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The progression of green technologies has driven higher future demands for valuable metals such as lithium, cobalt, nickel, and manganese, hence necessitating the recycling of lithium-containing energy storage systems. Restrategizing conventional metal recycling technologies with sustainable biological approaches can explore the potential to curtail expensive process costs and emissions of hazardous by-products. This article advocates the benefits and persuasion for future studies and applications to exploit current concepts of microbial-based metal recycling technologies as environmentally cleaner options. National Environmental Agency (NEA) National Research Foundation (NRF) Submitted/Accepted version This research/project is supported by the National Research Foundation, Singapore, and National Environment Agency, Singapore, under its Closing the Waste Loop Funding Initiative (award no. USSIF- 2018-4).

The antimicrobial resistance crisis is a global health issue requiring discovery and development of novel therapeutics. However, conventional screening of natural products or synthetic chemical libraries is uncertain. Combination therapy using approved antibiotics with inhibitors targeting innate resistance mechanisms provides an alternative strategy to develop potent therapeutics. This review discusses the chemical structures of effective β-lactamase inhibitors, outer membrane permeabilizers, and efflux pump inhibitors that act as adjuvant molecules of classical antibiotics. Rational design of the chemical structures of adjuvants will provide methods to impart or restore efficacy to classical antibiotics for inherently antibiotic-resistant bacteria. As many bacteria have multiple resistance pathways, adjuvant molecules simultaneously targeting multiple pathways are promising approaches to combat multi-drug-resistant bacterial infections. Ministry of Education (MOE) Submitted/Accepted version This work was funded and supported by the Singapore MOE Tier 3 grant (MOE2018-T3-1-003).

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections. Treatment of P. aeruginosa infections is difficult given its multiple virulence mechanisms, intrinsic antibiotic resistance mechanisms, and biofilm-forming ability. Auranofin, an approved oral gold compound for rheumatoid arthritis treatment, was recently reported to inhibit the growth of multiple bacterial species. Here, we identify P. aeruginosa's global virulence factor regulator Vfr as one target of auranofin. We report the mechanistic insights into the inhibitory mechanism of auranofin and gold(I) analogues to Vfr through structural, biophysical, and phenotypic inhibition studies. This work suggests that auranofin and gold(I) analogues have potential to be developed as anti-virulence drugs against P. aeruginosa. Ministry of Education (MOE) Published version This research is supported by the Singapore Ministry of Education under its Singapore Ministry of Education Academic Research Fund Tier 1 (2018-T1-002-010) and Tier 2 (2016-T2-2-097) to D.L.; Nanyang Presidential Graduate Scholarship to B.L.A.C.; the Guangdong Natural Science Foundation for Distinguished Young Scholar [2020B1515020003]; the National Key Research and Development Program of China (2022YFC2304700); the National Natural Science Foundation of China (32270196 and 32200155); the Guangdong Basic and Applied Basic Research Foundation [2019A1515110640]; and the Shenzhen Overseas High-level Talent Team (KQTD20200909113758004).

Enterococcus faecalis relies upon a number of cell wall-associated proteins for virulence. One virulence factor is the sortase-assembled endocarditis and biofilm associated pilus (Ebp), an important factor for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that the pilus sortase covalently links pilus monomers prior to recognition, while the housekeeping sortase cleaves at the LPXTG motif within the terminal pilin subunit, and subsequently attaches assembled pilus fiber to the growing cell wall at sites of new cell wall synthesis. While the cell wall anchoring mechanism and polymerization of Ebp is well characterized, less is known about the spatial and temporal deposition of this protein on the cell surface. We followed the distribution of Ebp and peptidoglycan (PG) throughout the E. faecalis cell cycle via immunofluorescence microscopy and fluorescent D-amino acids (FDAA) staining. Surprisingly, cell surface Ebp did not co-localize with newly synthesized PG. Instead, surface-anchored Ebp was localized to the cell hemisphere but never at the septum where new cell wall is deposited. In addition, the older hemisphere of the E. faecalis diplococcus were completely saturated with Ebp, while Ebp appeared as two foci directly adjacent to the nascent septum in the newer hemisphere. A similar localization pattern was observed for another cell wall anchored substrate by sortase A, aggregation substance (AS), suggesting that this may be a general rule for all SrtA substrates in E. faecalis. When cell wall synthesis was inhibited by ramoplanin, an antibiotic that binds and sequesters lipid II cell wall precursors, new Ebp deposition at the cell surface was not disrupted. These data suggest an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto un-crosslinked cell wall, independent of new PG synthesis.

Extracellular polymeric substances (EPS) are core biofilm components, yet how they mediate interactions within and contribute to the structuring of biofilms is largely unknown, particularly for non-culturable microbial communities that predominate in environmental habitats. To address this knowledge gap, we explored the role of EPS in an anaerobic ammonium oxidation (anammox) biofilm. An extracellular glycoprotein, BROSI_A1236, from an anammox bacterium, formed envelopes around the anammox cells, supporting its identification as a surface (S-) layer protein. However, the S-layer protein also appeared at the edge of the biofilm, in close proximity to the polysaccharide-coated filamentous Chloroflexi bacteria but distal to the anammox bacterial cells. The Chloroflexi bacteria assembled into a cross-linked network at the edge of the granules and surrounding anammox cell clusters, with the S-layer protein occupying the space around the Chloroflexi. The anammox S-layer protein was also abundant at junctions between Chloroflexi cells. Thus, the S-layer protein is likely transported through the matrix as an EPS and also acts as an adhesive to facilitate the assembly of filamentous Chloroflexi into a three-dimensional biofilm lattice. The spatial distribution of the S-layer protein within the mixed species biofilm suggests that it is a "public-good" EPS, which facilitates the assembly of other bacteria into a framework for the benefit of the biofilm community, and enables key syntrophic relationships, including anammox. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Published version This research was supported by Singapore Centre for Environmental Life Sciences Engineering (SCELSE), which is funded by Singapore’s Ministry of Education, National Research Federation, Nanyang Technological University, Singapore (NTU), and National University of Singapore (NUS), and hosted by NTU in partnership with NUS.

Addressing the growing volume of end-of-life lithium-ion battery (LIB) waste is one of the global challenges in tackling the electronic waste problem. In this study, the regeneration of LiNi0.3Co0.3Mn0.3O2 (NMC111) and Li- Ni0.6Co0.2Mn0.2O2 (NMC622) cathode-active materials from end of- life LIBs was accomplished through an environmentally friendly bioleaching process. In the bioleaching process mediated by Acidithiobacillus ferrooxidans, 85.5% of Ni, 91.8% of Mn, 90.4% of Co, and 89.9% of Li were leached out from NMC-based spent LIBs in 6 h at a pulp density of 100 g/L. One of the challenges in bioleaching-based metal recovery is the presence of impurities, including Cu, Al, and Fe (excess Fe3+ and Fe2+ from bacterial nutrients). The impurity removal was performed by air oxidation and pH adjustment without substantial losses of other metallic ions. Thereafter, ammonium oxalate coprecipitation effectively recovered the transition metal ions as metal oxalates from the bioleaching liquor. NMC111 and NMC622 were regenerated from the coprecipitated product. The electrochemical stability of the regenerated NMC111 and NMC622 was comparable to commercial NMC (∼85% of capacity retention after 50 cycles at 100 mA g−1). This regeneration approach appears promising in LIB recycling for long-term industrial development. National Environmental Agency (NEA) National Research Foundation (NRF) Submitted/Accepted version This research/project is supported by the National Research Foundation, Singapore, and National Environment Agency, Singapore, under its Closing the Waste Loop Funding Initiative (Award no. USS-IF-2018-4)

The assembly of macromolecules on the plasma membrane concentrates cell surface biomolecules into nanometer- to micrometer-scale clusters (nano- or microdomains) that help the cell initiate or respond to signals. In plant-microbe interactions, the actin cytoskeleton undergoes rapid remodeling during pathogen-associated molecular pattern-triggered immunity (PTI). The nanoclustering of formin-actin nucleator proteins at the cell surface has been identified as underlying actin nucleation during plant innate immune responses. Here, we show that the condensation of nanodomain constituents and the self-assembly of remorin proteins enables this mechanism of controlling formin condensation and activity during innate immunity in Arabidopsis thaliana. Through intrinsically disordered region-mediated remorin oligomerization and formin interaction, remorin gradually recruits and condenses formins upon PTI activation in lipid bilayers, consequently increasing actin nucleation in a time-dependent manner postinfection. Such nanodomain- and remorin-mediated regulation of plant surface biomolecules is expected to be a general feature of plant innate immune responses that creates spatially separated biochemical compartments and fine tunes membrane physicochemical properties for transduction of immune signals in the host. Ministry of Education (MOE) Nanyang Technological University This study was supported by the National Science Foundation of China (Grants 31870170) to Xu Chen, the Nanyang Technological University (NTU) Singapore startup grant (M4081533) and Singapore Ministry of Education (MOE) Tier 1 (RG32/20) and Tier 3 (MOE2019-T3-1-012) funding awarded to Yansong Miao.

Mixed species biofilms exhibit increased tolerance to numerous stresses compared to single species biofilms. The aim of this study was to examine the effect of grazing by the heterotrophic protist, Tetrahymena pyriformis, on a mixed species biofilm consisting of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Protozoan grazing significantly reduced the single species K. pneumoniae biofilm, and the single species P. protegens biofilm was also sensitive to grazing. In contrast, P. aeruginosa biofilms were resistant to predation. This resistance protected the otherwise sensitive members of the mixed species biofilm consortium. Rhamnolipids produced by P. aeruginosa were shown to be the primary toxic factor for T. pyriformis. However, a rhamnolipid-deficient mutant of P. aeruginosa (P. aeruginosa ΔrhlAB) maintained grazing resistance in the biofilm, suggesting the presence of at least one additional protective mechanism. P. aeruginosa with a deleted gene encoding the type III secretion system also resisted grazing. A transposon library was generated in the ΔrhlAB mutant to identify the additional factor involved in community biofilm protection. Results indicated that the Pseudomonas Quinolone Signal (PQS), a quorum sensing signaling molecule, was likely responsible for this effect. We confirmed this observation by showing that double mutants of ΔrhlAB and genes in the PQS biosynthetic operon lost grazing protection. We also showed that PQS was directly toxic to T. pyriformis. This study demonstrates that residing in a mixed species biofilm can be an advantageous strategy for grazing sensitive bacterial species, as P. aeruginosa confers community protection from protozoan grazing through multiple mechanisms. IMPORTANCE: Biofilms have been shown to protect bacterial cells from predation by protists. Biofilm studies have traditionally used single species systems, which have provided information on the mechanisms and regulation of biofilm formation and dispersal, and the effects of predation on these biofilms. However, biofilms in nature are comprised of multiple species. To better understand how multispecies biofilms are impacted by predation, a model mixed-species biofilm was here exposed to protozoan predation. We show that the grazing sensitive strains K. pneumonia and P. protogens gained associational resistance from the grazing resistant P. aeruginosa. Resistance was due to the secretion of rhamnolipids and quorum sensing molecule PQS. This work highlights the importance of using mixed species systems. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Published version We acknowledge the financial support from National Research Foundation and Ministry of Education Singapore under its Research Centre of Excellence Program. This work was also supported by a grant from the Singapore Ministry of Education (MOE2019-T2-1-050) and Australian Research Council Discovery Project DP170100453. Siew Herng Chan and Yu Fen Goh were supported by the Interdisciplinary Graduate School in conjunction with the Singapore Centre for Environmental Life Sciences Engineering. We also wish to thank the ESCMID Study Group for Biofilms–ESGB for supporting Henriette Lyng Røders participation in Eurobiofilms 2015 with a travel grant.

In the past two decades, the concept of microbial resources evolved from the conserved strains in culture collection centers and microbial genome data in databases to functional microbial consortia, strains with superior phenotypes, novel biochemical and signaling systems, highly efficient enzymes, secondary metabolites, and unique genetic traits. Natural and engineered water environments provide diverse ecological niches for microorganisms to evolve and thrive, serving as treasure troves of microbial resources. Compared with that of chemical and energy resources from water, mining of microbial resources is severely underexplored. In this review, we first summarize various approaches (e.g., cultivation, functional metagenomics, and single-cell genomics) that have been employed to mine microbial resources from water, and then discuss their distinctive advantages and limitations. Lastly, we explore future directions on the mining of microbial resources from water. Nanyang Technological University National Research Foundation (NRF) This research / project is supported by the National Research Foundation, Singapore, under its NERC-NRF Joint Grant Call (Award no. NRF-NERC-SEAP-2020-02). This research is also supported by the Accelerating Creativity and Excellence (ACE) grant (Award no. NTUACE2021-01), Nanyang Technological University, Singapore.

Probiotic functionalization of non-dairy beverages has been garnering interest to provide dairy-sensitive populations with greater probiotic product varieties. The addition of probiotics into popularly consumed beverages-carbonated sodas and beers, presents an interesting challenge as the presence of acidic pH, hops-derived compounds, and ethanol have highly deleterious effects. Herein, alginate encapsulation was proposed to improve probiotics viability within sodas and beers. Three probiotics, namely Lacticaseibacillus rhamnosus GG, Escherichia coli Nissle 1917, and Bifidobacterium longum were encapsulated in alginate spheres and exposed to Coca-Cola, 7-Up, Tiger Beer, and Guinness under refrigerated, room temperature and simulated gastric fluid conditions. Results demonstrate that alginate encapsulation significantly improved the viabilities of all three probiotics in various beverages and conditions. Refrigerated storage better preserved probiotic viabilities and reduced the formation of the probiotic metabolic by-product, L-lactate, than at room temperature storage. Findings here could provide beverage manufacturers with a novel way to develop probiotic-sodas and probiotic-beers through encapsulation. Ministry of Education (MOE) Singapore Food Agency Published version SCJ Loo received funding from the Singapore Centre of Environmental Life Sciences Engineering (SCELSE) MOE/RCE: M4330019.C70 (www.scelse.sg), Ministry of Education AcRF-Tier 1 grant RG19/18 and RT08/19 (www.moe.gov.sg), the Singapore National Biofilm Consortium SNBC/ 2021/SF2/P04 (www.snbc.sg), and the Singapore Food Agency SFS_RND_SUFP_001_06 (www.sfa.gov.sg).

New antimicrobials are urgently needed to combat Gram-negative bacteria, particularly multi-drug resistant (MDR) and phenotypically resistant biofilm species. At present, only sequence-defined alpha-peptides (e.g. polymyxin B) can selectively target Gram-negative bacterial lipopolysaccharides. We show that a copolymer, without a defined sequence, shows good potency against MDR Gram-negative bacteria including its biofilm form. The tapered blocky co-beta-peptide with controlled N-terminal hydrophobicity (#4) has strong interaction with the Gram-negative bacterial lipopolysaccharides via its backbone through electrostatic and hydrogen bonding interactions but not the Gram-positive bacterial and mammalian cell membranes so that this copolymer is non-toxic to these two latter cell types. The new #4 co-beta-peptide selectively kills Gram-negative bacteria with low cytotoxicity both in vitro and in a mouse biofilm wound infection model. This strategy provides a new concept for the design of Gram-negative selective antimicrobial peptidomimetics against MDR and biofilm species. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Submitted/Accepted version This research is supported by the Ministry of Education, Singapore, under its MOE AcRF Tier 3 Awards of MOE2018-T3-1-003 and MOE2013-T3-1-002. Zhangyong Si acknowledges the support of an NTU Ph.D. scholarship. The authors thank the NSCC and ASTAR ACRC for providing the computational resources to perform simulations and A*STAR for support (grant IDs H17/01/a0/010, IAF111213C). We also thank the A*STAR Wound Care Innovation for the Tropics IAF-PP (HBMS Domain) with grant number H17/01/a0/0M9, the A*STAR RIE2020 Advanced Manufacturing and Engineering (AME) IAP-PP Specialty Chemicals Programme (Grant No. A1786a0032) and A*STAR Career Development Award (CDA, Grant No. 202D8155), the Major Project of 2025 Sci &Tech Innovation of Ningbo, China (2018B10052) and NTU for funding support.

Complement Receptor Type 1 (CR1) is a malaria-associated gene that encodes a transmembrane receptor of erythrocytes and is crucial for malaria parasite invasion. The expression of CR1 contributes to the rosetting of erythrocytes in the brain bloodstream, causing cerebral malaria, the most severe form of the disease. Here, we study the history of adaptation against malaria by analyzing selection signals in the CR1 gene. We used whole-genome sequencing datasets of 907 healthy individuals from malaria-endemic and non-endemic populations. We detected robust positive selection in populations from the hyperendemic regions of East India and Papua New Guinea. Importantly, we identified a new adaptive variant, rs12034598, which is associated with a slower rate of erythrocyte sedimentation and is linked with a variant associated with low levels of CR1 expression. The combination of the variants likely drives natural selection. In addition, we identified a variant rs3886100 under positive selection in West Africans, which is also related to a low level of CR1 expression in the brain. Our study shows the fine-resolution history of positive selection in the CR1 gene and suggests a population-specific history of CR1 adaptation to malaria. Notably, our novel approach using population genomic analyses allows the identification of protective variants that reduce the risk of malaria infection without the need for patient samples or malaria individual medical records. Our findings contribute to understanding of human adaptation against cerebral malaria. Ministry of Education (MOE) Published version This research was supported by the Singapore Ministry of Education, Academic Research Fund Tier 1 (grant number 2017-T1-001-046 and grant number RG100/20).

Wastewater surveillance (WWS) has been globally recognised to be a useful tool in quantifying SARS-CoV-2 RNA at the community and residential levels without biases associated with case-reporting. The emergence of variants of concern (VOCs) have given rise to an unprecedented number of infections even though populations are increasingly vaccinated. This is because VOCs have been reported to possess higher transmissibility and can evade host immune responses. The B.1.1.529 lineage (Omicron) has severely disrupted global plans to return to normalcy. In this study, we developed an allele-specific (AS) RT-qPCR assay which simultaneously targets the stretch of deletions and mutations in the spike protein from position 24-27 for quantitative detection of Omicron BA.2. Together with previous assays that detect mutations associated with Omicron BA.1 (deletion at position 69 and 70) and all Omicron (mutation at position 493 and 498), we report the validation and time series of these assays from September 2021 to May 2022 using influent samples from two wastewater treatment plants and across four University campus sites in Singapore. Viral RNA concentrations at the treatment plants corroborate with locally reported clinical cases, AS RT-qPCR assays revealed co-incidence of Omicron BA.1 and BA.2 on 12 January 2022, almost two months after initial BA.1 detection in South Africa and Botswana. BA.2 became the dominant variant by the end of January 2022 and completely displaced BA.1 by mid-March 2022. University campus sites were similarly positive for BA.1 and/or BA.2 in the same week as first detection at the treatment plants, where BA.2 became rapidly established as the dominant lineage within three weeks. These results corroborate clinical incidence of the Omicron lineages in Singapore and indicate minimal silent circulation prior to January 2022. The subsequent simultaneous spread of both variant lineages followed strategic relaxation of safe management measures upon meeting nationwide vaccination goals. Ministry of Education (MOE) National Environmental Agency (NEA) National Research Foundation (NRF) Published version This research is supported by the National Research Foundation, Prime Minister's Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program funding to the Singapore Centre for Environmental Life Sciences Engineering (SCELSE) and the IntraCREATE Thematic Grant (Cities) grant NRF2019-THE001-0003a to SW & JT, the National Environmental Agency (NEA) of Singapore, and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to SCELSE.

Probiotics have been recognized as a bio-control measure in aquaculture against bacterial infections, especially with the surge of antibiotic-resistant pathogens. To fully exert the benefits of host-derived probiotics, which are well adapted to prevailing environmental conditions, microencapsulation is often needed to prevent them from gastric acidity, storage, and feed preparation processes before reaching the targeted sites and releasing in a desired manner. Common encapsulants include polysaccharides and proteins, immobilizing probiotics in a 3D matrix to maintain their viability and effectiveness. It is worth noticing that some of these encapsulants are seaweed-derived, which can be viable candidates as prebiotics when served as dietary supplements. Therefore, this work evaluated how four common polysaccharide encapsulants, alginate, carrageenan, xanthan, and gum arabic, can be utilized to modulate the host gut microbiota in an ex vivo model. Alginate and kappa-carrageenan were shortlisted for subsequent probiotic encapsulation based on their fermentability to produce short-chain fatty acids (SCFAs), which are energy sources for intestinal epithelial cells, and the bacterial composition change in the gut microflora after the co-incubation with the fish gut suspensions. Alginate is the most common encapsulant for probiotic encapsulation due to its ability to gel with di-valent ions to provide bacteria with good gastric protection and a lack of toxicity. Kappa-carrageenan can crosslink with mono-valent ions, and it produced the most abundant SCFAs and could not be selectively utilized by pathogenic bacteria. Both needle extrusion and spray drying were explored as encapsulation techniques. Extrusion is easy to set up and mild to bacteria. Extrusion parameters, including the needle size, the polysaccharide concentration, the flow rate, and the extrusion temperature, were optimized to yield microparticles with sufficient gastric protection. The survivability of the probiotics that underwent the simulated gastric fluid remained at the same 8 Log CFU/g magnitude as before the challenge, while no survival was found for naked bacteria. The robust protection of needle-extruded particles was accompanied by difficulty releasing bacteria in the intestine, with only around 5 Log CFU/g could be released in 2 hours. Considering fish’s short digestion time, needle extrusion may not be optimal for probiotic delivery in aquaculture. Spray drying was adopted to resolve the release problem. The host-derived strain can withstand the spray drying condition and readily release in the simulated intestinal fluid with uncompromised protection in the gastric environment. The ex vivo co-incubation study confirmed that introducing this encapsulated host-derived isolate could effectively suppress the growth of Aeromonas. Future work includes the in vivo challenge test in jade perch to assess whether manipulating the host gut microbiota through host-derived probiotic supplementation can increase disease resistance and how the bacteria persist in their native environment. Singapore National Biofilm Consortium

The increasing global population has put enormous stresses on food security. Aquaculture is a major source of food fish and is considered one of the key pillars of our global food supply. The increasing aquaculture production has led to a surge in demand for fishmeal and fish oil – key ingredients of fish feeds obtained from finite marine resources. Decreasing the dependence on marine resources could improve the sustainability of farmed fishes. One strategy is to replace fishmeal with alternative proteins. This review aims to provide a comprehensive overview of how industrial food wastes can be valorised into a sustainable protein source as a replacement for fishmeal. The direct substitution of fishmeal with food wastes and the challenges associated with it will first be discussed. Next, a section will be devoted to repurposing of industrial food waste as insect feeds for the bioconversion of waste into high value protein source. A special mention on the use of encapsulated micronutrients and probiotics for functionalised feeds will also be reviewed. The review concludes by summarising the existing regulations on the use of waste in aquaculture, its public perception, and future perspectives in converting food waste into aquaculture feed for a sustainable circular economy. Ministry of Education (MOE) Singapore Food Agency Published version The authors would like to acknowledge the financial support from the Singapore Centre for Environmental Life Sciences Engineering (SCELSE) (MOE/RCE: M4330019.C70), Ministry of Education AcRF-Tier 1 grant (RG19/18 and RT08/19), the Singapore National Biofilm Consortium (SNBC/2021/SF2/P04) and the Singapore Food Agency (SFS_RND_SUFP_001_06).

Marine cyanobacteria are known to produce structurally diverse bioactive specialized metabolites during bloom occurrence. These ecologically active allelochemicals confer chemical defense for the microalgae from competing microbes and herbivores. From a collection of a marine cyanobacterium, cf. Lyngbya sp., a small quantity of a new cyclopropane-containing molecule, benderadiene (2), and lyngbyoic acid (1) were purified and characterized using spectroscopic methods. Using live reporter quorum-sensing (QS) inhibitory assays, based on P. aeruginosa PAO1 lasB-gfp and rhlA-gfp strains, both compounds were found to inhibit QS-regulated gene expression in a dose-dependent manner. In addition to lyngbyoic acid being more active in the PAO1 lasB-gfp biosensor strain (IC50 of 20.4 µM), it displayed anti-biofilm activity when incubated with wild-type P. aeruginosa. The discovery of lyngbyoic acid in relatively high amounts provided insights into its ecological significance as a defensive allelochemical in targeting competing microbes through interference with their QS systems and starting material to produce other related analogs. Similar strategies could be adopted by other marine cyanobacterial strains where the high production of other lipid acids has been reported. Preliminary evidence is provided from the virtual molecular docking of these cyanobacterial free acids at the ligand-binding site of the P. aeruginosa LasR transcriptional protein. Nanyang Technological University Published version The facilities and equipment support were provided by the National Institute of Education, NTU and Singapore Centre for Environmental Life Sciences Engineering, NTU. This research is funded by NIE AcRF grant RI 2/20/TLT.

Ammonia-oxidizing archaea (AOA) can oxidize ammonia to nitrite for energy gain. They have been detected in chloraminated drinking water distribution systems (DWDS) along with the more common ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). To date, no members of the AOA have been isolated or enriched from drinking water environments. To begin the investigation of the role of AOA in chloraminated DWDS, we developed a selective approach using biofilm samples from a full-scale operational network as inoculum. A Nitrososphaera viennensis-like AOA taxon was enriched from a mixed community that also included Nitrosomonas-like AOB while gradually scaling up the culture volume. Dimethylthiourea (DMTU) and pyruvate at 100 μM were added to promote the growth of AOA while inhibiting AOB. This resulted in the eventual washout of AOB, while NOB were absent after 2 or 3 rounds of amendment with 24 μM sodium azide. The relative abundance of AOA in the enrichment increased from 0.2% to 39.5% after adding DMTU and pyruvate, and further to 51.6% after filtration through a 0.45-μm pore size membrane, within a period of approximately 6 months. IMPORTANCE Chloramination has been known to increase the risk of nitrification episodes in DWDS due to the presence of ammonia-oxidizing microorganisms. Among them, AOB are more frequently detected than AOA. All publicly available cultures of AOA have been isolated from soil, marine or surface water environments, meaning they are allochthonous to DWDS. Hence, monochloramine exposure studies involving these strains may not accurately reflect their role in DWDS. The described method allows for the rapid enrichment of autochthonous AOA from drinking water nitrifying communities. The high relative abundance of AOA in the resulting enrichment culture reduces any confounding effects of co-existing heterotrophic bacteria when investigating the response of AOA to varied levels of monochloramine in drinking water. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Published version The authors acknowledge financial support from the Singapore Centre for Environmental Life Sciences Engineering (SCELSE), whose research is supported by the National Research Foundation Singapore, Ministry of Education, Nanyang Technological University, and National University of Singapore, under its Research Centre of Excellence Program.

The biofilm matrix, with its diversity of extracellular polymeric substances (EPS), remains a poorly understood entity. It consists of a heterogeneous, multifunctional microenvironment that imparts a range of emergent properties to the biofilm, including social cooperation and resource sharing, adaptation to environmental changes, and resistance to harmful chemicals and antibiotics. Generally, studies of the biofilm matrix focus on the regulation of EPS gene expression and associated biofilm phenotypes (Flemming et al., 2022; Flemming & Wingender, 2010). For example, the differential regulation of exopolymers, which impart different mechanical properties, is an often-studied genetic marker for characterizing transitions between different stages of biofilm development (Chew, Kundukad, et al., 2014; Irie et al., 2012). New insights, however, suggest that the emergent properties of the matrix, which arise because of physical interactions between EPS molecules as well as those between EPS and bacterial cells, also play important roles in biofilm formation and organization (Liu et al., 2022; Rubinstein et al., 2012). For example, the secretion and accumulation of EPS components generate new physical forces, such as osmotic stresses, bridging interactions, and depletion effects within the crowded matrix (Liu et al., 2022). These forces alter the physical environment of the biofilm, affecting conformational and aggregational landscapes and dynamics, and thus functions, of matrix biopolymers. Together with the biological program, they stabilize extended structural, compositional, and morphological gradients in space and time, drive phase transitions, immobilize cells, and induce phase separation, creating spatial functional niches within the matrix (Worlitzer et al., 2022). Considering these insights, we highlight here the emerging perspective that understanding the competition and the collaboration between physical and biological factors is crucial for a more complete appreciation of biofilm formation, dynamics, organization, and function. Nanyang Technological University National Research Foundation (NRF) Published version The authors acknowledge funding grants fromNanyang Technological University, National Research Foundation Singapore and National University ofSingapore for conducting the research.

Arboviruses are viral pathogens transmitted by blood-borne vectors that impose a great social and economic burden globally. Most clinical surveillance of arbovirus outbreaks underestimates the true prevalence as a large proportion of cases exhibit no or only mild clinical symptoms (i.e., are subclinical). Reports of urinary shedding of several arboviruses such as Dengue (DENV), yellow fever (YFV), and Zika (ZIKV) viruses suggest the possibility of utilizing wastewater surveillance to assess the prevalence of arboviral outbreaks. To determine the feasibility of wastewater surveillance, we investigated the decay of representative arboviruses (including DENV-2, DENV-3, YFV, and ZIKV) along with murine hepatitis virus (MHV) as a surrogate for human coronavirus within a wastewater matrix at 6, 25, or 37 °C using RT-qPCR. DENV-2, DENV-3, YFV, ZIKV, and MHV experienced a one log10reduction within 3.95-6.21 days at 25 °C and within 2.60-5.12 days at 37 °C, while incubation at 6 °C did not indicate substantial decay within 21 days. Our work suggests that these arboviruses or their RNA could persist sufficiently in wastewater over a range of temperatures, supporting the potential for wastewater-based surveillance of arboviral outbreaks. Ministry of Education (MOE) National Research Foundation (NRF) Singapore-MIT Alliance for Research and Technology (SMART) This research was supported by the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program, through Intra-CREATE Thematic Grant (Cities) Grant NRF2019-THE001-0003 to J.T. and E.J.A., the Singapore-MIT Alliance for Research and Technology (SMART) Antimicrobial Resistance Interdisciplinary Research Group (AMR IRG), and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to the Singapore Centre for Environmental Life Sciences Engineering (SCELSE).

The ocular surface microbiome has implications for ocular surface inflammation and immunology. Previous shotgun metagenomics analyses were performed in China, showing results that differed according to environment and age. Patients with Sjogren's syndrome were reported to have altered conjunctival microbiome, but such studies have not been done in milder dry eye. The aim of this study is to describe the conjunctival microbiome in people with mild dry eye in Singapore. Samples were collected from 14 participants with mild dry eye and 10 age-matched comparison participants recruited from Singapore National Eye Centre (SNEC) clinics. Shotgun metagenomic sequencing analysis was employed to evaluate the conjunctival microbiome composition. Proteobacteria formed the predominant phylum in the conjunctiva. As in a study from a coastal city in China, Achromobacter spp. was numerically most abundant. Compared to age-matched controls, the conjunctival microbial composition in mild dry eye was similar. Several microorganisms, including Streptococcus spp. increased in representation with age, and the abundance of Staphylococcus correlated with Schirmer readings. In addition, when cultured corneal epithelial cells were exposed to three strains of Achromobacter xylosoxidans, cytokines such as TNF-α and IL-6 were upregulated in the cell lysates and supernatants. Ourresults suggest that age is an important factor that affects composition of the conjunctival microbiome, and relative abundance of specific microorganism may vary according to the environment of the human host. National Medical Research Council (NMRC) Published version This research was supported by the National Medical Research Council (NMRC) (NMRC/CSA/045/2012 and NMRC/CSA-SI/0017/2017).

The use of food-processing wastewaters to produce microbial biomass-derived single cell protein (SCP) is a sustainable way to meet the global food demand. Yet, despite the potential benefits of lower costs and greater resource recovery compared to pure cultures, bioconversion processes relying on microbial community-based approaches to SCP production have received scarce attention. Here, we evaluated SCP production from soybean-processing wastewaters under controlled reactor conditions using the existent microbial communities in these wastewaters. Six sequencing batch reactors of 4.5-L working volume were operated at 30 □ for 34 d in cycles consisting of 3-h anaerobic and 9-h aerobic phases. Four reactors received no microbial inoculum and the remaining two were amended with a 1.5 L of mixed culture from a prior microbial community-based SCP production. Microbial characterization was done via 16S rRNA gene metabarcoding. Influent wastewater batches had variable chemical characteristics but a similar microbial composition. Reactors produced more SCP when fed with wastewaters of higher soluble total Kjeldahl nitrogen (sTKN) content and a lower carbon-to-nitrogen ratio (sCOD:sTKN). The biomass protein yield ranged from 0.24 to 3.13 g protein/g sTKN, with a maximum protein content of 50%. An average of 92% of sCOD and 73% of sTN removal was achieved. Distinct microbial communities were enriched in all six bioreactors after 34 d, where the prevailing genera included Azospirillum, Rhodobacter, Lactococcus, Novosphingobium, and Acidipropionibacterium. In contrast, the microbial community of influent wastewaters was dominated by Lactococcus and Weissella. We showed that constituents in soybean wastewater can be converted to SCP through microbial community-based growth processes and demonstrated the effect of variable influent wastewater composition on SCP production.

The increasing occurrence of steroidal hormone micropollutant in the aquatic environment and their associated consequences have caused serious environmental concerns globally. Adsorptive removal of hormonal pollutants using polymeric membranes has been suggested but information on their performance in various environmental conditions is lacking. In this study, we examined the effect of salinity on the performance of polyethersulfone (PES) membrane to remove synthetic hormone 17α-ethinyl estradiol (EE2) from water. Our results show that an increase of salinity from 0 to 3% results in higher retention of EE2 onto PES membrane from 79.3 to 98.7%. The experimental results fit the Freundlich isotherm model better as compared to the Langmuir model. The Freundlich parameters n and Kf yielded the highest values at 3% salinity. The molecular simulation results suggest that a high salinity increases the binding energy between EE2 and PES membranes, promoting the PES-EE2 interaction through π–π interaction, hydrogen bonding and hydrophobic interaction. This study provides valuable information for improving design of specialised treatment facilities (in farming, pharmaceutical industries, etc.) to allow better removal of EE2 and other low-polar organic contaminants from water via a membrane-based sorption-elution method, and we recommend the inclusion of salinity as a factor in modelling the adsorption capacity of membranes to prevent the oversaturation of membrane and minimise the release of contaminants into the environment.

The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, we find here that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy. M. tuberculosis can metabolize oxygen efficiently at least two orders of magnitude below the concentration predicted to occur in hypoxic lung granulomas. Despite a difference in apparent affinity for oxygen, both the cytochrome bcc:aa3 and cytochrome bd oxidase respiratory branches are required for hypoxic respiration. Simultaneous inhibition of both oxidases blocks oxygen consumption, reduces ATP levels, and kills M. tuberculosis under hypoxia. The capacity of mycobacteria to scavenge trace levels of oxygen, coupled with the absence of complex regulatory mechanisms to achieve hierarchal control of the terminal oxidases, may be a key determinant of long-term M. tuberculosis survival in hypoxic lung granulomas. National Research Foundation (NRF) Published version This work was supported by the National Research Foundation (NRF) Singapore under its Investigatorship Program (grant NRF-NRFI06-2020-0004) and the NRF Competitive Research Program (project award numbers NRF-CRP18–2017–01 and NRF-CRP27-2021-0002, to K.P.) and by the National Institutes of Health USA (R01 AI139465, to M.B.). K.H. and G.M.C. were funded by Marsden grants awarded by the Royal Society of New Zealand and a Health Research Council of New Zealand grant.

Alopecia areata (AA) is an autoimmune skin disorder that affects any hair-bearing areas and results in localized nonscarring hair loss. It can occur in individuals at any age regardless of ethnicity with a lifetime risk of 1–2%.1 The loss of hair is caused by perifollicular inflammation, which is associated with cytokines and chemokines released by T cells.2, 3 Although spontaneous regrowth is possible in 80% of patchy AA, full resolution is slow.4 Patients with AA can be treated with various treatment options including a topical or intralesional steroid. While intralesional steroid injection is more effective and frequently used, it requires repeated injections every 4–6 weeks. In addition, patients may experience discomfort caused by the needle pricks during injection and the presence of steroid-induced atrophy at the site of injection. Ministry of Health (MOH) National Medical Research Council (NMRC) Published version Ministry of Health’s National Medical Research Council, Singapore,Grant Number: NMRC/CSA-INV/0023/2017.

Enterococcus faecalis (E. faecalis) biofilms are implicated in endocarditis, urinary tract infections, and biliary tract infections. Coupled with E. faecalis internalization into host cells, this opportunistic pathogen poses great challenges to conventional antibiotic therapy. The inability of ampicillin (Amp) to eradicate bacteria hidden in biofilms and intracellular niches greatly reduces its efficacy against complicated E. faecalis infections. To enhance the potency of Amp against different forms of E. faecalis infections, Amp was loaded into Lipid-Polymer hybrid Nanoparticles (LPNs), a highly efficient nano delivery platform consisting of a unique combination of DOTAP lipid shell and PLGA polymeric core. The antibacterial activity of these nanoparticles (Amp-LPNs) was investigated in a protozoa infection model, achieving a much higher multiplicity of infection (MOI) compared with studies using animal phagocytes. A significant reduction of total E. faecalis was observed in all groups receiving 250 µg/mL Amp-LPNs compared with groups receiving the same concentration of free Amp during three different interventions, simulating acute and chronic infections and prophylaxis. In early intervention, no viable E. faecalis was observed after 3 h LPNs treatment whereas free Amp did not clear E. faecalis after 24 h treatment. Amp-LPNs also greatly enhanced the antibacterial activity of Amp at late intervention, and boosted the survival rate of protozoa approaching 400%, where no viable protozoa were identified in the free Amp groups at the 40 h post-infection treatment timepoint. Prophylactic effectiveness with Amp-LPNs at a concentration of 250 μg/mL was exhibited in both bacteria elimination and protozoa survival towards subsequent infections. Using protozoa as a surrogate model for animal phagocytes to study high MOI infections, this study suggests that LPN-formulated antibiotics hold the potential to significantly improve the therapeutic outcome in highly complicated bacterial infections. Agri-Food and Veterinary Authority of Singapore (AVA) Ministry of Education (MOE) Accepted version The authors would like to acknowledge the financial support from the Singapore Centre for Environmental Life Sciences Engineering (SCELSE) (MOE/RCE: M4330019.C70), Ministry of Education AcRF-Tier 1 grant (RG19/18), Agri-Food & Veterinary Authority of Singapore (APF LCK102), Biomedical Research Council (BMRC) - Therapeutics Development Review (TDR-G-004-001), NTU-HSPH grant (NTU-HSPH 17002), and the Bill and Melinda Gates Foundation (OPP1199116).

Biofouling is recognized as one of the most problematic fouling types in reverse osmosis (RO) processes and lead to high energy requirements and operating costs. Over the past decade, many studies on membrane bioreactor (MBR) systems for wastewater applications demonstrated that disrupting cell-cell communications among bacteria, called quorum quenching (QQ), is a promising approach to inhibit biofouling of membranes. Here, we developed the QQ-column as a novel strategy to control biofouling in RO systems. The QQ-column was prepared by incorporating a recombinant bacterial QQ strain into hydrogel beads and embedding these beads inside a column. The QQ-column was installed upstream of the RO module to degrade N-acyl homoserine lactone, a quorum sensing (QS) signal, from the feed in a laboratory-scale RO system operating in total recycle mode. The QQ-column reduced the concentrations of signal molecules by ~29% in an RO system and mitigated biofilm formation (38.6% reduction of cell number) on the membrane, consequently reducing the transmembrane pressure by 50.1%. These results demonstrate that integrating QQ bacteria into columns is a practical method to control biofouling in RO systems. Economic Development Board (EDB) National Research Foundation (NRF) Accepted version This study was supported by the Research Fund for a new professor by the SeoulTech (Seoul National University of Science and Technology). We also acknowledge financial support from the Singapore Centre for Environmental Life Sciences Engineering, whose research is supported by the National Research Foundation Singapore, Ministry of Education, Nanyang Technological University and National University of Singapore, under its Research Centre of Excellence Programme. The funding support from the Economic Development Board (EDB) of Singapore to the Singapore Membrane Technology Centre (SMTC), Nanyang Environment & Water Research Institute (NEWRI), Nanyang Technological Univer- sity (NTU) is also acknowledged

Neisseria species are frequently identified in the bronchiectasis microbiome, but they are regarded as respiratory commensals. Using a combination of human cohorts, next-generation sequencing, systems biology, and animal models, we show that bronchiectasis bacteriomes defined by the presence of Neisseria spp. associate with poor clinical outcomes, including exacerbations. Neisseria subflava cultivated from bronchiectasis patients promotes the loss of epithelial integrity and inflammation in primary epithelial cells. In vivo animal models of Neisseria subflava infection and metabolipidome analysis highlight immunoinflammatory functional gene clusters and provide evidence for pulmonary inflammation. The murine metabolipidomic data were validated with human Neisseria-dominant bronchiectasis samples and compared with disease in which Pseudomonas-, an established bronchiectasis pathogen, is dominant. Metagenomic surveillance of Neisseria across various respiratory disorders reveals broader importance, and the assessment of the home environment in bronchiectasis implies potential environmental sources of exposure. Thus, we identify Neisseria species as pathobionts in bronchiectasis, allowing for improved risk stratification in this high-risk group. Published version

The age-associated alterations in microbiomes vary across populations due to the influence of genetics and lifestyles. To the best of our knowledge, the microbial changes associated with aging have not yet been investigated in Singapore adults. We conducted shotgun metagenomic sequencing of fecal and saliva samples, as well as fecal metabolomics to characterize the gut and oral microbial communities of 62 healthy adult male Singaporeans, including 32 young subjects (age, 23.1 ± 1.4 years) and 30 elderly subjects (age, 69.0 ± 3.5 years). We identified 8 gut and 13 oral species that were differentially abundant in elderly compared to young subjects. By combining the gut and oral microbiomes, 25 age-associated oral-gut species connections were identified. Moreover, oral bacteria Acidaminococcus intestine and Flavonifractor plautii were less prevalent/abundant in elderly gut samples than in young gut samples, whereas Collinsella aerofaciens and Roseburia hominis showed the opposite trends. These results indicate the varied gut-oral communications with aging. Subsequently, we expanded the association studies on microbiome, metabolome and host phenotypic parameters. In particular, Eubacterium eligens increased in elderly compared to young subjects, and was positively correlated with triglycerides, which implies that the potential role of E. eligens in lipid metabolism is altered during the aging process. Our results demonstrated aging-associated changes in the gut and oral microbiomes, as well as the connections between metabolites and host-microbe interactions, thereby deepening the understanding of alterations in the human microbiome during the aging process in a Singapore population. Nanyang Technological University Published version This work was supported by a seed fund from the Nanyang Technological University Integrated Medical, Biological & Environmental Life Sciences (NIMBELS), Singapore (NIM/ 06/2016), Shenzhen Basic Research Program (JCYJ20190808182402941) and Health and Medical Research Fund in Hong Kong (COVID190206).

Aspergillus terreus species complex is an opportunistic fungal pathogen increasingly implicated in invasive infection, as well as chronic respiratory disease. Currently, an understanding of A. terreus pathogenicity is impeded by a limited number of whole-genome sequences of this fungal pathogen. We here describe a high-quality whole-genome assembly of European A. terreus clinical isolate M6925, derived by single-molecule real-time sequencing with short-read polishing.

The opportunistic pathogen Pseudomonas aeruginosa is ubiquitous in the environment, and in humans, it is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when coincubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence for the nematode Caenorhabditis elegans was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and nonadapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoebae as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoebae resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water, where bacteria are constantly under threat of being consumed by bacterial predators, e.g., protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examined the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa. We show that long-term coincubation with protozoa gave rise to mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to those observed in isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types as well as similar adaptation strategies. Ministry of Education (MOE) National Research Foundation (NRF) Published version We acknowledge support from the iThree Institute at the University of Technology Sydney, Sydney, Australia, from the Australian Research Council Discovery Project (DP170100453), and from the National Research Foundation and Ministry of Education Singapore under its Research Centre of Excellence Program to the Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University.

Natural products derived from marine sponges have exhibited bioactivity and, in some cases, serve as potent quorum sensing inhibitory agents that prevent biofilm formation and attenuate virulence factor expression by pathogenic microorganisms. In this study, the inhibitory activity of the psammaplin-type compounds, psammaplin A (1) and bisaprasin (2), isolated from the marine sponge, Aplysinellarhax, are evaluated in quorum sensing inhibitory assays based on the Pseudomonas aeruginosa PAO1 lasB-gfp(ASV) and rhlA-gfp(ASV) biosensor strains. The results indicate that psammaplin A (1) showed moderate inhibition on lasB-gfp expression, but significantly inhibited the QS-gene promoter, rhlA-gfp, with IC50 values at 14.02 μM and 4.99 μM, respectively. In contrast, bisaprasin (2) displayed significant florescence inhibition in both biosensors, PAO1 lasB-gfp and rhlA-gfp, with IC50 values at 3.53 μM and 2.41 μM, respectively. Preliminary analysis suggested the importance of the bromotyrosine and oxime functionalities for QSI activity in these molecules. In addition, psammaplin A and bisaprasin downregulated elastase expression as determined by the standard enzymatic elastase assay, although greater reduction in elastase production was observed with 1 at 50 μM and 100 μM. Furthermore, the study revealed that bisaprasin (2) reduced biofilm formation in P. aeruginosa. National Research Foundation (NRF) Published version The facilities and equipment support were provided partially by the National Institute of Education, Singapore. This research is funded by the National Research Foundation, Prime Minister’s Office, Singapore under its Marine Science Research and Development Program (Award Nos. MSRDP-P15 and MSRDP-P34). In addition, we acknowledge the E.U. Seventh Framework Programme Project PharmaSea (grant agreement no-312184) for financial support for the collection of the marine sample.

Background: The biofilm way of life is a common strategy for bacteria to adapt and gain tolerance towards antibiotic treatments. Up to 80% of all infections are biofilm-mediated and they are often challenging to treat as the underlying bacterial cells can become 100 – 1000-fold more tolerant towards antibiotics. Antibiotic-loaded nanoparticles have gained traction as a potential drug delivery system to treat biofilm infections. Specifically, lipid-coated hybrid nanoparticles (LCHNPs) were investigated on their capability to deliver antibiotics into biofilms. In this study, LCHNPs composed of a poly(lactic-co-glycolic acid) (PLGA) core and dioleoyl-3-trimethylammonium propane (DOTAP) lipid shell were developed and loaded with vancomycin (Van). In vitro antibacterial and antibiofilm tests were performed to evaluate the antimicrobial efficacy of the LCHNPs.Results: LCHNPs were successfully fabricated with high vancomycin encapsulation efficiency of 48.66% and loading efficiency of 72.99 µg/mg. DOTAP was determined to be successfully coated on the PLGA core by measuring the ζ-potential of the nanoparticles. LCHNPs had a positive ζ-potential of +36.13 mV which contrasted significantly from the ζ-potential of -36.83 mV of bare PLGA nanoparticles (PLGANPs). LCHNPs exhibited enhanced antibacterial effects against planktonic Staphylococcus aureus USA300 cells, with at least 6-fold reduction in minimum inhibitory concentration when compared against Free-Van and Van-PLGANPs. When used to treat USA300 biofilms, Van-LCHNPs eradicated up to 99.99% of the underlying biofilm cells, an effect which was not observed for Free-Van and Van-PLGANPs. Finally, we showed that by possessing a robust DOTAP shell, LCHNPs were able to penetrate deeply into the biofilms. Conclusion: LCHNPs were shown to display remarkable antimicrobial efficacy towards both planktonic and biofilm cells as the presence of the lipid shell enhanced the interactions with bacterial cells and penetration into biofilms. More work could be done to understand nanoparticle-biofilm interactions; this would help to optimize the LCHNPs further to treat biofilm infections successfully.

As antimicrobial resistance (AMR) continues to pose an ever-growing global health threat, propelling us into a post-antibiotic era, novel alternative therapeutic agents are urgently required. Lysins are bacteriophage-encoded peptidoglycan hydrolases that display great potential as a novel class of antimicrobials for therapeutics. While lysins against Gram-positive bacteria are highly effective when applied exogenously, it is challenging for lysins to access and cleave the peptidoglycan of Gram-negative bacteria due to their outer membrane. In this study, we identify a novel phage lysin Abp013 against Acinetobacter baumannii. Abp013 exhibited significant lytic activity against multidrug-resistant strains of A. baumannii. Notably, we found that Abp013 was able to tolerate the presence of human serum by up to 10%. Using confocal microscopy and LIVE/DEAD staining, we show that Abp013 can access and kill the bacterial cells residing in the biofilm. These results highlight the intrinsic bacteriolytic property of Abp013, suggesting the promising use of Abp013 as a novel therapeutic agent. National Research Foundation (NRF) Singapore-MIT Alliance for Research and Technology (SMART) Published version This research was funded by the Intra-CREATE Seed Collaboration Grant, ITS-006-003, and the Antimicrobial Resistance IRG of the Singapore-MIT Alliance for Research and Technology Centre, supported by the National Research Foundation, Prime Minister’s Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) Program.

Kefir grains consist of complex symbiotic mixtures of bacteria and yeasts, and are reported to impart numerous health-boosting properties to milk and water kefir beverages. The objective of this work was to investigate the microbial communities in kefir grains, and explore the possibility of deriving useful probiotic strains from them. A total of 158 microbial strains, representing six fungal and 17 bacterial species, were isolated from milk and water kefir grains collected from a Singapore-based homebrewer. Based on 16S rRNA sequencing, isolated genera included Lactobacillus, Liquorilactobacillus, Lacticaseibacillus, Lentilactobacillus, Leuconostoc, Lactococcus, Acetobacter, Gluconobacter, Oenococcus, Clostridium, Zymomonas, Saccharomyces, Kluyveromyces, Pichia, Lachancea, Candida, and Brettanomyces. To characterize these isolates, a funnel approach, involving numerous phenotypic and genomic screening assays, was applied to identify kefir-derived microbial strains with the highest probiotic potential. Particular focus was placed on examining the pathogen inhibitory properties of kefir isolates toward enteric pathogens which pose a considerable global health burden. Enteric pathogens tested include species of Bacillus, Salmonella, Vibrio, Clostridium, Klebsiella, Escherichia, and Staphylococcus. Well diffusion assays were conducted to determine the propensity of kefir isolates to inhibit growth of enteric pathogens, and a competitive adhesion/exclusion assay was used to determine the ability of kefir isolates to out-compete or exclude attachment of enteric pathogens to Caco-2 cells. Seven bacterial strains of Lentilactobacillus hilgardii, Lacticaseibacillus paracasei, Liquorilactobacillus satsumensis, Lactobacillus helveticus, and Lentilactobacillus kefiri, were ultimately identified as potential probiotics, and combined to form a “kefir probiotics blend.” Desirable probiotic characteristics, including good survival in acid and bile environments, bile salt hydrolase activity, antioxidant activity, non-cytotoxicity and high adhesion to Caco-2 cells, and a lack of virulence or antimicrobial resistance genes. In addition, vitamin and γ-aminobutyric acid (GABA) synthesis genes, were identified in these kefir isolates. Overall, probiotic candidates derived in this study are well-characterized strains with a good safety profile which can serve as novel agents to combat enteric diseases. These kefir-derived probiotics also add diversity to the existing repertoire of probiotic strains, and may provide consumers with alternative product formats to attain the health benefits of kefir.

Current Genome-Wide Association Studies (GWAS) rely on genotype imputation to increase statistical power, improve fine-mapping of association signals, and facilitate meta-analyses. Due to the complex demographic history of Latin America and the lack of balanced representation of Native American genomes in current imputation panels, the discovery of locally relevant disease variants is likely to be missed, limiting the scope and impact of biomedical research in these populations. Therefore, the necessity of better diversity representation in genomic databases is a scientific imperative. Here, we expand the 1,000 Genomes reference panel (1KGP) with 134 Native American genomes (1KGP + NAT) to assess imputation performance in Latin American individuals of mixed ancestry. Our panel increased the number of SNPs above the GWAS quality threshold, thus improving statistical power for association studies in the region. It also increased imputation accuracy, particularly in low-frequency variants segregating in Native American ancestry tracts. The improvement is subtle but consistent across countries and proportional to the number of genomes added from local source populations. To project the potential improvement with a higher number of reference genomes, we performed simulations and found that at least 3,000 Native American genomes are needed to equal the imputation performance of variants in European ancestry tracts. This reflects the concerning imbalance of diversity in current references and highlights the contribution of our work to reducing it while complementing efforts to improve global equity in genomic research. Published version This work was supported by “The Mexican Biobank Project: Building Capacity for Big Data Science in Medical Genomics in Admixed Populations”, a binational initiative between Mexico and the UK co-funded by CONACYT (Grant number FONCICYT/50/ 2016), and The Newton Fund through The Medical Research Council (Grant number MR/N028937/1) awarded to AME and AVSH. It was also supported by the International Center for Genetic Engineering and Biotechnology (ICGEB, Italy) grant number CRP/MEX20-01. MS was partially supported by the Chicago Fellows program of the University of Chicago. DODV is supported by the UC MEXUS CONACYT collaborative program (Grant number CN-19-29), and the UNAM PAPIIT funding program (Grant number IA200620).

Massive amounts of data from nucleic acid sequencing have changed our perspective about diversity and dynamics of marine viral communities. Here, we summarize recent metatranscriptomic and metaviromic studies targeting predominantly RNA viral communities. The analysis of RNA viromes reaffirms the abundance of lytic (+) ssRNA viruses of the order Picornavirales, but also reveals other (+) ssRNA viruses, including RNA bacteriophages, as important constituents of extracellular RNA viral communities. Sequencing of dsRNA suggests unknown diversity of dsRNA viruses. Environmental metatranscriptomes capture the dynamics of ssDNA, dsDNA, ssRNA, and dsRNA viruses simultaneously, unravelling the full complexity of viral dynamics in the marine environment. RNA viruses are prevalent in large size fractions of environmental metatranscriptomes, actively infect marine unicellular eukaryotes larger than 3 µm, and can outnumber bacteriophages during phytoplankton blooms. DNA and RNA viruses change abundance on hourly timescales, implying viral control on a daily temporal basis. Metatranscriptomes of cultured protists host a diverse community of ssRNA and dsRNA viruses, often with multipartite genomes and possibly persistent intracellular lifestyles. We posit that RNA viral communities might be more diverse and complex than formerly anticipated and that the influence they exert on community composition and global carbon flows in aquatic ecosystems may be underestimated. Agency for Science, Technology and Research (A*STAR) National Research Foundation (NRF) Published version This work was supported by the National Research Foundation, Prime’s Minister’s Office, Singapore under its Intra-CREATE Seed Collaboration Grant (Award NRF2018-ITS004-0001) and Competitive Research Programme (Award CRP21-2018-0005). Sandra Kolundzija is supported by a Singapore International Graduate Award (SINGA) from the Agency for Science, Technology & Research (A*STAR).

Naturally occurring hydrazones are rare despite the ubiquitous usage of synthetic hydrazones in the preparation of organic compounds and functional materials. In this study, we discovered a family of novel microbial metabolites (tasikamides) that share a unique cyclic pentapeptide scaffold. Surprisingly, tasikamides A−C (1−3) contain a hydrazone group (CNN) that joins the cyclic peptide scaffold to an alkyl 5-hydroxylanthranilate (AHA) moiety. We discovered that the biosynthesis of 1−3 requires two discrete gene clusters, with one encoding a nonribosomal peptide synthetase (NRPS) pathway for assembling the cyclic peptide scaffold and another encoding the AHA-synthesizing pathway. The AHA gene cluster encodes three ancillary enzymes that catalyze the diazotization of AHA to yield an aryl diazonium species (diazo-AHA). The electrophilic diazo-AHA undergoes nonenzymatic Japp−Klingemann coupling with a β-keto aldehyde-containing cyclic peptide precursor to furnish the hydrazone group and yield 1− 3. The studies together unraveled a novel mechanism whereby specialized metabolites are formed by the coupling of two biosynthetic pathways via an unprecedented in vivo Japp−Klingemann reaction. The findings raise the prospect of exploiting the arylamine-diazotizing enzymes (AAD) for the in vivo synthesis of aryl compounds and modification of biological macromolecules. Nanyang Technological University National Research Foundation (NRF) Submitted/Accepted version This research was supported by an NIMBEL grant (NIM/03/ 2017, Z.-X.L.) from Nanyang Technological Universty and an NRF-SBP grant (SBP-01 Z.-X.L.) from the National Research Foundation of Singapore.

Monochloramine (MCA) is a widely used secondary disinfectant to suppress microbial growth in drinking water distribution systems. In monochloraminated drinking water, a significant amount of extracellular DNA (eDNA) has been reported, which has many implications ranging from obscuring DNA-based drinking water microbiome analyses to posing potential health concerns. To address this, it is imperative for us to know the origin of the eDNA in drinking water. Using Pseudomonas aeruginosa as a model organism, we report for the first time that MCA induces the release of nucleic acids from both biofilms and planktonic cells. Upon exposure to 2 mg/L MCA, massive release of DNA from suspended cells in both MilliQ water and 0.9% NaCl was directly visualized using live cell imaging in a CellASIC ONIX2 microfluidic system. Exposing established biofilms to MCA also resulted in DNA release from the biofilms, which was confirmed by increased detection of eDNA in the effluent. Intriguingly, massive release of RNA was also observed, and the extracellular RNA (eRNA) was also found to persist in water for days. Sequencing analyses of the eDNA revealed that it could be used to assemble the whole genome of the model organism, while in the water, certain fragments of the genome were more persistent than others. RNA sequencing showed that the eRNA contains non-coding RNA and mRNA, implying its role as a possible signaling molecule in environmental systems and a snapshot of the past metabolic state of the bacterial cells. Ministry of Education (MOE) National Research Foundation (NRF) This research was supported by the National Research Foundation (NRF), Singapore, under its NERC-NRF Joint Grant Call (award no. NRF-NERC-SEAP-2020-02 to B.C.) and by the NRF and Ministry of Education Singapore under its Research Centre of Excellence Programme, Singapore Centre for Environmental Life Sciences Engineering (M4330005.C70 to B.C.), Nanyang Technological University, Singapore.

A functioning anaerobic digestion (AD) microbiome is integral for sludge management to be successful. Comprehensive ecological insights, and a full accounting of important microbial species, can help to improve and validate operational strategies. We analysed a time-series of metagenome samples obtained from full-scale anaerobic digesters and performed genome-resolved analysis to gain insight into the microbial community structure and potential functions of the AD microbiome. Ninety samples from three full-scale digesters were collected over a period of nine months and their nucleic acids extracted and subjected to shotgun sequencing (Illumina HiSeq2500; average 70M PE reads/sample). The raw reads were quality controlled, trimmed, assembled, binned, and dereplicated to obtain metagenome-assembled genomes (MAGs). Genome quality was assessed using the MIMAG criteria. The taxonomic assignment of the recovered MAGs was conducted using GTDB-Tk, and gene-level functional annotations were obtained using the KEGG, and CAZy databases. From ninety metagenome assemblies, 14,236 MAGs were recovered, of which 37%, 16%, and 1% satisfied medium-quality (Completeness > 50% and contamination 90% and contamination 90% and contamination

The troposphere constitutes the final frontier of global ecosystem research due to technical challenges arising from its size, low biomass, and gaseous state. Using a vertical testing array comprising a meteorological tower and a research aircraft, we conducted synchronized measurements of meteorological parameters and airborne biomass (n = 480) in the vertical air column up to 3,500 m. The taxonomic analysis of metagenomic data revealed differing patterns of airborne microbial community composition with respect to time of day and height above ground. The temporal and spatial resolution of our study demonstrated that the diel cycle of airborne microorganisms is a ground-based phenomenon that is entirely absent at heights >1,000 m. In an integrated analysis combining meteorological and biological data, we demonstrate that atmospheric turbulence, identified by potential temperature and high-frequency three-component wind measurements, is the key driver of bioaerosol dynamics in the lower troposphere. Multivariate regression analysis shows that at least 50% of identified airborne microbial taxa (n = ∼10,000) are associated with either ground or height, allowing for an understanding of dispersal patterns of microbial taxa in the vertical air column. Due to the interconnectedness of atmospheric turbulence and temperature, the dynamics of microbial dispersal are likely to be impacted by rising global temperatures, thereby also affecting ecosystems on the planetary surface. Ministry of Education (MOE) Nanyang Technological University Published version This work was supported by the Singapore Ministry of Education Academic Research Fund tier 3 grant (MOE2013-T3-1-013). We thank the Singapore Centre for Life Sciences Engineering (SCELSE) for supporting the project with internal funds and infrastructure.

Owing to the relatively lower dimensional accuracy and poorer surface finish compared to other additive manufacturing (AM) technologies, directed energy deposition (DED) yields parts that often require extensive post-processing. Thus, it is well suited to be combined with subtractive or deformation processes into hybrid manufacturing strategies, which may enable microstructure engineering of near-net-shape parts directly upon production. In this work, we use a custom-made machine that combines DED and single point incremental forming (SPIF) processes to produce samples of stainless steel 316L which are amenable to undergo recrystallization upon heat treatment. After recrystallization, the microstructure exhibits a high density of twin boundaries, which are known to enhance the physical and mechanical properties of alloys. We investigate how different SPIF parameters affect the extent of recrystallization and find that our strategy may be used to produce both gradient and “sandwich” microstructures, which integrate dissimilar grain boundary character distributions and grain structures. We assess the corrosion resistance and mechanical properties of such samples and discuss the resulting performance enhancement. Our results showcase new opportunities for microstructure engineering of DED components and lay the groundwork for the design of AM processes that enable grain boundary engineering of metal alloys. National Research Foundation (NRF) Published version This work was supported by the National Research Foundation (NRF) Singapore, under the NRF Fellowship programme (NRF-NRFF2018-05).

Background As a consequence of precision medicine initiatives, genomic technologies have rapidly spread around the world, raising questions about genetic privacy and the ethics of data sharing. Previous scholarship in bioethics and science and technology studies has made clear that different nations have varying expectations about trust, transparency, and public reason in relation to emerging technologies and their governance. The key aims of this article are to assess genetic literacy, perceptions of genetic testing, privacy concerns, and governing norms amongst the Singapore population by collecting surveys. Methods This study investigated genetic literacy and broad public attitudes toward genetic tests in Singapore with an online public survey (n = 560). To assess potential changes in attitudes following receipt of results from a genetic test, we also surveyed undergraduate students who underwent a genetic screen as part of a university class before and after they received their test results (n = 25). Results Public participants showed broad support for the use of genetic tests; scored an average of 48.9% in genetic literacy; and expressed privacy concerns over data sharing and a desire for control over their genetic data. After taking a genetic test and receiving genetic test results, students reported less fear of genetic tests while other attitudes did not change significantly. Conclusion These findings highlight the potential of genetic education and active engagement with genetic testing to increase support and participation in genomic projects, PM, and biobanking initiatives; and they suggest that data privacy protections could potentially reduce discrimination by giving participants control over who can access their data. More specifically, these findings and the dataset we provide may be helpful in formulating culturally sensitive education programs and regulations concerning genomic technologies and data privacy.

Members of the genus Defluviicoccus occur often at high abundances in activated sludge wastewater treatment plants designed to remove phosphorus, where biomass is subjected to alternating anaerobic feed/aerobic famine conditions, believed to favor the proliferation of organisms like Ca. Accumulibacter and other phosphate-accumulating organisms (PAO), and Defluviicoccus. All have a capacity to assimilate readily metabolizable substrates and store them intracellularly during the anaerobic feed stage so that under the subsequent famine aerobic stage, these can be used to synthesize polyphosphate reserves by the PAO and glycogen by Defluviicoccus. Consequently, Defluviicoccus is described as a glycogen-accumulating organism or GAO. Because they share a similar anaerobic phenotype, it has been proposed that at high Defluviicoccus abundance, the PAO are out-competed for assimilable metabolites anaerobically, and hence aerobic P removal capacity is reduced. Several Defluviicoccus whole genome sequences have been published (Ca. Defluviicoccus tetraformis, Defluviicoccus GAO-HK, and Ca. Defluviicoccus seviourii). The available genomic data of these suggest marked metabolic differences between them, some of which have ecophysiological implications. Here, we describe the whole genome sequence of the type strain Defluviicoccus vanusT, the only cultured member of this genus, and a detailed comparative re-examination of all extant Defluviicoccus genomes. Each, with one exception, which appears not to be a member of this genus, contains the genes expected of GAO members, in possessing multiple copies of those for glycogen biosynthesis and catabolism, and anaerobic polyhydroxyalkanoate (PHA) synthesis. Both 16S rRNA and genome sequence data suggest that the current recognition of four clades is insufficient to embrace their phylogenetic biodiversity, but do not support the view that they should be re-classified into families other than their existing location in the Rhodospirillaceae. As expected, considerable variations were seen in the presence and numbers of genes encoding properties associated with key substrate assimilation and metabolic pathways. Two genomes also carried the pit gene for synthesis of the low-affinity phosphate transport protein, pit, considered by many to distinguish all PAO from GAO. The data re-emphasize the risks associated with extrapolating the data generated from a single Defluviicoccus population to embrace all members of that genus.

Summary Antibiotic-associated diarrhea (AAD) affects a significant proportion of patients receiving antibiotics. We sought to understand if differences in the gut microbiome would influence the development of AAD. We administered a 3-day course of amoxicillin-clavulanate to 30 healthy adult volunteers, and analyzed their stool microbiome, using 16S rRNA gene sequencing, at baseline and up to 4 weeks post antibiotic administration. Lower levels of gut Ruminococcaceae were significantly and consistently observed from baseline until day 7 in participants who developed AAD. Overall, participants who developed AAD experienced a greater decrease in microbial diversity. The probability of AAD could be predicted based on qPCR-derived levels of Faecalibacterium prausnitzii at baseline. Our findings suggest that a lack of gut Ruminococcaceae influences development of AAD. Quantification of F. prausnitzii in stool prior to antibiotic administration may help identify patients at risk of AAD, and aid clinicians in devising individualized treatment regimens to minimize such adverse effects., Graphical Abstract, Highlights • Antibiotic-associated diarrhea (AAD) affects a significant proportion of patients • 13/30 healthy volunteers who received oral amoxicillin-clavulanate developed AAD • Individuals who developed AAD had lower levels of gut Ruminococcaceae at baseline • Baseline levels of Faecalibacterium prausnitzii were predictive of risk of AAD, Health sciences; Pathophysiology; Microbiome

The optimal therapeutic efficiency of any treatment depends on patient adherence to the medication regimen. Medication adherence is the cornerstone of treatment outcomes that may consequently impact economic and healthcare costs. With the oral route being the preferred route of drug administration, slow or extended-release oral formulations can, therefore, be utilized. Here, a biocompatible oral delivery system that can be retained in the stomach for a week, while providing continuous release of the encased drug, is proposed. The fabrication of the delivery system was achieved using a simple mold casting technique. The hydrogel-based raft was able to float under simulated gastric conditions for seven days with pH switches to mimic the fasted and fed states of the stomach. The functionality of such a delivery system has been exemplified using two different model drugs—risperidone and metoprolol tartrate of varied solubilities and has been shown to effectively sustain the release of drugs under the tested conditions. Ministry of Education (MOE) Singapore Food Agency Submitted/Accepted version The authors would like to acknowledge the fnancial support from the Singapore Centre for Environmental Life Sciences Engineering (SCELSE) (MOE/RCE: M4330019.C70), Ministry of Education AcRF-Tier 1 Grant (RG19/18 & RT08/19 (S)), Singapore Food Agency (SFS_RND_SUFP_001_06), and the Singapore National Bioflm Consortium (SNBC/2021/SF2/P04).

ObjectiveEvidence indicates that multistrain probiotics benefit preterm infants more than single-strain (SS) probiotics. We assessed the effects of SS versus triple-strain (TS) probiotic supplementation (PS) in extremely preterm (EP) infants.DesignEP infants (gestational age (GA) Results173 EP (SS: 86, TS: 87) neonates with similar GA and birth weight (BW) were randomised. Median TFF was comparable (11 (IQR 8–16) vs 10 (IQR 8–16) days, p=0.92). Faecal propionate (SS, p0.05) between groups, whereas beta-diversity analysis revealed significant differences between PS and REF groups (both p=0.001). Actinobacteria were higher (both pConclusionTFF in EP infants was similar between SS and TS probiotics. Both probiotics were effective in reducing dysbiosis (higher bifidobacteria and lower Gammaproteobacteria). Long-term significance of increased propionate and butyrate needs further studies.Trial registration numberACTRN 12615000940572.

Vibrio vulnificus is an opportunistic human pathogen and autochthonous inhabitant of coastal marine environments, where the bacterium is under constant predation by heterotrophic protists or protozoans. As a result of this selection pressure, genetic variants with antipredation mechanisms are selected for and persist in the environment. Such natural variants may also be pathogenic to animal or human hosts, making it important to understand these defense mechanisms. To identify antipredator strategies, 13 V. vulnificus strains of different genotypes isolated from diverse environments were exposed to predation by the ciliated protozoan Tetrahymena pyriformis, and only strain ENV1 was resistant to predation. Further investigation of the cell-free supernatant showed that ENV1 acidifies the environment by the excretion of organic acids, which are toxic to T. pyriformis. As this predation resistance was dependent on the availability of iron, transcriptomes of V. vulnificus in iron-replete and iron-deplete conditions were compared. This analysis revealed that ENV1 ferments pyruvate and the resultant acetyl-CoA leads to acetate synthesis under aerobic conditions, a hallmark of overflow metabolism. The anaerobic respiration global regulator arcA was upregulated when iron was available. An ΔarcA deletion mutant of ENV1 accumulated less acetate and, importantly, was sensitive to grazing by T. pyriformis. Based on the transcriptome response and quantification of metabolites, we conclude that ENV1 has adapted to overflow metabolism and has lost a control switch that shifts metabolism from acetate excretion to acetate assimilation, enabling it to excrete acetate continuously. We show that overflow metabolism and the acetate switch contribute to prey-predator interactions. IMPORTANCE Bacteria in the environment, including Vibrio spp., interact with protozoan predators. To defend against predation, bacteria evolve antipredator mechanisms ranging from changing morphology, biofilm formation, and secretion of toxins or virulence factors. Some of these adaptations may result in strains that are pathogenic to humans. Therefore, it is important to study predator defense strategies of environmental bacteria. V. vulnificus thrives in coastal waters and infects humans. Very little is known about the defense mechanisms V. vulnificus expresses against predation. Here, we show that a V. vulnificus strain (ENV1) has rewired the central carbon metabolism, enabling the production of excess organic acid that is toxic to the protozoan predator T. pyriformis. This is a previously unknown mechanism of predation defense that protects against protozoan predators. Ministry of Education (MOE) National Research Foundation (NRF) Published version We acknowledge support from the iThree Institute at the University of Technology Sydney, Sydney, Australia, the Australian Research Council Discovery Project (DP170100453) and by the National Research Foundation and Ministry of Education Singapore under its Research Centre of Excellence Program to the Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University.

The analysis of metagenome data based on the recovery of draft genomes (so called metagenome-assembled genomes, or MAG) has assumed an increasingly central role in microbiome research in recent years. Microbial communities underpinning the operation of wastewater treatment plants are particularly challenging targets for MAG analysis due to their high ecological complexity, and remain important, albeit understudied, microbial communities that play ssa key role in mediating interactions between human and natural ecosystems. Here we consider strategies for recovery of MAG sequence from time series metagenome surveys of full-scale activated sludge microbial communities. We generate MAG catalogs from this set of data using several different strategies, including the use of multiple individual sample assemblies, two variations on multi-sample co-assembly and a recently published MAG recovery workflow using deep learning. We obtain a total of just under 9,100 draft genomes, which collapse to around 3,100 non-redundant genomic clusters. We examine the strengths and weaknesses of these approaches in relation to MAG yield and quality, showing that co-assembly may offer advantages over single-sample assembly in the case of metagenome data obtained from closely sampled longitudinal study designs. Around 1,000 MAGs were candidates for being considered high quality, based on single-copy marker gene occurrence statistics, however only 58 MAG formally meet the MIMAG criteria for being high quality draft genomes. These findings carry broader broader implications for performing genome-resolved metagenomics on highly complex communities, the design and implementation of genome recoverability strategies, MAG decontamination and the search for better binning methodology. Ministry of Education (MOE) National Research Foundation (NRF) National Supercomputing Centre (NSCC) Singapore Published version This research was supported by the Singapore National Research Foundation and Ministry of Education under the Research Centre of Excellence Programme and by program grants 1102- IRIS-10-02 (to SW, RW, and SS) from the National Research Foundation (NRF). The computational work was performed in part on resources of the National Supercomputing Centre (NSCC, Singapore) supported by Project 11000984.

The anti-inflammatory effect of different sourced honeys and the impact on elderly gut microbiota were studied in terms of chemical compositions, anti-inflammatory effect and gut microbiota modulating capacities. All four honeys suppressed the production of pro-inflammatory markers NO, IL-1β and IL-6 induced by lipopolysaccharide and promoted the expression of anti-inflammatory cytokines IL-10 in RAW 264.7 cells. Moreover, in the ex vivo batch gut model using elderly fecal microbiota (referred to as microcosm), it was showed that the addition of honeys increased the abundance of beneficial lactobacilli, decreased the abundance of potentially harmful Gram negative enteric bacteria, and exerted a beneficial effect on the production of short chain fatty acids. The concentration of gallic acid in honeys was positively correlated with the expression level of IL-10 and the abundance of lactobacilli. These findings indicate honeys with anti-inflammatory capacity have great potential for regulating the elderly gut microbiota which would lead to health benefits. Nanyang Technological University We sincerely thank the support from the funding of China Scholarship Council (grant no. 201906240301).

Environmental DNA, i.e., DNA extracted directly from environmental samples, has been used to understand microbial communities in the environment and to monitor contemporary biodiversity in the conservation context. Environmental DNA often contains both intracellular DNA (iDNA) and extracellular DNA (eDNA). eDNA can persist in the environment and complicate environmental DNA sequencing-based analyses of microbial communities and biodiversity. Although several studies acknowledged the impact of eDNA on DNA-based profiling of environmental communities, eDNA is still being neglected or ignored in most studies dealing with environmental samples. In this article, we summarize key findings on eDNA in environmental samples and discuss the methods used to extract and quantify eDNA as well as the importance of eDNA on the interpretation of experimental results. We then suggested several factors to consider when designing experiments and analyzing data to negate or determine the contribution of eDNA to environmental DNA-based community analyses. This field of research will be driven forward by (i) carefully designing environmental DNA extraction pipelines by taking into consideration technical details in methods for eDNA extraction/removal and membrane-based filtration and concentration; (ii) quantifying eDNA in extracted environmental DNA using multiple methods, including qPCR and fluorescent DNA binding dyes; (iii) carefully interpreting the effect of eDNA on DNA-based community analyses at different taxonomic levels; and (iv) when possible, removing eDNA from environmental samples for DNA-based community analyses. Ministry of Education (MOE) National Research Foundation (NRF) Submitted/Accepted version This research/project was supported by the National Research Foundation, Singapore, under its NERC-NRF Joint Grant Call (Award SEAP-2020-0004). This research is also supported by the National Research Foundation and MOE Singapore under its Research Centre of Excellence Program, Singapore Centre for Environmental Life Sciences Engineering (SCELSE) (M4330005.C70 to B.C.), Nanyang Technological University, Singapore.