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streptococci and other rare gram-positive
cocci
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Endocarditis: native valves, prosthetic valves

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

Immunocompromised:
septicemia,
pneumonia,
antibiotic resistance

Cytochrome and catalase content and cellular arrangement
of facultatively anaerobic gram-positive cocci

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______________________________________________________________________________

Genus

Cytochrome/
Cellular
catalase
arrangement
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Staphylococcus
Streptococcus
Enterococcus
Abiotrophia
Granulicatella
Gemella

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S
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Lactococcus
(Leuconostoc
Pediococcus
Aerococcus

+/+
-/-/-

Clusters
Chains
Chains

(1)
(3)
(7)

-/-/-/-

Chains
Chains
Pairs or tetrads

(5)
(4)
(3)

-/-/-/-

Chains
Chains)
Pairs or tetrads

(7)

-/+W

Pairs or tetrads

Other genera: Facklamia (6), (Rothia (6)

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MALDI-TOF MS applied on non-non hemolytic streptococci:
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Identification and use of complementary libraries.e
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Identification of ”fastidious” gram-positive cocci

Identification of ”fastidious” gram-positive cocci y r a r b i L e r u t c Which genera and species? (16 genera. 51 e r species) L o e h t n i u l a n O by D © I M •Abiotrophia •Granulicatella •Gemella Blood C S E Wound •Lactococcus •(Leuconostoc) •Pediococcus •Dolosigranulum •Globicatella •Facklamia •Vagococcus •Dolosicoccus •Helcococcus •Ignavigranum •Alloiococcus •Aerococcus •(Rothia) .

y r a r b i L e •Abiotrophia •Granulicatella •Gemella r u t c e r L o e h t n i u l a n O by D © I M Blood C S E •Lactococcus •(Leuconostoc) •Pediococcus kød grøntsager samt normalflora i tarm •Aerococcus .

2/8) n i u l a n • Lactococcus species (n=5) y O b • Globicatella species (n=5) D I © • Leuconostoc species (n=5) M C • Rothia species (n=3) S E • Facklamia.Challenge strains: n=90 y r a r b i L e r u t • Aerococcus species (n=35) c e r L • Gemella species (n=23) o e h t • Abiotrophia/Granulicatella(n=10. Helcococcus. Alloiococcus . Vagococcus.

y r a r b i L e C S E r u t c e r L o e h t n i u l a n O by D © I M .

urinae singly A. 29:1049-1053. Number of patients _________________________________________________________________ A. Clin. 1991 .y r a r b i L e Do they mean anything? r u t c e r L o e h t n i u l a n O by D © I M Parameters indicative of UTI in 92 patients whose urine yielded growth of 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 ______________________________________________________________________________ C S E TOTAL 41 5 38 6 ______________________________________________________________________________ Aerococcus-like organism. Microbiol. a newly recognized potential urinary tract pathogen. J. H Vibits. J Ursing and B Korner. urinae. JJ Christensen.

. urine Blood. J Færk. JJ Christensen. B Kristensen. IP Jensen. urinae in vegetation Died of A. urine Blood. urinae in heart valves Died. urine Blood. Dis. urine Blood Blood Blood Died with A. despite clearing of bacteremia Died with killed A.y r a r b i L e Clinical data on 17 patients in Denmark with septicemia/bacteremia caused by A. urine Blood. urine Blood Blood. B Korner and an ALO study group. 1995. urine Blood. urine Blood. urinae infection Recovered Recovered Recovered Survived infection (eventually died) Recovered Recovered Recovered Recovered Recovered Recovered Survived infection (eventually died) Recovered ____________________________________________________________________________________________ Bacteremia/septicemia due to Aerococcus-like organisms: Report of seventeen cases. Clin. urine Blood Blood Blood Blood* Blood. urinae Outcome ____________________________________________________________________________________________ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 78/M 81/M 73/M 81/M 55/F 78/M 63/M 76/M 77/M 79/M 86/F 73/M 40/M 90/M 37/M 82/M 80/M C S E r u t c e r L o e h t n i u l a n O by D © I M Endocarditis Endocarditis Endocarditis Endocarditis Endocarditis Endocarditis Urosepticemia Urosepticemia Urosepticemia Urosepticemia Urosepticemia Urosepticemia Urosepticemia Urosepticemia Septicemia Urosepticemia? Urosepticemia? Blood. Infect. 21:943-947. urinae Clinical Sources of Case no. Age/Sex presentation A. R Skov. urinae in vegetation Died with killed A.

Susceptibility patterns of A. 15: 78-80. JJ Christensen and B Korner. Antimicrobics and Infectious Disease Newsletter. 1996 . urinae y r a r b i L e r u t c e r L o e h t n i u l a n O by D © I M _________________________________________________________________________ C S E SENSITIVE TO: RESISTANT TO: Penicillin Ampicillin Cehalosporins Clavulanate Erythromycin Clindamycin Vancomycin Teicoplanin Mupirocin Tetracyclines Chloramphenicol Sulfonamides Trimethoprim Nalidixic acid Aminoglycocides Mecillinam Aerococcus urinae: A newcomer in clinical and micrbiological practice.

y r Instruments for identification a r b i L e • Semi-automated phenotypic systems r u t c • Molecular methods e r L o • MALDI-TOF MS e h t n i u l a n O by D © I M C S E .

M C S (T. Jensen et al. G. CMI. 1999) (G. Haanperä. Brigante et al. JCM. especially for the Mitis group. JCM. 2007) E . 2006) (M. et al.y r ??? Automated systems ??? ra b i L e r Rapid ID 32 Strep system u t c Phoenix automated system e r L o VITEK e h t n i u l a n All the automated identification systems have y O b difficulty in identification D © of NHS to the species I level..

Sensititre panels .y r a r b i L e C S E r u t c e r L o e h t n i u l a n O by D © I M API panels. Vitek cards. Phoenix panels. MicroScan conventional panels. Crystal panels.

Worst for the Mitis group. OK .5:417-423 122 strains: Pyogenic (n=29): 86% correct identification. Bruun B CMI: 1999. NOT OK C S E Enterococcal (n=27): 93% Correct identificatopn. Konradsen HB. OK Viridans (n=51): 65% to the species level.y r a r b i L e r u t c e r L o e h t n i u l a n O by D © I M Evaluation of the Rapid ID 32 Strep system Jensen TG.

gdh. sodA .  Multi-gene analysis . rpo.ddl.Gene analysis y r a r b i L e r u t c e r L o e h t n i u l a n O by D © I M  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) C S E No single gene analysis is sufficient for species identification.

B. et al. Clin. KMA. J. 36(9):2778-2781 Xiaohui Chen Nielsen. R.y r a r b i L e r u t c e r L o e h t n i u l a n O by D © I M C S E Berridge. 1998. Slagelse Sygehus . Microbiol.

1. Phylogenetic tree based on ITS sequences of the 11. mitis (Smit). S. mitis. S. pneumoniae (Spneu) strains using unrooted neighbor joining method. It clearly demonstrates that S. pneumoniae and S. No distinct cluster were formed according to the species. oralis (Soral) and 17 S. oralis are genetically closely related species and cannot be discriminated from each other based on ITS sequences.y r a r b i L e Oralis cluster r u t c e r L o e h t n i u l a n O by D © I M Mitis-pneumoniae cluster C S E FIG. . 11 S.

while S. oralis are the most dominant endocarditis pathogen. pseudopneumoniae cluster might evolved from the same ancester: pneumoncoccus-like bacteria. mitis. The memebers have strikingly different pathogenic potentials. pneumonae is among the most frequent microbial killers. pneumoniae. PlosOne 3: 1-11.y r a r b i L e What is so special about the Mitis group The Mitis group contains species that are important clinical pathogens and are geneticallly closely related. mitis and S. r u t c e r L o e h t n i u l a n O by D © I M The memebers are natural competent for genetic transformation and homologous recombination happened among species S. C S E Killian et al. 2008. . S. S. S.

encoding for glucose-6e r L phosphate dehydrogenase. 2005. 51:297-301. Diagnostic microbiology and infectious disease. . Kiratisin et al.y r Partial gdh sequencing a r b i L e r u t c Gdh gene: house keeping gene. O by D © I M C S E  P. …demonstrated that sequencing af gdh (zwf) gene could be discriminative enough for species identification in this group of Streptococcus with limited intraspecies variation. o e h t n  Use of housekeepinglgene sequencing for species identification i u a n of viridans streptococci.

The S. . mitis strains. 17 S. pneumoniae and 11 S. It shows that the three species forms three distinct clusters. oralis. indicating that S. pneumoniae cluster y r a r b i L e r u t c e r L o e h t n i u l a n O by D © I M S.S. oralis cluster S. pneumoniae and S. oralis cluster has longer distance to the two other clusters. mitis contains a heterogeneous group of strains. There are three subclusters within the S. mitis cluster C S E FIG. indicating that the species S. 2. based on partial gdh sequences of 11 S. mitis are genetically closer related based on gdh gene evolution. mitis cluster. Minimal evolution algorithm (suppressed) using the MEGA 4 program.

y r a r b i Streptococcus sp. Salivarius Gr. Mutans Gr. . Anginosus Gr. C S E Species ID Bovis Gr. Sanguinis Gr. L e r u t c e r L ITS sequencing o e h t n i u l a n O by D © I MPartial gdh sequencing Species ID Identification of NHS based on ITS and partial gdh sequencing Mitis Gr.

16: 1614–1619 . Greub Clin Microbiol Infect 2010. Bizzini and G. c e inr clinical microbial a revolution L o identification e h t n i u l a n O by D © I M C S E What is this Mass spectrometry about? A.y r a r b i Use of laser to find the ”dangerous” bacteria! L e r u t MALDI-TOF mass spectrometry.

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y r a r b i L e r u t c e r L o e h t n i u l a n O by D © I M Acceleration Drift + + + Electrodes Laser canon C S E Time-of-Flight Intensity m/z + + + Detector .

y r a r b i L e C S E r u t c e r L o e h t n i u l a n O by D © I M .

y r a r b i L e r u t c e r L o e h t n i u l a n O by D © I M MALDI NHS .enterococci C S E .

43.8))  Usefulness and benefits: 51 CCUG type strains  Usefulness and benefits: 90 ”challenge” strains .y r Identification of ”fastidious” gram-positive cocci a r b i L e r u t c e r L o e h t n i u l a n O by D © I M C S E  Database setups: 1) Maldi Biotyper Automation Control 2.0SR1 (build 223.1) 2) + homemade library (mass spectrum profiles (MSPs) for typestrains) (MALDI Biotyper 2.0.

Editing the spectra (deleting bad spectra) y r a r b i L e r u t c e r L o e h t n i u l a n O by D © I M C S E Rimtas Dargis 2011 .

0SR1 (build 223.43.0.8))  C S E  Usefulness and benefits: 51 CCUG type strains Usefulness and benefits: 90 ”challenge” strains .y r a r b i L e  r u t c e r L o e h t n i u l a n O by D © I M Database setups: 1) Maldi Biotyper Automation Control 2.1) 2) + homemade library (mass spectrum profiles (MSPs) for typestrains) (MALDI Biotyper 2.

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CCUG no.1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + y r a r b i L e r u t c e r L o e h t n i u l a n O by D © I M C S E CCUG: Received from Culture Collection. hordniae CCUG 32210 Lactococcus lactis subsp.0.0. 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 ____________________________________________________________________ in the BioTyper v.TABLE S1.1 software. cremoris CCUG 21953 Lactococcus lactis subsp. cremoris CCUG 21965 Leuconostoc mesenteroides subsp. 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. mesenteroides) CCUG 30066 Leuconostoc mesenteroides subsp. 2.43.2. University of of Göteborg (CCUG) . lactis CCUG 7980 Leuconostoc lactis CCUG 30064 Leuconostoc mesenteroides (subsp.43. Collection strains used for database extension and species included in the BioTyper v.

cheap and i u l a n of them secure make a O catch – at least with respect y b to what you shall call them! D © I M C S E Laser-diagnostics! • • ”but can they have a susceptibility testing with this terrifying machine?” .y r a (Mass spectrometry) r b i L There are many curious .and dangerous e r u t bacteria! c e r L o e h t n With laser diagnostics you can very quick.

You can pretty much eat whatever you feel comfortable with eating. If it hurts to eat something. then stop eating it. .y r a r b i L e C S E r u t c e r L o e h t n i u l a n O by D © I M I’ve had my tongue pierced 3 times. and split twice.

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