Labquality External Quality Assessment Programmes General Bacteriology 1 2/2013

Transcription

1 Labquality External Quality Assessment Programmes General Bacteriology 1 2/2013 Photos and text: Markku Koskela, M.D., Ph.D. Clinical microbiology specialist Oulu, Finland

2 Sample 11/2013 Pus sample from an infected wound of a Syrian refugee. Finding: Escherichia coli as a pathogen Lab exercises: Isolation and identification of the pathogen. Antimicrobial susceptibility tests.

3 Sample 11/2013 Pus from a wound. Growth on sheep blood agar and chocolated blood agar plates in 5% CO 2 at 35 C after 24 hours. ->Gray, glistening, convex bacillar colonies.

4 Sample 11/2013. Pus from a wound. Gram-negative rods Growth on sheep blood agar and chocolated blood agar plates in 5% CO 2 at 35 C after 48 hours. ->Gray, glistening, convex bacillar colonies.

5 Sample 11/2013. E coli 1. API20E Escherichia coli (99,5%) Oxidase + - +, + - -, - - +, - - +, + - +, + + +, Vitek V2C GN E. coli

6 Sample 12/2013 Middle ear effusion drawn from outer auditory canal. Acute otitis media in a 53-years-old male with spontaneous perforation of typanic membrane. Findings: Pneumococcus (Streptococcus pneumoniae ) as a pathogen. Normal flora of the outer auditory canal including Staphylococcus lugdunensis, (a co-pathogen??) and Micrococcus sp. Lab exercises: Isolation and identification of the pathogens. Interpretation of the findings.

7 Sample 12/2013 Middle ear effusion Growth on sheep blood agar and chocolated blood agar plates in 5% CO 2 at 35 C after 24 hours. ->1. Flat, gray colonies surrounded by a narrow alpha-haemolytic zone. ->2. Small colonies. How many different kinds?

8 Sample 12/2013 Middle ear effusion Growth on sheep blood agar and chocolated blood agar plates in 5% CO 2 at 35 C after 48 hours. ->1. Flat, gray alpha-haemolytic streptococcal colonies. ->2. Flat, yellowish, glistening, beta-haemolytic colonies. ->3. Convex, dry, yellow colonies.

9 Sample 12/2013 Middle ear effusion Growth on sheep blood agar and chocolated blood agar plates in 5% CO 2 at 35 C after 24 hours. ->1. Alpha-haemolytic colonies. ->2. Flat, yellowish colonies. ->3. Convex, dry, yellow colonies.

10 Sample 12/2013. (14/2011) Streptococcus pneumoniae Pneumococcal colonies The pneumococcus, an alpha-haemolytic streptococcus which is sensitive to optochin. (24 hours, 35 C) Gram-positive diplococci

11 Sample 12/2013. (3/2010) Staphylococcus lugdunensis Growth on a sheep blood agar plate after 24 hours at 35 C. Flat, yellowish, glistening and beta-haemolytic colonies which are similar to S aureus colonies. Gram-positive cocci in clusters

12 Sample 12/2013. (3/2010) Staphylococcus lugdunensis S.lugdunensis is giving a positive agglutination reaction in a Staphylococcal latex test like S.aureus, Staphaurex Remel Europe Ltd, UK Pasteurex TM Staph Plus, Bio-Rad, France but S.lugdunensis do not coagulate rabbit plasma ie. It is a coagulase negative staphylococcus species. Rabbit plasma coagulation test: S lugdunensis is negative S aureus is positive

13 Sample 12/2013. (3/2010) Staphylococcus lugdunensis S aureus S lugdunensis Growth on a selective S aureus identification plate (SAID, biomerieux, France) after 24 hours at 35 C. S aureus is green and S lugdunensis is white.

14 Sample 12/2013. (3/2010) Staphylococcus lugdunensis 1. API20Staph: Staphylococcus lugdunensis ( 97 %) - + +, + + -, + - -, - + -, + - -, + - +, Vitek-2 GP Staphylococcus lugdunensis

15 Sample 12/2013 (6/2011) Micrococcus sp. Furazolidone resistent growth Catalase positive Growth on a sheep blood agar plate after 24 hours at 35 C. Convex, dry, yellow colonies. Gram-positive tetracocci

16 Sample 13/2013 Tissue sample from a stab wound in the left leg of 47-years-old male construction worker taken after an accident at work. Findings: Clostridium novyi as a pathogen and Corynebacterium jeikeum as a co-pathogen. Lab exercises: Isolation and identification of the pathogens. Interpretation of the findings.

17 Sample 13/2013 Tissue sample from leg. Gram-staining of the native specimen Gram-positive coccobacilli and 2. long, Gram-positive, spore-forming bacilli (clostridia?)

18 Sample 13/2013. Tissue sample from leg. Aerobic primary culture Growth on sheep blood agar and chocolated blood agar plates in 5% CO 2 at 35 C after 24 hours. ->Small, graywhite colonies.

19 Sample 13/2013. Tissue sample from leg. Aerobic primary culture Growth on sheep blood agar and chocolated blood agar plates in 5% CO 2 at 35 C after 48 hours. ->Small, convex, graywhite colonies.

20 Sample 13/2013 (10/2011) Corynebacterium jeikeum Catalase+ (bubbles) Oxidase neg. (colourless) Aerobic growth on a sheep blood agar plate after 48 hours at 35 C. Small, graywhite to yellowish, convex colonies, which are catalase positive. Note! Streptococci are catalase negative. Short pleomorphic Gram-positive rods

21 Sample 13/2013 (10/2011) Corynebacterium jeikeum 1. RapID CB Plus Corynebacterium jeikeum (99,9%) Catalase + - +, - - -, - - -, + - -, - - -, + - -, + 2. Vitek-2 ANC Corynebacterium jeikeum

22 Sample 13/2013. Tissue sample from leg. Anaerobic primary culture Anaerobic growth on an anaerobic blood agar plate (FAA) after 48 hours at 35 C. A great inhibition zone around a metronidazole disk ->anaerobes! The colonies are growing close to a tobramycin disk. Note! The aerobic bacterium seen on aerobic plates is not growing on this anaerobic plate.

23 Sample 13/2013. Tissue sample from leg. Anaerobic primary culture Anaerobic growth on FAA-plate after 48 hours at 35 C. Cluster of colonies surrounded by several beta-haemolytic zones with variable intensity! ->Aerotolerance test and Gram staining

24 Sample 13/2013. Tissue sample from leg. Anaerobic primary culture on EYA plate. 2 1 Anaerobic growth and reactions on an egg-yolk-agar (EYA) -plate after 48 to 72 hours at 35 C. Flat, irregular, spreading colonies which 1. are surrounded by a pearly lipase reaction (Lipase +) and 2. by a lecithinase reaction. (Lecithinase+)

25 Sample 13/2013 Aerotolerance test on a FAA-plate. Blood agar: aerobic; Chocolated blood agar: 5% CO 2 ; FAA: anaerobic 1. Left quadrant: A Gram-positive bacillus, which is growing only in aerobic conditions on blood and chocolated blood agar plates. (Corynebacterium jeikeum). 2. Right quadrant: A Gram-positive, spore-forming bacillus, which is growing only on the anaerobic FAA-plate. It is an obligate anaerobe (Clostridium novyi).

26 Sample 13/2013. Clostridium novyi Catalase negative (No bubbles) Anaerobic growth on a FAA-plate after 72 hours at 35. Flat, irregular, spreading colonies surrounded by beta-haemolytic zones of different intensity. Spot indole test negative (Colourless)

27 Sample 13/2013. Clostridium novyi Anaerobic identification test (AI) Kanamycin S Vankomycin S Colistin R Metronidazole S. A Gram-positive anaerobe (because vankomysin is S and colistin R). A spore-forming bacillus -> suspicion of a Clostridium sp. Gram-positive spore-forming rods (subterminal spores)

28 Sample 13/2013. Clostridium novyi 1. RapID ANA II Clostridium tetani (99 %), But the positive indole reaction is not right. See the negative result in the spot indole test. (C tetani is lecithinase negative, etc.) When indole is negatíve -> Clostridium novyi (96%) - - -, - - -, - - -, + - -, - - +, (?)

29 Sample 14/2013. CT-mediated biopsy sample from a lumbar vertebra of a middle-aged male suffering from spondylodiscitis. Finding: Staphylococcus saccharolyticus as a pathogen. Lab exercises: Isolation and identification of the pathogen. Interpretation of the finding.

30 Sample 14/2013. CT-mediated biopsy sample from a lumbar vertebra. Gram-staining of a native specimen. Gram-positive cocci

31 Sample 14/2013. CT-mediated biopsy sample from a lumbar vertebra. Aerobic primary culture. Results on sheep blood agar and chocolated blood agar plates in 5% CO 2 at 35 C after seven days. ->No growth.

32 Sample 14/2013. CT-mediated biopsy sample from a lumbar vertebra. Anaerobic primary culture. Anaerobic growth on anaerobic blood agar (FAA) after 48 hours at 35 C. Flat, white, glistening coccal colonies : 1) which are growing around a metronidazole disk and 2) which are sensitive to tobramycin indicating that the bacterium is not a real anaerobe although it seems to grow only in anaerobic conditions.

33 Sample 14/2013 Aerotolerance test Blood agar: aerobic; Chocolated blood agar: 5% CO 2 ; FAA: anaerobic 1. Blood agar (left): No growth 2. Chocolated blood agar (in the middle): No growth. 3. Anaerobic blood agar (FAA): White coccal colonies. An anaerobe.

34 Sample 14/2013. Staphylococcus saccharolyticus Catalae: positive (Bubbles) Growth on anaerobic blood agar (FAA) after 48 hours at 35 C. Flat, white, glistening, staphylococcal colonies. Spot indole test: negative (Colourless)

35 Sample 14/2013. Staphylococcus saccharolyticus Anaerobic identification test (AI): Kanamycin S Vankomycin S Coilistin R. Metronidazole R. A Gram-positive bacterium (because vancomycin is S and colistin R). Middle-sized Gram-positive cocci in clusters

36 Sample 14/2013. Staphylococcus saccharolyticus 1. RapID ANA II: (Not present in the test panel) - - -, - - -, - - -, - - (+)/-, - - (+)/-, (+)/ Vitek V2C ANC Staphylococcus saccharolyticys

37 Have Nice Summerdays!