Surface-Enhanced Raman Scattering-Based Surface Chemotaxonomy: Combining Bacteria Extracellular Matrices and Machine Learning for Rapid and Universal Species Identification.

Rapid, universal, and accurate identification of bacteria in their natural states is necessary for on-site environmental monitoring and fundamental microbial research. Surface-enhanced Raman scattering (SERS) spectroscopy emerges as an attractive tool due to its molecule-specific spectral fingerprinting and multiplexing capabilities, as well as portability and speed of readout. Here, we develop a SERS-based surface chemotaxonomy that uses bacterial extracellular matrices (ECMs) as proxy biosignatures to hierarchically classify bacteria based on their shared surface biochemical characteristics to eventually identify six distinct bacterial species at >98% classification accuracy. Corroborating with in silico simulations, we establish a three-way inter-relation between the bacteria identity, their ECM surface characteristics, and their SERS spectral fingerprints. The SERS spectra effectively capture multitiered surface biochemical insights including ensemble surface characteristics, e.g., charge and biochemical profiles, and molecular-level information, e.g., types and numbers of functional groups. Our surface chemotaxonomy thus offers an orthogonal taxonomic definition to traditional classification methods and is achieved without gene amplification, biochemical testing, or specific biomarker recognition, which holds great promise for point-of-need applications and microbial research.

Structure vs. chemistry: Alternate mechanisms for controlling leaf microbiomes.

The analysis of phyllosphere microbiomes traditionally relied on DNA extracted from whole leaves. To investigate the microbial communities on the adaxial (upper) and abaxial (lower) leaf surfaces, swabs were collected from both surfaces of two garden plants, Rhapis excelsa and Cordyline fruticosa. Samples were collected at noon and midnight and at five different locations to investigate if the phyllosphere microbial communities change with time and location. The abaxial surface of Rhapis excelsa and Cordyline fruticosa had fewer bacteria in contrast to its adaxial counterpart. This observation was consistent between noon and midnight and across five different locations. Our co-occurrence network analysis further showed that bacteria were found almost exclusively on the adaxial surface while only a small group of leaf blotch fungi thrived on the abaxial surface. There are higher densities of stomata on the abaxial surface and these openings are vulnerable ports of entry into the plant host. While one might argue about the settling of dust particles and microorganisms on the adaxial surface, we detected differences in reactive chemical activities and microstructures between the adaxial and abaxial surfaces. Our results further suggest that both plant species deploy different defence strategies to deter invading pathogens on the abaxial surface. We hypothesize that chemical and mechanical defence strategies evolved independently for harnessing and controlling phyllosphere microbiomes. Our findings have also advanced our understanding that the abaxial leaf surface is distinct from the adaxial surface and that the reduced microbial diversity is likely a consequence of plant-microbe interactions.

Vertical stratification of the air microbiome in the lower troposphere.

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.

Airborne SARS-CoV-2 surveillance in hospital environment using high-flowrate air samplers and its comparison to surface sampling.

Reliable methods to detect the presence of SARS-CoV-2 at venues where people gather are essential for epidemiological surveillance to guide public policy. Communal screening of air in a highly crowded space has the potential to provide early warning on the presence and potential transmission of SARS-CoV-2 as suggested by studies early in the epidemic. As hospitals and public facilities apply varying degrees of restrictions and regulations, it is important to provide multiple methodological options to enable environmental SARS-CoV-2 surveillance under different conditions. This study assessed the feasibility of using high-flowrate air samplers combined with RNA extraction kit designed for environmental sample to perform airborne SARS-CoV-2 surveillance in hospital setting, tested by RT-qPCR. The success rate of the air samples in detecting SARS-CoV-2 was then compared with surface swab samples collected in the same proximity. Additionally, positive RT-qPCR samples underwent viral culture to assess the viability of the sampled SARS-CoV-2. The study was performed in inpatient ward environments of a quaternary care university teaching hospital in Singapore housing active COVID-19 patients within the period of February to May 2020. Two types of wards were tested, naturally ventilated open-cohort ward and mechanically ventilated isolation ward. Distances between the site of air sampling and the patient cluster in the investigated wards were also recorded. No successful detection of airborne SARS-CoV-2 was recorded when 50 L/min air samplers were used. Upon increasing the sampling flowrate to 150 L/min, our results showed a high success rate in detecting the presence of SARS-CoV-2 from the air samples (72%) compared to the surface swab samples (9.6%). The positive detection rate of the air samples along with the corresponding viral load could be associated with the distance between sampling site and patient. The furthest distance from patient with PCR-positive air samples was 5.5 m. The airborne SARS-CoV-2 detection was comparable between the two types of wards with 60%-87.5% success rate. High prevalence of the virus was found in toilet areas, both on surfaces and in air. Finally, no successful culture attempt was recorded from the environmental air or surface samples.

Effective design of barrier enclosure to contain aerosol emissions from COVID-19 patients.

Facing shortages of personal protective equipment, some clinicians have advocated the use of barrier enclosures (typically mounted over the head, with and without suction) to contain aerosol emissions from coronavirus disease 2019 (COVID-19) patients. There is, however, little evidence for its usefulness. To test the effectiveness of such a device, we built a manikin that can expire micron-sized aerosols at flow rates close to physiological conditions. We then placed the manikin inside the enclosure and used a laser sheet to visualize the aerosol leaking out. We show that with sufficient suction, it is possible to effectively contain aerosol from the manikin, reducing aerosol exposure outside the enclosure by 99%. In contrast, a passive barrier without suction only reduces aerosol exposure by 60%.

Experimental parameters defining ultra-low biomass bioaerosol analysis.

Investigation of the microbial ecology of terrestrial, aquatic and atmospheric ecosystems requires specific sampling and analytical technologies, owing to vastly different biomass densities typically encountered. In particular, the ultra-low biomass nature of air presents an inherent analytical challenge that is confounded by temporal fluctuations in community structure. Our ultra-low biomass pipeline advances the field of bioaerosol research by significantly reducing sampling times from days/weeks/months to minutes/hours, while maintaining the ability to perform species-level identification through direct metagenomic sequencing. The study further addresses all experimental factors contributing to analysis outcome, such as amassment, storage and extraction, as well as factors that impact on nucleic acid analysis. Quantity and quality of nucleic acid extracts from each optimisation step are evaluated using fluorometry, qPCR and sequencing. Both metagenomics and marker gene amplification-based (16S and ITS) sequencing are assessed with regard to their taxonomic resolution and inter-comparability. The pipeline is robust across a wide range of climatic settings, ranging from arctic to desert to tropical environments. Ultimately, the pipeline can be adapted to environmental settings, such as dust and surfaces, which also require ultra-low biomass analytics.

Microbial communities in the tropical air ecosystem follow a precise diel cycle.

The atmosphere is vastly underexplored as a habitable ecosystem for microbial organisms. In this study, we investigated 795 time-resolved metagenomes from tropical air, generating 2.27 terabases of data. Despite only 9 to 17% of the generated sequence data currently being assignable to taxa, the air harbored a microbial diversity that rivals the complexity of other planetary ecosystems. The airborne microbial organisms followed a clear diel cycle, possibly driven by environmental factors. Interday taxonomic diversity exceeded day-to-day and month-to-month variation. Environmental time series revealed the existence of a large core of microbial taxa that remained invariable over 13 mo, thereby underlining the long-term robustness of the airborne community structure. Unlike terrestrial or aquatic environments, where prokaryotes are prevalent, the tropical airborne biomass was dominated by DNA from eukaryotic phyla. Specific fungal and bacterial species were strongly correlated with temperature, humidity, and CO2 concentration, making them suitable biomarkers for studying the bioaerosol dynamics of the atmosphere.

Genome Sequence of the Tropical Atmosphere Bacterium Pontibacter sp. Strain SGAir0037.

The Pontibacter bacterial genus has been detected in marine and soil environments. Here, we report the genome sequence of Pontibacter sp. strain SGAir0037, which was isolated from outdoor air samples collected in Singapore. The genome comprises one chromosome of 5.26 Mb and one plasmid of 127 kb.

Exploring temporal patterns of bacterial and fungal DNA accumulation on a ventilation system filter for a Singapore university library.

INTRODUCTION: Ventilation system filters process recirculated indoor air along with outdoor air. This function inspires the idea of using the filter as an indoor bioaerosol sampler. While promising, there remains a need to investigate several factors that could limit the accuracy of such a sampling approach. Among the important factors are the dynamics of microbial assemblages on filter surfaces over time and the differential influence of outdoor versus recirculated indoor air. METHODS: This study collected ventilation system filter samples from an air handling unit on a regular schedule over a 21-week period and analyzed the accumulation patterns of biological particles on the filter both quantitatively (using fluorometry and qPCR) and in terms of microbial diversity (using 16S rDNA and ITS sequencing). RESULTS: The quantitative result showed that total and bacterial DNA accumulated monotonically, rising to 41 ng/cm2 for total DNA and to 2.8 ng/cm2 for bacterial DNA over the 21-week period. The accumulation rate of bacterial DNA correlated with indoor occupancy level. Fungal DNA first rose to 4.0 ng/cm2 before showing a dip to 1.4 ng/cm2 between weeks 6 and 10. The dip indicated a possible artifact of this sampling approach for quantitative analysis as DNA may not be conserved on the filter over the months-long service period. The sequencing results indicate major contributions from outdoor air for fungi and from recirculated indoor air for bacteria. Despite the quantitative changes, the community structure of the microbial assemblages was stable throughout the 21-week sampling period, highlighting the robustness of this sampling method for microbial profiling. CONCLUSION: This study supports the use of ventilation system filters as indoor bioaerosol samplers, but with caveats: 1) an outdoor reference is required to properly understand the contribution of outdoor bioaerosols; and 2) there is a need to better understand the persistence and durability of the targeted organisms on ventilation system filters.

Genome Sequence of Pantoea ananatis SGAir0210, Isolated from Outdoor Air in Singapore.

Pantoea ananatis SGAir0210 was isolated from outdoor air collected in Singapore. The genome was assembled from long reads generated by single-molecule real-time sequencing complemented with short reads. The genome size was approximately 4.81 Mb, with 4,303 protein-coding genes, 80 tRNAs, and 22 rRNAs identified.

DNA accumulation on ventilation system filters in university buildings in Singapore.

INTRODUCTION: Biological particles deposit on air handling system filters as they process air. This study reports and interprets abundance and diversity information regarding biomass accumulation on ordinarily used filters acquired from several locations in a university environment. METHODS: DNA-based analysis was applied both to quantify (via DNA fluorometry and qPCR) and to characterize (via high-throughput sequencing) the microbial material on filters, which mainly processed recirculated indoor air. Results were interpreted in relation to building occupancy and ventilation system operational parameters. RESULTS: Based on accumulated biomass, average DNA concentrations per AHU filter surface area across nine indoor locations after twelve weeks of filter use were in the respective ranges 1.1 to 41 ng per cm2 for total DNA, 0.02 to 3.3 ng per cm2 for bacterial DNA and 0.2 to 2.0 ng DNA per cm2 for fungal DNA. The most abundant genera detected on the AHU filter samples were Clostridium, Streptophyta, Bacillus, Acinetobacter and Ktedonobacter for bacteria and Aspergillus, Cladosporium, Nigrospora, Rigidoporus and Lentinus for fungi. Conditional indoor airborne DNA concentrations (median (range)) were estimated to be 13 (2.6-107) pg/m3 for total DNA, 0.4 (0.05-8.4) pg/m3 for bacterial DNA and 2.3 (1.0-5.1) pg/m3 for fungal DNA. CONCLUSION: Conditional airborne concentrations and the relative abundances of selected groups of genera correlate well with occupancy level. Bacterial DNA was found to be more responsive than fungal DNA to differences in occupancy level and indoor environmental conditions.

Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis.

INTRODUCTION: As bioaerosol research attracts increasing attention, there is a need for additional efforts that focus on method development to deal with different environmental samples. Bioaerosol environmental samples typically have very low biomass concentrations in the air, which often leaves researchers with limited options in choosing the downstream analysis steps, especially when culture-independent methods are intended. OBJECTIVES: This study investigates the impacts of three important factors that can influence the performance of culture-independent DNA-based analysis in dealing with bioaerosol environmental samples engaged in this study. The factors are: 1) enhanced high temperature sonication during DNA extraction; 2) effect of sampling duration on DNA recoverability; and 3) an alternative method for concentrating composite samples. In this study, DNA extracted from samples was analysed using the Qubit fluorometer (for direct total DNA measurement) and quantitative polymerase chain reaction (qPCR). RESULTS AND FINDINGS: The findings suggest that additional lysis from high temperature sonication is crucial: DNA yields from both high and low biomass samples increased up to 600% when the protocol included 30-min sonication at 65°C. Long air sampling duration on a filter media was shown to have a negative impact on DNA recoverability with up to 98% of DNA lost over a 20-h sampling period. Pooling DNA from separate samples during extraction was proven to be feasible with margins of error below 30%.