Clinical importance of viridans group streptococci and other rare gram-positive cocci © by Endocarditis:author native valves, prosthetic valves Immunocompromised: ESCMID Online Lecture septicemia, Library pneumonia, Jens Jørgen Christensen antibiotic resistance Dept. of clinical microbiology Slagelse Hospital, Slagelse Denmark 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 © by author Lactococcus (5) -/- Chains (Leuconostoc (4) -/- Chains) Pediococcus (3) -/- Pairs or tetrads AerococcusESCMID Online(7) -/+W Lecture Pairs orLibrary tetrads Other genera: Facklamia (6), (Rothia (6) Identification of ”fastidious” gram-positive cocci MALDI-TOF MS applied on non-non hemolytic streptococci: Identification and use of complementary libraries. © by author ESCMID Online Lecture Library Identification of ”fastidious” gram-positive cocci Which genera and species? (16 genera, 51 species) •Abiotrophia •Dolosigranulum •Granulicatella •Globicatella •Facklamia •Gemella •Vagococcus •Dolosicoccus Blood •Lactococcus © by author •Helcococcus •(Leuconostoc) •Ignavigranum •Pediococcus •Alloiococcus ESCMID Online•Aerococcus Lecture•(Rothia) Library Wound •Abiotrophia •Lactococcus •Granulicatella •(Leuconostoc) •Gemella •Pediococcus kød grøntsager samt normalflora i tarm •Aerococcus © byBlood author ESCMID Online Lecture Library 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© by author(n=5) • Rothia species (n=3) •ESCMIDFacklamia, Vagococcus, Online Helcococcus, Lecture Alloiococcus Library © by author ESCMID Online Lecture Library 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 5 1 6 - Dysuria + fever 2 1 3 - Dysuria + pyuria 13 1 9 3 Fever + pyuria - 1 1 1 Dysuria 3 - 4 - Fever 1 1 - - Pyuria 11 - 8 1 Incontinence - - 2 - No symptoms © 1 by - author 2 - No information 5 - 5 1 ______________________________________________________________________________ TOTAL 41 5 38 6 ______________________________________________________________________________ ESCMID Online Lecture Library Aerococcus-like organism, a newly recognized potential urinary tract pathogen. JJ Christensen, H Vibits, J Ursing and B Korner. J. Clin. Microbiol. 29:1049-1053, 1991 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 © byBlood, urineauthor Recovered 15 37/M Septicemia Blood Recovered 16 82/M Urosepticemia? Blood Survived infection (eventually died) 17 80/M Urosepticemia? Blood Recovered ____________________________________________________________________________________________ESCMID Online Lecture Library 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. 21:943-947, 1995. Susceptibility patterns of A. urinae _________________________________________________________________________ SENSITIVE TO: RESISTANT TO: Penicillin Sulfonamides Ampicillin Trimethoprim Cehalosporins Nalidixic acid Clavulanate Aminoglycocides Erythromycin Mecillinam Clindamycin Vancomycin Teicoplanin© by author Mupirocin Tetracyclines ESCMID Chloramphenicol Online Lecture Library Aerococcus urinae: A newcomer in clinical and micrbiological practice. JJ Christensen and B Korner. Antimicrobics and Infectious Disease Newsletter. 15: 78-80, 1996 Instruments for identification • Semi-automated phenotypic systems • Molecular methods • MALDI-TOF MS © by author ESCMID Online Lecture Library ??? 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 theby Mitis author group. ESCMID Online Lecture Library © by author API panels, Crystal panels, Vitek cards, ESCMID OnlineMicroScan conventional Lecture panels, Library Phoenix panels, Sensititre panels Evaluation of the Rapid ID 32 Strep system Jensen TG, Konradsen HB, Bruun B CMI: 1999,5:417-423 122 strains: Pyogenic (n=29): 86% correct identification. OK Viridans (n=51): 65% to the species level. Worst for the Mitis group. NOT OK © by authorEnterococcal (n=27): 93% Correct identificatopn. OK ESCMID Online Lecture Library Gene analysis . 16S rRNA gene . 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) © by author No single gene analysis is sufficient for species identification. ESCMID. Multi-gene analysis Online Lecture Library - ddl, gdh, rpo, sodA © by author ESCMID Online Lecture Library Berridge, B. R. et al. 1998. J. Clin. Microbiol. 36(9):2778-2781 Xiaohui Chen Nielsen, KMA, Slagelse Sygehus 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 © by authorunrooted neighbor joining method. No distinct cluster were formed according to the species. It clearly demonstrates that S. mitis, S. ESCMID Online Lecturepneumoniae Library and S. oralis are genetically closely related species and cannot be discriminated from each other based on ITS sequences. 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 by author different pathogenic potentials. S. pneumonae is among the most frequent microbial killers, while S. mitis and S. oralis are the most dominant endocarditis ESCMID pathogen . Online Lecture Library Killian et al. 2008. PlosOne 3: 1-11. 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© by and infectiousauthor disease. 2005. 51:297-301. …demonstrated that sequencing af gdh (zwf) gene could be discriminative enough for species identification in this ESCMIDgroup of Streptococcus Online with limitedLecture intraspecies Library variation. S. pneumoniae cluster S. oralis cluster S. mitis cluster © by author 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. pneumoniaeESCMID and S. mitis are genetically Online closer related Lecture based on gdh gene evolution. Library There are three sub- clusters within the S. mitis cluster, indicating that the species S. mitis contains a heterogeneous group of strains. Identification of NHS based on ITS and partial gdh sequencing Streptococcus sp. ITS sequencing Bovis Gr. Anginosus Gr. Mitis Gr. Salivarius Gr. Mutans Gr. © by author Sanguinis Gr. Partial gdh sequencing Species ID ESCMID Online Lecture Library Species ID 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: 1614–1619 © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Acceleration Drift + + + + + + Electrodes Detector Laser canon © by author Time-of-Flight ESCMIDIntensity Online Lecture Library m/z © by author ESCMID Online Lecture Library MALDI NHS -