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Bioaerosol biomonitoring: Sampling optimization for molecular microbial ecology.

ABSTRACT: Bioaerosols (or biogenic aerosols) have largely been overlooked by molecular ecologists. However, this is rapidly changing as bioaerosols play key roles in public health, environmental chemistry and the dispersal ecology of microbes. Due to the low environmental concentrations of bioaerosols, collecting sufficient biomass for molecular methods is challenging. Currently, no standardized methods for bioaerosol collection for molecular ecology research exist. Each study requires a process of optimization, which greatly slows the advance of bioaerosol science. Here, we evaluated air filtration and liquid impingement for bioaerosol sampling across a range of environmental conditions. We also investigated the effect of sampling matrices, sample concentration strategies and sampling duration on DNA yield. Air filtration using polycarbonate filters gave the highest recovery, but due to the faster sampling rates possible with impingement, we recommend this method for fine -scale temporal/spatial ecological studies. To prevent bias for the recovery of Gram-positive bacteria, we found that the matrix for impingement should be phosphate-buffered saline. The optimal method for bioaerosol concentration from the liquid matrix was centrifugation. However, we also present a method using syringe filters for rapid in-field recovery of bioaerosols from impingement samples, without compromising microbial diversity for high -throughput sequencing approaches. Finally, we provide a resource that enables molecular ecologists to select the most appropriate sampling strategy for their specific research question.

SUBMITTER: Ferguson RMW

PROVIDER: S-EPMC6850074 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Bioaerosol biomonitoring: Sampling optimization for molecular microbial ecology.

Ferguson Robert M W RMW Garcia-Alcega Sonia S Coulon Frederic F Dumbrell Alex J AJ Whitby Corinne C Colbeck Ian I

Molecular ecology resources 20190501 3

Bioaerosols (or biogenic aerosols) have largely been overlooked by molecular ecologists. However, this is rapidly changing as bioaerosols play key roles in public health, environmental chemistry and the dispersal ecology of microbes. Due to the low environmental concentrations of bioaerosols, collecting sufficient biomass for molecular methods is challenging. Currently, no standardized methods for bioaerosol collection for molecular ecology research exist. Each study requires a process of optimi ...[more]

PMID: 30735594

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Project description:IntroductionDetection of Mycobacterium tuberculosis (Mtb) in patient-derived bioaerosol is a potential tool to measure source case infectiousness. However, current bioaerosol sampling approaches have reported low detection yields in sputum-positive TB cases. To increase the utility of bioaerosol sampling, we present advances in bioaerosol collection and Mtb identification that improve detection yields.MethodsA previously described Respiratory Aerosol Sampling Chamber (RASC) protocol, or "RASC-1", was modified to incorporate liquid collection of bioaerosol using a high-flow wet-walled cyclone (RASC-2). Individuals with GeneXpert-positive pulmonary TB were sampled pre-treatment over 60-minutes. Putative Mtb bacilli were detected in collected fluid by fluorescence microscopy utilising DMN-Trehalose. Exhaled air and bioaerosol volumes were estimated using continuous CO2 monitoring and airborne particle counting, respectively. Mtb capture was calculated per exhaled air volume sampled and bioaerosol volume for RASC-1 (n = 35) and for RASC-2 (n = 21). Empty chamber samples were collected between patients as controls.ResultsThe optimised RASC-2 protocol sampled a median of 258.4L (IQR: 226.9-273.6) of exhaled air per patient compared with 27.5L (IQR: 23.6-30.3) for RASC-1 (p<0.0001). Bioaerosol volume collection was estimated at 2.3nL (IQR: 1.1-3.6) for RASC-2 compared with 0.08nL (IQR: 0.05-0.10) for RASC-1 (p<0.0001). The detection yield of viable Mtb improved from 43% (median 2 CFU, range: 1-14) to 95% (median 20.5 DMN-Trehalose positive bacilli, range: 2-155). These improvements represent a lowering of the limit of detection in the RASC-2 platform to 0.9 Mtb bacilli per 100L of exhaled air from 3.3 Mtb bacilli per 100L (RASC-1).ConclusionThis study demonstrates that technical improvements in particle collection together with sensitive detection enable rapid quantitation of viable Mtb in bioaerosols of sputum positive TB cases. Increased sampling sensitivity may allow future TB transmission studies to be extended to sputum-negative and subclinical individuals, and suggests the potential utility of bioaerosol measurement for rapid intervention in other airborne infectious diseases.

Project description:BackgroundTuberculosis (TB) is transmitted in bioaerosols containing Mycobacterium tuberculosis (Mtb). Despite being central to ongoing TB transmission, no routine diagnostic assay exists to measure Mtb in bioaerosols. Furthermore, published studies of Mtb in bioaerosol samples have been limited to individuals with sputum-positive pulmonary TB. Notably, TB diagnosis is based on clinical symptoms and sputum laboratory findings. This is despite the fact that approximately half of all patients commencing TB treatment are sputum-negative, resulting in a high proportion of presumptive treatments. Here, we propose to use a sensitive air sampling protocol to investigate the prevalence of Mtb-containing bioaerosols in both sputum-positive and sputum-negative TB suspects, at the same time evaluating the potential to identify unrecognized transmitters of TB.MethodsOur parallel-group design will identify viable Mtb in bioaerosols produced by individuals attending a TB clinic in South Africa. Sampling will be performed on eligible individuals presenting with symptoms indicative of TB and repeated at 14 days if initially positive. Participants will be prospectively classified into three distinct groups based on National TB Control Program (NTBCP) criteria: Group A, TB notification with sputum-based laboratory confirmation; Group B, TB notification with empiric diagnosis; and Group C, individuals not notified. Group C individuals with detectable Mtb bioaerosol will be monitored until resolution of clinical and laboratory status. Collection of bioaerosol specimens will be via two consecutive sampling modalities: (1) direct sampling following a specific respiratory manoeuvre; and (2) indirect sampling during passive respiratory activity. Bioaerosol specimens will be analyzed for viable Mtb using DMN-trehalose staining and live-cell fluorescence microscopy. Mtb genomes and mycobacterial and host lipids will be detected using droplet digital PCR and mass spectrometry analyses, respectively. The primary objective is to determine the prevalence of Mtb bioaerosols in all TB clinic attendees and in each of the groups. Secondary objectives are to investigate differences in prevalence of Mtb bioaerosol by HIV status and current isoniazid preventive therapy (IPT) use; we will also determine the impact of anti-TB chemotherapy on Mtb-containing bioaerosol production.DiscussionRespiratory bioaerosol has a potential role in non-invasive TB diagnosis, infectivity measurement and treatment monitoring.Trial registrationClinicalTrials.gov: NCT04241809 . Date of Registration: 27/1/2020.

Project description:As a bioaerosol sampling standard, Andersen type impactor is widely used since its invention in 1950s, including the investigation of the anthrax attacks in the United States in 2001. However, its related problems such as impaction and desiccation stress as well as particle bounce have not been solved. Here, we improved its biological collection efficiencies by plating a mineral oil layer (100 µL) onto the agar plate. An Andersen six-stage sampler and a BioStage impactor were tested with mineral-oil-spread agar plates in collecting indoor and outdoor bacterial and fungal aerosols. The effects of sampling times (5, 10 and 20 min) were also studied using the BioStage impactor when sampling environmental bioaerosols as well as aerosolized Bacillus subtilis (G+) and Escherichia coli (G-). In addition, particle bounce reduction by mineral-oil-plate was also investigated using an optical particle counter (OPC). Experimental results revealed that use of mineral-oil-spread agar plate can substantially enhance culturable bioaerosol recoveries by Andersen type impactors (p-values<0.05). The recovery enhancement was shown to depend on bioaerosol size, type, sampling time and environment. In general, more enhancements (extra 20%) were observed for last stage of the Andersen six-stage samplers compared to the BioStage impactor for 10 min sampling. When sampling aerosolized B. subtilis, E. coli and environmental aerosols, the enhancement was shown to increase with increasing sampling time, ranging from 50% increase at 5 min to ?100% at 20 min. OPC results indicated that use of mineral oil can effectively reduce the particle bounce with an average of 66% for 10 min sampling. Our work suggests that enhancements for fungal aerosols were primarily attributed to the reduced impaction stress, while for bacterial aerosols reduced impaction, desiccation and particle bounce played major roles. The developed technology can readily enhance the agar-based techniques including those high volume portable samplers for bioaerosol monitoring.

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Bioaerosol biomonitoring: Sampling optimization for molecular microbial ecology.

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Bioaerosol biomonitoring: Sampling optimization for molecular microbial ecology.

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