Dias Nummer 1

Clinical importance of viridans group streptococci and other rare gram-positive cocci

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.

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

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 Chloramphenicol ESCMID 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

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

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

. 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

+ + + + + +

Time-of-Flight ESCMIDIntensity Online Lecture Library

 Database setups: 1) Maldi Biotyper Automation Control 2.0.43.1) 2) + homemade library (mass spectrum profiles (MSPs) for typestrains) (MALDI Biotyper 2.0SR1 (build 223.8))

ESCMID Online Lecture Library Editing the spectra (deleting bad spectra)

Rimtas Dargis 2011

 Database setups: 1) Maldi Biotyper Automation Control 2.0.43.1) 2) + homemade library (mass spectrum profiles (MSPs) for typestrains) (MALDI Biotyper 2.0SR1 (build 223.8))

CCUG no. in the BioTyper v.2.0.43.1

Abiotrophia defectiva CCUG 27639 + Aerococcus christensenii CCUG 28831 + Aerococcus sanguinicola CCUG 4100 + Aerococcus suis CCUG 52530 - Aerococcus urinae CCUG 36881 + Aerococcus urinaeequi CCUG 28094 - Aerococcus urinaehominis CCUG 42038 B + Aerococcus viridans CCUG 4311 + Alloiococcus otitis CCUG 32997 + Dolosicoccus paucivorans CCUG 39307 - Dolosigranulum pigrum CCUG 33392 - Facklamia hominis CCUG 36813 + Facklamia ignava CCUG 37419 - Facklamia languida CCUG 37842 + Facklamia miroungae CCUG 42728 - Facklamia sourekii CCUG 28783ª - Facklamia tabacinasalis CCUG 30090 - Gemella asaccharolytica CCUG 57045 - Gemella bergeri CCUG 37817 + Gemella cuniculi CCUG 42726 - Gemella haemolysans CCUG 37985 + Gemella morbillorum CCUG 18164 - Gemella palaticanis CCUG 39489 - Gemella sanguinis CCUG 37820 + Globicatella sanguinis CCUG 32999 - Globicatella sulfidifaciens CCUG 44365 - Granulicatella adiacens CCUG 27809 + Granulicatella balaenopterae CCUG 37380 + Granulicatella elegans CCUG 38949 + Helcococcus kunzii CCUG 32213 + Helcococcus ovis CCUG 37441 - Helcococcus sueciensis CCUG 47334 - Ignavigranum ruoffiae CCUG 37658 - Lactococcus garvieae CCUG 32208 + Lactococcus lactis subsp. cremoris CCUG 21953© by author+ Lactococcus lactis subsp. hordniae CCUG 32210 - Lactococcus lactis subsp. lactis CCUG 7980 + Leuconostoc lactis CCUG 30064 + Leuconostoc mesenteroides (subsp. mesenteroides) CCUG 30066 + Leuconostoc mesenteroides subsp. cremoris CCUG 21965 + Leuconostoc mesenteroides subsp. dextranicum CCUG 30065 - Pediococcus acidilactici CCUG 32235 + Pediococcus parvulus CCUG 28439 - Pediococcus pentosaceus CCUG 32205 + Rothia aeria ESCMID OnlineCCUG 51932 Lecture+ Library Rothia amarae CCUG 47294 + Rothia dentocariosa CCUG 35437 + Rothia mucilaginosa CCUG 20962 + Rothia nasimurium CCUG 35957 + Rothia terrae CCUG 55855 - Vagococcus fluvialis CCUG 32704 + ______

CCUG: Received from Culture Collection, University of of Göteborg (CCUG)

Laser-diagnostics! (Mass spectrometry)

• There are many curious - and dangerous bacteria!

• 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!by author

I’ve had my tongue pierced 3 times, and split twice. You can ESCMID Online Lecturepretty muchLibrary eat whatever you feel comfortable with eating. If it hurts to eat something, then stop eating it. © by author ESCMID Online Lecture Library