laboratory diagnosis of staphylococcus

lab diagnosis of staphylococcus powerpoint

1. LABORATORY DIAGNOSIS OF STAPHYLOCOCCUS MADE BY: SHALINI BISHT Saturday, February 18, 2017 1

2. LABORATORY DIAGNOSIS Sample collection and Transportation Direct smear Microscopy Culture Biochemicals Typing of Staphylococcus aureus Antibiotic Sensitivity Testing (AST) Saturday, February 18, 2017 2

3. SAMPLE COLLECTION Type of sample depends on the site of infection. Saturday, February 18, 2017 3 Infection Specimen Suppurative lesion Pus, wound swab Respiratory infection Sputum UTI Mid stream urine PUO, Bacteremia Blood Food poisoning Feces, Vomitus, food Carriers Nasal and perianal swab

4. DIRECT SMEAR MICROSCOPY Staphylococcus appear as GPC measuring 0.5-1.5 microns Occur singly, in pairs, short chains or clusters Present within and outside PMNs Reporting of direct smears: quantitation of cell types and microorganisms Eg . Many pus cells along with moderate number of GPC seen Saturday, February 18, 2017 4

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6. CULTURE Specimens are inoculated onto the suitable media. Plates incubated for 18-24 hour at 37°C. On nutrient agar Colonies are golden yellow and opaque with smooth glistening surface, 2-4 mm in diameter, circular, convex, shiny & easily emusifiable . (Most strains produce non diffusible Golden yellow pigment) Nutrient Agar slope Confluent growth, Oil paint appearance. Saturday, February 18, 2017 6

7. Blood agar Colonies similar to those on Nutrient Agar Colonies are beta-hemolytic Liquid medium Uniform turbidity MacConkey Agar Small pink colonies due to Lactose fermentation Saturday, February 18, 2017 7

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9. Selective media MSA – 1% Mannitol + 7.5% NaCl + phenol red Salt milk agar – 6.5% NaCl + 10% skimmed milk Ludlam’s medium – Lithium chloride and tellurite Saturday, February 18, 2017 9

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11. BIOCHEMICAL REACTIONS Catalase : positive Coagulase test : positive Oxidase : negative (Except S.sciuri group i.e., S.sciuri , S.lentus , S.vitulinus ) Ferment glucose, lactose, maltose, sucrose and mannitol , with production of acid but no gas Indole : negative MR test : positive VP test : positive Gelatin liquefaction : positive Phosphatase : positive DNA- ase : positive Saturday, February 18, 2017 11

12. Catalase test Done to distinguish staphylococci from streptococci ( catalase negative) Saturday, February 18, 2017 12

13. Coagulase test Done to distinguish pathogenic strain ( S.aureus ) from non-pathogenic strains. 2 Methods of coagulase detection are: Slide coagulase test : detects bound coagulase Tube cogulase test : detects free coagulase (other coagulase + ive staphylococci are S.intermedius , S.hyicus ) Saturday, February 18, 2017 13

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15. Gelatin liquefaction Principle: this test is used to determine the ability of an organism to produce proteolytic enzyme ( gelatinase ) that liquefies the gelatin. Saturday, February 18, 2017 15

16. Methyl Red test Principle: this test detects the production of sufficient acid during fermentation of glucose by bacteria and sustained maintenance of ph below 4.5 Saturday, February 18, 2017 16

17. Voges-Proskauer (VP) test Principle: the test depends upon the production of acetoin from pyruvic acid in the media. In the presence of alkali & atmospheric oxygen, acetoin is oxidised to diacetyl which reacts with alpha naphthol to give red color. Saturday, February 18, 2017 17

18. DNA Hydrolysis Principle : this test is used to determine the ability of an organism to hydrolyze DNA. Green color of the medium is due to DNA-methyl green complex. If the organism growing on the medium hydrolyzes DNA, the green color fades & the colony is surrounded by a colorless zone. Saturday, February 18, 2017 18

19. Phosphatase test Principle: Staphylococci are grown on nutrient agar containing sodium phenolphthalein diphosphate and incubated overnight at 37°C. The plate is exposed to ammonia vapours . The pink color of the colonies indicate a positive result. Saturday, February 18, 2017 19

20. S.aureus v/s CONS Saturday, February 18, 2017 20

21. BACTERIOPHAGE TYPING Saturday, February 18, 2017 21

22. Epidemiological purpose to trace source of infection. Useful in outbreaks like food poisoning in a community. Typing methods: Phenotypic- bacteriophage typing: staphylococci are typed based on their to bacteriophages . Molecular typing: DNA finger-printing, ribotyping , PFGE etc. Saturday, February 18, 2017 22

23. Bacteriophage typing Method Test strain inoculated as lawn culture on NA. Drops of routine test dose of known set of different phages are spot inoculated & incubated. Zone of lysis will be produced in those areas where test strain is susceptible to phages applied. If strain lysed by phages 29, 52A, 79, but not other phages; it is designated as phage type 29/52A/79 National Reference Centre: MAMC, New Delhi Saturday, February 18, 2017 23

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25. ANTIBIOTIC SENSITIVITY TESTING This is important as staphylococci develop resistance to drugs readily. Saturday, February 18, 2017 25

26. Why AST has become a necessity ?? Bacteria have the ability to develop resistance following repeated or subclinical doses, so more advanced antibiotics are required to overcome them. Antibiotic sensitivity test: A laboratory test which determines how effective antibiotic therapy is against a bacterial infections. Testing will assist the clinicians in the choice of drugs for the treatment of infections. Helps in the local pattern of antibiotic prescribing. Saturday, February 18, 2017 26

27. Methods of AST Performed on MHA by Kirby-Bauer Disc diffusion method. Following antibiotics are employed for staphylococcus: Amoxyclav Clindamycin Cefoxitin Ciprofloxacin Erythromycin Gentamicin Linezolid Levofloxacin Penicilin -G Vancomycin Teicoplanin Saturday, February 18, 2017 27

28. Kirby-Bauer method Dry the agar plates (MHA) & label them. Dip a sterile swab into the broth and express any excess moisture by pressing the swab against the side of the tube. Swab is streaked as a lawn (lawn culture) onto a Mueller-Hinton agar (in 3 directions to ensure confluence). The anitibiotic (s) disk will be placed onto the MHA plate. The plate is incubated and is examined for resistance and sensitivity pattern the following day. Saturday, February 18, 2017 28

29. MRSA M ethicillin - r esistant S. a ureus . First reported in 1960s. May colonize mucosal or epithelial surfaces, (common : anterior nares ) Nosocomial pathogen. Shows Resistant to penicillins , cephalosporins , carbapenems , monobactams . Vancomycin resistance is rare – so far Hospital-acquired (HA MRSA) Community-acquired cases now (CA MRSA) Saturday, February 18, 2017 29

30. Predisposing factors for MRSA Prolonged & repeated hospitalization Indiscriminate use of antibiotics Intravenous drug abuse Presence of indwelling medical devices Saturday, February 18, 2017 30

31. MECHANISM MRSA contains the mecA gene which is responsible for the production of an altered plasma (cell) membrane-bound enzyme, penicillin-binding protein 2a (PBP- 2a.) The altered PBP 2a while able to perform its cell-wall synthesis function, has a lower affinity and does not bind to beta- lactam antibiotics Thus, the presence of the mecA gene confers resistance to all beta- lactam antibiotics such as methicillin . Saturday, February 18, 2017 31

32. Vancomycin remains the drug of choice for treatment of infections caused by MRSA, although it is intrinsically less active than the antistaphylococcal penicillins . Combinations of vancomycin with ss-lactam antibiotics may be synergistic in vivo against MRSA strains, including those with intermediate susceptibility to vancomycin . Given the increasing prevalence of MRSA in hospitals and in community settings, alternative approaches are needed for treatment of infections caused by MRSA. Saturday, February 18, 2017 32

33. Preventive measures Isolation & treatment of MRSA patients. Detection of carriers among hospital staff, their isolation & treatment. Avoid indiscriminate usage of antibiotics. Following strict aseptic technique Saturday, February 18, 2017 33

34. HAND WASHING STILL CONTINUES TO REDUCE SEVERAL INCIDENCES OF MRSA SPREAD IN HEALTH CARE Saturday, February 18, 2017 34

35. Detection of MRSA MRSA is determined by disc diffusion test using cefoxitin (30µg) disc on MHA with 2% NaCl & 10 4 cfu /ml inoculum and incubated at 33-35°C for 24 hour. As per CLSI guidelines inhibition zone of </= 21 mm was taken to be MRSA. Saturday, February 18, 2017 35

36. Coagulase Negative Staphylococci Two species of coagulase negative Staphylococci can cause human infections: 1 . Staphylococcus epidermidis 2. Staphylococcus saprophyticus Saturday, February 18, 2017 36

37. S. epidermidis : It is a common cause of stitch abscesses. It has predilection for growth on implanted foreign bodies such as artificial valves, shunts, intravascular catheters and prosthetic appliances leading to bacteremia . In persons with structural abnormalities of urinary tract, it can cause cystitis. Endocarditis may be caused, particularly in drug addicts. Saturday, February 18, 2017 37

38. S.saprophyticus : It causes urinary tract infections, mostly in sexually active young women. The infection is symptomatic and may involve the upper urinary tract also. Men are infected much less often. It is one of the few frequently isolated CoNS that is resistant to Novobiocin Saturday, February 18, 2017 38

39. Other coagulase negative staphylococci: S.haemolyticus S.saprophyticus   S.warneri , S.hominis , S.epidermidis S.caprae S.lugdunensis Saturday, February 18, 2017 39

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