Gram-Positive Anaerobic Cocci: Clinical Relevance, Changed Taxonomy, Identification and Antibiotic Resistance

Gram-positive anaerobic cocci: clinical relevance, changed taxonomy, identification and antibiotic resistance

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

New species and taxonomy

Identification © by author

Antibiotic resistance ESCMID Online Lecture Library 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 GPAC in general

Clinical relevance: ± 30% of all anaerobes recovered from human clinical specimens

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

Metronidazole-resistance: ESCMID- strict anaerobic Online cocci are Lecturegenerally sensitive Library - micro-aerophilic cocci are generally resistent

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 genus and species

Peptostreptococcus stomatis Peptostreptococcus anaerobius Peptostreptococcus canis Peptostreptococcus russellii Finegoldia magna Gallicola barnesae Parvimonas micra Murdochiella asaccharolytica Anaerococcus murdochii Anaerococcus lactolyticus Anaerococcus tetradius Anaerococcus prevotii Anaerococcus vaginalis Anaerococcus obensiensis Anaerococcus hydrogenalis Anaerococcus senegalensis Anaerococcus octavius Anaerococcus pacaensis Peptoniphilus indolicus Peptoniphilus asaccharolyticus ©Peptoniphilus by methioninivorax author Peptoniphilus ivorii Peptoniphilus coxii Candidatus Peptoniphilus Peptoniphilus tyrelliae(Peptoniphilus senegalensis) Peptoniphilus allenii ESCMID OnlinePeptoniphilus Lecture grossensis Library Peptoniphilus harei Peptoniphilus gorbachii Peptoniphilus timonensis Peptoniphilus olsenii Peptoniphilus lacrimalis Peptoniphilus koenoeniae Peptoniphilus duerdenii Peptoniphilus obesi Phenotypic identification

Enzymatic tests derived from the API 32A (BioMerieux) Table Wadsworth manual

Reliable identification: - F. magna - P. micra

P. harei – P. asaccharolyticus similar biochemical features

The addition of new species made© available by schemes author for the identification of GPAC less useful

ESCMID Online Lecture Library Identification GPAC using 16S rRNA sequencing

presented in NCBI

Species Nucleotide collection 16S rRNA sequences remark Bacteria and Archaea

A. murdochii only strain nr no A. tetradius species name yes A. lactolyticus species name yes A. octavius species name yes A. prevotii species name yes A. vaginalis species name yes A. hydrogenalis species name no sequence similarity with A. senegalensis is >99% A. pacaensis only strain nr no link with accession number yields species name A. senegalensis only strain nr no sequence similarity with A. hydrogenalis is >99% © by author link with accession number yields species name A. obesiensis species name no P. coxii species name no P. tyrelliae species name no sequence similarity with P. senegalensis is >99% P. harei species name yes P. ivorii species name yes also in NCBI under the species name P. ivoricus P. olsenii ESCMID only strain nr Online no Lecture type strain onlyLibrary mentioned with WAL number in NCBI clinical isolate with species name present in NCBI P. gorbachii only strain nr no type strain only mentioned with WAL number in NCBI clinical isolate with species name present in NCBI P. duerdenii species name no P. koenoeniae species name yes P. timonensis species name no Identification GPAC using 16S rRNA sequencing

presented in NCBI

Species Nucleotide collection 16S rRNA sequences remark Bacteria and Archaea P. grossensis species name no P. lacrimalis species name yes P. asaccharolyticus species name yes ………….. P. indolicus species name yes Candidatus P. massiliensis species name no this species is not officially described Candidatus P. allenii only strain nr no this species is not officially described P. rhinitidis no sequence of 16S rRNA gene available P. senegalensis only strain nr no sequence similarity with P. tyrelliae is >99% link with accession number yields species name P. obesi species name no P. micra species name © yes by author F. magna species name yes P. anaerobius species name yes P. stomatis species name yes M. asaccharolytica species name no ESCMID Online Lecture Library 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 GPAC by MALDI-TOF MS

Bruker system

• 75% of GPAC encountered in human clinical specimens is identified by MALDI- TOF MS (estimated)

• On target extraction with 70% formic acid is recommended

• A. vaginalis is misidentifed as A. hydrogenalis

• P. harei is correctly identified © by author • New species are not represented in de MALDI-TOF MS database ESCMID Online Lecture Library Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry Veloo et al. Syst Appl Microbiol 2011; 34:58-62

Shimatzu/BioMerieux MS system

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, 100 AF542233D14140 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 2002-2004

Veloo et al., Antimicrob Agent Chemother, 2011;55:1199-1203 24 64 24 90 MIC © by author ESCMID Online Lecture Library Resistance GPAC 2011-2013

Veloo et al., Anaerobe, 2014 in press

2011-2013 MIC90 >256

8 7 6 5 4 3 F. magna 2 P. micra 1 0 P. harei 2002-2004 MIC90

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