Complete nitrification by Nitrospira bacteria.

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

Poikilothermic Animals as a Previously Unrecognized Source of Fecal Indicator Bacteria in a Backwater Ecosystem of a Large River.

Quantitative information regarding the presence of Escherichia coli, intestinal enterococci, and Clostridium perfringens in poikilotherms is notably scarce. Therefore, this study was designed to allow a systematic comparison of the occurrence of these standard fecal indicator bacteria (SFIB) in the excreta of wild homeothermic (ruminants, boars, carnivores, and birds) and poikilothermic (earthworms, gastropods, frogs, and fish) animals inhabiting an alluvial backwater area in eastern Austria. With the exception of earthworms, the average concentrations of E. coli and enterococci in the excreta of poikilotherms were equal to or only slightly lower than those observed in homeothermic excreta and were 1 to 4 orders of magnitude higher than the levels observed in the ambient soils and sediments. Enterococci reached extraordinarily high concentrations in gastropods. Additional estimates of the daily excreted SFIB (E. coli and enterococcus) loads (DESL) further supported the importance of poikilotherms as potential pollution sources. The newly established DESL metric also allowed comparison to the standing stock of SFIB in the sediment and soil of the investigated area. In agreement with its biological characteristics, the highest concentrations of C. perfringens were observed in carnivores. In conclusion, the long-standing hypothesis that only humans and homeothermic animals are primary sources of SFIB is challenged by the results of this study. It may be necessary to extend the fecal indicator concept by additionally considering poikilotherms as potential important primary habitats of SFIB. Further studies in other geographical areas are needed to evaluate the general significance of our results. We hypothesize that the importance of poikilotherms as sources of SFIB is strongly correlated with the ambient temperature and would therefore be of increased significance in subtropical and tropical habitats and water resources.IMPORTANCE The current fecal indicator concept is based on the assumption that the standard fecal indicator bacteria (SFIB) Escherichia coli, intestinal enterococci, and Clostridium perfringens multiply significantly only in the guts of humans and other homeothermic animals and can therefore indicate fecal pollution and the potential presence of pathogens from those groups. The findings of the present study showed that SFIB can also occur in high concentrations in poikilothermic animals (i.e., animals with body temperatures that vary with the ambient environmental temperature, such as fish, frogs, and snails) in an alluvial backwater area in a temperate region, indicating that a reconsideration of this long-standing indicator paradigm is needed. This study suggests that poikilotherms must be considered to be potential primary sources of SFIB in future studies.

Linking antibiotic resistance gene patterns with advanced faecal pollution assessment and environmental key parameters along 2300 km of the Danube River.

The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX-M-1 group, blaCTX-M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.

Environmental microbiome diversity and stability is a barrier to antimicrobial resistance gene accumulation.

When antimicrobial resistant bacteria (ARB) and genes (ARGs) reach novel habitats, they can become part of the habitat's microbiome in the long term if they are able to overcome the habitat's biotic resilience towards immigration. This process should become more difficult with increasing biodiversity, as exploitable niches in a given habitat are reduced for immigrants when more diverse competitors are present. Consequently, microbial diversity could provide a natural barrier towards antimicrobial resistance by reducing the persistence time of immigrating ARB and ARG. To test this hypothesis, a pan-European sampling campaign was performed for structured forest soil and dynamic riverbed environments of low anthropogenic impact. In soils, higher diversity, evenness and richness were significantly negatively correlated with relative abundance of >85% of ARGs. Furthermore, the number of detected ARGs per sample were inversely correlated with diversity. However, no such effects were present in the more dynamic riverbeds. Hence, microbiome diversity can serve as a barrier towards antimicrobial resistance dissemination in stationary, structured environments, where long-term, diversity-based resilience against immigration can evolve.

MiDAS 5: Global diversity of bacteria and archaea in anaerobic digesters.

Anaerobic digestion of organic waste into methane and carbon dioxide (biogas) is carried out by complex microbial communities. Here, we use full-length 16S rRNA gene sequencing of 285 full-scale anaerobic digesters (ADs) to expand our knowledge about diversity and function of the bacteria and archaea in ADs worldwide. The sequences are processed into full-length 16S rRNA amplicon sequence variants (FL-ASVs) and are used to expand the MiDAS 4 database for bacteria and archaea in wastewater treatment systems, creating MiDAS 5. The expansion of the MiDAS database increases the coverage for bacteria and archaea in ADs worldwide, leading to improved genus- and species-level classification. Using MiDAS 5, we carry out an amplicon-based, global-scale microbial community profiling of the sampled ADs using three common sets of primers targeting different regions of the 16S rRNA gene in bacteria and/or archaea. We reveal how environmental conditions and biogeography shape the AD microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 692 genera and 1013 species. These represent 84-99% and 18-61% of the accumulated read abundance, respectively, across samples depending on the amplicon primers used. Finally, we examine the global diversity of functional groups with known importance for the anaerobic digestion process.

Have genetic targets for faecal pollution diagnostics and source tracking revolutionized water quality analysis yet?

The impacts of nucleic acid-based methods - such as PCR and sequencing - to detect and analyze indicators, genetic markers or molecular signatures of microbial faecal pollution in health-related water quality research were assessed by rigorous literature analysis. A wide range of application areas and study designs has been identified since the first application more than 30 years ago (>1100 publications). Given the consistency of methods and assessment types, we suggest defining this emerging part of science as a new discipline: genetic faecal pollution diagnostics (GFPD) in health-related microbial water quality analysis. Undoubtedly, GFPD has already revolutionized faecal pollution detection (i.e., traditional or alternative general faecal indicator/marker analysis) and microbial source tracking (i.e., host-associated faecal indicator/marker analysis), the current core applications. GFPD is also expanding to many other research areas, including infection and health risk assessment, evaluation of microbial water treatment, and support of wastewater surveillance. In addition, storage of DNA extracts allows for biobanking, which opens up new perspectives. The tools of GFPD can be combined with cultivation-based standardized faecal indicator enumeration, pathogen detection, and various environmental data types, in an integrated data analysis approach. This comprehensive meta-analysis provides the scientific status quo of this field, including trend analyses and literature statistics, outlining identified application areas, and discusses the benefits and challenges of nucleic acid-based analysis in GFPD.

Emergence of SARS-CoV-2 Alpha lineage and its correlation with quantitative wastewater-based epidemiology data.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gave rise to an international public health emergency in 3 months after its emergence in Wuhan, China. Typically for an RNA virus, random mutations occur constantly leading to new lineages, incidental with a higher transmissibility. The highly infective alpha lineage, firstly discovered in the UK, led to elevated mortality and morbidity rates as a consequence of Covid-19, worldwide. Wastewater surveillance proved to be a powerful tool for early detection and subsequent monitoring of the dynamics of SARS-CoV-2 and its variants in a defined catchment. Using a combination of sequencing and RT-qPCR approaches, we investigated the total SARS-CoV-2 concentration and the emergence of the alpha lineage in wastewater samples in Vienna, Austria linking it to clinical data. Based on a non-linear regression model and occurrence of signature mutations, we conclude that the alpha variant was present in Vienna sewage samples already in December 2020, even one month before the first clinical case was officially confirmed and reported by the health authorities. This provides evidence that a well-designed wastewater monitoring approach can provide a fast snapshot and may detect the circulating lineages in wastewater weeks before they are detectable in the clinical samples. Furthermore, declining 14 days prevalence data with simultaneously increasing SARS-CoV-2 total concentration in wastewater indicate a different shedding behavior for the alpha variant. Overall, our results support wastewater surveillance to be a suitable approach to spot early circulating SARS-CoV-2 lineages based on whole genome sequencing and signature mutations analysis.

Elucidating fecal pollution patterns in alluvial water resources by linking standard fecal indicator bacteria to river connectivity and genetic microbial source tracking.

A novel concept for fecal pollution analysis was applied at alluvial water resources to substantially extend the information provided by fecal indicator bacteria (FIB). FIB data were linked to river connectivity and genetic microbial source tracking (MST). The concept was demonstrated at the Danube River and its associated backwater area downstream of the city of Vienna, using a comprehensive 3-year data set (10 selected sites, n = 317 samples). Enumeration of Escherichia coli (ISO 16649-2), intestinal enterococci (ISO 7899-2) and Clostridium perfringens (ISO 14189) revealed a patchy distribution for the investigation area. Based on these parameters alone a clear interpretation of the observed fecal contamination patterns was not possible. Comparison of FIB concentrations to river connectivity allowed defining sites with dominating versus rare fecal pollution influence from the River Danube. A strong connectivity gradient at the selected backwater sites became obvious by 2D hydrodynamic surface water modeling, ranging from 278 days (25%) down to 5 days (<1%) of hydraulic connectivity to the River Danube within the 3-year study period. Human sewage pollution could be identified as the dominating fecal source at the highly connected sites by adding information from MST analysis. In contrast, animal fecal pollution proofed to be dominating in areas with low river connectivity. The selection of genetic MST markers was focusing on potentially important pollution sources in the backwater area, using human (BacHum, HF183II), ruminant (BacR) and pig (Pig2Bac) -associated quantitative PCR assays. The presented approach is assumed to be useful to characterize alluvial water resources for water safety management throughout the globe, by allocating fecal pollution to autochthonous, allochthonous, human or animal contamination components. The established river connectivity metric is not limited to bacterial fecal pollution, but can be applied to any type of chemical and microbiological contamination.

Low yield and abiotic origin of N2O formed by the complete nitrifier Nitrospira inopinata.

Nitrous oxide (N2O) and nitric oxide (NO) are atmospheric trace gases that contribute to climate change and affect stratospheric and ground-level ozone concentrations. Ammonia oxidizing bacteria (AOB) and archaea (AOA) are key players in the nitrogen cycle and major producers of N2O and NO globally. However, nothing is known about N2O and NO production by the recently discovered and widely distributed complete ammonia oxidizers (comammox). Here, we show that the comammox bacterium Nitrospira inopinata is sensitive to inhibition by an NO scavenger, cannot denitrify to N2O, and emits N2O at levels that are comparable to AOA but much lower than AOB. Furthermore, we demonstrate that N2O formed by N. inopinata formed under varying oxygen regimes originates from abiotic conversion of hydroxylamine. Our findings indicate that comammox microbes may produce less N2O during nitrification than AOB.

Global diversity and biogeography of bacterial communities in wastewater treatment plants.

Microorganisms in wastewater treatment plants (WWTPs) are essential for water purification to protect public and environmental health. However, the diversity of microorganisms and the factors that control it are poorly understood. Using a systematic global-sampling effort, we analysed the 16S ribosomal RNA gene sequences from ~1,200 activated sludge samples taken from 269 WWTPs in 23 countries on 6 continents. Our analyses revealed that the global activated sludge bacterial communities contain ~1 billion bacterial phylotypes with a Poisson lognormal diversity distribution. Despite this high diversity, activated sludge has a small, global core bacterial community (n = 28 operational taxonomic units) that is strongly linked to activated sludge performance. Meta-analyses with global datasets associate the activated sludge microbiomes most closely to freshwater populations. In contrast to macroorganism diversity, activated sludge bacterial communities show no latitudinal gradient. Furthermore, their spatial turnover is scale-dependent and appears to be largely driven by stochastic processes (dispersal and drift), although deterministic factors (temperature and organic input) are also important. Our findings enhance our mechanistic understanding of the global diversity and biogeography of activated sludge bacterial communities within a theoretical ecology framework and have important implications for microbial ecology and wastewater treatment processes.

Author Correction: Global diversity and biogeography of bacterial communities in wastewater treatment plants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cultivation and Genomic Analysis of "Candidatus Nitrosocaldus islandicus," an Obligately Thermophilic, Ammonia-Oxidizing Thaumarchaeon from a Hot Spring Biofilm in Graendalur Valley, Iceland.

Ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota are the only known aerobic ammonia oxidizers in geothermal environments. Although molecular data indicate the presence of phylogenetically diverse AOA from the Nitrosocaldus clade, group 1.1b and group 1.1a Thaumarchaeota in terrestrial high-temperature habitats, only one enrichment culture of an AOA thriving above 50°C has been reported and functionally analyzed. In this study, we physiologically and genomically characterized a newly discovered thaumarchaeon from the deep-branching Nitrosocaldaceae family of which we have obtained a high (∼85%) enrichment from biofilm of an Icelandic hot spring (73°C). This AOA, which we provisionally refer to as "Candidatus Nitrosocaldus islandicus," is an obligately thermophilic, aerobic chemolithoautotrophic ammonia oxidizer, which stoichiometrically converts ammonia to nitrite at temperatures between 50 and 70°C. "Ca. N. islandicus" encodes the expected repertoire of enzymes proposed to be required for archaeal ammonia oxidation, but unexpectedly lacks a nirK gene and also possesses no identifiable other enzyme for nitric oxide (NO) generation. Nevertheless, ammonia oxidation by this AOA appears to be NO-dependent as "Ca. N. islandicus" is, like all other tested AOA, inhibited by the addition of an NO scavenger. Furthermore, comparative genomics revealed that "Ca. N. islandicus" has the potential for aromatic amino acid fermentation as its genome encodes an indolepyruvate oxidoreductase (iorAB) as well as a type 3b hydrogenase, which are not present in any other sequenced AOA. A further surprising genomic feature of this thermophilic ammonia oxidizer is the absence of DNA polymerase D genes - one of the predominant replicative DNA polymerases in all other ammonia-oxidizing Thaumarchaeota. Collectively, our findings suggest that metabolic versatility and DNA replication might differ substantially between obligately thermophilic and other AOA.

Giant viruses with an expanded complement of translation system components.

The discovery of giant viruses blurred the sharp division between viruses and cellular life. Giant virus genomes encode proteins considered as signatures of cellular organisms, particularly translation system components, prompting hypotheses that these viruses derived from a fourth domain of cellular life. Here we report the discovery of a group of giant viruses (Klosneuviruses) in metagenomic data. Compared with other giant viruses, the Klosneuviruses encode an expanded translation machinery, including aminoacyl transfer RNA synthetases with specificities for all 20 amino acids. Notwithstanding the prevalence of translation system components, comprehensive phylogenomic analysis of these genes indicates that Klosneuviruses did not evolve from a cellular ancestor but rather are derived from a much smaller virus through extensive gain of host genes.

High abundance of genetic Bacteroidetes markers for total fecal pollution in pristine alpine soils suggests lack in specificity for feces.

Two frequently applied genetic Bacteroidetes markers for total fecal pollution (AllBac and BacUni) were found in high numbers in pristine soil samples of two alpine catchment areas casting doubt on their value as fecal indicators. This finding underlines the necessity to evaluate assays locally and against non-intestinal samples before application.