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Inga-maria Frick - One of the best experts on this subject based on the ideXlab platform.
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identification of pili on the surface of Finegoldia magna a gram positive anaerobic cocci
Anaerobe, 2014Co-Authors: Elizabeth C Murphy, Christofer Karlsson, Robert Janulczyk, Matthias Morgelin, Inga-maria FrickAbstract:Pili have only been discovered in the major Gram-positive pathogens in the past decade and they have been found to play an important role in colonisation and virulence. Pili have been shown to have many important functions including attachment to host tissues, mediating bacterial aggregation, biofilm formation and binding to proteins in the extracellular matrix. In this study, sortase-dependent pili have been found to be expressed on the surface of Finegoldia magna ALB8. F. magna is a Gram-positive anaerobic coccus that, primarily, is a commensal of the skin and mucous membranes, but has also been isolated from various clinical infection sites and is associated with soft-tissue abscesses, wound infections and bone and prosthetic joint infections. In this study, F. magna ALB8 was found to harbour three sortases at the pilus locus, two of which bear high similarity to class C sortases in Streptococcus pneumoniae. Two putative sortase-dependent pili proteins were found in the locus, with one being identified as the major pilus subunit, Fmp1 (F. magna pilus subunit 1), due to its high similarity to other major pilus proteins in prominent Gram-positive pathogens. The presence of sortase-dependent pili was confirmed experimentally through recombinant production of Fmp1 and production of antiserum. The Fmp1 antiserum was used in Western blot to show the presence of a high molecular weight protein ladder, characteristic of the presence of pili, in trypsin released cell wall surface proteins from F. magna. The presence of sortase-dependent pili was visually confirmed by transmission electron microscopy, which showed the binding of gold labelled anti-Fmp1 to individual pilus proteins along the pilus. Furthermore, pili could also be found to bind and interact with keratinocytes in the epidermal layer of human skin, suggesting an adhesive role for pili on F. magna. Our work represents the first description of pilus structures in F. magna. This discovery further elucidates F. magna physiology and allows for additional analysis of host-bacterial interactions in future studies.
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Substrate profiling of Finegoldia magna SufA protease, inhibitor screening and application to prevent human fibrinogen degradation and bacteria growth in vitro.
Biochimie, 2014Co-Authors: Ewa Burchacka, Inga-maria Frick, Marcin Sieńczyk, Magdalena Wysocka, Adam Lesner, Józef OleksyszynAbstract:SufA, which belongs to the subtilisin-like serine protease family, contains a non-canonical Asp-His-Ser catalytic triad. Under in vitro conditions, SufA is capable of human fibrinogen hydrolysis leading to inhibition of fibrin network formation, thus suggesting its important role in the development and progression of Finegoldia magna infections. In addition, it has been demonstrated that SufA can hydrolyze antibacterial peptides such as LL-37 and the chemokine MIG/CXCL 9, hence evading host defence mechanisms. Although the SufA protease from F. magna was discovered several years ago, its optimal substrate preference has not yet been identified. Considering the role of SufA, we have focused on the profiling of its substrate sequence preference spanning S1-S3 binding pockets using the FRET (fluorescence resonance energy transfer) approach. Next, based on the structure of the P1 residue of the developed substrate, we narrowed the inhibitor screening to the phosphonic analogues of amino acids containing an arginine-like side chain. Among all the compounds tested, only Cbz-6-AmNphth(P)(OPh)2 showed any inhibitory activity against SufA displaying k2/Ki value of 10,800 M(-1) s(-1). In addition, it prevented SufA-mediated human fibrinogen hydrolysis in vitro and exhibited potent antibacterial activity against F. magna, Staphylococcus aureus and Escherichia coli. Herein, we report on the substrate specificity, synthesis and kinetic evaluation of phosphonic inhibitors of SufA protease from F. magna which could help to establish its function in pathogenesis development and may lead to the elaboration of new antibacterial drugs.
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faf and sufa proteins of Finegoldia magna that modulate the antibacterial activity of histones
Journal of Innate Immunity, 2014Co-Authors: Elizabeth C Murphy, Tirthankar Mohanty, Inga-maria FrickAbstract:Many bacterial pathogens have developed methods to overcome the defences of the host innate immune system. One such defence is the release of antimicrobial peptides (AMPs). Histones have been found to function as AMPs, in addition to their main biological function of packaging and organising DNA into nucleosomes. In this study, the Gram-positive anaerobic coccus Finegoldia magna was found to bind histones by Western blot and immunoprecipitation analysis. F. magna, which is normally a commensal of the skin and mucous membranes, is also known to act as an opportunistic pathogen and has been isolated from various clinical infection sites. It was found to bind to histones extracted from human skin epidermis through its surface and extracellular adhesion protein FAF. Through FAF binding, F. magna was protected from histone bactericidal activity. Furthermore, the histones were found to be degraded by SufA, a subtilisin-like extracellular serine protease of F. magna. Hence, the results of the present study will give more insight into how F. magna persists both as a commensal organism at the basement membrane of the skin and as an opportunistic pathogen during infection.
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Constitutive and Inflammation-Dependent Antimicrobial Peptides Produced by Epithelium Are Differentially Processed and Inactivated by the Commensal Finegoldia magna and the Pathogen Streptococcus pyogenes
Journal of immunology (Baltimore Md. : 1950), 2011Co-Authors: Inga-maria Frick, Matthias Morgelin, Anders I. Olin, Sara L. Nordin, Maria Baumgarten, Ole E. Sørensen, Arne EgestenAbstract:Epithelial linings serve as physical barriers and produce antimicrobial peptides (AMPs) to maintain host integrity. Examples are the bactericidal proteins midkine (MK) and BRAK/CXCL14 that are constitutively produced in the skin epidermal layer, where the anaerobic Gram-positive coccoid commensal Finegoldia magna resides. Consequently, this bacterium is likely to encounter both MK and BRAK/CXCL14, making these molecules possible threats to its habitat. In this study, we show that MK expression is upregulated during inflammation, concomitant with a strong downregulation of BRAK/CXCL14, resulting in changed antibacterial conditions. MK, BRAK/CXCL14, and the inflammation-dependent antimicrobial β-defensins human β-defensin (hBD)-2 and hBD-3 all showed bactericidal activity against both F. magna and the virulent pathogen Streptococcus pyogenes at similar concentrations. SufA, a released protease of F. magna, degraded MK and BRAK/CXCL14 but not hBD-2 nor hBD-3. Cleavage was seen at lysine and arginine residues, amino acids characteristic of AMPs. Intermediate SufA-degraded fragments of MK and BRAK/CXCL14 showed stronger bactericidal activity against S. pyogenes than F. magna, thus promoting survival of the latter. In contrast, the cysteine-protease SpeB of S. pyogenes rapidly degraded all AMPs investigated. The proteins FAF and SIC, released by F. magna and S. pyogenes, respectively, neutralized the antibacterial activity of MK and BRAK/CXCL14, protein FAF being the most efficient. Quantitation and colocalization by immunoelectron microscopy demonstrated significant levels and interactions of the molecules in in vivo and ex vivo samples. The findings reflect strategies used by a permanently residing commensal and a virulent pathogen, the latter operating during the limited time course of invasive disease.
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sufa of the opportunistic pathogen Finegoldia magna modulates actions of the antibacterial chemokine mig cxcl9 promoting bacterial survival during epithelial inflammation
Journal of Biological Chemistry, 2009Co-Authors: Christofer Karlsson, Matthias Morgelin, Anders I. Olin, Mette Eliasson, Anna Karlsson, Martin Malmsten, Arne Egesten, Inga-maria FrickAbstract:The anaerobic bacterium Finegoldia magna is part of the human commensal microbiota, but is also an important opportunistic pathogen. This bacterium expresses a subtilisin-like serine proteinase, SufA, which partially degrade the antibacterial chemokine MIG/CXCL9. Here, we show that MIG/CXCL9 is produced by human keratinocytes in response to inflammatory stimuli. In contrast to the virulent human pathogen Streptococcus pyogenes, the presence of F. magna had no enhancing effect on the MIG/CXCL9 expression by keratinocytes, suggesting poor detection of the latter by pathogen-recognition receptors. When MIG/CXCL9 was exposed to SufA-expressing F. magna, the molecule was processed into several smaller fragments. Analysis by mass spectrometry showed that SufA cleaves MIG/CXCL9 at several sites in the COOH-terminal region of the molecule. At equimolar concentrations, SufA-generated MIG/CXCL9 fragments were not bactericidal against F. magna, but retained their ability to kill S. pyogenes. Moreover, the SufA-generated MIG/CXCL9 fragments were capable of activating the angiostasis-mediating CXCR3 receptor, which is expressed on endothelial cells, in an order of magnitude similar to that of intact MIG/CXCL9. F. magna expresses a surface protein called FAF that is released from the bacterial surface by SufA. Soluble FAF was found to bind and inactivate the antibacterial activity of MIG/CXCL9, thereby further potentially promoting the survival of F. magna. The findings suggest that SufA modulation of the inflammatory response could be a mechanism playing an important role in creating an ecologic niche for F. magna, decreasing antibacterial activity and suppressing angiogenesis, thus providing advantage in survival for this anaerobic opportunist compared with competing pathogens during inflammation.
John E Degener - One of the best experts on this subject based on the ideXlab platform.
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antimicrobial susceptibility of clinically relevant gram positive anaerobic cocci collected over a three year period in the netherlands
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Alida Veloo, G W Welling, John E DegenerAbstract:The susceptibility of 14 species of 115 Gram-positive anaerobic cocci (GPAC) was determined for 14 antibiotics. To assure correct identification, strains were genotypically identified by fluorescence in situ hybridization and sequencing. Susceptibility differences (MIC50 and MIC90) for penicillin G, clindamycin, tigecycline, levofloxacin, amoxicillin-clavulanic acid, cefoxitin, ertapenem, meropenem, metronidazole, and doxycycline were found for the three clinically most relevant GPAC species: Finegoldia magna, Parvimonas micra, and Peptoniphilus harei.
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identification of gram positive anaerobic cocci by maldi tof mass spectrometry
Systematic and Applied Microbiology, 2011Co-Authors: Alida Veloo, G W Welling, Marcel Erhard, Martin Welker, John E DegenerAbstract:Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 16S sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC.
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development of 16s rrna based probes for the identification of gram positive anaerobic cocci isolated from human clinical specimens
Clinical Microbiology and Infection, 2007Co-Authors: A C M Wildeboerveloo, Gjalt W. Welling, Hermie J. M. Harmsen, John E DegenerAbstract:Fluorescent probes targeted at 16S rRNA were designed for Peptostreptococcus anaerobius and Peptostreptococcus stomatis (Pana134), Parvimonas micra (Pamic1435), Finegoldia magna (Fmag1250), Peptoniphilus asaccharolyticus (Pnasa1254), Peptoniphilus ivorii (Pnivo731), Peptoniphilus harei (Pnhar1466), Anaerococcus vaginalis (Avag1280) and Anaerococcus lactolyticus (Alac1438), based on the 16S rRNA sequences of reference strains and 88 randomly chosen clinical isolates. These strains were also used for validation of the probes. Application of the probes to an additional group of 100 clinical isolates revealed that 87% of Gram-positive anaerobic cocci (GPAC) could be identified with this set of probes. The 16S rRNAs of 13 clinical isolates that could not be identified were sequenced. Most of these isolates were GPAC that were not targeted by the probes. No clinical isolates of Pn. asaccharolyticus were encountered. Near full-length sequences were obtained from 71 of 101 (n = 88 + 13) sequenced clinical isolates. Of these, 25 showed <98% similarity with the homologues of the closest established species. The Fmag1250, Pamic1435, Pnhar1466, Pana134, Pnasa1254 and Pnivo731 probes allowed reliable identification and hybridised with all corresponding isolates. The Avag1280 and Alac1438 probes failed to hybridise with two isolates and one isolate, respectively, because of intra-species variation. However, overall, the set of probes yielded fast and reliable identification for the majority of clinical isolates.
Pierre-edouard Fournier - One of the best experts on this subject based on the ideXlab platform.
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burden of emerging anaerobes in the maldi tof and 16s rrna gene sequencing era
Anaerobe, 2011Co-Authors: Bernard La Scola, Pierre-edouard FournierAbstract:The isolation of anaerobes from patients has declined in recent years, whereas their detection by molecular techniques has increased. In the present work, we analyzed the application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing to routine identification of anaerobes in clinical microbiology laboratory. We identified 544 isolates of 79 species by routine culture from deep samples in our hospital. MALDI-TOF MS allowed identification of 332 isolates (61%). The remaining 212 (39%) were identified by 16S rRNA gene sequencing, allowing identification of 202 at the species level. The most common anaerobes were Propionibacterium spp. (12%), Finegoldia magna (4%), Fusobacterium spp. (6%) and Bacteroides spp. (6%). However, among the 79 identified species, seven were new species or genera, including two Prevotella conceptionensis, a species previously detected by our team by amplification and sequencing, five Anaerococcus sp. and one Prevotella sp. Beyond the identification of these new species, we also identified several uncommon or previously not described associations between species and specific pathologic conditions. MALDI-TOF MS-based identification, which will become more effective with future spectra database improvement, will be likely responsible of a burden of emerging anaerobes in clinical microbiology.
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Finegoldia magna, an early post-operative cause of infectious endocarditis: report of two cases and review of the literature.
Anaerobe, 2008Co-Authors: Pierre-edouard Fournier, My Van La, Jean-paul Casalta, Hervé Richet, Frédéric Collart, Didier RaoultAbstract:We report two cases of infectious endocarditis (IE) on prosthetic valves caused by Finegoldia magna. The diagnosis was obtained by detection of the bacterium in valvular biopsies using 16S rRNA PCR amplication and sequencing, and prolonged culture. Five other cases were previously published in the literature. Following analysis of these seven cases, F. magna endocarditis presented as a subacute endocarditis, developing early (60 days) following valvular replacement (85%), with an elevated mortality (28%). Our report highlights the potential role of F. magna in early post-surgical endocarditis on prosthetic valves.
Ivan Mitov - One of the best experts on this subject based on the ideXlab platform.
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virulence arsenal of the most pathogenic species among the gram positive anaerobic cocci Finegoldia magna
Anaerobe, 2016Co-Authors: Lyudmila Boyanova, Rumyana Markovska, Ivan MitovAbstract:Abstract This review focuses on the virulence arsenal of the most pathogenic species among Gram positive anaerobic cocci, Finegoldia magna according to recently published data from 2012 to 2016. Virulence factors like sortase dependent pili and F. magna adhesion factor (FAF) facilitate the start of the infection. Albumin binding protein (PAB) enhances F. magna survival. FAF, subtilisin-like extracellular serine protease (SufA) and superantigen protein L protect the bacteria from factors of innate defense system. SufA, capsule and tissue-destroying enzymes provide a deep penetration or spread of the infections and the protein L is associated with infection severity. Biofilm production results in infection chronification and complicated treatment as well as to persistence of multi-species biofilms. Resistance rates to quinolones (13.0->70%) and clindamycin (0–40.0%) are important, and resistance to penicillins (
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anaerobic microbiology in 198 cases of pleural empyema a bulgarian study
Anaerobe, 2004Co-Authors: Lyudmila Boyanova, Vladimir Djambazov, Galina Gergova, Dragomir Iotov, Danail Petrov, Dencho Osmanliev, Zvetan Minchev, Ivan MitovAbstract:Abstract The aim of the study was to evaluate the incidence of anaerobic bacteria in 198 patients with pleural empyema and the susceptibility of isolates to eight antibacterial agents. Isolates were identified by the Crystal anaerobes identification system, API System rapid ID 32 A and/or routine methods. Susceptibility was tested by Sceptor MIC system for anaerobic bacteria and limited agar dilution method. Anaerobic bacteria were found in 74.2% of the patients and included 247 strains within 21 genera. The predominant anaerobes were Gram-positive anaerobic cocci (52 isolates), Fusobacterium (51), microaerophilic streptococci (24), Prevotella (19) and Bacteroides species (11). Common species/groups were Fusobacterium nucleatum (in 27.2% of specimens yielding anaerobes), Micromonas micros (8.2%), Finegoldia magna (7.5%), Bacteroides fragilis group (6.8%), Peptostreptococcus anaerobius (6.1%) and F. necrophorum (5.4%). No resistance to chloramphenicol and ampicillin/sulbactam was detected. The susceptibility rates of Gram-negative anaerobic isolates to penicillin, cefoxitin, clindamycin, clarithromycin, metronidazole and tetracycline were 63.8%, 90.2%, 87.8%, 58.6%, 98.8% and 71%, and those of Gram-positive anaerobes were 79.2%, 100%, 84.3%, 68.4%, 41.9% and 75%, respectively. The wide diversity of isolated anaerobic genera and species and the susceptibility patterns of the isolates emphasize the role of the anaerobic microbiology in cases of pleural empyema.
Alida Veloo - One of the best experts on this subject based on the ideXlab platform.
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the influence of incubation time sample preparation and exposure to oxygen on the quality of the maldi tof ms spectrum of anaerobic bacteria
Clinical Microbiology and Infection, 2014Co-Authors: Alida Veloo, P E Elgersma, Alexander W Friedrich, Elisabeth Nagy, Van Arie WinkelhoffAbstract:With matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), bacteria can be identified quickly and reliably. This accounts especially for anaerobic bacteria. Because growth rate and oxygen sensitivity differ among anaerobic bacteria, we aimed to study the influence of incubation time, exposure to oxygen and sample preparation on the quality of the spectrum using the Bruker system. Also, reproducibility and inter-examiner variability were determined. Twenty-six anaerobic species, representing 17 genera, were selected based on gram-stain characteristics, growth rate and colony morphology. Inter-examiner variation showed that experience in the preparation of the targets can be a significant variable. The influence of incubation time was determined between 24 and 96 h of incubation. Reliable species identification was obtained after 48 h of incubation for gram-negative anaerobes and after 72 h for gram-positive anaerobes. Exposure of the cultures to oxygen did not influence the results of the MALDI-TOF MS identifications of all tested gram-positive species. Fusobacterium necrophorum and Prevotella intermedia could not be identified after >24 h and 48 h of exposure to oxygen, respectively. Other tested gram-negative bacteria could be identified after 48 h of exposure to oxygen. Most of the tested species could be identified using the direct spotting method. Bifidobacterium longum and Finegoldia magna needed on-target extraction with 70% formic acid in order to obtain reliable species identification and Peptoniphilus ivorii a full extraction. Spectrum quality was influenced by the amount of bacteria spotted on the target, the homogeneity of the smear and the experience of the examiner.
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antimicrobial susceptibility of clinically relevant gram positive anaerobic cocci collected over a three year period in the netherlands
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Alida Veloo, G W Welling, John E DegenerAbstract:The susceptibility of 14 species of 115 Gram-positive anaerobic cocci (GPAC) was determined for 14 antibiotics. To assure correct identification, strains were genotypically identified by fluorescence in situ hybridization and sequencing. Susceptibility differences (MIC50 and MIC90) for penicillin G, clindamycin, tigecycline, levofloxacin, amoxicillin-clavulanic acid, cefoxitin, ertapenem, meropenem, metronidazole, and doxycycline were found for the three clinically most relevant GPAC species: Finegoldia magna, Parvimonas micra, and Peptoniphilus harei.
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identification of gram positive anaerobic cocci by maldi tof mass spectrometry
Systematic and Applied Microbiology, 2011Co-Authors: Alida Veloo, G W Welling, Marcel Erhard, Martin Welker, John E DegenerAbstract:Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 16S sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC.