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.
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.
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.
Background
Wastewater treatment plants are known as major sources for the release of micropollutants and bacteria into surface waters. To reduce this contaminant and microbial input, new technologies for effluent treatment have become available. The present paper reports the chemical, microbiological, biochemical, and biological effects of upgrading a wastewater treatment plant (WWTP) with a powdered activated carbon stage in the catchment area of the Schussen River, the largest German tributary of Lake Constance. Data were obtained prior to and after the upgrade between 2011 and 2017.
Results
After the upgrading, the release of antibiotic resistant and non-resistant bacteria, micropollutants, and their effect potentials was significantly lower in the effluent. In addition, in the Schussen River downstream of the wastewater treatment plant, reduced concentrations of micropollutants were accompanied by both a significantly improved health of fish and invertebrates, along with a better condition of the macrozoobenthic community.
Conclusions
The present study clearly provides evidence for the causality between a WWTP upgrade by powdered activated carbon and ecosystem improvement and demonstrates the promptness of positive ecological changes in response to such action. The outcome of this study urgently advocates an investment in further wastewater treatment as a basis for decreasing the release of micropollutants and both resistant and non-resistant bacteria into receiving water bodies and, as a consequence, to sustainably protect river ecosystem health and drinking water resources for mankind in the future.