Staphylcoccus. Objective

Transcription

1 Staphylococcus

2 Staphylcoccus Objective Cell morphology Importance Characteristics Classification natural habitat pathogenicity Pathogenesis Diseases Laboratory Diagnosis

3 Cell morphology and Important characteristic μ Gram positive cocci arranged in irregular clusters or bunches of grapes formation Average diameter of cocci is 1.0 micrometer Facultative anaerobe catalase positive, Oxidase Negative Non motile Growth occur on NA and Blood agar but not on MacConkey agar coagulase positive and coagulase negative

4 Sputum smear Staphylococcus & Streptococcus pneumoniae

5 Staphylococci, EM.

6 Classification Family Micrococcaceae Change in normenclature and New species Previous name present name Significant S. hyicus S. hyicus causes exudative subsp. hyicus epidermitis in pig S. hyicus S. chromogenes causes exudative subsp. chromogenes isolate from bovine mastitic milk most of species name rely on species of animal host. Coagulase positive ( pathogenic ) and coagulase negative ( non pathogenic )

7 Classification S. caprae S. gallinarum S. lentus S.equarum S. simulans, S.felis S.delphiini Goat s milk Skin of chicken skin of goats and sheep Skin of horses clinical specimen in cats skin of dolphins

8 Natural habitat Nasal cavity (man) Skin and mucous membranes of mammals can be transient in the G.I. Tract

9 Pathogenesis and Pathogenicity Antigenic structures and virulence factors *Cell wall component peptidoglycan Teicoic acid Protein A Clumbing factors Capsule * Enzyme catalase coagulase hyaluronidase beta-lactamase nuclease staphylokinase lipase * Toxin Hemolysin Leucocidin Exfoliatin Enterotoxin

10 Pathogenesis and Pathogenicity *Cell wall component peptidoglycan Teicoic acid Protein A Clumbing factors Capsule

11 Pathogenesis and Pathogenicity Important of Cell wall component peptidoglycan - rigid exoskelton - endotoxin like activity *induce local Swartzman partway (hypersensitivity) *Alternative complement pathway ( Staphylococcal protein A) Teicoic acid - Activate complement - Antiphagocytic ability

12 Pathogenesis and Pathogenicity Important of cell wall component Protein A - binding to Fc terminal of IgG - activate complement - Antiphagocytosis Clumping factors (cell bound coagulase) - Antiphagocytosis Capsule - Antiphagocytosis

13 Pathogenesis and Pathogenicity * Enzyme catalase coagulase hyaluronidase beta-lactamase nuclease staphylokinase lipase Urease Collagenase Proteases

14 Pathogenesis and Pathogenicity Enzymes catalase - reduce killing by phagocytic cells ( reduce H 2 O 2 produced by phagocytic cells) Coagulase which causes plasma coagulation in vitro - Cell bound coagulase ( clumping factor ) - Soluble (Free) coagulase coagulase reacting factor ( in Plasma) + free coagulase coagulase - thrombin + fibrinogen Fibrin ( clot )

15 Pathogenesis and Pathogenicity Hyaluronidase ( spreading factor ) is cause of cell dead by acts on intercellular bridge. Staphylokinase ( Fibrinolysin )is plasminogen activator. Nucleases : DNAase ( Deoxyribonuclease ) RNAase ( Ribonuclease )

16 Pathogenesis and Pathogenicity Toxins Hemolysin Leukocidin Epidermolytic toxin Exfoliatin or Demonecrotic toxin Enterotoxin

17 Pathogenesis and Pathogenicity Toxins The alpha hemolytic toxin ( hemolysin ) is associated with gangrenous mastitis in cattle Leukocidin kills neutrophils and macrophages of cattle, rabbit and humans. Epidermolytic toxins are implicated in porcine exudative epidermitis

18 Pathogenesis and Pathogenicity The stapylococcal haemolysin Haemolysin Type of Characteristics haemolysis Alpha Complete Cytotoxic, Acts on membrane lipids and causes of spasm of smooth muscle Beta Incomplete Basis of the CAMP test ( Partial ) Delta Complete Lethal and necrotising effect in rabbit Gamma causes oedema in rabbit

19 Pathogenesis and Pathogenicity Exfoliatin produces staphylococcal scalded skin syndrome ( SSSS ) in human infants and possibly in dogs ( pustular dermatitis ). Enterotoxin ( A-E ) involved in human food poisoning, they act by reflex stimulation of the emetic center.

20 Skin

21 Summary of virulence factors of Staphylococcus aureus

22 Pathogenesis and Pathogenicity Pathogenicity of staphylococci depend on cell wall component, toxins and enzymes produced. Pyogenic associated with abscesses formation and suppuration PUS = debris of dead leukocytes living and dead bacteria Intact Phagocytic cells and fibrin strand Fibrous capsule will be formed around the abscesses (capsule)

23 Host Defenses Antibodies are produced which neutralize toxins and promote opsonization. Phagocytosis is the major mechanism for combatting staphylococcal infection. The capsule and protein A may interfere with phagocytosis.

24 Diseases Staphylococcus aureus Many animal species Cattle Sheep,Goat Pigs Horse Abscesses and suppurative conditions Mastitis Udder impetigo Mastitis Stapylococcal Dermatitis Mastitis endometritis Udder impetigo ( after arations of teeth of piglets) Mastitis Botryomycosis after castration

25 Diseases Staphylococcus aureus Rabbits Poultry Dogs, Cat Exudative dermatitis Bumble-foot ( pyogranulomatous ) arthritis and septicemia in turkeys omphalitis Suppurative condition Pyoderma pustular dermatitis pyometra Otitis externa wound infection

26 Diseases Staphylococcus aureus subsp. anaerobius Sheep lesion similar to those of caseous lymphadenitis (Colynebacterium pseudotuberculosis) Staphylococcus intermedius Dogs, Cats Canine (Feline) pyoderma Pyometra Otitis externa Infection of respiratory tract, bones, joints, wounds, eyelid and conjunctiva

27 Diseases Staphylococcus hycus Pigs Cattle Exudative epidermitis ( greasy pig disease ) Usually in pigs under 7 weeks old ( high mortality rate ) rare case of mastitis

28 Laboratory diagnosis Specimens - Exudates - Pus from abscesses -mastitic milk - Skin Scrapings -Urine - Other affected tissues

29 Laboratory diagnosis Direct morphoscopy Gram-stained smear of pus or exudates ( Gram positive cocci in the typical of bunches of grapes formation )

30 Laboratory diagnosis Isolation Medium for isolation Sheep or Ox blood agar ( MacConkey agar plate is inoculated in parallel to detect any gram negative Selective medium for gram Positive Sheep or Ox blood agar with 15 mg. Nalidixic acid and 10 mg. Colistin sulphate per litre of medium inoculate plate are incubated aerobically at 37 0 C for hes.

31 Laboratory diagnosis Identification Colonial characteristics Microscopic appearance Test for the pathogenicity of staphylococcal isolates Biochemical test Antibiotic susceptibility testing Phage Typing

32 Laboratory diagnosis Identification Colonial characteristics - 4 mm. In diameter -round - smooth - glistening -hemolysis - pigmentation

33 Laboratory diagnosis Identification ( continue) Microscopic appearance

34 Laboratory diagnosis Identification ( continue) Tests for the pathogenicity of staphylococcal isolate Coagulase test slide coagulase test tube coagulase test Dnase test Test for protein A

35 Laboratory diagnosis Coagulase test slide coagulase test 1. A loop full of staphylococcal culture is emulsified in a drop of water on microscopic slide. 2. A loop full of rabbit plasma is added and mixed well with bacterial suspension. 3. A positive reaction is indicates by clumping within 1-2 minute.

36 Laboratory diagnosis Coagulase test Tube coagulase test ml. of rabbit plasma is placed in a small test tube. 2. Two drop of an overnight broth culture of the staphylococcus or heavy suspension made from the culture on the agar plate in sterile water, are added. 3. The tube is rotated gently to mix the contents and then incubate at 37 0 C. 4. A positive test with clotting of the plasma can occur in 2-4 hrs.

37 Coagulase positive S. aureus S. intermedius S. hyicus

38 Laboratory diagnosis DNase test Commercially available Dnase Agar This test is not entirely reliable as an indicator of pathogenicity ( 18 % of coagulase-negative stapylococci have Dnase activity ).

39 Laboratory diagnosis Test for protein A Commercially available Kits -SeroSTAT ( Scott labs Inc., Fiskeille, RI.)

40 Laboratory diagnosis Biochemical test Antibiotic Susceptibility test Phage typing ( by staphylococcus bacteriophage ) - for epidemiological purposes - Particularly for S. aureus from human cases of food poisoning. - Animal staphylococcal phage types are usually different from those of human origin.

41 Biochemical test Catalase O/F Mannitol fermentation Maltose fermentation Aesculin hydrolusis Novobiocin susceptibility test Polymyxin susceptibility test Bacitracin susceptibility test

42 Biochemical test MANNITOL SALTS AGAR A common medium used for the isolation of staphylococci is the Mannitol Salts Agar. The high salt concentration of this medium inhibits the growth of most other organisms. Pathogenic staphylococci not only grow on the medium, but they also produce acid from it. This acid production turns the ph indicator from red to yellow.

43 MANNITOL SALTS AGAR

44 Laboratory diagnosis Commercial Identification Systems ส าหร บแบคท เร ยแกรมบวกใน family Micrococcaceae ( ใช modified carbohydrate fermentation tests) a. API Staph-IDENT b. API Staph c. ID32 Staph d. Minitek Gram-Positive e. Vitek Gram Positive Identification Card f. MicroScan Rapid Pos Combo Panel g Microscan Positive ID Panel h. Minitek Gram Positive Panel i. Sceptor Gram Positive MIC / ID Panel j. Staf-Sistem 18-R k. Staph-ZYM l. Microbial Identification System m. Biolog Microplate Identification System

45 Cluster of cocci

46 Staphylococcus epidermidis Staphylococcus saprophyticus Coagualse positive Staphylococcus Micrococcus species

47 Treatment Infections can usually be treated with penicillinaseresistant bata-lactams antibiotics antibiotic resistant strains and can only be treated with vancomycin.

48 The knowledge is not only in the classroom. THE END