Clinically relevant Gram- positive anaerobic cocci (GPAC), new names and their antibiotic susceptibility © by author ESCMID Online Lecture Library Linda Veloo Contents 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 authorAnaerococcus 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 New species © 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 © byS authorS 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 © by author ESCMID Online Lecture Library F. magna Phenotypic identification Most GPAC can be identified using enzyme reactions Present in Rapid ID 32A © by author ESCMIDObtained code Onlinenot suitable forLecture identification Library Use table Wadsworth manual © by author ESCMID Online Lecture Library Phenotypic identification 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 © by author ESCMID Online Lecture Library 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