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1 Jens Jørgen Christensen Dept. of clinical microbiology Slagelse Hospital, Slagelse Denmark Clinical importance of viridans group streptococci and other rare gram-positive cocci Endocarditis: native valves, prosthetic valves Immunocompromised: septicemia, pneumonia, antibiotic resistance
2 Cytochrome and catalase content and cellular arrangement of facultatively anaerobic gram-positive cocci Genus Cytochrome/ Cellular catalase arrangement Staphylococcus +/+ Clusters Streptococcus -/- Chains Enterococcus -/- Chains Abiotrophia (1) -/- Chains Granulicatella (3) -/- Chains Gemella (7) -/- Pairs or tetrads Lactococcus (5) -/- Chains (Leuconostoc (4) -/- Chains) Pediococcus (3) -/- Pairs or tetrads Aerococcus (7) -/+W Pairs or tetrads Other genera: Facklamia (6), (Rothia (6)
3 Identification of fastidious gram-positive cocci MALDI-TOF MS applied on non-non hemolytic streptococci: Identification and use of complementary libraries.
4 Identification of fastidious gram-positive cocci Blood Wound Which genera and species? (16 genera, 51 species) Abiotrophia Granulicatella Gemella Lactococcus (Leuconostoc) Pediococcus Aerococcus Dolosigranulum Globicatella Facklamia Vagococcus Dolosicoccus Helcococcus Ignavigranum Alloiococcus (Rothia)
5 Abiotrophia Granulicatella Gemella Blood Lactococcus (Leuconostoc) Pediococcus kød grøntsager samt normalflora i tarm Aerococcus
6 Challenge strains: n=90 Aerococcus species (n=35) Gemella species (n=23) Abiotrophia/Granulicatella(n=10; 2/8) Lactococcus species (n=5) Globicatella species (n=5) Leuconostoc species (n=5) Rothia species (n=3) Facklamia, Vagococcus, Helcococcus, Alloiococcus
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8 Do they mean anything? Parameters indicative of UTI in 92 patients whose urine yielded growth of A. urinae. Number of patients A. urinae singly A. urinae in admixture Indwelling catheter Dysuria + fever + pyuria Dysuria + fever Dysuria + pyuria Fever + pyuria Dysuria Fever Pyuria Incontinence No symptoms No information TOTAL Aerococcus-like organism, a newly recognized potential urinary tract pathogen. JJ Christensen, H Vibits, J Ursing and B Korner. J. Clin. Microbiol. 29: , 1991
9 Clinical data on 17 patients in Denmark with septicemia/bacteremia caused by A. urinae Clinical Sources of Case no. Age/Sex presentation A. urinae Outcome 1 78/M Endocarditis Blood, urine Died with A. urinae in heart valves 2 81/M Endocarditis Blood, urine Died, despite clearing of bacteremia 3 73/M Endocarditis Blood Died with killed A. urinae in vegetation 4 81/M Endocarditis Blood Died with killed A. urinae in vegetation 5 55/F Endocarditis Blood Died of A. urinae infection 6 78/M Endocarditis Blood* Recovered 7 63/M Urosepticemia Blood, urine Recovered 8 76/M Urosepticemia Blood, urine Recovered 9 77/M Urosepticemia Blood, urine Survived infection (eventually died) 10 79/M Urosepticemia Blood Recovered 11 86/F Urosepticemia Blood, urine Recovered 12 73/M Urosepticemia Blood, urine Recovered 13 40/M Urosepticemia Blood, urine Recovered 14 90/M Urosepticemia Blood, urine Recovered 15 37/M Septicemia Blood Recovered 16 82/M Urosepticemia? Blood Survived infection (eventually died) 17 80/M Urosepticemia? Blood Recovered Bacteremia/septicemia due to Aerococcus-like organisms: Report of seventeen cases. JJ Christensen, IP Jensen, J Færk, B Kristensen, R Skov, B Korner and an ALO study group. Clin. Infect. Dis. 21: , 1995.
10 Susceptibility patterns of A. urinae SENSITIVE TO: Penicillin Ampicillin Cehalosporins Clavulanate Erythromycin Clindamycin Vancomycin Teicoplanin Mupirocin Tetracyclines Chloramphenicol RESISTANT TO: Sulfonamides Trimethoprim Nalidixic acid Aminoglycocides Mecillinam Aerococcus urinae: A newcomer in clinical and micrbiological practice. JJ Christensen and B Korner. Antimicrobics and Infectious Disease Newsletter. 15: 78-80, 1996
11 Instruments for identification Semi-automated phenotypic systems Molecular methods MALDI-TOF MS
12 ??? Automated systems??? Rapid ID 32 Strep system (T. G. Jensen et al. CMI, 1999) Phoenix automated system (G. Brigante et al., JCM. 2006) VITEK (M. Haanperä, et al. JCM, 2007) All the automated identification systems have difficulty in identification of NHS to the species level, especially for the Mitis group.
13 API panels, Crystal panels, Vitek cards, MicroScan conventional panels, Phoenix panels, Sensititre panels
14 Evaluation of the Rapid ID 32 Strep system Jensen TG, Konradsen HB, Bruun B CMI: 1999,5: strains: Pyogenic (n=29): 86% correct identification. OK Viridans (n=51): 65% to the species level. Worst for the Mitis group. NOT OK Enterococcal (n=27): 93% Correct identificatopn. OK
15 16S rrna gene Gene analysis SodA gene (Magnganese-dependent superoxide dismutase) RnpB gene (RNase P RNA) Tuf gene (elongation factor Tu) GroESL gene (heat shock protein) Rpo gene (beta-subunit of RNA polymearse) No single gene analysis is sufficient for species identification. Multi-gene analysis - ddl, gdh, rpo, soda
16 Berridge, B. R. et al J. Clin. Microbiol. 36(9): Xiaohui Chen Nielsen, KMA, Slagelse Sygehus
17 Oralis cluster Mitis-pneumoniae cluster FIG. 1. Phylogenetic tree based on ITS sequences of the 11. S. mitis (Smit), 11 S. oralis (Soral) and 17 S. pneumoniae (Spneu) strains using unrooted neighbor joining method. No distinct cluster were formed according to the species. It clearly demonstrates that S. mitis, S. pneumoniae and S. oralis are genetically closely related species and cannot be discriminated from each other based on ITS sequences.
18 What is so special about the Mitis group The Mitis group contains species that are important clinical pathogens and are geneticallly closely related. The memebers are natural competent for genetic transformation and homologous recombination happened among species S. pneumoniae, S. mitis, S. pseudopneumoniae cluster might evolved from the same ancester: pneumoncoccus-like bacteria. The memebers have strikingly different pathogenic potentials. S. pneumonae is among the most frequent microbial killers, while S. mitis and S. oralis are the most dominant endocarditis pathogen. Killian et al PlosOne 3: 1-11.
19 Partial gdh sequencing Gdh gene: house keeping gene, encoding for glucose-6- phosphate dehydrogenase. Use of housekeeping gene sequencing for species identification of viridans streptococci. P. Kiratisin et al. Diagnostic microbiology and infectious disease : demonstrated that sequencing af gdh (zwf) gene could be discriminative enough for species identification in this group of Streptococcus with limited intraspecies variation.
20 S. pneumoniae cluster S. mitis cluster S. oralis cluster FIG. 2. Minimal evolution algorithm (suppressed) using the MEGA 4 program, based on partial gdh sequences of 11 S. oralis, 17 S. pneumoniae and 11 S. mitis strains. It shows that the three species forms three distinct clusters. The S. oralis cluster has longer distance to the two other clusters, indicating that S. pneumoniae and S. mitis are genetically closer related based on gdh gene evolution. There are three subclusters within the S. mitis cluster, indicating that the species S. mitis contains a heterogeneous group of strains.
21 Identification of NHS based on ITS and partial gdh sequencing Mitis Gr. Partial gdh sequencing Species ID Streptococcus sp. ITS sequencing Bovis Gr. Anginosus Gr. Salivarius Gr. Mutans Gr. Sanguinis Gr. Species ID
22 What is this Mass spectrometry about? Use of laser to find the dangerous bacteria! MALDI-TOF mass spectrometry, a revolution in clinical microbial identification A. Bizzini and G. Greub Clin Microbiol Infect 2010; 16:
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24 Intensity Acceleration Laser canon Electrodes Drift Detector Time-of-Flight m/z
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26 MALDI NHS - enterococci
27 Identification of fastidious gram-positive cocci Database setups: 1) Maldi Biotyper Automation Control ) 2) + homemade library (mass spectrum profiles (MSPs) for typestrains) (MALDI Biotyper 2.0SR1 (build 223.8)) Usefulness and benefits: 51 CCUG type strains Usefulness and benefits: 90 challenge strains
28 Editing the spectra (deleting bad spectra) Rimtas Dargis 2011
29 Database setups: 1) Maldi Biotyper Automation Control ) 2) + homemade library (mass spectrum profiles (MSPs) for typestrains) (MALDI Biotyper 2.0SR1 (build 223.8)) Usefulness and benefits: 51 CCUG type strains Usefulness and benefits: 90 challenge strains
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33 TABLE S1. Collection strains used for database extension and species included in the BioTyper v software. CCUG no. in the BioTyper v Abiotrophia defectiva CCUG Aerococcus christensenii CCUG Aerococcus sanguinicola CCUG Aerococcus suis CCUG Aerococcus urinae CCUG Aerococcus urinaeequi CCUG Aerococcus urinaehominis CCUG B + Aerococcus viridans CCUG Alloiococcus otitis CCUG Dolosicoccus paucivorans CCUG Dolosigranulum pigrum CCUG Facklamia hominis CCUG Facklamia ignava CCUG Facklamia languida CCUG Facklamia miroungae CCUG Facklamia sourekii CCUG 28783ª - Facklamia tabacinasalis CCUG Gemella asaccharolytica CCUG Gemella bergeri CCUG Gemella cuniculi CCUG Gemella haemolysans CCUG Gemella morbillorum CCUG Gemella palaticanis CCUG Gemella sanguinis CCUG Globicatella sanguinis CCUG Globicatella sulfidifaciens CCUG Granulicatella adiacens CCUG Granulicatella balaenopterae CCUG Granulicatella elegans CCUG Helcococcus kunzii CCUG Helcococcus ovis CCUG Helcococcus sueciensis CCUG Ignavigranum ruoffiae CCUG Lactococcus garvieae CCUG Lactococcus lactis subsp. cremoris CCUG Lactococcus lactis subsp. hordniae CCUG Lactococcus lactis subsp. lactis CCUG Leuconostoc lactis CCUG Leuconostoc mesenteroides (subsp. mesenteroides) CCUG Leuconostoc mesenteroides subsp. cremoris CCUG Leuconostoc mesenteroides subsp. dextranicum CCUG Pediococcus acidilactici CCUG Pediococcus parvulus CCUG Pediococcus pentosaceus CCUG Rothia aeria CCUG Rothia amarae CCUG Rothia dentocariosa CCUG Rothia mucilaginosa CCUG Rothia nasimurium CCUG Rothia terrae CCUG Vagococcus fluvialis CCUG CCUG: Received from Culture Collection, University of of Göteborg (CCUG)
34 There are many curious - and dangerous bacteria! Laser-diagnostics! (Mass spectrometry) With laser diagnostics you can very quick, cheap and secure make a catch of them at least with respect to what you shall call them! but can they have a susceptibility testing with this terrifying machine?
35 I ve had my tongue pierced 3 times, and split twice. You can pretty much eat whatever you feel comfortable with eating. If it hurts to eat something, then stop eating it.
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