Real-time monitoring for bioaerosols—flow cytometry

Abstract

Bioaerosol detection in real time is an urgent civilian and military requirement. In this article, bioaerosol mass spectrometry, an instrument for real-time detection of bioaerosols using simultaneous measurement of particle aerodynamic size and intrinsic fluorescence, real-time qPCR, and FCM/FL were discussed. Although, challenging work remains to determine the interfering substances (e.g. particulates) of different environments, distinguish the specific species with specific probe, and overcome the high detection limit of FCM (10⁴–10⁸ cells ml⁻¹), literature reports suggested that FCM/FL has a great potential for real-time monitoring of bioaerosols.

Recently, exposure assessment of bioaerosols has become an important issue. Airborne microorganisms in indoor and outdoor environments, from either nature or industrial sources, may produce ill effects on human ranging from mild irritation to diseases. Therefore, it is important to accurately measure concentration of bioaerosols in different environments to ensure the health of the workers and the public. Bioaerosols are those airborne particles that are living or originate from living organisms. Bioaerosols include microorganisms (i.e., culturable, viable but nonculturable, and dead microorganisms) and fragments, toxins, and particulate waste products from all varieties of living things. In regard to living bioaerosols, fungi (2–10 µm), bacteria (1–5 µm) and virus (20–300 nm) are the target microorganisms of great concern.

Bioaerosol detection in real time is an urgent civilian and military requirement. However, measurement and detection of biological aerosols in real time has been a difficult task. Conventional approaches involving growth of biological samples by culture methods or enumeration of biological samples by microscopic techniques take days to produce results. For overcoming this disadvantage, several techniques were developed, optimized, and validated to bioaerosol monitoring.¹ Among these techniques, bioaerosol mass spectrometry, real-time quantitative polymerase chain reaction (real-time qPCR), and FCM in combination with fluorescent technique (FCM/FL) were the potential methods.

Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time.² Characteristic mass spectra from individual bacterial endospores of Bacillus subtilis var. niger were obtained in a bipolar aerosol time-of-flight mass spectrometer. In addition, the mass special signature of individual Bacillus spore species was characterized and the ability to distinguish two Bacillus spore species, B. thuringiensis and B. atrophaeus, was also demonstrated.³ This example demonstrated that the chemical differences between these two Bacillus spore species are consistently and easily detected within single cells in second. Furthermore, bioaerosol mass spectrometry was applied to detect the biochemical and morphological changes of Bacillus atrophaeus cells during the sporulation process.⁴ However, it is necessary to characterize the mass spectra of other species.

In 1997, a prototype instrument has been constructed to measure individual airborne particles based on their aerodynamic size and intrinsic fluorescence (excited a wavelength of 325 nm and emitted from 420 to 580 nm).⁵ A biomolecule in live bacteria, reduced nicotiamide adenine dinucleotide phosphate [NAD(P)H], was considered as the primary biomolecule associated with the fluorescent signals. Bacillus subtilis var niger spores were generated and detected by this proposed instrument in a chamber study. Initial tests have indicated that a low level of fluorescence is detected from 17% of the aerosolized Bacillus subtilis var niger spores. This technique showed potential ability for real-time indication of airborne bacterial concentrations; however, further investigation is needed to validate this method to environmental samples.

Recently, rapid identification methods using molecular techniques have recently been developed and utilized in clinical laboratories.^6–11 One such method is the real-time qPCR system, which is a commercially available system designed to decrease the time required for PCR assays by using fluorescent probes to monitor the amplification of the target sequences in real time.¹² The advantages of this real-time qPCR system are its high specificity, high sensitivity and broad liner range over several orders of magnitude.^6–11 The analytical time of real-time qPCR is around two hours. In addition, this method has already validated to field study for several important bioaerosol, such as airborne Mycobacterium tuberculosis.¹² However, this technology for bioaerosol evaluation needs to be developed for specific species since its high specificity. Furthermore, it requires 2 h of DNA extraction and 2 h of real-time qPCR analysis after environmental sampling.

The application of FCM to the analysis of microbial systems has been a slow and difficult process due to the following reasons.¹³ Firstly, the differences in size and volume between microbial and mammalian cells are numerous. Because of the lack of interest from microbiologists and the difficulty of the engineering concept, the FCM instrument is not designed to measure microorganisms (1–5 µm), but rather cells in the range of 5 to 15 µm. A second problem is that the fluorescent dyes used by FCM were better understood in mammalian systems and relatively poorly understood in the microbial system. Thirdly, FCM was conventionally a relative quantification, however, analysis in bioaerosol or environmental microbiology always needs the absolute quantification. Although it has taken microbiologists a longer time to adopt FCM as a useful tool than was initially expected, there has been some progress. In a previous bacterial investigation,¹⁴ FCM demonstrated tremendous potential in total cell determinations in comparison with traditional culture and microscopy methods.

Recently, the development of various fluorescent stains has improved the measurement of total cell concentration and viability of microorganisms by FCM. It has not only improved the differentiation between biotic particles and abiotic particles, but also improved the determination of the different physiological status of bioaerosols.^15,16 In addition, bacteria and fungi can be successfully distinguished from debris by using the three parameters of FSC (Forward Scatter Characteristic), SSC (Side Scatter Characteristic), and fluorescence intensity in acridine orange (AO) or SYTO-13 stained samples.¹⁵ It was believed that FSC is related to cell size, and SSC is related to the granule properties of cells. In regard to the quantification problem, a known amount of fluorescent beads was added to enable the absolute quantification.^14,15 In addition, the concentrations obtained by using FCM were proved to be correlated to those obtained by culture method, epifluorescence microscopy (EFM) and real-time qPCR.¹Therefore, the problems mentioned above were all solved.^15,16

Fluorescent dyes for microbial application can be categorized as total and vital dyes. Total dyes stain the nucleic acid of all microorganisms. The so-called vital stains that have been used in attempts to estimate microbial viability fall into three broad categories.¹³ (1) Some dyes, such as propidium iodide (PI), are excluded by the intact membrane of viable cells. Therefore, the presence of the dye within the cells indicates disruption of cell membrane and may be expected to be correlated with cell death. (2) other dyes, such as rhodamine123, are actively accumulated by viable cells; thus, the number of brightly stained cells reflects the viability of the samples. (3) In the case of dyes such as fluorescein diacetate (FDA), a membrane-permeant nonfluorescent precursor is converted to a membrane-impermeant fluorescent molecule by the activity of intracellular enzymes, and thus is an indicator of metabolically active cells. Therefore, basic cell functions such as reproductive ability, metabolic activity, and membrane integrity, to characterize the physiological state or degree of viability of bacteria can be determined via various fluorescent dyes.

From the previous investigations, FCM with fluorescent brighter Calcofluor white M2R successfully differentiated P. infestans sporangia and powdery mildew conidia,¹⁷ as well as Aspergillus fumigatus and Penicillium brevicompactum conidia.¹⁸ In the field environments of composting tunnel of a compost plant, woodland in a town area, a polluted dwelling house,¹⁸ detection and quantification of airborne fungi by FCM was obtained combining light scatter and PI red fluorescence parameters. A close agreement between FCM and epifluorescence microscopy counts was demonstrated. Moreover, the data indicated that FCM can be considered more precise and reliable at any of the tested concentrations.

There were more investigations undertaken in chamber and field samples for understanding the potential of FCM/FL for quantification of total, viability and culturability of bioaerosols.^15,16 The optimal conditions of five fluorescent dyes [AO, SYTO-13, PI, YOPRO-1, and 5-cyano-2, 3-ditolytetrazolium chloride (CTC)] were determined for bacterial (Escherichia coli, and endospore of Bacillus subtilis) and fungal aerosols (Candida famata and Penicillium citrinum spores). Furthermore, field samples by impinger device were assessed from aeration process of activated sludge pool. By using cell concentration, fluorescence intensity, and staining efficiency as indicators for dye performance evaluation, it was demonstrated that SYTO-13 and YOPRO-1 were the most suitable fluorescent dyes for determining total concentration and viability of bioaerosols, respectively. The fluorescent techniques in combination with FCM for assessment of total concentration and viability for bacterial and fungal bioaerosols were demonstrated to successfully apply for environmental field samples.

The great advantage of FCM is the automatically rapid counting nature. It can count 1000 cells s⁻¹. In combination with the fluorescent stain incubation, a sample can be analyzed within 7 min. It makes FCM a great potential technique for real-time monitoring of bioaerosols. Although bacteria and fungi can be distinguished without fluorescent dyes staining in some environment, dyes are still suggested to obtained better separation from abiotic particles and other physiological information. A connection of a sampler and FCM makes it possible to obtain total bacterial and fungal concentration as well as the viabilities of bacteria and fungi in real-time mode. After sampling in a liquid suspension, a 5 min hold is needed for dye staining. Then, this liquid can transfer to FCM for analysis less than 1 min. However, there are still some challenges for developing a real-time monitoring technique for bioaerosols by using FCM/FL.

First of all, the interfering substances (e.g. particulates) of different environments need to be characterized. Interfering substances may deplete the fluorescent dyes, so that the optimal concentration of fluorescent dyes determined in chamber study may be insufficient for environmental bioaerosol staining. In addition, the FSC, SSC and even FL characteristics of interfering substances may overlap with those of target bioaerosols. The overlapping may lead to failure of determination. Fortunately, the biotic component could be recognized by fluorescent dyes, since inorganic debris is unstainable with fluorescent dyes such as PI.¹⁸ Furthermore, it was observed that the natural fluorescence was not interfering with that of the target bioaerosols, so that the problem of interferance from air samples was not so significant.^15,16,18

Secondly, it is not possible to distinguish the species of the airborne microorganisms without using specific probes for specific microbes. If a specific species is to be determined, a probe hybridization process must be developed before applying FCM. On the other hand, other analytical methods with high specificity such as real-time qPCR and bioaerosol mass spectrometry should be considered as alternatives. Lastly, the detection limit of FCM (10⁴–10⁸ cells ml⁻¹) may be a limitation for monitoring bioaerosols in some environments with low bioaerosol concentrations. However, this limitation might be overcome by connecting with high volume sampler since the situation was observed by using a sampler at 12.5 l min⁻¹ (All Glass Impinger, AGI-30).^15,16 In conclusion, a connection of a sampler and FCM/FL provide the great possibility for real-time monitoring of bioaerosols.

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