Medical Bacteriology Lecture 1 - Staphylococci PDF

460 MBIO Manal M. Alkhluaifi Medical Bacteriology Lecture 1 Staphylococci M edical Bacteriology - Lecture 5 Staphylococci 0 Dr. M anal Mohammed Alkhulaifi 1  Gram p...

460 MBIO Manal M. Alkhluaifi Medical Bacteriology Lecture 1 Staphylococci M edical Bacteriology - Lecture 5 Staphylococci 0 Dr. M anal Mohammed Alkhulaifi 1  Gram positive cocci facultative Aerobic cocci Anaerobic cocci anaerobic Streptococci Staphylococci Micrococci peptostreptococci 2  How do you differentiate between Staphylococcus, Streptococcus and Micrococcus? Morphology Grapes like clusters chain tretrads 3  How do you differentiate between Staphylococcus, Streptococcus and Micrococcus? Biochemical test 1. Catalase test Catalase test is important in distinguishing streptococci (catalase-negative) staphylococci which are catalase positive. 4 2. Oxidative – fermentation test The oxidative-fermentative test is used to determine if bacteria metabolise carbohydrates oxidatively, or by fermentation, or are non-saccharolytic and therefore have no ability to use the carbohydrate in the media. Using 2 test tube containing glucose media 1st cultivate microbe &seal tube with paraffin layer 2nd cultivate microbe &let the tube opened After cultivation of the test tubes notice the glucose fermentation (media turn to yellow color) 5  3. Furazalidone sensitivity: FTO test It is the ability of organism to grow in the presence of antibacterial agent termed furazolidone 20µg/ml Furazalidone sensitivity: Staphylococci are sensitive, but micrococci are resistant 4. Bacitracin inhibition test Incubate of the microbe with bacitracin Bacitracin inhibition test: disc 0.04 micro disc micrococci are bacitracin-susceptible, staphylococci are resistant. 6  G:Staphylococcus G:Streptococcus G:Micrococcus 1-Type Facultative Facultative Aerobic cocci anaerobic anaerobic 2-Morphology Grapes like clusters chain tretrads 3- biochemical identification catalase + - + Oxidative-fermentative test open fermentation + + + close fermentation + + - Furazalidone sensitivity S(can't grow) S/R R Bacitracin R S/R S Table 1 7  Staphylococci General Characteristics Gram-positive Cocci, which appears as grape-like clusters when viewed through a microscope. Catalase-positive Grow at 15 % NaCl concentrations. Facultative anaerobes (respiration or fermentation), fermentation of glucose produces mainly lactic acid. Optimum temperature at 37°C, can grow at a temperature range (15 to 45 °C). 8  Classification of genus Staphylococcus 1-According to pigment produced by microorganism S. aureus...........golden yellow S. citrus.............yellow pigment S. albus............white pigment This classification is old and failed because there is no strict demarcation between species where S. aureus may give white colonies 2-According to the pathogenicity of the species Mostly pathogenic (S. aureus ) Cause disease in many organs invade it as osteomyelitis, Pneumonia, food poisoning within 6 hours. Moderate pathogenic (S.epidermidis) Universal skin pathogen and cause human acnes Less pathogenic (S. saprophaticus) 9 Cause urinary tract infection ( pylonephritis, cystitis....).  S. aureus S. epidermidis S. saprophaticus 1-Coagulase -free + _ _ + _ _ -bound 2-Mannitol fermentation + - Or different- 3-Tellurite reduction + - - 4-Phosphatase Test + + _ 5-Hemolysis of blood agar + _ _ 6-cell wall composition-SPA + - _ 7-Novabiocin sensitivity S S R 10  Medically Important Staphylococci Species Coagulase Positive Staphylococci (CoPS) S. aureus colonizes mainly nasal passage as normal flora, but it may be found in other sites (skin, mucous membranes, oral cavity & gastrointestinal tract). always considered a potential pathogen. causes nosocomial infections. Coagulase Negative Staphylococci (CoNS) are part of normal flora of human skin and mucous membranes. relatively low virulence. Frequently involved in nosocomial and opportunistic infections. Cause clinically significant infection associated with endocarditis, joint infection, wound infections, Bacteremia, Urinary tract infections (UTI). S. epidermidis is an inhabitant of the skin and mucous membranes, mostly nonpathogenic and may play a protective role in humans as normal flora. The production of biofilm, a significant determinant of virulence for S. epidermidis. S. saprophyticus is a leading cause of cystitis in young women. And shares in urinary tract infection. 11  Staphylococcus aureus General characteristics Pathogenicity in human The most common pyogenic organism May cause abscess, pyemia or septicemia depending individual immunity-pneumonia – conjunctivitis- pylonephritis- osteomylitis- endocarditis - impetigo– food poisoning within 6 hours- Toxic shock syndrome )TSS(-nosocomial infection- Staphylococcal scalded skin syndrome (SSSS) or Ritter's disease. S. aureus is a major cause of hospital acquired (nosocomial) infections, surgical wounds and infections associated with medical devices. 50% of normal individuals S. aureus present in the nose Morphology Gram positive Cocci arranged either (grape like cluster) Non motile organism- non sporulating. 12 Capsulated or not.  Culture characteristics Facultative anaerobic optimum temp.37○C Produce golden yellow colonies In broth media ; uniform turbidity On blood agar; hemolysis Selective media - Mannitol salt agar (salt7.5%) - Phenol ethyl alcohol agar (PEAagar) Biochemical reaction Catalase test positive Coagulase test positive Mannitol fermentation. Resistant character 1. Resist dryness for a month. 2. Highly sensitive to gentian violet so, it used for selective isolation in case of 13 mastitis.  Virulence factors Cell surface of protein (help adherence between S. aureus and host cell). ) Capsules and protein A (antiphagocytosis) Staphyloxanthin: carotenoid pigment which responsible for golden colonies, and it has an antioxidant action. Extracellular product e.g. Toxins , Enzyme Toxins. Leucocidins Kill leucocytes, macrophages, pleomorphic cells. Heamolysin Cause lysis of RBCs. Enterotoxins Produced by 30-50% of staphylococcus Caused food poisoning Resist boiling and digestive enzyme (trypsin ,pepsin) Symptoms appear after 6-8 hours as diarrhea and vomiting 4. Exofoliatin or epidermolytic toxin Causes lysis of the epidermis leading to Staphylococcus scalded skin syndrome (SSSS) also known as ritter's disease 5. Toxic shock syndrome toxin (TSST) Systemic disturbance as heart failure, lower blood pressure with diarrhea and vomiting 14  Enzyme 1. Hyaluridase Known as spreading factor. It cause lysis of the cement substance between cells helping spreading of infection. 2. Coagulase Marker of pathogenic strains Cause coagulation of plasma Its two types free and bound coagulase lead to deposit of fibrin around the microorganism, so inhibit the ability of phagocytic cell to engulf it. 3. Staphylokinase (fibrinolysin ) Cause lysis of fibrin Used for treatment of recent thrombi in the first 5 hrs after thrombus formation 15 4. DNase Cause digestion of DNA leading to impairment the biological function of the leukocytes and polymorph cells. Detection: Using DNA agar media commercially prepared (straw yellow colour ) If DNase positive: digest DNA surrounding the colony (clear zone appear around the colony) and this is confirmed by flooding the plate with diluted HCL which cause precipitation of undigested DNA. 5. Phosphatase Cause digestion of phosphate Detection: Detected by phenol phthalein phosphate agar Some non-pathogenic strains are phosphatase test positive. 6. Pencillinase (β-lactamase ) Cause inactivation of penicillin Called β-lactamase because of the destruction of β –lactam ring of the penicillin. 16  Laboratory diagnosis 1. Sampling According to the site of infection e.g. Blood in cause of pyemia and septicemia , Abcess (pus) 2. Film preparation (direct microscopic examination) See morphology 3. Cultivation see culture character 4. Biochemical identification 5. Commercial kits using depending on SPA 6. Bacteriophage typing Bacteriophage are group of virus attacking staph. Due the high specific relation between the virus and the bacteria causing lysis of the colony. There are 24 bacteriophage. Uses of bacteriophage 1-used in epidemic causes such as food poisoning. 2-not important in lab. Diagnosis. Method : Using glass plate after bottom with 24 squares Cultivat staph on the plate colony Add drop of the bacteriophage of each type on the specific square Then observe lysis after 24 hrs. 17  Staphylococcus protein A (SPA): only restricted to the most pathogenic species of Staphylococci especially Staphylococcus aureus SPA is a major cell component of Staph. aureus mainly human strains Importance of SPA 1. Marking of antibodies SPA+Ab …..SPA attached to the Fc portion of the antibody so it is important in marking appear in detection of infection 2. Has antitumor activity SPA inhibit growth of tumor through inhibition of the immunocomplexes given a chance for Ab to attach tumor cell. SPA antitumor activity nearly equal to that of the interferon Detection of protein A Protein A present in 95% of Staph. aureus 1. AGP test White line of precipitation indicate positive result 2. Hemoagglutination If the sensitized RBCs which carry antibodies attached to the Staphylococci it indicates positive result. Types of protein A 1. Extracellular 1/3. 18 2. Bound SPA to cell wall 2/3  Treatment Hospital acquired infection of S. aureus is often caused by antibiotic resistant strains (e.g. MRSA) and can be treated with vancomycin or an alternative. The term MRSA refers to Methicillin resistant S. aureus and related beta- lactam antibiotics (e.g. penicillin, oxacillin, amoxacillin). Some MRSA are resistant to vancomycin (VRSA). The infections have been treated with combination therapy using sulfa drugs (Sulfonamide) and or rifampin. Coagulase Negative Staphylococci (CoNS); produce an enzyme called beta- lactamase that makes them resistant to methicillin. Vancomycin is the most common antibiotic used to treat infections caused by CoNS; if they not resistant. Rifampin and gentamicin may be added to prevent antibiotic resistance. 19 20