Sampling strategy and assessment options for environmental antimicrobial resistance in airborne microorganisms

Executive summary The appearance and spread of antimicrobial resistant (AMR) microorganisms and their genes in the environment are a major concern. While little is known about these microorganisms within the atmosphere, recent studies report of their presence in the air covering the UK. This report aims at summarizing sampling options for airborne microorganisms including assessing their potential for containing antimicrobial resistance genes and whether the microorganisms possess the capability for transmission through the atmosphere to other parts of the environment. The review extends previous works on antimicrobial resistant microorganisms in the atmosphere by • Assessing the composition of the atmospheric microbiome, where AMR organisms occur. • Determining the specification for bioaerosol samples suitable for analysis for AMR. • Reviewing methods available for bioaerosol sampling and compare them with the sample specification. The work was used to identify the most suitable approach for identifying antimicrobial resistant microorganisms in the UK atmosphere and finds the following: • Airborne fungal spores and bacteria with the potential to contain antimicrobial resistant genes may be present all year round, but the highest concentrations should be expected in the summer and autumn. • Sources of antimicrobial resistant microorganisms are expected to be mainly anthropogenic. Some sources (e.g., crop fields) will peak in summer or early autumn, while other sources (e.g., agricultural buildings or waste sites) will be linked to activities and can be more or less constant throughout the year. • It is not known if antimicrobial resistant microorganisms have spread to the wider environment and if the atmosphere contains a non-trivial, expectedly low, concentration of these harmful microorganisms. • There are two main analytical approaches to quantify biodiversity and antimicrobial resistant microorganisms. One approach is based on culturing and a second is based on molecular methods. Both have advantages and disadvantages, and it is recommended to use both approaches in campaigns and long-term monitoring. • There is no superior device for the collection of antimicrobial resistant microorganisms and the type of device depends on the objectives of the study. Many available instruments have been developed for one specific purpose. The best sampling strategy is often to combine at least two types of instruments: One type that samples directly onto growing media such as a cascade impactor and a second type that uses a set of filters such as a high-volume cascade sampler. In some cases, a cost-effective solution for long term campaigns or monitoring can be the application of semi-automatic mini cyclones. 6 of 66 • Guidelines for storing and processing of fungal spores and bacteria have been produced based on general knowledge on fungal spores, bacteria and how to handle genetic material. It is important to apply a common set of protocols, partly to allow for robust intercomparison of studies and partly to protect the samples against loss of material during transport, storage, or handling. • A decision tree and a set of questions that typically need addressing for developing a campaign has been produced, where the aim is the detection of airborne microorganisms, suspected to contain antimicrobial resistant genes. This is supported by two practical examples on how to develop a campaign at several locations addressing fungal spores or a single site campaign addressing both fungal spores and bacteria. • A number of data sets as well as models are needed for further understanding and potential mitigation. Basic atmospheric models from air quality studies are already available, while more advanced models handling viability and potential transmission have not yet been developed. The most import data sets are meteorological data supported by specific vegetation variables with land cover and land use data. Activity data around anthropogenic activities such as harvesting, handling of waste sites or animal productivity may also be important. Until now, it has not been possible to identify studies on antimicrobial resistant microorganisms covering the UK atmosphere. Consequently, it is not possible to assess the extent of the problem and whether this causes a significant risk to humans, animals, or the environment. Neither is it known if there is a trend such as increased concentrations of specific harmful microorganisms or if there is an overall increase in biodiversity of microorganisms with antimicrobial resistant genes

Spatial Bi-hourly Variation of Alternaria Spore Concentration in Worcester, UK

Alternaria species are ubiquitous fungi affecting food security and human health. They are pathogenic on many economically important crops and allergenic to many sensitive people worldwide. Studies from Worcester, UK have shown high a concentration of Alternaria spores, most likely caused by agricultural activities. However, it is unknown whether Alternaria spore concentrations vary geographically throughout Worcestershire. An investigation on the spatial variation in bi-hourly concentration of Alternaria spores in Worcestershire during 2016 and 2017 was conducted. Spores were sampled using two Hirst-type Burkard spore traps at the University of Worcester. One on the rooftop of a building at St John’s Campus and another at Lakeside Campus approximately 7 km away. St John’s Campus is located in the centre of Worcester (52.1970, -2.2421), while Lakeside Campus is located in an agricultural environment (52.2537, -2.2535) with regularly cut grass in the near surroundings. Slides were counted using bi-hourly traverse at x 400 magnification. The total number of spores per slide were converted to the daily mean of spores m¯³ of air. There was a highly positive correlation in the concentration of Alternaria spores between the two sites in both 2016 and 2017. St John’s had the highest peak of spore concentration (213 m¯³) in 2016 and Lakeside had the peak concentration in 2017 (184 m¯³). Concentrations above 100 m¯³ of air were observed more frequently at Lakeside. The study revealed that Alternaria spore concentrations were higher at Lakeside than at St John’s. This could be attributed to spores released from either crops or agricultural activities (e.g. haying or harvesting) or from decomposed grass since the surrounding area is routinely managed. Further work in 2018 will include spore correlations with weather variables from a pair of weather stations located at each site, enabling studies caused by variations in weather and climate. Spatial variation in bi-hourly spore concentrations is useful information to atopic subjects, health experts and crop pathologists. Keywords: Harvesting. Allergy. Fungal Spores

Aerobiology Meets Ecology: Development of Low-Cost Passive Gravitational Samplers

Access to proper and reliable research equipment is crucial in all natural science disciplines. This is especially true in biological research since experiments and observations require equipment that will show the same thing consistently. One method often utilized is called sampling, and it means collecting something by using a strict protocol. Our research is focused around sampling pollen and other biological particles from the air around us. If we want to sample particles in the air from locations where there is no electricity, the type of devices we can use limits us. This has led us to develop a sampler that does not require any electrical power; this technique is often referred to as passive sampling. We have created samplers with the design Sigma-2. It works by using a double wall and apertures with strategic placement to deflect the wind, and particles it carries, which then fall into a collection container within the sampler. In this poster we detail the construction, use and analysis using this passive sampler along with methods used to conduct quality control, to ensure that the results we acquire are reliable, replicable and comparable to other samplers

Summer pollen flora in rural and urban central England dominated by nettle, ryegrass and other pollen missed by the national aerobiological network

Abundance and diversity of airborne pollen are important to human health and biodiversity. The UK operational network collects airborne pollen from 8 flowering trees, grasses and three weeds using Hirst traps and microscopic identification from urban areas. Knowledge of total pollen diversity and differences between rural and urban zones is limited. We collect environmental DNA (eDNA) from air during summer and autumn over 3 years with mini cyclones from one urban and one rural site. Data are analysed using next generation sequencing and metabarcoding. We find the most common genus, Urtica (57%), is also identified by the national network. The grasses Lolium (10%), Agrostis (2%) and Holcus (1%) are in the national network grouped at family level, while Brassica (2%), Chenopodium (1%), Impatiens (2%), Plantago (4%) and Tilia (7%) are not part of the UK operational network. DNA from 138 genera was identified, where 2% of the sample could not be associated with specific genera. 40% of the sample was classified better using eDNA methods at the genus level, than by optical methods. We calculate Bray–Curtis dissimilarity for the rural and urban zones and find a systematic difference in biodiversity. Overall, this shows airborne DNA reveals more information than methods based on morphological differences. The results also suggest data from sites located in large urban areas will be less representative for less populated rural areas. This presents a dilemma in balancing a network and the associated costs delivering health relevant information to the most populated areas vs. a nation-wide approach

Using qPCR and microscopy to assess the impact of harvesting and weather conditions on the relationship between Alternaria alternata and Alternaria spp. spores in rural and urban atmospheres.

Alternaria is a plant pathogen and human allergen. Alternaria alternata is one of the most abundant fungal spores in the air. The purpose of this study was to examine whether Alternaria spp. spore concentrations can be used to predict the abundance and spatio-temporal pattern of A. alternata spores in the air. This was investigated by testing the hypothesis that A. alternata dominates airborne Alternaria spp. spores and varies spatio-temporally. Secondarily, we aimed at investigating the relationship between airborne Alternaria spp. spores and the DNA profile of A. alternata spores between two proximate (~ 7 km apart) sites. These were examined by sampling Alternaria spp. spores using Burkard 7-day and cyclone samplers for the period 2016-2018 at Worcester and Lakeside campuses of the University of Worcester, UK. Daily Alternaria spp. spores from the Burkard traps were identified using optical microscopy whilst A. alternata from the cyclone samples was detected and quantified using quantitative polymerase chain reaction (qPCR). The results showed that either A. alternata or other Alternaria species spores dominate the airborne Alternaria spore concentrations, generally depending on weather conditions. Furthermore, although Alternaria spp. spore concentrations were similar for the two proximate sites, A. alternata spore concentrations significantly varied for those sites and it is highly likely that the airborne samples contained large amounts of small fragments of A. alternata. Overall, the study shows that there is a higher abundance of airborne Alternaria allergen than reported by aerobiological networks and the majority is likely to be from spore and hyphal fragments. [Abstract copyright: © 2023. The Author(s).

Spatial and Temporal Variance of Bi-hourly Grass Pollen Concentrations in the Local Surroundings of Worcester, UK

B: Grass pollen is the most important aeroallergen worldwide and the health outcome among sensitive individuals is closely related to exposure. It has been argued that grass pollen concentrations can be expected to vary substantially within the urban environment, partly due to source distribution and partly due to dispersion and deposition mechanisms. Most studies conducted on local spatial and temporal variance of pollen concentrations are from one season. Only a few studies include multiple seasons and the results are inconclusive. The patterns and factors responsible for local spatial and temporal pollen variance are still largely unknown. Bi-hourly pollen data provides finer temporal resolution than the standardized daily data otherwise used. Bi-hourly data collected from two sampling sites are used to investigate local spatial and temporal patterns of grass pollen concentrations in Worcester. M: Grass pollen was sampled from two locations in Worcester during the years 2016 and 2017 using a Burkard sampler. Daily and bi-hourly grass pollen concentrations were investigated for temporal and spatial variance using statistical methods by comparing years and locations. The investigation is being repeated for the year 2018. R: Preliminary results from 2016 and 2017 suggest that there is a high spatial correlation for the bi-hourly concentrations in 2016 but not in 2017. In 2016, all recorded peaks except one coincide with a corresponding peak. In 2017, the highest peak coincided with a corresponding peak but the rest of the data did not. Results from 2018 are currently unknown. C: Spatial and temporal variance in grass pollen concentrations fluctuates between years and locations. Peak concentrations tend to have the highest correlations compared to low concentrations. The results show that at least two years of data are needed to establish potential autocorrelation between nearby sites. Future work needs to include longer time-series, more locations and local grass source maps to understand key underlying factors of localized grass pollen concentrations

Microscale pollen release and dispersal patterns in flowering grass populations

Characterizing pollen release and dispersion processes is fundamental for knowledge advancement in ecological, agricultural and public health disciplines. Understanding pollen dispersion from grass communities is especially relevant due to their high species-specific allergenicity and heterogeneously distributed source areas. Here, we aimed to address questions concerning fine level heterogeneity in grass pollen release and dispersion processes, with a focus on characterizing the taxonomic composition of airborne grass pollen over the grass flowering season using eDNA and molecular ecology methods. High resolution grass pollen concentrations were compared between three microscale sites (<300 m apart) in a rural area in Worcestershire, UK. The grass pollen was modelled with local meteorology in a MANOVA (Multivariate ANOVA) approach to investigate factors relevant to pollen release and dispersion. Simultaneously, airborne pollen was sequenced using Illumina MySeq for metabarcoding, analysed against a reference database with all UK grasses using the R packages DADA2 and phyloseq to calculate Shannon's Diversity Index (α-diversity). The flowering phenology of a local Festuca rubra population was observed. We found that grass pollen concentrations varied on a microscale level, likely attributed to local topography and the dispersion distance of pollen from flowering grasses in local source areas. Six genera (Agrostis, Alopecurus, Arrhenatherum, Holcus, Lolium and Poa) dominated the pollen season, comprising on average 77 % of the relative abundance of grass species reads. Temperature, solar radiation, relative humidity, turbulence and wind speeds were found to be relevant for grass pollen release and dispersion processes. An isolated flowering Festuca rubra population contributed almost 40 % of the relative pollen abundance adjacent to the nearby sampler, but only contributed 1 % to samplers situated 300 m away. This suggests that most emitted grass pollen has limited dispersion distance and our results show substantial variation in airborne grass species composition over short geographical scales

Environmental DNA reveals links between abundance and composition of airborne grass pollen and respiratory health

This is the final version. Available on open access from Elsevier via the DOI in this recordData and Code Availability Statement: Data collected using qPCR is archived and on NERC EIDC [https://doi.org/10.5285/28208be4-0163-45e6-912c-2db205126925]. Standard pollen monitoring ‘count’ data were sourced from the MEDMI database, with the exception of data from Bangor which were produced as part of the present study and are available on request. Prescribing datasets are publicly available, as are weather, air pollution, deprivation (IMD) and rural-urban category data. Hospital episode statistics (HES) datasets are sensitive, individual-level health data, which are subject to strict privacy regulations and are not publicly available. The study did not generate any unique codeGrass (Poaceae) pollen is the most important outdoor aeroallergen, exacerbating a range of respiratory conditions, including allergic asthma and rhinitis (‘hay fever’). Understanding the relationships between respiratory diseases and airborne grass pollen with view to improving forecasting has broad public health and socioeconomic relevance. It is estimated that there are over 400 million people with allergic rhinitis and over 300 million with asthma, globally, often comorbidly . In the UK, allergic asthma has an annual cost of around US$ 2.8 billion (2017). The relative contributions of the >11,000 (worldwide) grass species to respiratory health have been unresolved, as grass pollen cannot be readily discriminated using standard microscopy. Instead, here we used novel environmental DNA (eDNA) sampling and quantitative PCR (qPCR) , to measure the relative abundances of airborne pollen from common grass species, during two grass pollen seasons (2016 and 2017), across the UK. We quantitatively demonstrate discrete spatiotemporal patterns in airborne grass pollen assemblages. Using a series of generalised additive models (GAMs), we explore the relationship between the incidences of airborne pollen and severe asthma exacerbations (sub-weekly) and prescribing rates of drugs for respiratory allergies (monthly). Our results indicate that a subset of grass species may have disproportionate influence on these population-scale respiratory health responses during peak grass pollen concentrations. The work demonstrates the need for sensitive and detailed biomonitoring of harmful aeroallergens in order to investigate and mitigate their impacts on human health.Natural Environment Research Council (NERC)National Institute for Health Research (NIHR)Public Health EnglandUniversity of ExeterUniversity College LondonMet Offic

Linking Grass Pollen Biodiversity and Human Health: an Environmental Genomic Approach

In Europe and the UK, grass pollen is the single most important outdoor aeroallergen; 27% of the population are sensitised to grass pollen. Grass pollen allergy has been linked to increased risk of allergic asthma exacerbations, which can lead to hospitalisation and fatalities. Sensitivity towards grass pollen varies between species, of which there are over 150 in the UK. However, due to few unique morphological features, grass pollen from different species cannot be discriminated using traditional observational methods. Currently, there is no way of detecting, modelling or forecasting the aerial-dispersion of pollen from the biodiversity of UK grasses. Consequently, grasses are coalesced into a single group in the UK forecast. PollerGEN is an interdisciplinary NERC project with the aim of revolutionising the way that pollen dispersion is measured and forecast, with concomitant synergies for understanding the ecology of aerial dispersed pollen. In collaboration with the UK Met Office, a key goal is to build a more accurate forecast of individual grass pollen species. Using environmental genomics, we will identify which species of grass pollen are present during the summer months across 16 specific collection sites in the UK, and measure the abundance of the different allergenic species of grass. The information will be used to model the spatial and temporal deposition of different species of grass pollen and identify linkages to human health. The project therefore aims to provide a paradigm shift in our understanding of the ecology of windborne pollen in time and space and inform the public about the timing and environmental factors that put them at risk of exposure to pollen they are allergic to; a key strategy in the prevention of allergy and asthma attacks