Background: As global travel resumed in coronavirus disease 2019 (COVID-19) endemicity, the potential of aircraft wastewater monitoring to provide early warning of disease trends for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and other infectious diseases, particularly at international air travel hubs, was recognized. We therefore assessed and compared the feasibility of testing wastewater from inbound aircraft and airport terminals for 18 pathogens including SARS-CoV-2 in Singapore, a popular travel hub in Asia. Methods: Wastewater samples collected from inbound medium- and long-haul flights and airport terminals were tested for SARS-CoV-2. Next Generation Sequencing was carried out on positive samples to identify SARS-CoV-2 variants. Airport and aircraft samples were further tested for 17 other pathogens through quantitative reverse transcription polymerase chain reaction. Results: The proportion of SARS-CoV-2-positive samples and the average virus load was higher for wastewater samples from aircraft as compared with airport terminals. Cross-correlation analyses indicated that viral load trends from airport wastewater led local COVID-19 case trends by 2–5 days. A total of 10 variants (44 sub-lineages) were successfully identified from aircraft wastewater and airport terminals, and four variants of interest and one variant under monitoring were detected in aircraft and airport wastewater 18–31 days prior to detection in local clinical cases. The detection of five respiratory and four enteric viruses in aircraft wastewater samples further underscores the potential to expand aircraft wastewater to monitoring pathogens beyond SARS-CoV-2. Conclusion: Our findings demonstrate the feasibility of aircraft wastewater testing for monitoring infectious diseases threats, potentially detecting signals before clinical cases are reported. The triangulation of similar datapoints from aircraft wastewater of international travel nodes could therefore serve as a useful early warning system for global health threats.

Quorum sensing (QS) can function to shape the microbial community interactions, composition, and function. In wastewater treatment systems, acylated homoserine lactone (AHL)-based QS has been correlated with the conversion of floccular biomass into microbial granules, as well as EPS production and the nitrogen removal process. However, the role of QS in such complex communities is still not fully understood, including the QS-proficient taxa and the functional QS genes involved. To address these questions, we performed a metagenomic screen for AHL genes in an activated sludge microbial community from the Ulu Pandan wastewater treatment plant (WWTP) in Singapore followed by functional validation of luxI activity using AHL biosensors and LC–MSMS profiling. We identified 13 luxI and 30 luxR homologs from the activated sludge metagenome. Of those genes, two represented a cognate pair of luxIR genes belonging to a Nitrospira spp. and those genes were demonstrated to be functionally active. The LuxI homolog synthesized AHLs that were consistent with the dominant AHLs in the activated sludge system. Furthermore, the LuxR homolog was shown to bind to and induce expression of the luxI promoter, suggesting this represents an autoinduction feedback system, characteristic of QS circuits. Additionally, a second, active promoter was upstream of a gene encoding a protein with a GGDEF/EAL domain, commonly associated with modulating the intracellular concentration of the secondary messenger, c-di-GMP. Thus, the metagenomic approach used here was demonstrated to effectively identify functional QS genes and suggests that Nitrospira spp. maybe QS is active in the activated sludge community.

It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in Pseudomonas aeruginosa PAO1. These behaviours are linked to the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homologue of the P2 phage repressor C and was recently named Pf4r. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show a critical site in repressor C (Pf4r) where a mutation in the site, 788799A>G (Ser4Pro), causes Pf4r to lose its function as the immunity factor against reinfection by Pf4. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the E.coli bacteriophage P2 RepC protein. A mutation, Pf4r*, associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by multi-angle light scattering (MALS) analysis, where the Pf4r* protein only forms monomers. The loss of dimerisation also explains the loss of Pf4r’s immunity function. Phenotypic assays showed that Pf4r increased LasB activity and was also associated with a slight increase in the percentage of morphotypic variants. ChIPseq and transcriptomic analyses suggest that Pf4r also likely functions as a transcriptional regulator for other host genes. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in P. aeruginosa biofilm development.

Numerical simulations that are conducted on a transport-type airfoil, NACA 64-210, at a Reynolds number of2.6×106and LWC of 25 g/m³explore the aerodynamic penalties and mechanisms that affect airfoil performance in heavy rain conditions. Our simulation results agree well with the experimental data and show significant aerodynamic penalties for the airfoil in heavy rain. The maximum percentage decrease inCLis reached by 13.2% and the maximum percentage increase inCDby 47.6%. Performance degradation in heavy rain at low angles of attack is emulated by an originally creative boundary-layer-tripped technique near the leading edge. Numerical flow visualization technique is used to show premature boundary-layer separation at high angles of attack and the particulate trajectories at various angles of attack. A mathematic model is established to qualitatively study the water film effect on the airfoil geometric changes. All above efforts indicate that two primary mechanisms are accountable for the airfoil aerodynamic penalties. One is to cause premature boundary-layer transition at low AOA and separation at high AOA. The other occurs at times scales consistent with the water film layer, which is thought to alter the airfoil geometry and increase the mass effectively.

Methods for the study of member species in complex microbial communities remain a high priority, particularly for rare and/or novel member species that might play an important ecological role. Specifically, methods that link genomic information of member species with its spatial structure are lacking. This study adopts an integrative workflow that permits the characterisation of previously unclassified bacterial taxa from microbiomes through: (1) imaging of the spatial structure; (2) taxonomic classification and (3) genome recovery. Our study attempts to bridge the gaps between metagenomics/metatranscriptomics and high-resolution biomass imaging methods by developing new fluorescence in situ hybridisation (FISH) probes—termed as R-Probes—from shotgun reads that harbour hypervariable regions of the 16S rRNA gene. The sample-centric design of R-Probes means that probes can directly hybridise to OTUs as detected in shotgun sequencing surveys. The primer-free probe design captures larger microbial diversity as compared to canonical probes. R-Probes were designed from deep-sequenced RNA-Seq datasets for both FISH imaging and FISH–Fluorescence activated cell sorting (FISH–FACS). FISH–FACS was used for target enrichment of previously unclassified bacterial taxa prior to downstream multiple displacement amplification (MDA), genomic sequencing and genome recovery. After validation of the workflow on an axenic isolate of Thauera species, the techniques were applied to investigate two previously uncharacterised taxa from a tropical full-scale activated sludge community. In some instances, probe design on the hypervariable region allowed differentiation to the species level. Collectively, the workflow can be readily applied to microbiomes for which shotgun nucleic acid survey data is available.