Corynebakterier - mikrobiologi og klinik Kristian Schønning

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1 Corynebakterier - mikrobiologi og klinik Kristian Schønning Department of Clinical Microbiology 445 Hvidovre Hospital DK-265 Hvidovre Klinisk mikrobiologisk afdeling Navn (Sidehoved/fod)

2 Corynebakterier difterigene og non-difterigene Corynebacterium (>5 arter): Gram+, irregulære stave Katalase+ Normal hud og slimhindeflora Difterigene: C. diphteriae (4 biovarer) C. ulcerans C. pseudotuberculosis Alle kan indeholde det fag-bårne difteri-toxin gen Alle er Pyrazinamidase-negative. Non-difterigene: Corynebacterium spp. Bakteriæmi, endocardit, Opportunistiske patogener Fremmedlegeme-infektion Med undtagelse af C. macginleyi er alle Pyrazinamidase-positive. Dept of Clinical Microbiology 445 Hvidovre Hospital DK-265 Denmark Kristian Schønning Maj 13

3 Difteri i England From Wagner et al. (); Epidemiol. Infect. 13: Dept of Clinical Microbiology 445 Hvidovre Hospital DK-265 Denmark Kristian Schønning Maj 13

4 2 toxigene isolater fra UK: 42 C. diphteriae, 59 C. ulcerans, 1 C. pseudotuberculosis From Wagner et al. (); Epidemiol. Infect. 13: Dept of Clinical Microbiology 445 Hvidovre Hospital DK-265 Denmark Kristian Schønning Maj 13

5 Symptomatologi og vaccinationsstatus Dept of Clinical Microbiology 445 Hvidovre Hospital DK-265 Denmark Kristian Schønning Maj 13

6 Corynebakterier difterigene og non-difterigene Corynebacterium (>5 arter): Gram+, irregulære stave Katalase+ Normal hud og slimhindeflora Difterigene: C. diphteriae (4 biovarer) C. ulcerans C. pseudotuberculosis Alle kan indeholde det fag-bårne difteri-toxin gen Alle er Pyrazinamidase-negative. Non-difterigene: Corynebacterium spp. Bakteriæmi, endocardit, Opportunistiske patogener Fremmedlegeme-infektion Med undtagelse af C. macginleyi er alle Pyrazinamidase-positive. Dept of Clinical Microbiology 445 Hvidovre Hospital DK-265 Denmark Kristian Schønning Maj 13

7 Identifikation af Corynebakterier Fænotypiske metoder, e.g. API coryne panel; i et studium >% misidentifikationer VITEK 2 ANC kort. Genotypiske metoder rrn (S rrna) (og evt. rpob). Flere species tæt relaterede; e.g. C. ulcerans og C. pseudotuberculosis <1% afstand. MALDI-TOF-MS Diskriminerer difterogene spp. (99.1%; Konrad et al., ). Identificerer non-difterogene spp. korrekt med undtagelse af C. aurimocosum/minutissimum og C. pseudodiphteriticum/propinquum (Bruker Biotyper; Alatoom et al., 12). Fuldgenomsekventering Dept of Clinical Microbiology 445 Hvidovre Hospital DK-265 Denmark Kristian Schønning Maj 13

8 C. urealyticum: Et overset urinvejspatogen? Langsomt voksende; lipofil; inkubation >24h ofte nødvendig Kraftig urease-danner; alkaniserer urin; danner struvit/apatit-sten Ofte multiresistent Asymptomatisk bakteriuri (%). Typisk bestående urinvejspatologi og immunsuppression (e.g. RTX) Akut UTI Kronisk UTI Encrusted cystit/pyelit Dept of Clinical Microbiology 445 Hvidovre Hospital DK-265 Denmark Kristian Schønning Maj 13

9 Corynebakterier som luftvejspatogener? Dept of Clinical Microbiology 445 Hvidovre Hospital DK-265 Denmark Nhan et al. (12) identificerede 27 corynebakterier isoleret fra luftvejssekreter genetisk 15/27 (56%) skønnedes inficerede /15 (6%) C. pseudodiphteriticum 4/15 (27%) C. striatum 2/15 (13%) C. accolens Diez-Aguilar et al. (13) identificerede ptt. (MALDI-TOF-MS) 4/ (%) C. pseudodiphteriticum 4/ (%) C. propinquum 2/ (%) C. striatum C. pseudodiphteriticum adhærerer, internaliseres og overlever intracellulært i humane epithel-cellelinier Kristian Schønning Maj 13

10 Konklusioner Corynebakterier kan være årsag til klinisk infektion; specielt hos disponerede individer Artsidentifikation kan være vejledende for klinisk tolkning af fund og behandling MALDI-TOF-MS giver mikrobiologiske laboratorier mulighed for korrekt identifikation Resistensforhold er artsafhængige og er ofte komplicerede. Dept of Clinical Microbiology 445 Hvidovre Hospital DK-265 Denmark Kristian Schønning Maj 13

11 Resistensbestämning av Corynebakterier Erika Matuschek NordicAST Workshop 13

12 Corynebacterium spp. Inga brytpunkter Få artspecifika MIC-distributioner Problem med artidentifiering Ingen lappdiffusionsmetod

13 Resistensbestämning och artidentifiering av Corynebakterier Kliniska isolat av olika geografiskt ursprung SENTRY-kollektionen (R. Jones, JMI Labs, USA) Spanien (L. Martinez-Martinez, Spanien) Kronobergs län Artidentifiering med Maldi-tof Resistensbestämning Buljongspädning (BMD) Etest Lappdiffusion

14 Validering av Maldi-tof MS EUCAST Laboratory for AST Organism* ID med Maldi-tof Växjö Score-värde C. amycolatum C. amycolatum (12/12) 2. C. jeikeium C. jeikeium (12/12) 2. C. pseudodiphtheriticum C. pseudodiphtheriticum (11/12) C. lipophile grpe F1 (1/12) C. striatum C. striatum (12/12) 2. C. urealyticum C. urealyticum (12/12) 2. (/12) (2/12) *Enligt API Coryne och/eller SrRNA eller tidigare utförd Maldi-tof. Meaning of Score Values Range Description highly probable species identification secure genus identification, probable species identification probable genus identification not reliable identification

15 Corynebacterium spp. No C. striatum 76 C. amycolatum 63 C. jeikeium 35 C. pseudodiphtheriticum C. urealyticum 1 C. afermentans 9 C. aurimucosum C. propinquum 5 C. singulare 4 C. coyleae 3 C. imitans 3 C. striatum 2 C. glucuronolyticum 2 C. minutissimum 2 C. xerosis 2 C. diphtheriae 1 C. freneyi 1 C. mucifaciens 1 C. riegelii 1 C. simulans 1 C. ulcerans 1 Total olika species % score 2. ID med score bedömdes som riktiga om fyra upprepningar gav identiska resultat Score < 2. vanligast för C. afermentans och C. amycolatum

16 Resistensbestämning Metod Medium Inokulat Inkubering BMD 5 x 5 cfu/ml - h (44 h) 35±1 C Luft (förseglade paneler) Etest MH-F McFarland.5 - h (44 h) 35±1 C 5% CO 2 MH-Fbuljong Lappdiffusion MH-F McFarland.5 - h (44 h) 35±1 C 5% CO 2 MH-F-buljong: Mueller-Hintonbuljong med 5% lyserat hästblod och mg/l β-nad

17 Inkuberingstid Lappdiffusion % av isolaten inkuberades 44 h C. jeikeium (6/35) C. pseudodiphtheriticum (6/) C. urealyticum (9/1) MIC (broth microdilution) % av isolaten inkuberades 44 h C. amycolatum (1/63) C. jeikeium (22/35) C. pseudodiphtheriticum (/) C. urealyticum (1/1)

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19 Testade antibiotika Bensylpenicillin Ciprofloxacin Moxifloxacin Gentamicin Vankomycin Klindamycin Tetracyklin Linezolid Rifampicin Trimetoprim-sulfametoxazol

20 No of isolates Benzylpenicillin MIC vs. species Corynebacterium spp., 25 clinical isolates C. striatum C. amycolatum C. jeikeium C. pseudodiphtheriticum C. urealyticum C. afermentans C. aurimucosum C. propinquum C. singulare Other MIC (mg/l)

21 No of isolates 1 Benzylpenicillin 1 unit vs. species Corynebacterium spp., 25 clinical isolates 6 C. striatum C. amycolatum C. jeikeium C. pseudodiphtheriticum C. urealyticum C. afermentans C. aurimucosum C. propinquum C. singulare Other Inhibition zone diameter (mm)

22 No of isolates 1 6 Benzylpenicillin 1 unit vs. MIC Corynebacterium spp., 25 clinical isolates Benzylpenicillin breakpoints Staphylococci.12/.12 Strep A,B,C,G.25/.25 S. pneumoniae.6/2 Viridans strep.25/2 PK/PD.25/2 MIC (mg/l) Inhibition zone diameter (mm) Suggested breakpoints: S.12, R>.12 mg/l and S 29, R<29 mm

23 No of isolates 6 Moxifloxacin MIC vs. species Corynebacterium spp., 25 clinical isolates 5 C. striatum C. amycolatum C. jeikeium C. pseudodiphtheriticum C. urealyticum C. afermentans C. aurimucosum C. propinquum C. singulare Other MIC (mg/l)

24 No of isolates 1 6 Moxifloxacin 5 µg vs. MIC Corynebacterium spp., 25 clinical isolates Moxifloxacin breakpoints Staphylococci.5/1 Strep A,B,C,G.5/1 S. pneumoniae.5/.5 Viridans strep - PK/PD.5/1 MIC (mg/l) Inhibition zone diameter (mm) Suggested breakpoints: S.5, R>.5 mg/l and S 25, R<25 mm

25 No of isolates Vancomycin MIC vs. species Corynebacterium spp., 25 clinical isolates C. striatum C. amycolatum C. jeikeium C. pseudodiphtheriticum C. urealyticum C. afermentans C. aurimucosum C. propinquum C. singulare Other MIC (mg/l)

26 No of isolates 5 Vancomycin 5 µg vs. species Corynebacterium spp., 259 clinical isolates C. striatum C. amycolatum C. jeikeium C. pseudodiphtheriticum C. urealyticum C. afermentans C. aurimucosum C. propinquum C. singulare Other Inhibition zone diameter (mm)

27 No of isolates Vancomycin 5 µg vs. MIC Corynebacterium spp., 25 clinical isolates Vancomycin breakpoints S. aureus 2/2 CoNS 4/4 Strep A,B,C,G 2/2 S. pneumoniae 2/2 Viridans strep 2/2 PK/PD IE MIC (mg/l) Inhibition zone diameter (mm) Suggested breakpoints: S 2, R>2 mg/l and S 17, R<17 mm

28 No of isolates 1 Tetracycline MIC vs. species Corynebacterium spp., 25 clinical isolates 6 C. striatum C. amycolatum C. jeikeium C. pseudodiphtheriticum C. urealyticum C. afermentans C. aurimucosum C. propinquum C. singulare Other MIC (mg/l)

29 No of isolates Tetracycline µg vs. species Corynebacterium spp., 259 clinical isolates C. striatum C. amycolatum C. jeikeium C. pseudodiphtheriticum C. urealyticum C. afermentans C. aurimucosum C. propinquum C. singulare Other Inhibition zone diameter (mm)

30 No of isolates Tetracycline µg vs. MIC Corynebacterium spp., 25 clinical isolates Tetracycline breakpoints Staphylococci 1/2 Strep A,B,C,G 1/2 S. pneumoniae 1/2 Viridans strep - PK/PD IE MIC (mg/l) Inhibition zone diameter (mm) Suggested breakpoints: S 2, R>2 mg/l and S 24, R<24 mm

31 No of isolates No of isolates No of isolates No of isolates Artspecifika MIC-distributioner C. striatum with benzypenicillin 7 clinical isolates C. amycolatum with benzylpenicillin 63 clinical isolates MIC (mg/l) MIC (mg/l) C. jeikeium with benzylpenicillin 35 clinical isolates C. pseudodiphtheriticum with benzylpenicillin clinical isolates MIC (mg/l) MIC (mg/l)

32 No of isolates Artspecifika zondistributioner 25 Benzylpenicillin 1 unit vs. MIC C. striatum, 7 clinical isolates 15 5 MIC (mg/l) Inhibition zone diameter (mm)

33 No of isolates Artspecifika zondistributioner Benzylpenicillin 1 unit vs. MIC C. amycolatum, 63 clinical isolates MIC (mg/l) Inhibition zone diameter (mm)

34 No of isolates Artspecifika zondistributioner Benzylpenicillin 1 unit vs. MIC C. jeikeium, 35 clinical isolates MIC (mg/l) Inhibition zone diameter (mm)

35 No of isolates Artspecifika zondistributioner 4 Benzylpenicillin 1 unit vs. MIC C. pseudodiphtheriticum, clinical isolates MIC (mg/l) Inhibition zone diameter (mm)

36 No of readings Variation mellan laboratorier Moxifloxacin 5 µg vs. MIC Corynebacterium spp., 6 clinical isolates tested at 3 sites Växjö (EUCAST), Linköping, Santander (Spanien) MIC (mg/l) Inhibition zone diameter (mm)

37 No of readings Variation mellan laboratorier Tetracycline µg vs. MIC Corynebacterium spp., 6 clinical isolates tested at 3 sites Växjö (EUCAST), Linköping, Santander (Spanien) 12 mm 13 mm 24 mm MIC (mg/l) > Inhibition zone diameter (mm)

38 Skillnader i spädningssteg mellan Etest och BMD (referens) Benzylpenicillin > = >+2 EA 3% Corynebacterium amycolatum Corynebacterium jeikeium 11 1 Corynebacterium pseudodiphtheriticum Corynebacterium striatum Corynebacterium urealyticum 12 Sum Moxifloxacin > = >+2 EA 52% Corynebacterium amycolatum 1 11 Corynebacterium jeikeium Corynebacterium pseudodiphtheriticum Corynebacterium striatum 7 5 Corynebacterium urealyticum Sum Vancomycin > = >+2 EA % Corynebacterium amycolatum 6 6 Corynebacterium jeikeium 6 6 Corynebacterium pseudodiphtheriticum 5 7 Corynebacterium striatum Corynebacterium urealyticum 9 3 Sum Tetracycline > = >+2 EA 53% Corynebacterium amycolatum Corynebacterium jeikeium Corynebacterium pseudodiphtheriticum Corynebacterium striatum Corynebacterium urealyticum Sum

39 Corynebacterium spp. EUCASTs föreslagna brytpunkter Antibiotika S R> Lappstyrka S R< Bensylpenicillin unit Ciprofloxacin Moxifloxacin Gentamicin Klindamycin Tetracyklin Rifampicin Vankomycin Linezolid Trim-Sulfa

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