Estimating downwind concentrations of viable airborne microorganisms in dynamic atmospheric conditions

A Gaussian plume model has been modified to include an airborne microbial survival term that is a best-fit function of laboratory experimental data of weather variables. The model has been included in an algorithm using microbial source strength and local hourly mean weather data to drive the model through a summer- and winter-day cycle. For illustrative purposes, a composite airborne "virus" (developed using actual characteristics from two viruses) was used to show how wind speed could have a major modulating effect on near-source viable concentrations. For example, at high wind speeds such as those occurring during the day, or with short travel times, near-source locations experience high viable concentrations because the microorganisms have not had time to become inactivated. As the travel time increases, because of slow wind speed or longer distances, die-off modulation by sunshine, relative humidity, temperature, etc., potentially becomes increasingly predominant.     

Estimation of viable airborne microbes downwind from a point source.

Modification of the Pasquill atmospheric diffusion equations for estimating viable microbial airborne cell concentrations downwind form a continuous point source is presented. A graphical method is given to estimate the ground level cell concentration given (i) microbial death rate, (ii) mean wind speed, (iii) atmospheric stability class, (iv) downwind sample distance from the source, and (v) source height.     

Size distribution of viable, cultivable, airborne microbes and their relationship to particulate matter concentrations and meteorological conditions in a Mediterranean site

The biological loading of viable, cultivable airborne microbes (heterotrophic bacteria, actinobacteria and fungi) in 6 size fractions as well as the three different fractions of respirable particulate matter (PM₁, PM_2.5 and PM₁₀) and their relationship to meteorological conditions were studied in the ambient air due to health-related interests. An Andersen six stage viable particle impactor and a MAS 100 sampler were used for microbial measurements. 82 measurements were performed at three different periods (41 days) at a suburban, residential site in the city of Chania (Crete, Greece) during the period from April 2008 to June 2009. The concentrations of the viable, cultivable airborne microbes (bioaerosols) as well as of the PM₁, PM_2.5 and PM₁₀ were highly variable during the whole measurement period. Among the airborne microbes, fungi presented the most abundant taxonomic group in the ambient air. A characteristic profile of the mean size distribution of biological loading in different PM fractions was obtained for every measured microbial taxonomic group. Although, the highest concentrations of the airborne fungi and actinobacteria were determined at aerodynamic diameters between 2.1 and 3.3 μm, a nearly equal distribution of the mean concentrations of the airborne heterotrophic bacteria was observed in the six different size fractions. However, two small maxima were observed at the airborne heterotrophic bacteria distribution, one at the fraction with aerodynamic diameters between 1.1 and 2.1 μm, and at other at the coarse fraction with aerodynamic diameter larger than 7 μm. A considerable part of the airborne microbes Cycloheximide per mL of growth medium of bacteriwere resistant to drugs. Between 10 and 40 % of the viable, cultivable airborne microbes were resistant to low concentrations of drugs (5–10 μg of Streptomycin or a or fungi, respectively). Furthermore, multiple linear regression of the data showed that the variation in fungi concentrations depends on the variation in PM₁₀ mass concentration, PM₁ number concentration, relative humidity and solar radiation. Likewise, the concentration of heterotrophic bacteria was found proportional to the values of relative humidity and fungal concentration, whereas was negatively correlated to the solar radiation.     

Observations on the use of membrane filtration and liquid impingement to collect airborne microorganisms in various atmospheric environments

The influence of sample-collection-time on the recovery of culturable airborne microorganisms using a low-flow-rate membrane-filtration unit and a high-flow-rate liquid impinger were investigated. Differences in recoveries were investigated in four different atmospheric environments, one mid-oceanic at an altitude of ~10.0 m, one on a mountain top at an altitude of ~3,000.0 m, one at ~1.0 m altitude in Tallahassee, Florida, and one at ~1.0 m above ground in a subterranean-cave. Regarding use of membrane filtration, a common trend was observed: the shorter the collection period, the higher the recovery of culturable bacteria and fungi. These data also demonstrated that lower culturable counts were common in the more remote mid-oceanic and mountain-top atmospheric environments with bacteria, fungi, and total numbers averaging (by sample time or method categories) <3.0 colony-forming units (CFU) m⁻³. At the Florida and subterranean sites, the lowest average count noted was 3.5 bacteria CFU m⁻³, and the highest averaged 140.4 total CFU m⁻³. When atmospheric temperature allowed use, the high-volume liquid impinger utilized in this study resulted in much higher recoveries, as much as 10× greater in a number of the categories (bacterial, fungal, and total CFU). Together, these data illustrated that (1) the high-volume liquid impinger is clearly superior to membrane filtration for aeromicrobiology studies if start-up costs are not an issue and temperature permits use; (2) although membrane filtration is more cost friendly and has a ‘typically’ wider operational range, its limits include loss of cell viability with increased sample time and issues with effectively extracting nucleic acids for community-based analyses; (3) the ability to recover culturable microorganisms is limited in ‘extreme’ atmospheric environments and thus the use of a ‘limited’ methodology in these environments must be taken into account; and (4) the atmosphere culls, i.e., everything is not everywhere.     

Chemiluminescent assay for detection of viable microorganisms

The redox reaction between quinone and viable microorganisms produces active oxygen species. In this study, the production rates of active oxygen species were determined by a luminol chemiluminescent assay, and the luminescence intensity was found to be proportional to the viable cell number. The high sensitivity of the luminol chemiluminescent assay was achieved with Mo-ethylenediaminetetraacetate complex and menadione or coenzyme Q1. The detectable cell densities of bacteria and yeasts were found to be approximately several thousand colony-forming units (CFU/ml) when assays were performed with a 96-well microplate luminometer. The chemiluminescent assay requires 10 min for incubation of quinone and microorganisms and 2s for photon counting. Single Escherichia coli was detected after 4h of cultivation and centrifugation (5 min x 2). This simple chemiluminescent assay is expected to be useful for the rapid detection of viable bacteria and yeast.     

Measurement of culturable airborne staphylococci downwind from a naturally ventilated broiler house

The objective of this study was to estimate the possible travel distance of airborne bacteria emitted from a naturally ventilated broiler house by using staphylococci as indicator organisms. Air samples were taken during the second half of three fattening periods with Impinger (AGI-30) in the barn and simultaneously upwind and downwind from the building. Staphylococci concentrations varied between 1 × 10⁶ and 1 × 10⁷ cfu m⁻³ in the barn. No Staphylococci were detected in air samples at the upwind side. A strong exponential decrease of these bacteria was observed at three sampling heights (1.5, 4.0 and 9.5 m) in the main wind direction downwind of the barn. Staphylococci concentrations up to 5.9 × 10³ cfu m⁻³ were detected at the farthest sampling point (333 m) downwind. Identification to the species level by means of a 16S–23S ITS PCR confirmed that Staphylococcus spp. from downwind samples originated from the barn. Staphylococci served as an useful indicator to demonstrate the travel distance of bacterial emissions originating from a naturally ventilated broiler house. These findings indicate that airborne transmission of viable bacteria from this type of housing system to adjacent residential dwellings or animal houses several hundred metres away is possible.     

Use of solid-phase PCR for enhanced detection of airborne microorganisms.

The solid-phase PCR (SP-PCR) was compared with a culture-based technique for the detection of aerosolized Escherichia coli DH1. Results with SP-PCR showed an increase in detection sensitivity over that of culture methods. Therefore, SP-PCR may be useful for the detection of airborne microorganisms which may be nonculturable because of aerosolization or sampling stress.     

Real-time detection of viable microorganisms by intracellular phototautomerism

Background  

To date, the detection of live microorganisms present in the environment or involved in infections is carried out by enumeration of colony forming units on agar plates, which is time consuming, laborious and limited to readily cultivable microorganisms. Although cultivation-independent methods are available, they involve multiple incubation steps and do mostly not discriminate between dead or live microorganisms. We present a novel generic method that is able to specifically monitor living microorganisms in a real-time manner.     

Suitability of poor medium in counting total viable airborne bacteria

The purpose of this study was to test the effect of incubation temperature and culture medium on viable counts of airborne bacteria. The incubation temperature had different effect on indoor and outdoor air bacteria. Indoor air bacteria grew as well at 20°C as 37°C, but less at 10°C. Outdoor air bacteria grew equally well at 10°C and 20°C, but less at 37°C. Both indoor and outdoor air bacteria grew differently on poor and rich media. The counts of both indoor and outdoor air bacteria were higher on poor R2A medium (low nutrient concentration) than on rich TYG and blood media (high nutrient concentration). The results indicate that a poor medium incubated at 20°C is adequate for counting viable airborne bacteria.     

Survey of viable airborne fungal propagules in French wine cellars

A study was carried out of the viable airbornemycological flora of 12 wine cellars in the Arboisregion in France. Results show that there wereconsiderable variations in the quantities ofpropagules: from 57 to 2547 CFU m^–3. There was arelatively large number of species (48), which aremore frequently recovered from certain cellar types.The most common were Cladosporium sphaerospermum,Aspergillus versicolor, A. restrictus, Penicilliumglabrum, P. dierckxii, P. implicatum and P.brevicompactum. So the risk of allergies subsequentto inhalation of large quantities of spores is farfrom minimal.     

ATP as a biomarker of viable microorganisms in clean-room facilities

A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.     

Radioactive fingerprinting of microorganisms that oxidize atmospheric methane in different soils.

Microorganisms that oxidize atmospheric methane in soils were characterized by radioactive labelling with (14)CH(4) followed by analysis of radiolabelled phospholipid ester-linked fatty acids ((14)C-PLFAs). The radioactive fingerprinting technique was used to compare active methanotrophs in soil samples from Greenland, Denmark, the United States, and Brazil. The (14)C-PLFA fingerprints indicated that closely related methanotrophic bacteria were responsible for the oxidation of atmospheric methane in the soils. Significant amounts of labelled PLFAs produced by the unknown soil methanotrophs coeluted with a group of fatty acids that included i17:0, a17:0, and 17:1omega8c (up to 9.0% of the total (14)C-PLFAs). These PLFAs are not known to be significant constituents of methanotrophic bacteria. The major PLFAs of the soil methanotrophs (73.5 to 89.0% of the total PLFAs) coeluted with 18:1 and 18:0 fatty acids (e.g., 18:1omega9, 18:1omega7, and 18:0). The (14)C-PLFAs fingerprints of the soil methanotrophs that oxidized atmospheric methane did not change after long-term methane enrichment at 170 ppm CH(4). The (14)C-PLFA fingerprints of the soil methanotrophs were different from the PLFA profiles of type I and type II methanotrophic bacteria described previously. Some similarity at the PLFA level was observed between the unknown soil methanotrophs and the PLFA phenotype of the type II methanotrophs. Methanotrophs in Arctic, temperate, and tropical regions assimilated between 20 and 54% of the atmospheric methane that was metabolized. The lowest relative assimilation (percent) was observed for methanotrophs in agricultural soil, whereas the highest assimilation was observed for methanotrophs in rain forest soil. The results suggest that methanotrophs with relatively high carbon conversion efficiencies and very similar PLFA compositions dominate atmospheric methane metabolism in different soils. The characteristics of the methane metabolism and the (14)C-PLFA fingerprints excluded any significant role of autotrophic ammonia oxidizers in the metabolism of atmospheric methane.     

Seasonal variation of outdoor airborne viable microfungi in Copenhagen,Denmark

Registration of viable microfungi in the Copenhagen outdoor air has been done since 1978 as a routine procedure for a service of the allergic patients.

Of the thirty-four genera of moulds and yeasts recorded Cladosporium, Alternaria, Penicillium, Aspergillus represent 84.1% of the total viable flora.

During the years the registrations have served as an actual information to allergic patients concerning the outdoor load of potential microfungal allergens. However, this long-term recordings may also serve as a database for comparison with actual measurement from the air over potential risk-areas such as open-air compost-plants or open air waste deposits with accumulation of organic debris. In many cases there will be a need for documentation of presence or absence of an additional microbial load.     

Exposure of workers to airborne microorganisms in open-air swine houses

This study quantified the levels of airborne microorganisms in six swine farms with more than 10,000 pigs in subtropical Taiwan. We evaluated breeding, growing, and finishing stalls, which were primarily open-air buildings, as well as partially enclosed farrowing and nursery piggeries. Airborne culturable bacteria, gram-negative bacteria, and fungi were placed on appropriate media by using an all-glass impinger or single-stage Andersen microbial sampler. Results showed that mean concentrations of culturable bacteria and gram-negative bacteria were 3.3 x 10(5) and 143.7 CFU/m(3), respectively. The concentration of airborne culturable fungi was about 10(3) CFU/m(3), with Cladosporium the predominant genus. The highest airborne levels of culturable bacteria and gram-negative bacteria were identified in the finishing units. The air of the nursery stalls was the least contaminated with culturable and gram-negative bacteria. Irregular and infrequent cleaning, high pig density, no separation of wastes from pen floors, and accumulation of water as a result of the processes for cleaning and reducing pig temperature possibly compromise the benefits of the open characteristic of the finishing units with respect to airborne bacterial concentration.     

Survey of viable airborne fungi in wine cellars of Tokaj,Hungary

The composition of fungal biota and air quality of five traditional subterranean wine cellars and one store building of a modern wine production facility were examined in the Tokaj wine region (northeastern Hungary). Air samples were collected with SAS IAQ sampler onto PDA, MEA and RBA. Strains representing morphotypes were isolated from colonies formed on agar plates from either air or surface samples. The internal transcribed spacer (ITS) region of the rRNA gene cluster was amplified with primers ITS1 and ITS4. Altogether 90 morphotypes were isolated, 48 and 12 strains (43 species) from the air and surfaces, respectively. The number of spore-forming species generated high diversity of indoor fungi and differences between the cellars’ fungal compositions; however, their dominant species were proved to be the same. Among the isolated strains Penicillium spp. were the most frequent. The walls of cellars were covered by colonies of Zasmidium (Cladosporium) cellare often referred to as a noble mold. Even so, this mold has been found only at a small concentration in the air samples (10–30 CFU/m³). The walls of the modern store were free of molds. Diversity of fungi of the examined wine cellars was influenced by environmental conditions to a certain degree, such as elevation (height above sea level), age, reconstruction time of cellars, indoor ethanol concentration and the number of chimneys. The location of cellars poorly influenced the concentration of fungi of the air inside cellars, contrary to outdoors where the air of the municipal area contained more CFUs than that of rural spaces.     

Some special problems in the determination of viable counts of groundwater microorganisms

Factors affecting viable cell counts in groundwater or sediments were studied with samples from the Segeberg Forest test area in northern Germany. There was very little variation in results with the season (April, August, November) or depth of sampling; generally there were 10³–10⁴ aerobic cells per ml or g sediment. Long incubation times resulted in higher cell counts; groundwater samples required 4–5 weeks, and sediment extracts had to be cultured for 7 weeks. Total cell counts in sediment were 10²–10⁴ cell/g higher than viable cell counts of aerobes. This was explained partly by the additional presence of anaerobes and partly by the observation that some morphotypes may not have grown under our conditions. Viable cell counts were not influenced by cell extraction from the sediment with either Na-pyrophosphate or groundwater extracts. However, iron-precipitating or manganese-oxidizing bacteria were better extracted with sterile groundwater. The microflora of wells was more numerous than that of the free aquifer; consequently it was better to pump off all well water before aquifer water was sampled. The diameter of the well was also important; thinner tubes had higher cell counts than those with wider diameter. For sampling, wells should be at least 1 year old, since young wells contain higher numbers of microorganisms due to underground disturbances from the drilling. Turbid water samples could be clarified by filtration, but this reduced the viable counts by 1–2 orders of magnitude. Two different media inoculated with a sample dilution resulted in the same cell counts, but their microbial diversity was different. Storage of groundwater samples before processing resulted in up to 17-fold increases in cell counts and loss of diversity in the first 24 hours. Cell numbers decreased slowly during longer storage.     

Effects of low concentrations of bisulfite-sulfite and nitrite on microorganisms.

A wide range of microorganisms was tested to determine their sensitivity to low concentrations of bisulfite-sulfite and nitrite, solubility products of SO2 and NO2, respectively. Photosynthesis by blue-green algae (cyanobacteria) was more strongly inhibited by 0.1 mM bisulfite-sulfite and 1 mM nitrite at pH 6.0 than photosynthesis by eucaryotic algae and respiration of bacteria, fungi, and protozoa. At pH 7.7, blue-green algae were still more sensitive to bisulfite-sulfite and nitrite than eucaryotic algae, but the toxicity of bisulfite-sulfite and nitrite decreased as the pH increased. Photosynthesis by Anabaena flos-aquae at pH 6.0 was inhibited 25% by a bisulfite-sulfite concentration of 10 micrometer and 15% by a nitrite concentration of 50 micrometer. Photosynthesis by the blue-green alga, Lyngbya sp., was not exceptionally sensitive to chlorate and thiosulfate. Acetylene-reducing activity of Beijerinckia indica was completely inhibited by 0.1 mM bisulfite-sulfite at pH 4.0, the suppression being decreased with increasing pH.