Anniek E. E. de Jong

Background: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a global health risk, yet there is insufficient knowledge about its sources and transmission routes within an integrated One Health framework. This study aimed to investigate CRPA presence and dynamics at the human-water interface inside and outside hospitals in three major cities located in different climatic zones. Methods: This cross-sectional study was conducted in Rotterdam (The Netherlands), Rome (Italy), and Jakarta (Indonesia) between March 2022 and June 2024. Samples were collected from humans, hospital environments, and various water sources. CRPA isolates underwent sequencing to determine carbapenemase gene presence and their genetic relatedness. Primary outcomes included CRPA carriage in healthy individuals and newly hospitalized patients, CRPA prevalence in hospital environments, CRPA concentrations in aquatic environments, and the frequency and transmission likelihood within epidemiological clusters. Findings: CRPA carriage was detected in 0·5% (1/194) of healthy individuals from Rotterdam, 0·0% (0/245) from Rome, and 0·8% (2/250) from Jakarta. Among patients, CRPA carriage differed significantly across cities (P<0·001), with 0·4% (2/469) in Rotterdam, 0·3% (1/363) in Rome, and 4·8% (28/584) in Jakarta. In wet hospital environments, CRPA prevalence ranged between 0·0% (Rome) and 13·4% (Jakarta). CRPA concentrations were highest in hospital wastewater and remained detectable in treated effluent of municipal wastewater treatment plants (mWWTP) and the receiving river in all cities. Carbapenemase genes were detected in 25·9% (100/386) of isolates. CRPA isolates identified in the clinical setting clustered with those from mWWTP influent or effluent (Rotterdam) or the river (Jakarta). High genetic diversity, overall and within reservoirs, was observed among isolates. Interpretation: The substantial presence of CRPA in environmental reservoirs, along with the cross-over between them, may pose a public health risk, and warrant mitigation strategies. To prevent CRPA transmission within hospitals and limit cross-sectoral transmission, robust infection prevention and control measures, alongside effective wastewater treatment, are essential.

Background/Objectives: The global spread of carbapenem-resistant Pseudomonas aeruginosa (CRPA) warrants collaborative action. Guidance should come from integrated One Health surveillance; however, a surveillance strategy is currently unavailable due to insufficient knowledge on the sources and transmission routes of CRPA. The aim of the SAMPAN study (“A Smart Surveillance Strategy for Carbapenem-resistant Pseudomonas aeruginosa”) is to develop a globally applicable surveillance strategy. Methods: First, an international cross-sectional study will be conducted to investigate CRPA in clinical and environmental settings in Rotterdam (The Netherlands), Rome (Italy), and Jakarta (Indonesia). Screening cultures and risk factor questionnaires will be taken from healthy individuals and patients upon hospital admission. Clinical CRPA isolates will also be included. Additionally, samples will be taken twice from wet hospital environments and monthly from the hospitals’ (drinking) water system, hospital and municipal wastewater treatment plants, and receiving rivers. Whole-genome sequencing will be performed to characterize CRPA isolates and determine the genetic relatedness among the isolates from different reservoirs. Findings from the cross-sectional study, combined with expert elicitation using a Delphi method, will serve as the input for the surveillance strategy. Conclusions: The SAMPAN study will provide a broader understanding of the sources and transmission routes of CRPA. Therewith, the development of a globally applicable smart surveillance strategy will be made possible, delivering information that is needed to guide actions against the spread of CRPA.

Microbial methane oxidation is a major biofilter preventing larger emissions of this powerful greenhouse gas from marine coastal areas into the atmosphere. In these zones, various electron acceptors such as sulfate, metal oxides, nitrate, or oxygen can be used. However, the key microbial players and mechanisms of methane oxidation are poorly understood. In this study, we inoculated a bioreactor with methane‐ and iron‐rich sediments from the Bothnian Sea to investigate microbial methane and iron cycling under low oxygen concentrations. Using metagenomics, we investigated shifts in microbial community composition after approximately 2.5 years of bioreactor operation. Marker genes for methane and iron cycling, as well as respiratory and fermentative metabolism, were identified and used to infer putative microbial metabolism. Metagenome‐assembled genomes representing novel Verrucomicrobia, Bacteroidetes, and Krumholzibacteria were recovered and revealed a potential for methane oxidation, organic matter degradation, and iron cycling, respectively. This work brings new hypotheses on the identity and metabolic versatility of microorganisms that may be members of such functional guilds in coastal marine sediments and highlights that microorganisms potentially composing the methane biofilter in these sediments may be more diverse than previously appreciated.

Microbial methane oxidation is a major biofilter preventing larger emissions of this powerful greenhouse gas from marine coastal areas into the atmosphere. In these zones, various electron acceptors such as sulfate, metal oxides, nitrate or oxygen can be utilized. However, the key microbial players and mechanisms of methane oxidation are poorly understood. In this study, we inoculated a bioreactor with methane- and iron-rich sediments from the Bothnian Sea in order to investigate microbial methane and iron cycling under low oxygen concentrations. Using metagenomics, we observed shifts in the microbial community over approximately 2.5 years of bioreactor operation. Marker genes for methane and iron cycling, as well as respiratory and fermentative metabolism, were investigated. Metagenome-assembled genomes representing novel Verrucomicrobia, Bacteroidetes and Krumholzibacteria were recovered and revealed potential for methane oxidation, organic matter degradation, and iron cycling, respectively. This work brings new insights into the identity and metabolic versatility of microorganisms that may be members of such functional guilds in coastal marine sediments and highlights that the methane biofilter in these sediments may be more diverse than previously appreciated. Importance: Despite the essential role of microorganisms in preventing most methane in the ocean floor to reach the atmosphere, comprehensive knowledge on the identity and the mechanisms employed by these microorganisms is still lacking. This is problematic because such information is needed to understand how the ecosystem functions in the present and how microorganisms may respond to climate change in the future. Here, we enriched and identified novel taxa potentially involved in methane and iron cycling in an oxygen-limited bioreactor inoculated with methane- and iron-rich coastal sediments. Metagenomic analyses provided hypotheses about the mechanisms they may employ, such as the use of oxygen at very low concentrations. The implication of our results is that in more shallow sediments, where oxygen-limited conditions are present, the methane biofilter is potentially composed of novel, metabolically versatile Verrucomicrobia that could contribute to mitigating methane emissions from coastal marine zones.

Summary: Arctic permafrost soils store large amounts of organic matter that is sensitive to temperature increases and subsequent microbial degradation to methane (CH ₄ ) and carbon dioxide (CO ₂ ). Here, we studied methanogenic and methanotrophic activity and community composition in thermokarst lake sediments from Utqiag˙vik (formerly Barrow), Alaska. This experiment was carried out under in situ temperature conditions (4°C) and the IPCC 2013 Arctic climate change scenario (10°C) after addition of methanogenic and methanotrophic substrates for nearly a year. Trimethylamine (TMA) amendment with warming showed highest maximum CH ₄ production rates, being 30% higher at 10°C than at 4°C. Maximum methanotrophic rates increased by up to 57% at 10°C compared to 4°C. 16S rRNA gene sequencing indicated high relative abundance of Methanosarcinaceae in TMA amended incubations, and for methanotrophic incubations Methylococcaeae were highly enriched. Anaerobic methanotrophic activity with nitrite or nitrate as electron acceptor was not detected. This study indicates that the methane cycling microbial community can adapt to temperature increases and that their activity is highly dependent on substrate availability.

Pollution of water resources is a major risk to human health and water quality throughout the world. Here, we studied the effect of river pollution on the microbial community composition in the Citarum River Basin, West Java, Indonesia. Sediment was collected at six sampling points along a gradient of pollution, from the pristine source of the river to the heavily polluted downstream site in the densely populated urban area of Bandung. After DNA extraction, microbial diversity and potential for nitrogen cycling were analyzed based on 16S rRNA gene amplicon sequencing and metagenomics. Comparing the pristine sediment to the polluted site showed a lower microbial diversity and higher dominance of anaerobic processes than at the polluted site. The most dominant phylum within the Bacteria were the Proteobacteria , which shifted from Beta - and Deltaproteobacteria in the pristine site to Alpha - and Gammaproteobacteria in the polluted sediment. With pollution Actinobacteria, Bacteroidetes, and Firmicutes increased in relative abundance. Relative high abundance of Soil Crenarchaeotic group was found in all sample sites, whilst the methanogenic archaea became more abundant with increased pollution and anaerobicity. The study of the nitrogen cycling potential revealed that ammonium oxidation and denitrification appeared to be abundant processes in the pristine site, whereas ammonification seemed to be more important in the polluted site. Increased water treatment would restore water quality and microbial diversity in Citarum river sediments.

Microorganisms are the drivers of biogeochemical methane and nitrogen cycles. Essential roles of chemolithoautotrophic microorganisms in these cycles were predicted long before their identification. Dedicated enrichment procedures, metagenomics surveys and single-cell technologies have enabled the identification of several new groups of most-wanted spookmicrobes, including novel methoxydotrophic methanogens that produce methane from methylated coal compounds and acetoclastic ‘Candidatus Methanothrix paradoxum’, which is active in oxic soils. The resultant energy-rich methane can be oxidized via a suite of electron acceptors. Recently, ‘Candidatus Methanoperedens nitroreducens’ ANME-2d archaea and ‘Candidatus Methylomirabilis oxyfera’ bacteria were enriched on nitrate and nitrite under anoxic conditions with methane as an electron donor. Although ‘Candidatus Methanoperedens nitroreducens’ and other ANME archaea can use iron citrate as an electron acceptor in batch experiments, the quest for anaerobic methane oxidizers that grow via iron reduction continues. In recent years, the nitrogen cycle has been expanded by the discovery of various ammonium-oxidizing prokaryotes, including ammonium-oxidizing archaea, versatile anaerobic ammonium-oxidizing (anammox) bacteria and complete ammonium-oxidizing (comammox) Nitrospira bacteria. Several biogeochemical studies have indicated that ammonium conversion occurs under iron-reducing conditions, but thus far no microorganism has been identified. Ultimately, iron-reducing and sulfate-dependent ammonium-oxidizing microorganisms await discovery.

Methane (CH₄) is produced in many natural systems that are vulnerable to change under a warming climate, yet current CH₄ budgets, as well as future shifts in CH₄ emissions, have high uncertainties. Climate change has the potential to increase CH₄ emissions from critical systems such as wetlands, marine and freshwater systems, permafrost, and methane hydrates, through shifts in temperature, hydrology, vegetation, landscape disturbance, and sea level rise. Increased CH₄ emissions from these systems would in turn induce further climate change, resulting in a positive climate feedback. Here we synthesize biological, geochemical, and physically focused CH₄ climate feedback literature, bringing together the key findings of these disciplines. We discuss environment‐specific feedback processes, including the microbial, physical, and geochemical interlinkages and the timescales on which they operate, and present the current state of knowledge of CH₄ climate feedbacks in the immediate and distant future. The important linkages between microbial activity and climate warming are discussed with the aim to better constrain the sensitivity of the CH₄ cycle to future climate predictions. We determine that wetlands will form the majority of the CH₄ climate feedback up to 2100. Beyond this timescale, CH₄ emissions from marine and freshwater systems and permafrost environments could become more important. Significant CH₄ emissions to the atmosphere from the dissociation of methane hydrates are not expected in the near future. Our key findings highlight the importance of quantifying whether CH₄ consumption can counterbalance CH₄ production under future climate scenarios.