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Prophage Carriage and Genetic Diversity within Environmental Isolates of Clostridioides difficile
(2023)
Clostridioides difficile is an important human pathogen causing antibiotic-associated diarrhoea worldwide. Besides using antibiotics for treatment, the interest in bacteriophages as an alternative therapeutic option has increased. Prophage abundance and genetic diversity are welldocumented in clinical strains, but the carriage of prophages in environmental strains of C. difficile has not yet been explored. Thus, the prevalence and genetic diversity of integrated prophages in the genomes of 166 environmental C. difficile isolates were identified. In addition, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems were determined in the genomes of prophage regions. Predicted prophages and CRISPR-Cas systems were identified by using the
PHASTER web server and CRISPRCasFinder, respectively. Phylogenetic relationships among predicated prophages were also constructed based on phage-related genes, terminase large (TerL) subunits and LysM. Among 372 intact prophages, the predominant prophages were phiCDHM1, phiCDHM19, phiMMP01, phiCD506, phiCD27, phiCD211, phiMMP03, and phiC2, followed by phiMMP02, phiCDKM9, phiCD6356, phiCDKM15, and phiCD505. Two newly discovered siphoviruses, phiSM101- and phivB_CpeS-CP51-like Clostridium phages, were identified in two C. difficile genomes. Most prophages were found in sequence types (STs) ST11, ST3, ST8, ST109, and ST2, followed by ST6, ST17, ST4, ST5, ST44, and ST58. An obvious correlation was found between prophage types and STs/ribotypes. Most predicated prophages carry CRISPR arrays. Some prophages carry several gene products, such as accessory gene regulator (Agr), putative spore protease, and abortive infection (Abi) systems. This study shows that prophage carriage, along with genetic diversity and their CRISPR arrays, may play a role in the biology, lifestyle, and fitness of their host strains.
Anaerobic digestion (AD) is an important technology for the effective conversion of waste and wastewater to methane. Here, syntrophic bacteria transfer molecular hydrogen (H2), formate, or directly supply electrons (direct interspecies electron transfer, DIET) to the methanogens. Evidence is accumulating that the methanation of short-chain fatty acids can be enhanced by the addition of conductive material to the anaerobic digester, which has often been attributed to the stimulation of DIET. Since little is known about the transcriptional response of a complex AD microbial community to the addition of conductive material, we added magnetite to propionate-fed laboratory-scale reactors that were inoculated with wastewater sludge. Compared to the control reactors, the magnetite-amended reactors showed improved methanation of propionate. A genome-centric metatranscriptomics approach identified the active SCFA-oxidizing bacteria that affiliated with Firmicutes, Desulfobacterota and Cloacimonadota. The transcriptional profiles revealed that the syntrophic bacteria transferred acetate, H2 and formate to acetoclastic and hydrogenotrophic methanogens, whereas transcription of potential determinants for DIET such as conductive pili and outer-membrane cytochromes did not significantly change with magnetite addition. Overall, changes in the transcriptional profiles of syntrophic Bacteria and Archaea in propionate-fed lab-scale reactors amended with magnetite refute a major role of DIET in the studied system.
Anaerobic digestion of biowaste not only reduces environmental burden but also plays an important role for sustainable energy supply. For process optimization simulation based on the Anaerobic Digestion Model No. 1 (ADM1) is commonly used.
The ADM1 was extended to include the known three genera of propionate oxidizing bacteria (POB) and the two routes of propionate degradation (methyl-malonyl CoA and C6-dismutation pathway). Kinetic parameters for anaerobic propionate oxidation by single strains of the three propionate oxidizing genera were determined from defined tri-cultures of the POB with hydrogenotrophic and acetotrophic methanogens and implemented into ADM1. The such improved model ADM1xpro was evaluated with operational data from a full scale wet biowaste digestion plant. Predicted amounts of biogas and composition with ADM1xpro (2201 m³ d−1, 68.1 % CH4 and 31.9 % CO2) correlated well with full-scale process data (2171 m³ d−1, 67.5 % CH4 and 31.9 % CO2).
Die anaerobe Vergärung von Bioabfällen erzeugt nachhaltig Bioenergie und verringert Umweltbelastungen. Die acetogene Gärphase wurde mit dem Anaerobic Digestion Model No. 1 (ADM1) modelliert, das um drei Gattungen Propionat-oxidierender Bakterien und zwei Varianten des Propionatabbaus, den Methyl-Malonyl-CoA- und C6-Dismutase-Weg, erweitert wurde. Die kinetischen Parameter für die anaerobe Propionat-Oxidation wurden mit definierten Trikulturen aus den drei Propionat-oxidierenden Gattungen mit hydrogenotrophen und acetotrophen methanogenen Archaea bestimmt und in das ADM1 implementiert, woraus das verbesserte Modell ADM1xpro resultierte.
Clostridioides difficile is the most important pathogen causing antimicrobial-associated diarrhea and has recently been recognized as a cause of community-associated C. difficile infection (CA-CDI). This study aimed to characterize virulence factors, antimicrobial resistance (AMR), ribotype (RT) distribution and genetic relationship of C. difficile isolates from diverse fecally contaminated environmental sources. C. difficile isolates were recovered from different environmental samples in Northern Germany. Antimicrobial susceptibility testing was determined by E-test or disk diffusion method. Toxin genes (tcdA and tcdB), genes coding for binary toxins (cdtAB) and ribotyping were determined by PCR. Furthermore, 166 isolates were subjected to whole genome sequencing (WGS) for core genome multi-locus sequence typing (cgMLST) and extraction of AMR and virulence-encoding genes. Eighty-nine percent (148/166) of isolates were toxigenic, and 51% (76/148) were positive for cdtAB. Eighteen isolates (11%) were non-toxigenic. Thirty distinct RTs were identified. The most common RTs were RT127, RT126, RT001, RT078, and RT014. MLST identified 32 different sequence types (ST). The dominant STs were ST11, followed by ST2, ST3, and ST109. All isolates were susceptible to vancomycin and metronidazole and displayed a variable rate of resistance to moxifloxacin (14%), clarithromycin (26%) and rifampicin (2%). AMR genes, such as gyrA/B, blaCDD-1/2, aph(3′)-llla-sat-4-ant(6)-la cassette, ermB, tet(M), tet(40), and tetA/B(P), conferring resistance toward fluoroquinolone, beta-lactam, aminoglycoside, macrolide and tetracycline antimicrobials, were found in 166, 137, 29, 32, 21, 72, 17, and 9 isolates, respectively. Eleven “hypervirulent” RT078 strains were detected, and several isolates belonged to RTs (i.e., RT127, RT126, RT023, RT017, RT001, RT014, RT020, and RT106) associated with CA-CDI, indicating possible transmission between humans and environmental sources pointing out to a zoonotic potential.
Linking community composition and ecosystem function via the cultivation-independent analysis of marker genes, e.g., the 16S rRNA gene, is a staple of microbial ecology and dependent disciplines. The certainty of results, independent of the bioinformatic handling, is imperative for any advances made within the field. In this work, thermophilic anaerobic co-digestion experimental data, together with primary and waste-activated sludge prokaryotic community data, were analyzed with two pipelines that apply different principles when dealing with technical, sequencing, and PCR biases. One pipeline (VSEARCH) employs clustering methods, generating individual operational taxonomic units (OTUs), while the other (DADA2) is based on sequencing error correction algorithms and generates exact amplicon sequence variants (ASVs). The outcomes of both pipelines were compared within the framework of ecological-driven data analysis. Both pipelines provided comparable results that would generally allow for the same interpretations. Yet, the two approaches also delivered community compositions that differed between 6.75% and 10.81% between pipelines. Inconsistencies were also observed linked to biologically driven variability in the samples, which affected the two pipelines differently. These pipeline-dependent differences in taxonomic assignment could lead to different conclusions and interfere with any downstream analysis made for such mis- or not-identified species, e.g., network analysis or predictions of their respective ecosystem service.
Clostridioides difficile (C. difficile) is the most common pathogen causing antibiotic-associated intestinal diseases in humans and some animal species, but it can also be present in various environments outside hospitals. Thus, the objective of this study was to investigate the presence and the characteristics of toxin-encoding genes and antimicrobial resistance of C. difficile isolates from different environmental sources. C. difficile was found in 32 out of 81 samples (39.50%) after selective enrichment of spore-forming bacteria and in 45 samples (55.56%) using a TaqMan-based qPCR assay. A total of 169 C. difficile isolates were recovered from those 32 C. difficile-positive environmental samples. The majority of environmental C. difficile isolates were toxigenic, with many (88.75%) positive for tcdA and tcdB. Seventy-four isolates (43.78%) were positive for binary toxins, cdtA and cdtB, and 19 isolates were non-toxigenic. All the environmental C. difficile isolates were susceptible to vancomycin and metronidazole, and most isolates were resistant to ciprofloxacin (66.86%) and clindamycin (46.15%), followed by moxifloxacin (13.02%) and tetracycline (4.73%). Seventy-five isolates (44.38%) showed resistance to at least two of the tested antimicrobials. C. difficile strains are commonly present in various environmental sources contaminated by feces and could be a potential source of community-associated C. difficile infections.
Die mikrobielle Umsetzung von organischem Material zu dem erneuerbaren Energieträger Methan ist eine bewährte und verbreitete Strategie der effektiven Abfallwirtschaft. Dennoch können in Biogasanlagen Prozessstörungen und im Extremfall Prozessausfälle auftreten und erhebliche Kosten verursachen. Forschende der Hochschule Emden/Leer untersuchen die Mikroorganismen und ihr Zusammenleben, um die Prozessstabilität und Produktivität zu steigern.
Background: Anaerobic digestion (AD) is a globally important technology for effective waste and wastewater management. In AD, microorganisms interact in a complex food web for the production of biogas. Here, acetoclastic methanogens and syntrophic acetate-oxidizing bacteria (SAOB) compete for acetate, a major intermediate in the mineralization of organic matter. Although evidence is emerging that syntrophic acetate oxidation is an important pathway for methane production, knowledge about the SAOB is still very limited.
Results: A metabolic reconstruction of metagenome-assembled genomes (MAGs) from a thermophilic solid state biowaste digester covered the basic functions of the biogas microbial community. Firmicutes was the most abundant phylum in the metagenome (53%) harboring species that take place in various functions ranging from the hydrolysis of polymers to syntrophic acetate oxidation. The Wood-Ljungdahl pathway for syntrophic acetate oxidation and corresponding genes for energy conservation were identified in a Dethiobacteraceae MAG that is phylogenetically related to known SAOB. 16S rRNA gene amplicon sequencing and enrichment cultivation consistently identified the uncultured Dethiobacteraceae together with Syntrophaceticus, Tepidanaerobacter, and unclassified Clostridia as members of a potential acetate-oxidizing core community in nine full-scare digesters, whereas acetoclastic methanogens were barely detected.
Conclusions: Results presented here provide new insights into a remarkable anaerobic digestion ecosystem where acetate catabolism is mainly realized by Bacteria. Metagenomics and enrichment cultivation revealed a core community of diverse and novel uncultured acetate oxidizing bacteria and point to a particular niche for them in dry fermentation of biowaste. Their genomic repertoire suggests metabolic plasticity besides the potential for syntrophic acetate oxidation.
Propionate is an important intermediate in the anaerobic mineralization of organic matter. In methanogenic environments, its degradation relies on syntrophic associations between syntrophic propionate-oxidizing bacteria (SPOB) and Archaea. However, only 10 isolated species have been identified as SPOB so far. We report syntrophic propionate oxidation in thermophilic enrichments of Candidatus Syntrophosphaera thermopropionivorans, a novel representative of the candidate phylum Cloacimonetes. In enrichment culture, methane was produced from propionate, while Ca. S. thermopropionivorans contributed 63% to total bacterial cells. The draft genome of Ca. S. thermopropionivorans encodes genes for propionate oxidation via methymalonyl-CoA. Phylogenetically, Ca. S. thermopropionivorans affiliates with the uncultured Cloacimonadaceae W5 and is more distantly related (86.4% 16S rRNA gene identity) to Ca. Cloacimonas acidaminovorans. Although Ca. S. thermopropionivorans was enriched from a thermophilic biogas reactor, Ca. Syntrophosphaera was in particular associated with mesophilic anaerobic digestion systems. 16S rRNA gene amplicon sequencng and a novel genus-specific quantitative PCR assay consistently identified Ca. Syntrophosphaera/Cloacimonadaceae W5 in 9 of 12 tested full-scale biogas reactors thereby outnumbering other SPOB such as Pelotomaculum, Smithella and Syntrophobacter. Taken together the ubiquity and abundance of Ca. Syntrophosphaera, those SPOB might be key players for syntrophic propionate metabolism that have been overlooked before.