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Keywords:

  • Staphylococcus aureus ;
  • Air sampling;
  • Sample storage;
  • Tween mixture;
  • Andersen;
  • BioSampler

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Staphylococcus aureus has been detected in indoor air and linked to human infection. Quantifying S. aureus by efficient sampling methods followed by appropriate sample storage treatments is essential to characterize the exposure risk of humans. This laboratory study evaluated the effects of sampler type (all-glass impinger (AGI-30), BioSampler, and Andersen one-stage sampler (Andersen 1-STG)), collection fluid (deionized water (DW), phosphate-buffered saline (PBS), and Tween mixture (TM)), and sampling time (3–60 min) on cell recovery. Effects of storage settings on bacterial concentration were also assessed over 48 h. Results showed BioSampler performed better than Andersen 1-STG and AGI-30 (P < 0.05) and TM was superior to PBS and DW (P < 0.05). An increase in sampling time negatively affected the recoveries of cells in PBS of BioSampler and AGI-30 (P < 0.05), whereas cell recoveries in TM were increased at sampling of 6–15 min compared with 3 min. Concentrations of cells collected in PBS were decreased with storage time at 4 and 23°C (P < 0.05), while cells stored in TM showed stable concentrations at 4°C (P > 0.05) and increased cell counts at 23°C (P < 0.05). Overall, sampling by BioSampler with TM followed by sample transportation and storage at 4°C is recommended.

Practical Implications

Staphylococcus aureus, present in the air of residences, hospitals, and livestock facilities, is considered a health threat to humans indoor. This study explores the factors governing the recovery of S. aureus in the bioaerosol sampling and sample storage processes. The suitable sampling technique and storage treatment are recommended, which may facilitate the residents and building owners to collect reliable data and further characterize the exposure risk and the efficacy of the interventions implemented against S. aureus.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Staphylococcus aureus, a gram-positive bacterium, may cause a variety of infections in humans from mild skin disorders to lethal pneumonia, sepsis, and toxic shock syndrome (Weber, 2005). In particular, methicillin-resistant S. aureus (MRSA) is known as one of the most common causes of nosocomial infections (Zetola et al., 2005). Recently, S. aureus has become a serious public health problem within communities (Perez et al., 2011) as MRSA infections have occurred in households (Davis et al., 2012), children day care centers, and correctional facilities (Elston, 2007). The impact of S. aureus on humans working in livestock farms and living in the vicinity is also increasingly emphasized considering S. aureus and MRSA present in the air of these facilities at substantial levels (103–104 cfu/m3, Friese et al., 2013; Schulz et al., 2012).

Staphylococcus aureus may be transmitted through person-to-person contact. In addition, airborne spread of S. aureus has been demonstrated in hospitals (Gehanno et al., 2009; Hsiao et al., 2012; Mirzaii et al., 2012; Shiomori et al., 2001, 2002), residences (Davis et al., 2012; Gandara et al., 2006; Perez et al., 2011), and livestock-housing barns (Friese et al., 2012, 2013; Schulz et al., 2012; Zhong et al., 2009), highlighting the importance of S. aureus transmission via airborne routes (Friese et al., 2012, 2013; Shiomori et al., 2001, 2002). Indeed, the prevention of airborne transmission of S. aureus has been addressed in many studies (Gehanno et al., 2009; Mirzaii et al., 2012; Schulz et al., 2012; Shiomori et al., 2001, 2002) and in the Dutch guideline for nosocomial infection controls (Bernards et al., 1998). Therefore, accurately quantifying airborne S. aureus is essential to characterize the extent of microbial exposure and further prevent its risk posed to human health, which demands efficient air sampling techniques and appropriate sample storage treatments.

Airborne S. aureus has been collected by agar impaction with the Andersen one-stage sampler (Andersen 1-STG) (Gandara et al., 2006; Hsiao et al., 2012; Huang et al., 2013; Mirzaii et al., 2012; Perez et al., 2011; Zhong et al., 2009) and by liquid impingement in the all-glass impinger (AGI-30) (Friese et al., 2012, 2013; Schulz et al., 2012). It is noted that the sampling with liquid collection allows a longer sampling time than the agar impaction method, which is important for the assessment of environmental S. aureus aerosols as it increases the volume of air sampled and thereby decreases the lower limit of detection and minimizes the false-negative result. However, the operational parameters of liquid impingers, including sampling time and type of collection fluid and sampler, may significantly affect the recovery efficiency of bioaerosols (Chang and Chou, 2011a,b; Ishimatsu et al., 2001; Rule et al., 2007), which have not been evaluated for S. aureus collection. Moreover, the efficiency of different kinds of liquid-type samplers [e.g., AGI-30 (Ace Glass Inc., Vineland, NJ, USA) and BioSampler (SKC Inc., Eighty Four, PA, USA)] for recovering airborne S. aureus has not been determined and compared with the commonly used agar impactor. In addition to the sampling method, the quantity of bioaerosols could be significantly biased due to inappropriate sample storage time and temperature (Li and Lin, 2001), which has also not been assessed for S. aureus. Thus, the suitable sampling and storage methods for quantifying airborne S. aureus remain unclear.

To address these issues, three kinds of fluids being used in bioaerosol collection, that is, deionized water (DW) (Chang and Hung, 2012a; Deloge-Abarkan et al., 2007), phosphate-buffered saline (PBS) (Chang et al., 2013; Friese et al., 2012, 2013), and Tween mixture (TM) (Chang and Chou, 2011a; Li et al., 2003), were evaluated for S. aureus recovery in a bioaerosol generation system. The loss of collection fluids due to sampling was also quantified. The AGI-30 and BioSampler filled with TM and PBS were further tested at five sampling durations. The collection efficiency of Andersen 1-STG was also characterized for comparison purpose. Moreover, concentrations of S. aureus stored in TM and PBS at 4 and 23°C were determined for 48 h after the air sampling.

Methodology

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial suspension

Staphylococcus aureus (ATCC 29213) was inoculated in 50 ml Luria-Bertani (LB) broth (Difco., Detroit, MI, USA) with 125 rpm shaking at 37°C for 14 h. Cells were harvested by centrifugation at 1000 × g for 5 min at 4°C and resuspended in sterile and filtered DW (Millipore, Bedford, MA, USA). S. aureus suspension was diluted with DW and loaded into a Collison three-jet nebulizer (BGI Collison Nebulizer, BGI Inc., Waltham, MA, USA) for aerosolization.

Bioaerosol generation system

A bioaerosol generation system (Figure 1) was set in a class II biological safety cabinet placed in a biosafety level II laboratory. HEPA-filtered compressed air was passed through a Collison three-jet nebulizer (10 psi) at 3 l/min (coefficient of variation (CV) ≤0.6%) to aerosolize S. aureus. Aerosolized cells were diluted with filtered air at 57 l/min (humidified air: 30 l/min; dry air: 27 l/min; CV ≤ 0.6%) and sampled in a test chamber (29.5 cm × 29.5 cm × 29.5 cm) by test samplers, one at a time. The sampling inlet of each sampler was located inside the test chamber at the same position (indicated as Sampling inlet site in Figure 1) where the size distribution of airborne S. aureus was also measured in triplicate by an aerodynamic particle sizer (APS, TSI Inc., Shoreview, MN, USA) for 150 min. The geometric mean of the aerodynamic diameter of S. aureus was revealed 0.77 μm with a geometric standard deviation of 1.17, indicating a monodisperse characteristic of bioaerosol. The relative humidity in test chamber was maintained at 59% (CV = 1.6%), and the temperature was set at 23°C (CV = 2.9%).

image

Figure 1. A schematic representation of bioaerosol generation system

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Collection fluid

The DW, TM, and PBS (20 ml each) were, respectively, filled in an autoclaved AGI-30 (Ace Glass Inc., Vineland, NJ, USA) and tested in this study. The PBS was the DW containing 0.82% NaCl, 0.02% KCl, 0.115% Na2HPO4, and 0.02% KH2PO4. The TM was the DW containing 1% peptone (Oxoid, Thermo Fisher Scientific Inc., Basingstoke Hampshire, UK), 0.01% Tween 80 (J.T. Backer, Phillipsburg, NJ, USA), and 0.005% Antifoam Y-30 (Sigma Chemical Co., St. Louis, MO, USA). Literature indicates the TM may help in reducing osmotic shock to bacteria and improving cell suspension in liquid (Chang et al., 2010).

To assess the recovery efficiency of three collection fluids, S. aureus (1.2 × 108 cfu/ml, CV = 10%, 40 ml) was loaded in the nebulizer, aerosolized, and stabilized for 30 min. The system was temporarily turned off to retrieve an aliquot (0.1 ml) from the nebulizer, serially diluted, and plated in duplicate (0.1 ml/plate) on tryptic soy agar (TSA, Difco) (Gandara et al., 2006; Mirzaii et al., 2012) and mannitol salt agar (MSA, Difco) (Schulz et al., 2011), respectively. The AGI-30 containing a specified collection fluid was then operated at 12.5 l/min for 3 min to sample airborne S. aureus from the test chamber after equilibrating the system for 10 min. At the end of sampling, 0.1 ml of cell suspension was taken again from the nebulizer, diluted, and plated on TSA and MSA. A 10-min equilibration was performed again prior to a next sampling trial. The experiments were conducted in two nebulizer runs. In each run, the TM, PBS, and DW were tested in a random order and two repeats were obtained for each fluid, resulting in 6 samples per run. At the end of each run, the bioaerosol generation system was sterilized by nebulizing 70% ethanol (40 ml) for 30 min.

The volume of collection fluid remaining in the AGI-30 was measured at the end of air sampling. The concentration of S. aureus in the liquid of AGI-30 was determined by serial dilutions of cell suspensions and plating in duplicate on TSA and MSA, respectively, followed by an incubation at 37°C for 24 h. The number of S. aureus (Nc) collected in the AGI-30 was calculated based on the residual volume (ml) and cell concentration (cfu/ml). With the known Nc, sampling time (T), and sampling flow rate (Q), the concentration of airborne S. aureus (Cair, cfu/m3) determined with a specified collection fluid was calculated as Nc/QT. The cell concentration in the liquid of the nebulizer before the sampling and at the end of each sampling trial was also quantified by the culture assay described above and presented as Csusp (cfu/ml). A decrease in Csusp with the operation time of the bioaerosol generation system was revealed. The Cair was thus divided by the average of Csusp values obtained before and after each sampling trial, resulting in a ratio (R) to adjust the variation of cell concentration generated between trials. The R value was used to represent the efficiency of collection fluid to recovering S. aureus.

Test sampler and sampling time

AGI-30, BioSampler (SKC Inc., Eighty Four, PA, USA), and Andersen 1-STG (Andersen Samplers Inc., Atlanta, GA, USA) with 50% cut-off diameter (d50) of 0.31, 0.3, and 0.65 μm, respectively (Chang and Hung, 2012b), were tested in this study. The BioSampler was chosen because it has been identified as the most efficient sampler for recovering culturable Legionella pneumophila (Chang and Chou, 2011a; Chang and Hung, 2012b; Chang et al., 2010), while the AGI-30 and Andersen 1-STG samplers are widely used for collection of S. aureus aerosols (Friese et al., 2012, 2013; Huang et al., 2013; Mirzaii et al., 2012; Perez et al., 2011). Tested samplers were autoclaved before use on the evaluation day.

Staphylococcus aureus in the nebulizer was adjusted to 1.0 × 108 cfu/ml (CV = 16.7%, 40 ml) for testing AGI-30 and BioSampler. The BioSampler and AGI-30 were filled with 20 ml of PBS or TM and operated at 12.5 l/min for 3, 6, 15, 30, and 60 min, respectively. The PBS and TM in samplers were replenished every 15 min during 30- and 60-min samplings to improve the recovery of S. aureus as such advantage has been shown in Pantoea agglomerans (Rule et al., 2007) and L. pneumophila (Chang et al., 2010). Four repeated experiments were performed for each sampler with a specified collection medium and a given sampling time. The sampler type and collection medium were tested in a random order on different evaluation days. Tested AGI-30 and BioSampler were not reused after the sampling in each nebulizer run, and the bioaerosol generation system was sterilized by nebulizing 70% ethanol between runs.

The Andersen 1-STG loaded with TSA plate (20 ml/plate) was also run for 3, 6, 15, 30, and 60 min at 28.3 l/min. Four repeated experiments were conducted for each sampling time. To obtain countable numbers of colonies from TSA plates, the testing of various sampling times was performed in different runs with various initial cell concentrations in the nebulizer (3.8 × 103–8.5 × 104 cfu/ml), decreasing with extending the sampling time. In each run, the Andersen 1-STG was sterilized using cotton wool immersed in 70% ethanol and air dry prior to loading a TSA plate. The generation system was also sterilized with nebulization of 70% ethanol between runs.

After sampling, cell suspensions in the nebulizer, AGI-30, and BioSampler were diluted, inoculated in duplicate on TSA, and incubated for colony counting as described in the section of collection fluid. The Cair was then calculated for AGI-30 and BioSampler with specified collection fluid and sampling time. The Csusp was also determined for each of sampling combinations and used to calculate R value. As for Andersen 1-STG, TSA plates were directly incubated at 37°C for 24 h. The number of colony-forming units was then corrected by the positive-hole correction factor indicated in the report of Macher (1989), which was used to determine Nc, Cair, and R. The R value represented the recovery efficiency of various sampling combinations. Relative R value was further calculated by taking the mean R of 3-min sampling as the reference to assess the effects of sampling time on cell recovery.

Loss of collection fluid during sampling

The AGI-30 and BioSampler filled with DW, PBS, and TM, respectively, were weighed at the beginning and end of 3-, 6-, and 15-min samplings. The difference in liquid mass at the beginning and end of sampling was calculated. The percentage of liquid loss relative to the initial quantity was further determined to represent the loss of collection fluid due to sampling. Experiments were repeated four times.

Storage effect

Staphylococcus aureus cells in the nebulizer (1.0 × 108 cfu/ml, CV = 10.4%, 40 ml) were aerosolized and sampled for 3 min by an autoclaved AGI-30 containing 20 ml of TM and PBS, respectively. Afterward, an aliquot (0.1 ml) of collection fluid was serially diluted and plated on TSA to obtain an initial concentration (C0, cfu/ml). To determine the storage effects, the remaining collection fluid was separated into two equal parts and stored at 4°C and 23°C, respectively. The stored suspension was appropriately diluted and inoculated on TSA at an interval of 1 h at the first 8 h and at 20, 24, 28, 32, 44, and 48 h. The Ct (cfu/ml in fluids stored for t h) was then determined after an incubation at 37°C for 24 h. Effects of storage time were presented as Ct/C0. Four trials were conducted for each combination of selected fluid, storage time, and temperature.

Statistical analysis

The Wilcoxon rank-sum test (Mann–Whitney) was conducted to examine the difference in (1) the R value and liquid loss percentage between BioSampler and AGI-30, (2) R value between TSA and MSA and between PBS and TM, and (3) log cell concentration between two storage temperatures. The Kruskal–Wallis test and post hoc analysis by Scheffe's test were also performed to compare (1) the R value among three collection fluids, five sampling times, and five combinations of sampling techniques, (2) liquid loss percentage among three sampling times and three kinds of collection fluids, and (3) log cell concentration among various storage times. All statistical analyses were performed with SAS software version 9.1 (SAS Institute Inc., Cary, NC, USA). Statistical significance was considered as P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Recovery efficiency and liquid loss of collection fluid

Regardless of agar type, Figure 2 shows the TM had the greatest recovery of S. aureus, followed by PBS and DW. Statistically significant differences were detected in any of two kinds of fluids (P < 0.05). A greater recovery of airborne S. aureus on TSA than MSA was also noted (P = 0.02). As for liquid deprivation (Table 1), the loss percentage in BioSampler and AGI-30 was significantly increased with the sampling time from 3 min to 15 min (P < 0.05). There was no significant difference of liquid loss between AGI-30 and BioSampler (P = 0.13) and among three kinds of fluids (P = 0.09). However, the PBS presented the least loss percentage at 15-min sampling compared with DW and TM.

Table 1. Liquid loss in TM, DW, and PBS after operating BioSampler and AGI-30 for 3, 6, and 15 min
Sampling time (min)Collection fluidaLiquid loss (%)Scheffe testb
BioSamplerAGI-30
  1. a

    TM (Tween mixture): sterile deionized water containing 1% peptone, 0.01% Tween 80, and 0.005% Antifoam Y-30; PBS: phosphate-buffered saline; DW: sterile deionized water.

  2. b

    Sampling times with the different letter have liquid loss percentages that are statistically different (P < 0.05).

  3. c

    Standard deviation of the liquid loss percentage in parentheses (n = 4).

3TM4.3 (0.4)c3.7 (0.1)A
DW4.5 (0.3)3.4 (0.1)
PBS3.3 (0.2)2.8 (0.1)
6TM8.7 (0.5)7.0 (0.5)B
DW8.0 (0.4)6.5 (0.5)
PBS6.2 (0.8)5.0 (0.5)
15TM20.9 (1.3)16.6 (1.0)C
DW18.1 (0.5)15.0 (1.2)
PBS11.6 (0.7)10.8 (0.7)
image

Figure 2. Performance of Tween mixture (TM, ■), phosphate-buffered saline (PBS, □), and deionized water (DW, image) for recovering airborne Staphylococcus aureus by sampling with AGI-30 and plating on TSA and MSA plates. R represents a ratio of cell concentration in air of test chamber to that in liquid of a nebulizer of a bioaerosol generation system. Each error bar represents one standard deviation from a mean of four repeated samples

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Collectively, the PBS and TM showed greater cell recoveries while the DW retained low amounts of S. aureus with a noticeable liquid loss (Figure 2 and Table 1). Moreover, the TSA preserved cell culturability better than MSA (Figure 2). Thus, the PBS and TM were adopted for further tests on S. aureus, followed by TSA plating, to determine the effects of sampling time, sampling method, and storage condition.

Effects of sampling time on AGI-30

Airborne S. aureus was quantified by the AGI-30 operated sequentially for 3, 6, 15, 30, and 60 min (Cair, gray bars, Figure 3). The concentration of S. aureus in the liquid of the nebulizer was also determined at the beginning and each end of air samplings (Csusp, lines with circles, Figure 3). A gradual decline of Csusp was observed along with the operation time of the bioaerosol generation system (Figure 3a and b). A decrease in Cair with operation time was expected and indeed observed when sampling S. aureus into the PBS of AGI-30 (Figure 3a). However, it was not the case when the TM was adopted (Figure 3b), for which the Cair was increased at 6-, 15-, and 30-min samplings compared with that of 3 min.

image

Figure 3. Concentration of Staphylococcus aureus in liquid of a nebulizer (Csusp) and in air of test chamber (Cair) in an operated bioaerosol generation system. Cair was determined by sampling bacteria for 3–60 min using AGI-30 filled with (a) PBS and (b) TM. Each error bar represents one standard deviation from a mean of four repeated samples. *Air sampling time (min)

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By calculating the R value to adjust Csusp variation and further determining the relative R (Table 2), a decrease in cell recovery efficiency with the sampling time was found when collecting cells into the PBS of AGI-30 (P < 0.05). In contrast, sampling with TM for 6 and 15 min recovered S. aureus significantly greater than that of 3-min sampling by a factor of 2.75 and 1.67 (P < 0.05). The lowest cell recovery was observed at 60-min sampling irrespective of fluid type, which was significantly different from that of 3–15 min sampling (P < 0.05).

Table 2. Effects of sampling time on efficiency of AGI-30 filled with PBS and TM for recovering Staphylococcus aureus
Sampling time (min)PBSTM
Relative R aScheffe testcRelative RScheffe test
Means.d.bMeans.d.
  1. a

    R, defined as a ratio of the concentration of bioaerosols to that of bacteria suspended in liquid of a nebulizer, was used to represent the recovery efficiency of a sampling method. Relative R was further calculated using the mean R of 3-min sampling as the reference (n = 4).

  2. b

    Standard deviation.

  3. c

    Sampling times with the different letter have the recovery efficiencies that are statistically different (P < 0.05).

31.000.01A1.000.02C
60.370.05B2.750.39A
150.300.05B1.670.24B
300.110.01C1.410.09BC
600.080.01C0.240.02D

Effects of sampling time on BioSampler

Sampling by the BioSampler for various durations showed similar trends in relative R as observed in the AGI-30. In detail, the recovery efficiency of the BioSampler with PBS was continuously decreased with extending the sampling time, and a statistical difference was detected between any two of sampling durations (P < 0.05, Table 3). With the TM, a greater efficiency was revealed at the samplings for 6 and 15 min compared with 3 min by a factor of 1.13–1.16. Sampling for 60 min resulted in the lowest efficiency in both PBS and TM (P < 0.05). Collective analyses of all R values from BioSampler and AGI-30 further showed significantly greater cell recoveries with TM than PBS (P < 0.0001) and with BioSampler than AGI-30 (P < 0.0001).

Table 3. Effects of sampling time on efficiency of BioSampler filled with PBS and TM for recovering Staphylococcus aureus
Sampling time (min)PBSTM
Relative R aScheffe testcRelative RScheffe test
Means.d.bMeans.d.
  1. a

    R, defined as a ratio of the concentration of bioaerosols to that of bacteria suspended in liquid of a nebulizer, was used to represent the recovery efficiency of a sampling method. Relative R was further calculated using the mean R of 3-min sampling as the reference (n = 4).

  2. b

    Standard deviation.

  3. c

    Sampling times with the different letter have the recovery efficiencies that are statistically different (P < 0.05).

31.000.01A1.000.01BC
60.780.03B1.160.09A
150.700.02C1.130.05AB
300.400.03D0.900.07C
600.260.00E0.540.03D

Effects of sampling time on Andersen 1-STG

The capability of Andersen 1-STG to retrieving S. aureus onto TSA plates was determined over 3–60 min sampling (Figure 4). Comparable efficiencies were found at the samplings for 6–60 min (P > 0.05), which were 62–77% relative to that of 3-min sampling (P < 0.05).

image

Figure 4. Efficiency of five sampling methods operated for 3 (image), 6 (image), 15 (image), 30 (image), and 60 (image) min for recovering airborne Staphylococcus aureus (n = 4). Sampling methods with the different letter have the recovery efficiencies that are statistically different (P < 0.05)

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Recovery efficiency among Andersen 1-STG, AGI-30, and BioSampler

The performance of Andersen 1-STG was compared with that of AGI-30 and BioSampler containing PBS and TM. Figure 4 shows the Andersen 1-STG was less efficient than the BioSampler filled with TM or PBS (P < 0.05), comparable to the AGI-30 with TM (P > 0.05) but significantly greater than the AGI-30 with PBS (P < 0.05). Overall, the highest recovery efficiencies were obtained by the BioSampler with TM among five sampling methods (P < 0.05), whereas the AGI-30 with PBS presented the lowest recovery regardless of sampling time (P < 0.05, Figure 4).

Storage effects after sampling

When collecting airborne S. aureus into TM and stored at 4°C (Figure 5a), cell concentrations were found stable over 48 h (P > 0.05) with the Ct/C0 ratio between 0.9 and 1.4. For those stored at 23°C in TM, the Ct/C0 ratio was increased to over 104 after 28 h and the log Ct of any storage time was significantly greater than the log C0 (P < 0.05, Figure 5a). Cell counts between two temperatures were statistically different (P < 0.0001). As for the cells stored in PBS (Figure 5b), cell concentrations were continuously decreased with time in similar trends between 4°C and 23°C (P = 0.23). Significant differences between log C0 and log Ct occurred at the storages for 7–48 h (Ct/C0 ≤ 0.58, P < 0.05).

image

Figure 5. Effects of storage time on concentration of Staphylococcus aureus sampled by AGI-30 into (a) TM and (b) PBS at 12.5 l/min for 3 min and stored at 4°C (image) and 23°C (image). C0 and Ct represent the cell concentration right after sampling and at various storage times, respectively. Each error bar represents one standard deviation from a mean of four repeated samples

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study shows airborne S. aureus collected and stored in TM multiplied rapidly at 23°C (Figure 5a), consistent with the literature on Escherichia coli and Bacillus subtilis spores being stored at 25°C (Li and Lin, 2001). This finding is probably attributed to the peptone and Tween 80 in TM. The peptone, including a variety of polypeptides, has been identified as the pivotal nutrients for S. aureus growth (Gray et al., 2008), while the Tween 80 (C64H124O26) can be degraded by the lipase of S. aureus (Gould et al., 2009) and has been demonstrated to support the growth of Staphylococcus warneri (Prema et al., 2006). Interestingly, such beneficial effects of TM occurred not only during the sample storage but also at the air sampling, evident by increased airborne cell concentrations after 6- and 15-min sampling into TM of AGI-30 even though the cell counts in the nebulizer were continuously decreased (Figure 3b) and 16.6% of TM was lost at the end of 15-min sampling (Table 1). Liquid loss could cause cell bounce and reaerosolization from the AGI-30, resulting in decreased cell counts (Willeke et al., 1998). Even so, our results strongly suggest that the culturability of S. aureus was preserved in TM during the bioaerosol sampling process.

The PBS, composed of inorganic compounds, had the least percentage of liquid loss in BioSampler and AGI-30 (Table 1); however, its recovery for S. aureus was lower than TM (Figures 2 and 4) and significantly decreased with sampling time (Tables 2 and 3). These results suggest that the culturability of S. aureus was adversely affected by the shear force of airflows passing through the PBS in the magnitude related to the sampling duration. A lack of organic compounds in PBS induced a further loss of cell culturability during the storage, evident by continuously decreased cell concentrations at both 4°C and 23°C (Figure 5b). Detrimental influences of PBS have also been noted in P. agglomerans (Rule et al., 2007).

The least recovery of S. aureus was observed with DW (Figure 2), partly ascribed to neither nutrient nor buffer available in DW to support bacterial survival. A lower efficiency with DW than PBS has also been revealed in collecting P. agglomerans (Rule et al., 2009). However, the present result disaccords with our previous finding that a greater collection of culturable L. pneumophila in DW than TM (Chang and Chou, 2011a; Chang et al., 2010). Different susceptibilities of bacteria to 0.01% of Tween 80 may be one of the explanations for this inconsistency as the literature shows Tween 80 should be limited to 0.001% to avoid its interference on Legionella growth (Mondello et al., 2009), whereas Tween 80 at ≥0.1% facilitates Staphylococcus growth and its enzyme activity (Gould et al., 2009; Prema et al., 2006).

In addition to the type of collection fluid, the samplers with different design in liquid impingement also showed an impact on S. aureus recovery. With increasing sampling time, a greater reduction of cell recovery was observed in AGI-30 than BioSampler filled with PBS (Tables 2 and 3). A greater variability of recovery efficiency was also revealed with AGI-30 filled with TM (relative R = 0.24–2.75) than BioSampler (0.54–1.16). BioSampler and AGI-30 are expected to have similar physical efficiency to capture airborne S. aureus as their d50 values are similar (~0.3 μm). Thus, different abilities of the samplers to retain the collected cells and maintain their culturability may contribute to the differences in S. aureus recovery. The airflow in AGI-30 impinged vertically downward into the liquid, causing shear force and violent bubbling that could damage and reaerosolize the collected cells (Lin et al., 2000), thereby decreasing the recovery of S. aureus. However, turbulent bubbling in AGI-30 might also increase the accessibility of collected cells to the nutrients (e.g., Tween 80 and peptone), enhancing cell culturability in TM. Compared with the AGI-30, fewer bubbles were generated from a swirling motion of liquid in the BioSampler, which possibly minimized the reentrainment of collected cells and the sampling stresses they suffered (Lin et al., 2000) but could also reduce the chance of cells to access nutrients. Consequently, the BioSampler presented a lower reduction and fluctuation in cell recovery with PBS and TM, respectively, relative to the AGI-30. In terms of sampling time, sampling for 60 min resulted in the lowest recovery efficiencies regardless of sampler type and collection fluid used (Tables 2 and 3). This consistent finding suggests that a long sampling of 60 min by liquid impingement might not be suitable for quantifying S. aureus even with a refilling of fluid every 15 min.

Our results show the Andersen 1-STG was less efficient than BioSampler filled with TM or PBS for retaining S. aureus (P < 0.05, Figure 4). This finding, in line with previous studies on culturable L. pneumophila (Chang et al., 2010; Deloge-Abarkan et al., 2007), may be partly due to a greater d50 of the Andersen 1-STG (0.65 μm) than the BioSampler (0.3 μm) and a lower sampling stress with BioSampler than Andersen 1-STG (Deloge-Abarkan et al., 2007).

The BioSampler with TM presented the greatest recovery efficiencies among five tested methods (Figure 4), suggesting the applicability of this sampling method for recovery of environmental S. aureus aerosols. This is the first report to demonstrate the performance of the BioSampler for S. aureus collection. Considering S. aureus may significantly replicate in TM at 23°C but remain stable at 4°C (Figure 5), the sampling of S. aureus by the BioSampler with TM should be followed by sample transportation and storage at 4°C.

This study was conducted under controlled situations for adequate comparison of the samplers; however, the laboratory conditions might not be the same as those occurred in real environments. For example, airborne S. aureus in fields could aggregate or attach to larger particles, increasing their aerodynamic diameter and cell recovery by inertial sampling devices. The organic matter of particles could be abundant in places such as livestock farms, which might compensate for use of non-enriching PBS or DW in retaining cell culturability. Thus, future research on field comparison of sampling methods is warranted as it is difficult to simulate complicated environments under experimental conditions.

In conclusion, this study demonstrates that the BioSampler was efficient to recover airborne S. aureus and the TM was superior to PBS and DW to preserve cell culturability. However, S. aureus collected in TM needs to be stored at 4°C to preventing cells from noticeable replication.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors are grateful to Dr. Sheng-Hsiu Huang for his technical assistance. This study was supported by the Institute of Occupational Safety and Health, Council of Labor Affairs, Taiwan.

References

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  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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