12 Christensen

Clinical importance of viridans group

streptococci and other rare gram-positive cocci

Endocarditis: native valves, prosthetic valves Immunocompromised: septicemia, pneumonia, antibiotic resistance

Jens Jørgen Christensen Dept. of clinical microbiology Slagelse Hospital, Slagelse Denmark

ESCMID Online Lecture Library

© by author

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)


© by author

Identification of ”fastidious” gram-positive cocci

MALDI-TOF MS applied on non-non hemolytic streptococci: Identification and use of complementary libraries.


© by author

http://nml.dbio.dk/neobuilder.php?id=2011070510435341800027331


Which genera and species? (16 genera, 51

species)

•Dolosigranulum •Globicatella •Facklamia •Vagococcus •Dolosicoccus •Helcococcus •Ignavigranum

•Alloiococcus

•(Rothia)

•Abiotrophia •Granulicatella •Gemella •Lactococcus •(Leuconostoc) •Pediococcus •Aerococcus

Wound

Blood


© by author

http://resource.nhi.no/resource/1534-12-res.jpg?size=800

Blood

•Lactococcus •(Leuconostoc) •Pediococcus

•Abiotrophia •Granulicatella •Gemella

•Aerococcus

kød grøntsager samt normalflora i tarm


© by author

http://resource.nhi.no/resource/1534-12-res.jpg?size=800

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


© by author

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 - 2 - No information 5 - 5 1 ______________________________________________________________________________ TOTAL 41 5 38 6 ______________________________________________________________________________

Do they mean anything?

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


© by author

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. 21:943-947, 1995.


© by author

Susceptibility patterns of A. urinae

_________________________________________________________________________ SENSITIVE TO: RESISTANT TO: Penicillin Sulfonamides Ampicillin Trimethoprim Cehalosporins Nalidixic acid Clavulanate Aminoglycocides Erythromycin Mecillinam Clindamycin Vancomycin Teicoplanin Mupirocin Tetracyclines Chloramphenicol

Aerococcus urinae: A newcomer in clinical and micrbiological practice. JJ Christensen and B Korner. Antimicrobics and Infectious Disease Newsletter. 15: 78-80, 1996


© by author

Instruments for identification

• Semi-automated phenotypic systems • Molecular methods • MALDI-TOF MS


© by author

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.

??? Automated systems ???


© by author

API panels, Crystal panels, Vitek cards, MicroScan conventional panels, Phoenix panels, Sensititre panels


© by author

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 Enterococcal (n=27): 93% Correct identificatopn. OK


© by author

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)

No single gene analysis is sufficient for species identification. Multi-gene analysis

- ddl, gdh, rpo, sodA


© by author

Xiaohui Chen Nielsen, KMA, Slagelse Sygehus

Berridge, B. R. et al. 1998. J. Clin. Microbiol. 36(9):2778-2781


© by author

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.


© by author

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.

What is so special about the Mitis group

Killian et al. 2008. PlosOne 3: 1-11.


© by author

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. 2005. 51:297-301.

…demonstrated that sequencing af gdh (zwf) gene could be discriminative enough for species identification in this group of Streptococcus with limited intraspecies variation.

Partial gdh sequencing


© 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. pneumoniae and S. mitis are genetically closer related based on gdh gene evolution. There are three sub-clusters within the S. mitis cluster, indicating that the species S. mitis contains a heterogeneous group of strains.

S. mitis cluster

S. oralis cluster

S. pneumoniae cluster


© by author

Streptococcus sp.

ITS sequencing

Mitis Gr.

Partial gdh sequencing

Bovis Gr. Anginosus Gr. Salivarius Gr. Mutans Gr. Sanguinis Gr.

Species ID

Species ID

Identification of NHS based on ITS and partial gdh sequencing


© by author

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

What is this Mass spectrometry about?


© by author

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))

Usefulness and benefits: 51 CCUG type strains

Usefulness and benefits: 90 ”challenge” strains



© by author

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))

Usefulness and benefits: 51 CCUG type strains

Usefulness and benefits: 90 ”challenge” strains ESCMID Online Lectu

re Library

© by author

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 + 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 CCUG 51932 + 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)

TABLE S1. Collection strains used for database extension and species included in the BioTyper v. 2.0.43.1 software.


© by author

• 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!

Laser-diagnostics! (Mass spectrometry)

”but can they have a susceptibility testing with this terrifying machine?” ESCMID Online Lectu

re Library

© by author

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.


© by author

12 Christensen

Documents

Transcript of 12 Christensen