Timely Monitoring of SARS-CoV-2 RNA Fragments in Wastewater Shows the Emergence of JN.1 (BA.2.86.1.1, Clade 23I) in Berlin, Germany.

The importance of COVID-19 surveillance from wastewater continues to grow since case-based surveillance in the general population has been scaled back world-wide. In Berlin, Germany, quantitative and genomic wastewater monitoring for SARS-CoV-2 is performed in three wastewater treatment plants (WWTP) covering 84% of the population since December 2021. The SARS-CoV-2 Omicron sublineage JN.1 (B.2.86.1.1), was first identified from wastewater on 22 October 2023 and rapidly became the dominant sublineage. This change was accompanied by a parallel and still ongoing increase in the notification-based 7-day-hospitalization incidence of COVID-19 and COVID-19 ICU utilization, indicating increasing COVID-19 activity in the (hospital-prone) population and a higher strain on the healthcare system. In retrospect, unique mutations of JN.1 could be identified in wastewater as early as September 2023 but were of unknown relevance at the time. The timely detection of new sublineages in wastewater therefore depends on the availability of new sequences from GISAID and updates to Pango lineage definitions and Nextclade. We show that genomic wastewater surveillance provides timely public health evidence on a regional level, complementing the existing indicators.

SARS-CoV-2 infection dynamics revealed by wastewater sequencing analysis and deconvolution.

The use of RNA sequencing from wastewater samples is a valuable way for estimating infection dynamics and circulating lineages of SARS-CoV-2. This approach is independent from testing individuals and can therefore become the key tool to monitor this and potentially other viruses. However, it is equally important to develop easily accessible and scalable tools which can highlight critical changes in infection rates and dynamics over time across different locations given sequencing data from wastewater. Here, we provide an analysis of lineage dynamics in Berlin and New York City using wastewater sequencing and present PiGx SARS-CoV-2, a highly reproducible computational analysis pipeline with comprehensive reports. This end-to-end pipeline includes all steps from raw data to shareable reports, additional taxonomic analysis, deconvolution and geospatial time series analyses. Using simulated datasets (in silico generated and spiked-in samples) we could demonstrate the accuracy of our pipeline calculating proportions of Variants of Concern (VOC) from environmental as well as pre-mixed samples (spiked-in). By applying our pipeline on a dataset of wastewater samples from Berlin between February 2021 and January 2022, we could reconstruct the emergence of B.1.1.7(alpha) in February/March 2021 and the replacement dynamics from B.1.617.2 (delta) to BA.1 and BA.2 (omicron) during the winter of 2021/2022. Using data from very-short-reads generated in an industrial scale setting, we could see even higher accuracy in our deconvolution. Lastly, using a targeted sequencing dataset from New York City (receptor-binding-domain (RBD) only), we could reproduce the results recovering the proportions of the so-called cryptic lineages shown in the original study. Overall our study provides an in-depth analysis reconstructing virus lineage dynamics from wastewater. While applying our tool on a wide range of different datasets (from different types of wastewater sample locations and sequenced with different methods), we show that PiGx SARS-CoV-2 can be used to identify new mutations and detect any emerging new lineages in a highly automated and scalable way. Our approach can support efforts to establish continuous monitoring and early-warning projects for detecting SARS-CoV-2 or any other pathogen.

Quantification of pathogenic microorganisms and microbial indicators in three wastewater reclamation and managed aquifer recharge facilities in Europe.

Managed Aquifer Recharge (MAR) is becoming an attractive option for water storage in water reuse processes as it provides an additional treatment barrier to improve recharged water quality and buffers seasonal variations of water supply and demand. To achieve a better understanding about the level of pathogenic microorganisms and their relation with microbial indicators in these systems, five waterborne pathogens and four microbial indicators were monitored over one year in three European MAR sites operated with reclaimed wastewater. Giardia and Cryptosporidium (oo)cysts were found in 63.2 and 36.7% of the samples respectively. Salmonella spp. and helminth eggs were more rarely detected (16.3% and 12.5% of the samples respectively) and Campylobacter cells were only found in 2% of samples. At the Belgian site advanced tertiary treatment technology prior to soil aquifer treatment (SAT) produced effluent of drinking water quality, with no presence of the analysed pathogens. At the Spanish and Italian sites amelioration of microbiological water quality was observed between the MAR injectant and the recovered water. In particular Giardia levels decreased from 0.24-6.14 cysts/L to 0-0.01 cysts/L and from 0.4-6.2 cysts/L to 0-0.07 cysts/L in the Spanish and Italian sites respectively. Salmonella gene copies and Giardia cysts were however found in the water for final use and/or the recovered groundwater water at the two sites. Significant positive Spearman correlations (p<0.05, r(s) range: 0.45-0.95) were obtained, in all the three sites, between Giardia cysts and the most resistant microbial markers, Clostridium spores and bacteriophages.

Interactions between hyphosphere-associated bacteria and the fungus Cladosporium herbarum on aquatic leaf litter.

We investigated microbial interactions of aquatic bacteria associated with hyphae (the hyphosphere) of freshwater fungi on leaf litter. Bacteria were isolated directly from the hyphae of fungi from sedimented leaves of a small stream in the National Park "Lower Oder," Germany. To investigate interactions, bacteria and fungi were pairwise co-cultivated on leaf-extract medium and in microcosms loaded with leaves. The performance of fungi and bacteria was monitored by measuring growth, enzyme production, and respiration of mono- and co-cultures. Growth inhibition of the fungus Cladosporium herbarum by Ralstonia pickettii was detected on leaf extract agar plates. In microcosms, the presence of Chryseobacterium sp. lowered the exocellulase, endocellulase, and cellobiase activity of the fungus. Additionally, the conversion of leaf material into microbial biomass was retarded in co-cultures. The respiration of the fungus was uninfluenced by the presence of the bacterium.

Quantitative PCR monitoring of antibiotic resistance genes and bacterial pathogens in three European artificial groundwater recharge systems.

Aquifer recharge presents advantages for integrated water management in the anthropic cycle, namely, advanced treatment of reclaimed water and additional dilution of pollutants due to mixing with natural groundwater. Nevertheless, this practice represents a health and environmental hazard because of the presence of pathogenic microorganisms and chemical contaminants. To assess the quality of water extracted from recharged aquifers, the groundwater recharge systems in Torreele, Belgium, Sabadell, Spain, and Nardò, Italy, were investigated for fecal-contamination indicators, bacterial pathogens, and antibiotic resistance genes over the period of 1 year. Real-time quantitative PCR assays for Helicobacter pylori, Yersinia enterocolitica, and Mycobacterium avium subsp. paratuberculosis, human pathogens with long-time survival capacity in water, and for the resistance genes ermB, mecA, blaSHV-5, ampC, tetO, and vanA were adapted or developed for water samples differing in pollutant content. The resistance genes and pathogen concentrations were determined at five or six sampling points for each recharge system. In drinking and irrigation water, none of the pathogens were detected. tetO and ermB were found frequently in reclaimed water from Sabadell and Nardò. mecA was detected only once in reclaimed water from Sabadell. The three aquifer recharge systems demonstrated different capacities for removal of fecal contaminators and antibiotic resistance genes. Ultrafiltration and reverse osmosis in the Torreele plant proved to be very efficient barriers for the elimination of both contaminant types, whereas aquifer passage followed by UV treatment and chlorination at Sabadell and the fractured and permeable aquifer at Nardò posed only partial barriers for bacterial contaminants.

Complexity of ultrafiltration membrane fouling caused by macromolecular dissolved organic compounds in secondary effluents.

Recent investigations indicate the relevance of extracellular polymeric substances (EPS) in terms of fouling of low-pressure membranes in advanced wastewater treatment. In this study, the high impact of the macromolecular fraction of effluent organic matter on fouling was confirmed in cross-flow ultrafiltration experiments using secondary effluent with and without autochthonous biopolymers. A method for the extraction of a natural mixture of EPS derived from the bacterium Sinorhizobium sp. is presented. Ultrafiltration of solutions of this bacterial EPS extract revealed a correlation between the concentration of EPS and the loss of permeate flux. However, in ultrafiltration tests using extracted bacterial EPS in a model solution as well as in secondary effluent without autochthonous biopolymers, the extent of membrane fouling was not identical with the fouling provoked by secondary effluent organic matter, although the biopolymer concentrations were comparable. The differences in the fouling behaviour of the extracted bacterial EPS and effluent organic matter are considered to be due to different compositions of the biopolymer fraction in terms of proteins, polysaccharides, and other organic colloids, indicating a particular impact of proteins on ultrafiltration membrane fouling.

Environmental influences on the partitioning and diffusion of hydrophobic organic contaminants in microbial biofilms.

A biofilm reactor was used to investigate kinetic and thermodynamic aspects of the sorption of polycyclic aromatic hydrocarbons (PAH) as model compounds for hydrophobic organic contaminants (HOC) to intact microbial biofilms. Effective diffusion coefficients are in the range of 10(-10) cm2 x s(-1) resulting in equilibration times of more than 3 days for a biofilm of 100 microm thickness. Diffusion in the biofilm was strongly temperature-dependent and increased by a factor of 3 (phenanthrene) to 6 (fluoranthene, pyrene) between 5 and 35 degrees C. Drying and rewetting of the biofilm as well as the inclusion of Ca2+ ions and of humic acids all strengthened the biofilm rigidity and slowed down the diffusion of PAH. The later two factors also influenced the thermodynamics of the process as they supported the partitioning of PAH into the biofilm. Humic acid inclusion from solution into the biofilm illustrates that a microbial biofilm can act as a primer allowing for the buildup of a particulate organic phase from dissolved organic matter. PAH metabolites (3-hydroxy-phenanthrene and 1-hydroxy-2-naphthoic acid) showed lower partition coefficients as compared to their parent compounds and 3-hydroxy-phenanthrene also showed a higher diffusion constant, indicating that these transformation products would be easily released into the water phase upon formation during PAH biodegradation in a biofilm. These results allow the quantification of the influence of environmental conditions on a biofilm's function as a sink or as a diffusion barrier for PAH from aqueous solution, and they indicate the importance of kinetic aspects of this partitioning process.

Detection of conjugative plasmids and antibiotic resistance genes in anthropogenic soils from Germany and India.

PCR typing methods were used to assess the presence of plasmids of the incompatibility (Inc) groups IncP, IncN, IncW, IncQ and rolling circle plasmids of the pMV158 type in total DNA extracts from anthropogenic soils from India and Germany. Ten different soils from two different locations in Germany, the urban park Berlin Tiergarten and the abandoned sewage field Berlin-Buch, and from four different locations in India were analysed. PCR amplification of the total DNA extracts revealed the prevalence of IncP-specific sequences in Berlin Buch and Indian soil samples. The detected IncP plasmids contained at least one transfer function, the origin of transfer, oriT. In contrast to IncP-specific sequences, IncQ, IncN, IncW and pMV158-specific sequences were never detected. The presence of ampC, tet (O), ermB, SHV-5, mecA, and vanA antibiotic resistance genes was also tested. Three Indian soil samples irrigated with wastewater contained the ampC gene, whereas the other resistance genes were not found in any of the samples. Detection of IncP trfA2 and oriT sequences by PCR amplification and hybridization is a clear indication that IncP plasmids are prevalent in these habitats. Exogenous plasmid isolation revealed conjugative plasmids belonging to the IncPbeta group encoding resistance to ampicillin.

Ultrastructural and electron energy-loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate.

Strain F8, a bacterial isolate from 'river snow', was found to produce extracellular fibres in the form of a filamentous network. These extracellular filaments, which were previously shown to be composed of DNA, have been studied for the first time by ultrastructural and electron energy-loss spectroscopy in the present work. 'Whole mount' preparations of strain F8 indicate these polymers are ultrastructurally homogeneous and form a network of elemental filaments, which have a width of 1.8-2.0 nm. When incubated at pH 3.5 with colloidal cationic ThO(2) tracers they become intensely stained (electron dense), affording direct evidence that the fibres are negatively charged and thus acidic chemically. Elemental analysis of the extracellular filaments by Energy-filtered Transmission Electron Microscopy revealed phosphorus to be the main element present and, because pretreatment of F8 cells with DNase prevented thorium labelling, the fibres must be composed of extracellular DNA (eDNA). Neither ultrathin sections nor 'whole mount negative stain' caused DNA release by general cell lysis. Additionally, cells infected with phages were never observed in ultrathin sections and phage particles were never detected in whole mount samples, which rules out the possibility of phages being directly involved in eDNA release.

Experimental and modeling approach to study sorption of dissolved hydrophobic organic contaminants to microbial biofilms.

A biofilm reactor was developed to investigate the sorption of polycyclic aromatic hydrocarbons (PAH) as model compounds for hydrophobic organic contaminants (HOC) to intact microbial biofilms at environmentally realistic concentrations. When operated as a differential column batch reactor, the system can be used to study the thermodynamics as well as the kinetics of the exchange of HOC between an aqueous phase and microbial biofilms. Organic carbon normalized partition coefficients (K(oc)) for phenanthrene, fluoranthene and pyrene were at the lower end of those known for other organic sorbents. Intra-biofilm diffusion coefficients (D) were calculated from decrease in solute concentration over time using a model for diffusion through a plane sheet and ranged from 0.23 to 0.45x10(-9)cm(2)s(-1) for the three PAH. These diffusion coefficients are about four orders of magnitude lower than those reported in literature for free aqueous solution. These data and the experimental approach presented here are useful to assess the importance of microbial biofilms for exchange processes of HOC in heterogeneous systems such as water distribution systems, membranes and aquifers, sewer systems or surface soils.

Bacterial extracellular DNA forming a defined network-like structure.

It is generally assumed that nucleic acids are localized inside of living cells and that their primary function is the storage of information. In contrast, extracellular DNA is mainly considered as a remnant of lysed cells. Here, we report the formation of extracellular bacterial DNA as a spatial structure. An aquatic bacterium, strain F8, was isolated, which produced a stable filamentous network of extracellular DNA. Different staining and enzymatic techniques confirmed that it was DNA. We were able to amplify the 16S rRNA gene from the extracellular DNA. Restriction endonuclease cleavage and randomly amplified polymorphic DNA analysis of extracellular and genomic DNAs revealed major similarities, but also some differences in both sequences. Our data demonstrate a new function and relevance for extracellular DNA.

A new enzymatic method for the detachment of particle associated soil bacteria.

A new enzymatic technique for the detachment of bacteria from soil particles was developed and applied to different soil samples taken at various sampling sites and depths. Many soil microorganisms are closely associated with the organic matrix of soil particles. They produce extracellular polymeric substances (EPS), which promote the irreversible adhesion of cells to soil particulates. To characterize the EPS, a prestaining of the soil samples with different lectins was performed. Samples from a sewage field, an urban park, a farmland, a mixed forest and garden mold were stained with a set of FITC-labelled lectins from Triticum vulgaris, Ulex europaeus, Concanavalin A and Pseudomonas aeruginosa. Based on the results, a combination of alpha-glucosidase, beta-galactosidase and a lipase was chosen for degradation of the EPS structures, followed by gentle mechanical and chemical dispersion in a modified sodium pyrophosphate buffer. The samples were fixed with formaldehyde and total cell counts were determined by DAPI staining. With the exception of the wheat field sample, this technique revealed up to 22-fold higher total cell counts for all investigated soil samples compared to the conventional detachment method, a simple dispersion with sodium pyrophosphate buffer. Efficiency of the technique was assessed by scanning electron microscopy. These images showed convincingly that the enzymatic treatment followed by sonication efficiently detached the bacteria and left the soil particles almost blank.

Investigation of lotic microbial aggregates by a combined technique of fluorescent in situ hybridization and lectin-binding-analysis.

A technique combining fluorescent in situ hybridization and lectin-binding-analysis (FISH-LBA) was developed and applied for the simultaneous detection of cellular components and glycoconjugates in lotic microbial aggregates (river snow). River snow aggregates were directly collected from the bulk water phase into coverslip chambers, in which the complete procedure including fixation, fluorescent in situ hybridization, lectin-binding and optical analysis by confocal laser scanning microscopy was performed. Neither autofluorescence originating from phyotosynthetic organisms nor inorganic particles did negatively interfere with the FISH-LBA technique. In river snow samples obtained from the river Elbe, Germany, distinct compartments of the river snow structure could be visualized with FITC-labelled lectins from Triticum vulgaris, Limulus polyphemus, Arachis hypogaea, Phaseolus vulgaris and Pseudomonas aeruginosa, binding to frequently occurring saccharide residues in the river snow matrix. The analysis could be performed on different levels of complexity. The combined technique visualized bacteria of different phylogenetic groups in the entire river snow structure as well as glycoconjugate components linked with various microcolonies. Different lectins stained slime layers and cell-envelopes of individual eukaryotic and prokaryotic cells. Consequently, application of the FISH-LBA technique allows the linkage between cellular and glycoconjugate identity in complex microbial communities.