nutrient agar slope – oil paint appearance STAPHYLOCOCCI
- 7. Universities Press © Universities Press (India) Private Limited CULTURAL CHARACTERISTICS • Blood agar – hemolytic, rabbit or sheep blood • MacConkey agar – small pink colonies, lactose fermentation • Liquid media – uniform turbidity • Selective media – media containing 8–10% NaCl (salt milk agar, salt broth), lithium chloride and tellurite (Ludlam’s medium) and polymyxin STAPHYLOCOCCI
- 8. Universities Press © Universities Press (India) Private Limited STAPHYLOCOCCI • Blood agar Growth on blood agar
- 9. Universities Press © Universities Press (India) Private Limited BIOCHEMICAL REACTIONS • Catalase positive (unlike Streptococci) • Hydrolyse urea • Reduce nitrates to nitrites • MR and VP positive STAPHYLOCOCCI
- 10. Universities Press © Universities Press (India) Private Limited STAPHYLOCOCCI Differences between S.aureus and S.epidermidis
- 11. Universities Press © Universities Press (India) Private Limited RESISTANCE • Thermal death point – 62°C for 30 minutes • Resist 1% phenol for 15 minutes • Aniline dyes – strongly bactericidal • Resistant to lysozymes • Sensitive to lysostaphin – a mixture of enzymes produced by a particular strain of S. epidermidis STAPHYLOCOCCI
- 12. Universities Press © Universities Press (India) Private Limited PENICILLIN RESISTANCE • Uniformly sensitive to penicillin in the pre- antibiotic era • Penicillin used clinically – resistant strains emerged • Penicillin resistance – three types: – Production of beta lactamase – Alteration in the penicillin binding protein – Tolerance to penicillin STAPHYLOCOCCI
- 13. Universities Press © Universities Press (India) Private Limited PENICILLIN RESISTANCE • Beta lactamase (penicillinase) – inactivates penicillin by splitting the beta lactam ring • Inducible enzyme • Production controlled by plasmids transmitted by transduction or conjugation STAPHYLOCOCCI
- 14. Universities Press © Universities Press (India) Private Limited PENICILLIN RESISTANCE • Alterations in the penicillin binding protein (PBP 2a) and changes in bacterial surface receptors reduce binding of beta lactam antibiotics to cells • Resistance also covers beta lactamase – resistant penicillins such as methicillin, methicillin resistant Staphylococcus aureus (MRSA) STAPHYLOCOCCI
- 15. Universities Press © Universities Press (India) Private Limited • Some strains show resistance to other antibiotics like erythromycin, tetracycline and aminoglycosides • These strains have been called ‘epidemic methicillin resistant Staphylococcus aureus’ (EMRSA) • Tolerance to penicillin, by which the bacterium is only inhibited but not killed STAPHYLOCOCCI
- 16. Universities Press © Universities Press (India) Private Limited PATHOGENICITY AND VIRULENCE • Two types of disease: – Infections – Intoxications STAPHYLOCOCCI
- 17. Universities Press © Universities Press (India) Private Limited INFECTIONS • Cocci gain access to damaged skin, mucosal or tissue sites • Colonise by adhering to cells or extracellular matrix • Evade host defence mechanisms • Multiply and cause tissue damage STAPHYLOCOCCI
- 18. Universities Press © Universities Press (India) Private Limited INTOXICATION • Caused by bacterial toxins – produced either in the infected host or preformed in vitro PATHOGENICITY AND VIRULENCE
- 19. Universities Press © Universities Press (India) Private Limited CELL-ASSOCIATED POLYMERS • Cell wall polysaccharide peptidoglycan • Techoic acid – antigenic component of the cell wall • Capsular polysaccharide VIRULENCE FACTORS
- 20. Universities Press © Universities Press (India) Private Limited CELL SURFACE PROTEINS • Protein A • Clumping factor –‘bound coagulase’ VIRULENCE FACTORS
- 21. Universities Press © Universities Press (India) Private Limited EXTRACELLULAR ENZYMES 1) Coagulase 2) Lipid hydrolases or lipases 3) Hyaluronidase 4) Nuclease 5) Protein receptors VIRULENCE FACTORS
- 22. Universities Press © Universities Press (India) Private Limited TOXINS 1) Cytolytic toxins: alpha hemolysin, beta hemolysin, gamma hemolysin, delta hemolysin 2) Leucocidin (Panton Valentine toxin) 3) Enterotoxin 4)Toxic shock syndrome toxin (TSST) 5) Exfoliative toxin (epidermolysin) toxin VIRULENCE FACTORS
- 23. Universities Press © Universities Press (India) Private Limited CELL-ASSOCIATED POLYMERS • The cell wall polysaccharide peptidoglycan confers rigidity and structural integrity. It activates complement and induces release of inflammatory cytokines. VIRULENCE FACTORS
- 24. Universities Press © Universities Press (India) Private Limited CELL-ASSOCIATED POLYMERS • Teichoic acid – facilitates adhesion of cocci to host cell surface and protects them from complement-mediated opsonisation • Capsular polysaccharide surrounding the cell wall inhibits opsonisation VIRULENCE FACTORS
- 25. Universities Press © Universities Press (India) Private Limited CELL SURFACE PROTEINS • Protein A – present in most S. aureus strains • Biological properties – chemotactic, anti- phagocytic and anti-complementary effects • Induces platelet damage and hypersensitivity VIRULENCE FACTORS
- 26. Universities Press © Universities Press (India) Private Limited Co-agglutination • Protein A binds to the Fc terminal of the IgG molecules, leaving the Fab region free to combine with its specific antigen • Protein A bearing staphylococci coated with any IgG antiserum will be agglutinated if mixed with corresponding antigen CELL SURFACE PROTEINS
- 27. Universities Press © Universities Press (India) Private Limited Co-agglutination • Applications – Streptococcal grouping and gonococcal typing CELL SURFACE PROTEINS
- 28. Universities Press © Universities Press (India) Private Limited Clumping factor • ‘Bound coagulase’ responsible for the slide coagulase test CELL SURFACE PROTEINS
- 29. Universities Press © Universities Press (India) Private Limited Coagulase • An enzyme that brings about clotting of human or rabbit plasma • Acts with a coagulase reacting factor (CRF) present in plasma • Binds to prothrombin • Converts fibrinogen to fibrin • Basis of tube coagulase test EXTRACELLULAR ENZYMES
- 30. Universities Press © Universities Press (India) Private Limited Lipid hydrolases or lipases • Help infect skin and subcutaneous tissues Hyaluronidase • Breaks down connective tissue • Staphylokinase (fibrinolysin) and proteases help in initiation and spread of infection EXTRACELLULAR ENZYMES
- 31. Universities Press © Universities Press (India) Private Limited Nuclease • Heat stable nuclease is characteristic feature of S. aureus Protein receptors • Staphylococci possess protein receptors for many mammalian proteins such as fibronectin, fibrinogen, IgG and Clq • Facilitate staphylococcal adhesion to host cells and tissues EXTRACELLULAR ENZYMES
- 32. Universities Press © Universities Press (India) Private Limited CYTOLYTIC TOXINS • Alpha hemolysin – alpha toxin, lysin • Leucocidal, cytotoxic, dermonecrotic, neurotoxic and lethal • Toxic to macrophages, lysosomes, muscle tissues, the renal cortex and the circulatory system TOXINS
- 33. Universities Press © Universities Press (India) Private Limited CYTOLYTIC TOXINS • Beta hemolysin – a sphingomyelinase, exhibits hot-cold phenomenon, hemolytic activity initiated at 37°C but becomes evident after chilling TOXINS
- 34. Universities Press © Universities Press (India) Private Limited CYTOLYTIC TOXINS • Gamma hemolysin – two separate proteins, both necessary for hemolytic activity • Delta hemolysin – detergent-like effect on cell membrane of erythrocytes, leucocytes, macrophages and platelets TOXINS
- 35. Universities Press © Universities Press (India) Private Limited LEUCOCIDIN • Panton Valentine Toxin or PVL, named after its discoverers • Two components: S and F TOXINS
- 36. Universities Press © Universities Press (India) Private Limited ENTEROTOXIN • Responsible for manifestations of Staphylococcal food poisoning – nausea, vomiting and diarrhea • 2–6 hours after consuming food contaminated by preformed toxin TOXINS
- 37. Universities Press © Universities Press (India) Private Limited ENTEROTOXIN • Meat, fish, milk and milk products contaminated with staphylococci, toxin accumulates • Source of infection – food handler • Disease – self-limited • Tests to detect toxin – ELISA, latex agglutination TOXINS
- 38. Universities Press © Universities Press (India) Private Limited TOXIC SHOCK SYNDROME (TSS) – a potentially fatal multisystem disease, presenting with fever, hypotension, myalgia, vomiting, diarrhea, mucosal hyperemia and an erythematous rash TOXINS
- 39. Universities Press © Universities Press (India) Private Limited • Associated with infection of the mucosal or sequestered sites by toxic shock syndrome toxin produced by S. aureus • TSST antibodies seen in convalescent • Tampon-related TSS is now rare TOXIC SHOCK SYNDROME (TSS)
- 40. Universities Press © Universities Press (India) Private Limited • Syndrome occurs in other infections of skin, mucosa and other sites • Staphylococcal enterotoxins and TSST-1 are superantigens – potent activators of T lymphocytes TOXIC SHOCK SYNDROME (TSS)
- 41. Universities Press © Universities Press (India) Private Limited • Leads to excessive and dysregulated immune response, release of cytokines, interleukins, tumour necrosis factor and interferon gamma • Explains multisystem involvement and florid manifestations in staphylococcal food poisoning and TSS TOXIC SHOCK SYNDROME (TSS)
- 42. Universities Press © Universities Press (India) Private Limited EXFOLIATIVE TOXIN • Exfoliative (epidermolytic) toxin, also known as ET or ‘exfoliatin’, responsible for staphylococcal scalded skin syndrome (SSS) • SSS is an exfoliative skin disease • The outer layer of the epidermis becomes separated from the underlying tissues TOXINS
- 43. Universities Press © Universities Press (India) Private Limited EXFOLIATIVE TOXIN • Severe form – Ritter’s disease in newborn, toxic epidermal necrolysis in older patients • Milder forms – pemphigus neonatorum and bullous impetigo TOXINS
- 44. Universities Press © Universities Press (India) Private Limited Localised pyogenic lesions • Skin and soft tissue – folliculitis, furuncle (boil), abscess (particularly breast abscess), wound infection, carbuncle, impetigo, paronychia, less often cellulitis • Musculoskeletal – osteomyelitis, arthritis, bursitis, pyomyositis STAPHYLOCOCCAL DISEASES
- 45. Universities Press © Universities Press (India) Private Limited • Respiratory – tonsillitis, pharyngitis, sinusitis, otitis, bronchopneumonia, lung abscess, empyema, rarely pneumonia • Central nervous system – abscess, meningitis, intracranial thrombophlebitis • Endovascular – bacteremia, septicemia, pyemia, endocarditis STAPHYLOCOCCAL DISEASES
- 46. Universities Press © Universities Press (India) Private Limited • Urinary – infection in association with instrumentation, implants or diabetes • Toxin mediated staphylococcal diseases: – Food poisoning – Toxic shock syndrome – Scalded skin syndrome STAPHYLOCOCCAL DISEASES
- 47. Universities Press © Universities Press (India) Private Limited Phenotypic–Bacteriophage typing • Based on susceptibility to an internationally accepted set of phages Molecular typing • DNA fingerprinting, ribotyping and PCR • Outbreak investigation – pulse field gel electrophoresis (PFGE) and sequence-based typing methods TYPING METHODS
- 48. Universities Press © Universities Press (India) Private Limited BACTERIOPHAGE TYPING Bacteriophage typing of staphylococci
- 49. Universities Press © Universities Press (India) Private Limited • Staphylococci – primary parasites, human beings and animals • Colonise – skin, skin glands and mucous membranes • Source of infection – human patients and carriers • Patients with superficial infections and respiratory infections disseminate large number of staphylococci into the environment EPIDEMIOLOGY
- 50. Universities Press © Universities Press (India) Private Limited • Healthy persons • 10–30% carry staphylococci in the nose • 5–10% in the vagina • Colonisation of umbilical stump – common • Some carriers, called shedders, disseminate large numbers of cocci for prolonged periods EPIDEMIOLOGY
- 51. Universities Press © Universities Press (India) Private Limited • Staphylococcal disease may follow endogenous or exogenous infection. • Mode of transmission: – Contact – direct/fomites – Dust – Airborne droplets EPIDEMIOLOGY
- 52. Universities Press © Universities Press (India) Private Limited Methicillin resistant S.aureus (MRSA) • Hospital infections – frequency – strains resistant to a variety of antibiotics • Post-operative wound infections and hospital cross-infections • Resistant to all beta lactam antibiotics and other antimicrobial agents EPIDEMIOLOGY
- 53. Universities Press © Universities Press (India) Private Limited Hospital strains • Present in hospital environment • Belong to a limited number of phages • Resistant to penicillin and other antibiotics used in the hospital MRSA
- 54. Universities Press © Universities Press (India) Private Limited Epidemic strains • Cause epidemics of hospital cross-infection • Cause of concern – emergence of CA MRSA earlier restricted to hospital setting HA MRSA MRSA
- 55. Universities Press © Universities Press (India) Private Limited • Isolation of patient with open staphylococcal lesion • Detection of staphylococcal lesions among surgeons, nurses and other hospital staff and keeping them away from work till lesions heal OUTBREAK CONTROL MEASURES
- 56. Universities Press © Universities Press (India) Private Limited • Strict asepsis in operation theatre • Hand washing – simplest and most effective method • Search for carriers among hospital staff • Carriers treated with local application of mupirocin or chlorhexidine OUTBREAK CONTROL MEASURES
- 57. Universities Press © Universities Press (India) Private Limited 1) Specimen 2) Microscopy 3) Culture 4) Identification 5) Serological tests LABORATORY DIAGNOSIS
- 58. Universities Press © Universities Press (India) Private Limited SPECIMEN • Type of lesion • Pus – suppurative lesions • Sputum – respiratory infections • Feces, remains of food – food poisoning • Carriers – nasal swab • Swabs from perineum, umbilical stump, pieces of hair may be necessary in special situations LABORATORY DIAGNOSIS
- 59. Universities Press © Universities Press (India) Private Limited LABORATORY DIAGNOSIS MICROSCOPY • Gram-stained smear - useful Staphylococcus aureus in Gram stain
- 60. Universities Press © Universities Press (India) Private Limited LABORATORY DIAGNOSIS CULTURE • Blood agar – colonies appear after overnight incubation Growth on blood agar
- 61. Universities Press © Universities Press (India) Private Limited CULTURE • Selective media – swabs from carriers, feces in food poisoning • Salt milk agar, Ludlam’s media • Staphylococci isolated – smear from culture, coagulase test done LABORATORY DIAGNOSIS
- 62. Universities Press © Universities Press (India) Private Limited IDENTIFICATION • Tube coagulase test – detects free coagulase • 0.1 ml of broth culture added to 0.5 ml of human or rabbit plasma, positive and negative controls are set up • Incubated – water bath at 37°C for 3–6 hours • If positive, plasma clots and does not flow when the tube is tilted LABORATORY DIAGNOSIS
- 63. Universities Press © Universities Press (India) Private Limited TUBE COAGULASE TEST Tube coagulase test: positive, negative
- 64. Universities Press © Universities Press (India) Private Limited • Slide coagulase test – detects bound coagulase • The isolate is emulsified in a drop of saline on a slide • A drop of human or rabbit plasma is added and mixed • Positive and negative controls are set up • Prompt clumping indicates a positive test LABORATORY DIAGNOSIS
- 65. Universities Press © Universities Press (India) Private Limited SLIDE COAGULASE TEST Slide coagulase test: negative and positive
- 66. Universities Press © Universities Press (India) Private Limited LABORATORY DIAGNOSIS IDENTIFICATION • Antibiotic sensitivity tests –important –drug resistance Zone of inhibition around antibiotic discs on the lawn culture of test bacteria
- 67. Universities Press © Universities Press (India) Private Limited LABORATORY DIAGNOSIS IDENTIFICATION • Typing – methods include antibiogram pattern, plasmid profile, DNA fingerprinting, ribotyping, and PCR based analysis
- 68. Universities Press © Universities Press (India) Private Limited IDENTIFICATION • Serological tests • Help in diagnosis of deep infections • Anti-staphylococcal titres of more than two units per ml – value in diagnosis of deep- seated infections – bone abscess LABORATORY DIAGNOSIS
- 69. Universities Press © Universities Press (India) Private Limited • Drug resistance – appropriate antibiotic based on antibiotic sensitivity tests • Benzyl penicillin – most effective if strain is sensitive • Cloxacillin used against penicillinase- producing strains TREATMENT
- 70. Universities Press © Universities Press (India) Private Limited • MRSA – resistant to all beta lactam antibiotics • Life-threatening staphylococcal infections – vancomycin – drug of choice • Mild superficial infections – topical applications – bacitracin, chlorhexidine or mupirocin • Carriers – local application – mupirocin TREATMENT
- 71. Universities Press © Universities Press (India) Private Limited • S.intermedius • S.hyicus OTHER COAGULASE-POSITIVE STAPHYLOCOCCI
- 72. Universities Press © Universities Press (India) Private Limited • Major component of normal flora • Some species can produce human infections – S.epidermidis, S.haemolyticus and S.saprophyticus COAGULASE-NEGATIVE STAPHYLOCOCCI
- 73. Universities Press © Universities Press (India) Private Limited • S.epidermidis – stitch abscess • Predilection for growth on implants – artificial heart valves, shunts, intravascular catheters – leading to bacteremia COAGULASE-NEGATIVE STAPHYLOCOCCI
- 74. Universities Press © Universities Press (India) Private Limited • Hospital strains – multidrug resistance • Can cause cystitis and central line associated blood tream infection (BSI) • Endocarditis in drug addicts COAGULASE-NEGATIVE STAPHYLOCOCCI
- 75. Universities Press © Universities Press (India) Private Limited Biofilm production • Important factor in the pathogenesis of infection by S.epdermidis • An extracellular polysaccharide matrix which protects the bacteria from antibacterial agents COAGULASE-NEGATIVE STAPHYLOCOCCI
- 76. Universities Press © Universities Press (India) Private Limited S. saprophyticus • Present in normal flora of skin –periurethral area – causes urinary tract infection in sexually active females • Sensitive to most antibiotics • Resistant to nalidixic acid and novobiocin COAGULASE-NEGATIVE STAPHYLOCOCCI
- 77. Universities Press © Universities Press (India) Private Limited • Gram-positive cocci – tetrads or irregular clusters • Non-pathogenic • Hugh–Leifson oxidation fermentation test – to differentiate micrococci and staphylococci MICROCOCCI
- 78. Universities Press © Universities Press (India) Private Limited Micrococci MICROCOCCI