Clinically Relevant Gram- Positive Anaerobic Cocci (GPAC), New Names and Their Antibiotic Susceptibility

Clinically relevant Gram- positive anaerobic cocci (GPAC), new names and their antibiotic susceptibility

Introduction Gram-positive anaerobic cocci Peptostreptococcus sp. (formerly)

New species and taxonomy

Identification : - Phenotypic - Vitek ANC© bycard author - Fluorescent in situ hybridisation (FISH) - MALDI-TOF MS ESCMID Online Lecture Library Antibiotic resistance

Virulence F. magna

Introduction

Gram-positive anaerobic cocci (GPAC):

Peptococcus P.niger Seldomly isolated from clinical material

Ruminococcus Faecel microbiota Seldomly ©isolated by fromauthor clinical material Insufficient culture methods?

ESCMIDCoprococcus Online Lecture Library Isolated from faecal samples

Gram positive anaerobic cocci, D.A. Murdoch Clinical Microbiology reviews, 1998

Introduction

Atopobium Faecal microbiota and clinical material

Sarcina Soil, abdominal samples, faecal samples from vegetarians and patients with gastro-intestinal abnormalities

Unique cell© morphology: by author Groups of 8 cells which might produce spores ESCMID Online Lecture Library Peptostreptococcus

The genus Peptostreptococcus contained 13 species.

Clinically most relevant (literature): P. anaerobius P. asaccharolyticus P. magnus P. micros © by author ESCMID Online Lecture Library New species, changed taxonomy (in chronological order)

In 1997 3 new species were added to the genus Peptostreptococcus

P. harei P. ivorii P. octavius

Clinical relevance unknown© by author ESCMID Online Lecture Library Description of Three New Species of the Genus Peptostreptococcus from Human Clinical Specimens: P. harei sp. nov., P. ivorii sp. nov., and P. octavius sp. nov., D. A. Murdoch et al., Int. J. Syst. Bact. 1997 New taxonomy

Reclassification of Peptostreptococcus magnus (Prevot 1933) Holdeman and Moore 1972 as Finegoldia magna comb. nov. and Peptostreptococcus micros (Prevot 1933) Smith 1957 as Micromonas micros comb. nov. Murdoch et al., Anaerobe,5, 1999

Proposal of the genera© Anaerococcus by author gen. nov., Peptoniphilus gen. nov. and Gallicola gen. nov. for members of the genus Peptostreptococcus EzakiESCMID et al., Int J syst Online Evol Microbiol, Lecture 51, 2001 Library

A. murdochii Anaerococcus spp.

Anaerobic cocci: anaerobic cocci Finegoldia magna MicromonasParvimonas micramicros Gallicola barnesea Helcococcus kunzii Peptoniphilus spp. P. gorbachii P. olsenii © by author

ESCMID Online LecturePeptostreptoccus Library stomatis sp. P. stomatis nov., isolated from the human oral cavity. J. Downes et al. Int. J. P. anaerobius Syst. Evol. Microbiol. 2006 New names

Old name New name

P. micros Parvimonas micra P. magna Finegoldia magna P. asaccharolyticus Peptoniphilus asaccharolyticus P. prevotii © by author Anaerococcus prevotii ESCMID Online Lecture Library New genus

Murdochia asaccharolytica gen. nov., sp. nov., a gram- stain-positive, anaerobic coccus isolated from human wound specimens Ulger-Toprak et al., Int J Syst Evol Microbiol, 60, 2010

© by author ESCMID Online Lecture Library Peptoniphilus coxii sp. nov. and Peptoniphilus tyrrelliae sp. nov. isolated from human clinical infections D.M. Citron et al. Anaerobe 2012; 18:244-248 GPAC in general

Most GPAC: - mixed infections - susceptible for antibiotics usually used to treat anaerobic infections

Metronidazole-resistance: - strict anaerobic cocci© byare generallyauthor sensitive - micro-aerophilic cocci are generally resistent

ESCMID Online Lecture Library Phenotypic identification

Level II:

Ka Va Ct SPS

Most GPAC S S R R P. anaerobius RS S R S P. stomatis S Pa. micra © by S author S R V Pn. ivorii S S R V

ESCMID Online Lecture Library SPS: S, take a good look at the cell morphology P. anaerobius Pa. micra

F. magna

Phenotypic identification

Most GPAC can be identified using enzyme reactions

Present in Rapid ID 32A

ESCMIDObtained code Onlinenot suitable forLecture identification Library

Use table Wadsworth manual

No differentation possible between Pn. asaccharolyticus and Pn. harei.

Pn. harei has an irregular colony- and cellmorphology, Pn. asaccharolyticus not.

In practice very difficult© by to achieve.author

ESCMID Online Lecture Library Phenotypic identification

Development of a flow chart for the identification of gram- positive anaerobic cocci in the clinical laboratory Song et al. J. Clin. Microbiol. 2007; 45: 512-516

Using sequence and phenotypic data of 13 reference strains and 111 clinical isolates,© by a flow author scheme was developed for the identification of GPAC ESCMID Online Lecture Library (8) (1 ref + 10)

(1 ref + 1) (1 ref + 4)

(1 ref + 15) (1 ref + 16) (1 ref + 12) (1 ref)

(1 ref) (6) © by author (1 ref +3) P. gorbachii (6) P. olsenii (1ref + 21) (2 ref + 3) A. murdochii ESCMID Online (4)Lecture Library (1ref + 3)

Song et al. J. Clin. Microbiol. 2007; 45: 512-516 Vitek ANC kaart

GPAC (103) correct Vitek ID (n) species (n) species ID (n) genus ID (n)

F. magna (33) 33 33 P. micra (18) 16 16 P. anaerobius (4) 4 4 overig (48) 12 32 © by author ESCMID Online Lecture Library

F. magna P. micra P. anaerobius Identification using fluorescent in situ hybridization

Species-specific probes were developed for:

F. magna Pa. micra Pn. asaccharolyticus A. vaginalis A. lactolyticus© by author Pn. ivorii Pn. harei ESCMID P.Online anaerobius/stomatis Lecture Library Application probes

Probes were used to identify 100 unknown clinical isolates:

29 % F. magna 28 % Pa. micra 17 % Pn. harei 1 % A. lactolyticus 2 % A. vaginalis © by author 6 % Pn. ivorii 4 % P. anaerobius ESCMID13 % no identification Online Lecture Library Probe ID vs. pheno ID (summary)

Reliability phenotypic identification:

Reliable: F. magna Pa. micra P. anaerobius Reasonably: A. vaginalis © byPn. ivoriiauthor Unreliable : Pn. harei ESCMID Online A. lactolyticusLecture Library Direct application on clinical material

Pa. micra in pus derived from an intra-oral abscess Matrix Assisted Laser Desorption/Ionization time-of-flight Mass Spectrometry (MALDI-TOF MS)

Vacuum flight tube

Target at 15-25 kV Detector

Ion source © by author

ESCMID- “Time of flight” Online of individual Lecture proteins is converted Library into mass information. - Spectrum is produced - Database is built Veloo et al. Anaerobe 2011; 17:211-212

Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry Veloo et al. Syst Appl Microbiol 2011; 34:58-62

Construction database: sequenced clinical isolates (n=77) and reference strains (n=12) reference strain P. harei is different compared to the clinical isolates P. harei © by author

ValidationESCMID of constructed Online database: Lecture Library

107 genotypically identified clinical isolates of GPAC Validation database

MALDI-TOF ID (n) Strain (n) correct ID no ID

F. magna (32) 29 3 1 strain sequence similarity <98 % A. vaginalis (3) 2 1 P. ivorii (3) 1 2 sequence similarity <98 % P. octavius (1) 1 Bacterium N14-24 (1) 1 Not present in database GPAC (3) 3 © by author

All other GPAC were correctly identified: P. micra (29) P. gorbachii (3) A. tetradius (1) ESCMIDP. harei (15) Online A. Lectureparvulum (3) LibraryP. lacrimalis (1) A. murdochii (6) P. niger (1) P. anaerobius (4) A. lactolyticus (1) Correctly identified: 96 strains

No identification: 11 strains - 2 strains, no reference spectra present in database - 6 strains, <98 % sequence similarity with closest relative - 3 strains, unknown © by author

90 % correctly identified ESCMID Online Lecture Library Clustering

P. anaerobius

P. niger © by author P. micra ESCMID Online Lecture Library

P. ivorii 100 Atopobium parvulum, AF292372 Dendrogram MALDI-TOF Peptococcus niger, X55797 100 RuminococcusPhylogeneticAtopobium gnavus, treeparvulum,X94967 AF292372 100 PeptostreptococcusPeptococcus niger, anaerobius,X55797 L04168 PeptostreptococcusRuminococcus gnavus, stomatis,X94967DQ160208 100 gpac228Peptostreptococcus94.8 % anaerobius, L04168 100Peptostreptococcus stomatis,Atopobium parvulum, AF292372 gpac216 94.4Peptococcus % niger, DQ160208 gpac228 X55797 97 gpac103 94.694.8 Ruminococcus %% gnavus, X94967 gpac216 10094.4Peptostreptococcus % anaerobius, L04168 gpac003 95.0Peptostreptococcus % stomatis, DQ160208 gpac103 94.6 % 97 gpac007 94.7gpac228 % 94.8 % gpac003 95.0gpac216 % 94.4 % gpac0749794.1gpac103 % 94.6 % 95 gpac007gpac018A94.7gpac003 % 95.0 % 100 gpac074 94.3 % Peptoniphilus94.1gpac007 %ivorii,94.7Y07840 % 100 95 gpac018A 94.3gpac074 % 94.1 % 100 Peptoniphilus100 95 gpac018A harei,94.3 % Y07839 Peptoniphilus ivorii, Y07840 gpac090Peptoniphilus97.8 % ivorii, Y07840 100 Peptoniphilus100 Peptoniphilus harei, Y07839harei, Y07839 Peptoniphilusgpac090 gorbachii,97.8 % DQ911241 gpac090 97.8 % gpac055 97.4Peptoniphilus % gorbachii, DQ911241 Peptoniphilusgpac055 gorbachii,97.4 % DQ911241 gpac018B 97.8 % gpac055 97.4gpac018B % 97.8 % 100 gpac121100 gpac12196.3 % 96.3 % gpac018B 97.8Peptoniphilus % olsenii, 100 Peptoniphilus100 olsenii, DQ911242 100 gpac121100 96.3gpac077 % 96.0 DQ911242% gpac077Peptoniphilus96.0 %lacrimalis, 100 100 Peptoniphilus olsenii, DQ911242AF542230 Peptoniphilus100 Peptoniphilus lacrimalis, asaccharolyticus,AF542230AF542228 100 gpac077Peptoniphilus96.0 indolicus, % AY153431 100 PeptoniphilusPeptoniphilusgpac015 asaccharolyticus, lacrimalis,97.8 % AF542230AF542228 Peptoniphilusgpac131 indolicus,97.9 % 100 Peptoniphilus gpac170asaccharolyticus,97.9 %AY153431AF542228 gpac01595 Peptoniphilus97.8 gpac022indolicus, % 97.7 %AY153431 gpac049 97.6 % gpac015gpac1319897.897.9 %% gpac170100 97.9gpac134 % 97.7 % 95 gpac131 97.9Parvimonas % micra, AF542231 gpac170gpac022 97.7 gpac072% 97.8 % 95 10097.9 % © by author98 gpac049 97.6 Finegoldia % magna, AF542227 gpac022 97.7gpac202 % 100gpac134 97.7 % 10098 gpac049 97.6 % Anaerococcus hydrogenalis, D14140 gpac047 100 Parvimonasgpac134 97.7 micra,100 % AF542231 gpac072 Anaerococcus vaginalis, AF542229 100Parvimonas100 micra,97.8 AF542231%gpac215 100 Finegoldiagpac072 magna,97.8gpac104 % AF542227 gpac202 Anaerococcus murdochii, DQ911243 100 Finegoldia100 magna,gpac126AF542227 ESCMID Online Lecturegpac202Anaerococcus LibraryAnaerococcus hydrogenalis, lactolyticus, AF542233 100 D14140 100 Anaerococcusgpac047Anaerococcus hydrogenalis, prevotii, AF542232 Anaerococcus tetradius, AF542234D14140 100 Anaerococcusgpac047Anaerococcus vaginalis, octavius,AF542229Y07841 100 Anaerococcusgpac215bacterium N14-24, vaginalis, AY880043AF542229 100 gpac104gpac215 10 Anaerococcus%gpac104 murdochii, DQ911243 100 Anaerococcusgpac126 murdochii, DQ911243 100 Anaerococcusgpac126 lactolyticus, AF542233 AnaerococcusAnaerococcus prevotii,lactolyticus,AF542232AF542233 Anaerococcus prevotii,tetradius,AF542232AF542234 AnaerococcusAnaerococcus tetradius, octavius,AF542234Y07841 bacteriumAnaerococcus N14-24, octavius,AY880043Y07841 bacterium N14-24, AY880043 10 % 10 % Resistance GPAC

MIC50 MIC50

8 7

1 0 ESCMID Online Lecture Library

Cefotetan Cefoxitin Linezolid Ertapenem Tigecycline Penicillin G MeropenemLevofloxacinMoxifloxacinClindamycin Doxycycline ErythromycinMetronidazole F. magna (n=31) Chloramphenicol P. micra (n=27) P. harei (n=16) Amoxicillin-clavulanic acid Resistance GPAC

MIC90 16 64 >256 24 8 7 6

Cefoxitin Linezolid Penicillin G Cefotetan Ertapenem ESCMID MeropenemOnlineClindamycin LectureDoxycycline LibraryTigecycline LevofloxacinMoxifloxacin ErythromycinMetronidazole Chloramphenicol F. magna (n=31) P. micra (n=27) Amoxicillin-clavulanic acid P. harei (n=16) Resistance GPAC

% resistance (EUCAST)

5 © by author

ESCMID0 Online Lecture Library F. magna P. micra P. harei F. magna

Infections in which anaerobic bacteria play a role:

30 % GPAC 30 % F. magna

Can be isolated in pure culture. Only Bacteroides fragilis is more often encountered in pure culture.

Not present in the oral microbiota Pathogenesis F. magna

Virulence factor Function

Collagenase breakdown collagen PAB binding to human serum albumin Protein L Immunoglobulin (Ig)-binding protein release© by of author “de novo-synthesized mediators” SufAESCMID Online degradation Lecture of fibrinogen Library degradation of antibacterial peptides release of FAF from bacterial cell wall Pathogenesis F. magna

Virulence factor Function

FAF mediation of bacterial aggregation binding with BM40, present on the skin blocking of an antibacterial peptide inactivation© by author of MIG/CXCL9 (=antibacterial peptide)

ESCMID Online Lecture Library Virulence and “site of infection”

Correlation between collagenase production and site of infection Krepel et al.(1991, 1992): F. magna (n) - 222 intra-abdominal samples 11 - 58 non-puerperal breast abscesses 21 - 56 diabetic foot infections 18

ESCMIDF. magna is the Online most common Lecture anaerobe isolatedLibrary from non- puerperal breast abscesses

Virulence and “site of infection”

Correlation between protein L producing F. magna strains and bacterial vaginosis Kastern et al. Infect immun 58: 1217, 1990

Total: 30 F. magna strains

- 19 from healthy© by women author - 4 from healthy men ESCMID - 7 from Online women with Lecture bacterial vaginosis Library 4 strains express protein L Virulence and “site of infection”

de Château et al. J Biol chem 271: 26609, 1996 Expression of protein L and PAB in 48 F. magna strains

- 30 strains from suppurative infections

1 strain protein L, 16 strains PAB

- 10 commensal strains ESCMID No expression Online of proteinLecture L or PAB Library

Protein L and PAB were not expressed at the same time. Message