Characterization of Biological Aerosol Exposure Risks from

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Characterization of Biological Aerosol Exposure Risks from Automobile Air Conditioning System Jing Li,†,§ Mingzhen Li,†,§ Fangxia Shen,† Zhuanglei Zou,† Maosheng Yao,*,† and Chang-yu Wu‡ †

State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China ‡ Department of Environmental Engineering Sciences, University of Florida, Gainesville, Florida 32611, United States S Supporting Information *

ABSTRACT: Although use of automobile air conditioning (AC) was shown to reduce in-vehicle particle levels, the characterization of its microbial aerosol exposure risks is lacking. Here, both AC and engine filter dust samples were collected from 30 automobiles in four different geographical locations in China. Biological contents (bacteria, fungi, and endotoxin) were studied using culturing, high-throughput gene sequence, and Limulus amebocyte lysate (LAL) methods. Invehicle viable bioaerosol concentrations were directly monitored using an ultraviolet aerodynamic particle sizer (UVAPS) before and after use of AC for 5, 10, and 15 min. Regardless of locations, the vehicle AC filter dusts were found to be laden with high levels of bacteria (up to 26 150 CFU/mg), fungi (up to 1287 CFU/mg), and endotoxin (up to 5527 EU/mg). More than 400 unique bacterial species, including human opportunistic pathogens, were detected in the filter dusts. In addition, allergenic fungal species were also found abundant. Surprisingly, unexpected fluorescent peaks around 2.5 μm were observed during the first 5 min use of AC, which was attributed to the reaerosolization of those filter-borne microbial agents. The information obtained here can assist in minimizing or preventing the respiratory allergy or infection risk from the use of automobile AC system.

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INTRODUCTION An automobile becomes a necessity in modern life, especially in developed nations; and a significant fraction of the population spends a considerable amount of time in passenger cars on a daily basis. For taxi drivers, such a time could be up to 10 h or more per day. Apart from particulate matter (PM), volatile organic compounds (VOCs), hydrocarbons, and tobacco smoke, bioaerosol exposure inside a vehicle has attracted great attention in recent years.1−4 The in-vehicle maximum bacterial aerosol concentration was shown to be up to 2550 CFU/m3, which was about 46 times higher than the in-vehicle background;1 and Cladosporium, Penicillium, Aspergillus, and Alternaria were found to be the dominant fungal genera.1 Exposure to biological aerosols can cause numerous adverse health effects including lung impairment, respiratory allergies, or infections. Thus, ensuring biologically good air quality inside the vehicle is of vital importance to the health of the in-vehicle occupants. A number of control strategies in terms of the vehicle evaporator surface condensate or cooling process induced microbial growth have been investigated, e.g., integration of ultraviolet light,5 biocide layer,6 and method of drying the condensate on the heat exchanger.7 In addition to providing thermal comfort, the cabin air conditioning system employs an air filter to capture airborne particles from outside. Therefore, © 2013 American Chemical Society

outside pollutants could be effectively prevented from getting inside the vehicle, thus acting as a pollution protection measure. Rudell et al. showed that the use of automotive cabin air filters can clearly reduce the acute health effects of diesel exhaust in human subjects due to its practical filtration effects.8 Use of air conditioning system in the cabin has also been shown to reduce more than 80% of the total number of microorganisms, including bacterial and fungal spores.1,3 In another separate study, use of air conditioner was also shown to effectively suppress the bioaerosol levels.4 Similar to indoor AC, those bacteria and fungi filtered from the air stream by the automobile air conditioner filter could proliferate under high humidity conditions, i.e., under raining or snowing conditions, thus presenting a source of biological exposure risk. When the air conditioning system is turned on, the air stream passing through the vehicle filtration system could reaerosolize AC filter-borne bacteria and fungi and subsequently carry them into the vehicle. Depending on the microbial types, the inhalation exposure can cause infections or allergic reactions. Such a possible risk is evidenced by a Received: Revised: Accepted: Published: 10660

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culturable concentration levels in the dust samples, 100 μL of each extraction was plated on Trypticase Soy Agar (TSA) (Becton, Dickson and Company, Sparks, MD) plate for bacteria culturing at 26 °C for 2−3 days and three replicates were performed. Likewise, 100 μL of the extraction was pipetted onto a Malt Extract Agar (Becton, Dickson and Company, Sparks, MD) plate for fungal culturing at 26 °C for 3−5 days with triplicates. Use of 26 °C would closely represent an average temperature in an indoor setting, while providing necessary warmth needed for microbial growth. The colony forming units (CFUs) developed were manually counted and the bacterial species were further identified by a highthroughput gene sequence technology using the 454 GS-FLX pyrosequencing platform (Majorbio, Inc., Shanghai, China). The entire procedure similar to the protocol reported by Hospodsky et al. was followed.10 Fungal genera were identified based on their morphological characteristics by comparing both CFU and microscopic images according to a reference book.11 For each city, the dust suspensions from all AC or engine filter samples were pooled separately for microbial species analysis. For endotoxin content, the dust sample suspension was further diluted 1000 times for analysis using Limulus amebocyte lysate (LAL) (Associates of Cape Cod Inc., East Falmouth, MA) according to the manufacturer’s instructions and the procedure documented in a previous study.12 To investigate the biological particle size distributions in the filter dusts collected, those dust suspensions prepared from the same city were first mixed, and then aerosolized using a Collison nebulizer (BGI, Inc.) at a flow rate of 2.5 L/min in a biological safety cabinet. The fluorescent aerosol particles (viable bioaerosols) in the resultant aerosols were monitored using the UVAPS (TSI, Inc.). The UVAPS detects the size distributions and concentration levels of viable bioaerosol particles by measuring the intrinsic fluorescence level in viable bioaerosol particles. The intensity of fluorescence directly reflects the stage of bacterial growth. The specific characteristics and applications of UVAPS are detailed elsewhere.13 To study the possibility of the bioparticle reaerosolization by the blowing cool air during the use of the vehicle cabin AC system, we also directly monitored the viable bioaerosol particle concentration levels inside the automobile with the AC turned on for 0, 5, 10, and 15 min, respectively, using the UVAPS. Statistical Analysis. One-way ANOVA analysis was used to analyze the differences in bacterial and fungal concentration levels in automobile AC filter dust samples. A p-value of 0.05 indicates a statistically significant difference. In addition, various statistics including Ace, Chao, Shannon, and Simpson were calculated for bacterial community richness and diversity based on a similarity confidence of 97%.

previous study in which a 40-year-old man got sick after exposure to a contaminated vehicle air conditioning system.9 Accordingly, it is highly possible that many such exposures associated with the use of automobile AC could have gone undetected or simply neglected in our daily life. Unfortunately, information about microbial species distribution in the AC filter and the risk of their reaerosolization is not well characterized. This work was designed to study the potential biological exposure risks associated with an automobile air conditioning system that is coupled with filtration. In brief, automobile AC filters and engine filter dusts were collected from vehicles in four different Chinese cities. The bacterial, fungal species, endotoxin, and their concentration levels in the dusts were studied using culturing, high-throughput gene sequence, and LAL methods. The bioaerosol levels inside the vehicle were also monitored using an ultraviolet aerodynamic particle sizer (UVAPS) without and with AC turned on for different periods of time. This work presented evidence that use of automobile AC, if not cleaned or disinfected properly, would predispose the in-vehicle occupants to respiratory allergies or infections.

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MATERIALS AND METHODS To characterize microbial aerosol exposure risks, automobile AC filter dusts were collected from randomly selected vehicles in several different geographical locations such as Beijing, Shanghai, Guangzhou, and Haikou as depicted in Figure 1

Figure 1. Vehicle AC/engine filter dust collection map (n = 11 for Beijing, n = 5 for Guangzhou, n = 12 for Haikou, and n = 2 for Shanghai), where n indicates the sample size.

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during March−April, 2013. According to China Meteorological Administration, the average temperatures and humidity levels for the time period indicated above were 0.8−11.6 °C, 38.6% for Beijing; 11.6−20.6 °C, 56.7% for Shanghai; 18.4−24 °C, 80.5% for Guangzhou; and 22.7−29 °C, 81.1% for Haikou. A total of 53 AC and engine filter dust samples were collected from 30 automobiles, including 11 in Beijing, 5 in Guangzhou, 12 in Haikou, and 2 in Shanghai. For most of these vehicles, both AC and engine filter dust samples were collected and subsequently stored using 50-mL corning tubes at 4 °C. To analyze the biological contents such as bacteria, fungi, and endotoxin, the filter dust samples were first extracted by DI water using the ratio of 1 mg of dust per 1 autoclaved mL of DI water by vigorous vortexing for 20 min at a vortex rate of 3200 rpm (Vortex genie-2, Scientific Industries Co., Ltd.). For

RESULTS AND DISCUSSION Although use of automobile cabin AC system could lead to decreased bioaerosol concentration levels inside the vehicle, the results from this study indicated that the biologically contaminated AC filter could pose a significant health hazard through reaerosolizing opportunistic pathogenic or allergenic aerosols. As observed in Figure 2, culturable bacterial concentrations in the AC filter dust collected from the automobiles were shown to vary significantly from vehicle to vehicle for all cities. The differences observed could arise from the characteristics of a particular vehicle such as the frequency of use of the AC system, the maintenance, and age and quality of the filter. The variations observed from Figure 2 certainly 10661

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Figure 2. Culturable bacterial concentrations (box plot) in the dusts collected from vehicle-borne air conditioner and engine filters in different Chinese cities. Black circle is the outlier; the lines from top to the bottom are for maximum, upper quartile, median, mean (black bold line), lower quartile, and minimum. n indicates the sample size.

Figure 3. Endotoxin concentrations in the dusts collected from vehicle-borne air conditioner and engine filters in different Chinese cities. Data points represent the values of the mixture from all dusts collected for a specific city. n indicates the sample size (endotoxin was not measured for Shanghai dust).

reflect the influences of these parameters discussed. However, such information was not available for each automobile at the time of the study. This is largely due to the fact that vehicle maintenance records are not well kept or centralized either in China. These factors to some extent could affect the total amount of dusts on the vehicle filters, but they would have fewer effects on the microbial contents per unit of dust which largely depended on the local air quality. Accordingly, these factors would have limited impact on our major research objectives. On average, vehicle filter dusts from Beijing were found to contain the highest culturable bacteria, up to 26 150 CFU/mg, followed by Shanghai, Haikou, and Guangzhou as can be observed in Figure 2. ANOVA tests show that there is a statistically significant difference in bacterial concentration levels in the AC filter samples among different cities (p-value = 0.003), except that between Guangzhou and Haikou (Tukey HSD test of post AVOVA analysis). Similar findings were found for the engine filter dust samples as shown in Figure 2. Overall, the averages of bacterial concentrations (CFU/mg) in AC filter dusts were found higher than those in the engine filter dust samples regardless of dust sources as observed in Figure 2. The pore sizes of AC and engine filters might be different; however, after the use of AC for certain amount of time the filter pores might be clogged by the dusts, accordingly effects of pore size should be diminished. Herein, samples from Shanghai were not involved in the statistical analysis due to its small sample size (n = 2). Different bacterial concentrations in the filter dust samples could to some extent reflect the ambient bioparticle concentration for a specific location given a similar automobile condition. In future studies, sampling sites such as inland and western parts of China can be further investigated and compared. Elucidation of specific microbial types in the AC filter dusts would be helpful to evaluate the exposure risks to the in-vehicle occupants. Figure 3 displays endotoxin levels in the AC filter dust samples. Similar to the bacterial concentration, the endotoxin level detected in Beijing vehicle filter dusts was found the highest with an average of 5527 EU/mg, followed by Haikou and Guangzhou. A recommended inhalation exposure dose for endotoxin is 614 EU/m3,14 thus if 1 mg could be reaerosolized into the air the exposure risk would exceed the recommend

value given the size of automobile cabin. In this work, all filter dust samples from the same geographical location were pooled together for endotoxin analysis, thus its variations among dust samples were not obtained. Similar to bacterial concentrations, AC filter dusts were found to contain higher endotoxin levels than the engine filter dusts as observed in Figure 3. By comparing data in Figure 2 and Figure 3, the high endotoxin level detected in Beijing dusts corresponded to the high level of culturable bacteria concentration as shown in Figure 2. Endotoxin, also called lipopolysaccharide (LPS), is a major constituent of the outer cell membrane of Gram-negative bacteria. Inhalation of endotoxin could cause various respiratory problems such as fever, shivering, or arthralgia.15 A good correlation was obtained between positive tests for endotoxin using LAL method and bacteremia due to Gram-negative organisms.16 The key effects of endotoxin are mediated by their interaction with specific receptors on immune cells such as monocytes, macrophages, dendritic cells, and others. Accordingly, exposure to high endotoxin levels in the filter dusts could be very toxic to human health. Overall, more than 400 unique bacterial types including dominant species such as Pseudomonas and Staphylococcus spp. were detected in the automobile filter dusts in this work, and the complete list of bacterial species and genera identified are shown in excel file S1 (Supporting Information (SI)). The top 20 bacterial genera identified in the automobile AC and engine filter dusts using the 454 GS-FLX pyro-sequencing platform with multiplex identifiers (MIDs) are shown in Table 1 (for Beijing filter dusts) and SI Figures S1−S4 (for Guangzhou, Haikou, and Shanghai filter dusts). The statistics regarding the bacterial community richness and diversity of filter dust samples including Ace, Chao, Coverage, Shannon, and Simpson are shown in SI Table S1. As observed from values of Coverage in the table, generally close to or higher than 90% of the bacterial species were sequenced for the samples collected. All estimators shown in Table S1 indicated that dusts from both AC and engine filters in Haikou had the highest bacterial community richness and diversity, followed by Shanghai, Beijing, and Guangzhou. It was also found that dust samples from AC filters had higher bacterial richness and diversity than those of engine filter for the cities studied. For all dust samples, the dominant phyla were Firmicutes, Actinobacteria, and Proteobacteria as 10662

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Table 1. Relative Abundances of Top Twenty Most Abundant Bacterial Genera Identified in the Vehicle AC and Engine Filter Dust Samples Collected in Beijing Based on a Similarity Confidence Level of 97% (10 AC Filter Samples and 11 Engine Filter Samples Were Pooled Together, Respectively, for Sequencing) Beijing AC filter dusts

Beijing engine filter dusts

top 20 bacterial genera

relative abundance (%)

top 20 bacterial genera

relative abundance (%)

Exiguobacterium Microbacterium Bacillus Sporosarcina no_rank_Planococcaceae Micrococcus Kocuria Lysinibacillus Arthrobacter Massilia Bhargavaea Pseudomonas Psychrobacter Alicycliphilus unclassif ied_Bacillales Acinetobacter Stenotrophomonas Carnobacterium Dietzia Curtobacterium

63.9019 10.3154 8.9720 3.6098 3.4463 1.8925 1.7874 1.6472 0.7944 0.5140 0.3154 0.2921 0.2336 0.2220 0.2220 0.2103 0.2103 0.1869 0.1285 0.1168

Exiguobacterium Bacillus Microbacterium Sporosarcina no_rank_Planococcaceae Arthrobacter Kocuria Solibacillus Micrococcus unclassif ied_Bacillales Citricoccus Dietzia unclassif ied_Actinobacteria unclassif ied_Bacteria Streptomyces Georgenia unclassif ied_Planococcaceae Brevibacillus Bhargavaea Lysinibacillus

81.6344 8.3026 4.0680 1.6891 0.9992 0.6780 0.5828 0.4282 0.3687 0.2141 0.1546 0.1427 0.1189 0.1071 0.0833 0.0714 0.0595 0.0595 0.0476 0.0238

shown in SI Figure S2. For samples from Haikou, more than 99% were Firmicutes as observed in the figure. Among the bacterial genera detected, Exiguobacterium, Bacillus, Staphylococcus, Pseudomonas, Lysinibacillus, Microbacterium, and Pantoea were found to dominate the dust samples as observed from Table 1 and SI Figures S1−S4. Overall, filter samples from different locations were shown to have distinctive bacterial population compositions. In addition to bacteria and endotoxin, high level fungal concentrations up to 1377 CFU/mg were also detected in the dust samples collected. In contrast, no statistically significant differences were observed among the culturable fungal concentration levels for different cities (p-value = 0.43 and 0.79 for AC and engine filters, respectively) as shown in Figure 4. Compared to the bacterial concentration levels shown in Figure 2, the culturable fungal concentrations seemed to be much lower as observed in Figure 4. This observation is in line with our previous studies, in which ambient culturable bacterial concentration in general exceeded that of fungal species.17,18 In this work, fungal species were identified by comparing CFU and microscopic images according to a reference book and are listed in Table 2. As shown in the table, the dominant fungal genera were Aspergillus, Penicillium, Cladosporium, and Alternaria. All of these fungal types could cause respiratory problems when inhaled. It seems Beijing had the highest diversity of fungi detected in the vehicle filter dusts. Differences in dominant fungal types could be a result of different temperature and humidity in different climate zones as implicated in four different cities. Thus, information about the microbial structures in the automobile filter dusts might reveal, in addition to the exposure risk, the climate conditions for a specific geographical location. Apart from the culturable microbial concentration levels, we also studied the biological agent size distributions in the dust samples using UVAPS. Figure 5 shows the size distribution patterns of fluorescent particles (viable biological agent) in the

Figure 4. Culturable fungal concentrations (box plot) in the dusts collected from vehicle-borne air conditioner and engine filters in different Chinese cities. Black circle is the outlier; the lines from top to the bottom are for maximum, upper quartile, median, mean (black bold line), lower quartile, and minimum. n indicates the sample size.

aerosols generated from vehicle dusts collected from the automobiles in different cities. For the automobile filter dusts from different cities, they exhibited distinctive fluorescent size distributions as revealed in the figure. For AC filter dusts, Haikou seemed to have more fluorescent peaks, and for engine filter Beijing seemed to have more fluorescent peaks. Because of variations in the aerosolization process, it was difficult to assess the absolute fluorescent particle concentration in the dust samples. The differences observed in microbial contents between the AC and engine filter dusts were likely due to the different temperature levels on the filter. Engine filters close to the engine had much higher temperatures than AC filters, thus theculturable bacteria detected on engine filters tended to be more thermophilic, and were able to survive temperatures between 45 and 122 °C. Regardless of dust sources, all 10663

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infections. Data from Figures 2−5, Table 1, and SI Figures S1− S6 clearly revealed that automobile AC filters harbored significant amounts of biological agents including diverse bacteria and fungi, and high levels of endotoxin; and some of these agents could reproduce under high humidity conditions. It was highly possible that yeasts were also present in the dust samples, but given the project’s scope only bacterial gene sequence was performed for the dust samples, and identification of yeasts was not performed. To investigate the possibility of reaerosolization of these biological agents during the use of automobile AC, we monitored the airborne fluorescent particle size distribution inside an automobile with and without turning on the AC. As observed in Figure 6, the concentration levels of fluorescent

Table 2. Fungal Species Identified from the Vehicle AC Filter Dusts for Three Different Chinese Cities (+ Means “Detected”, and − means “Non-detected”) fungal species

Beijing

Guangzhou

Haikou

Alternaria alternata Aspergillus niger Aspergillus f umigatus Aspergillus ustus Aspergillus oryzae Aspergillus ochraceus Aspergillus terreus Aspergillus restrictus Aspergillus versicolor Aspergillus sydowii Aspergillus amstalodami Cladsporium cladosporioides Penicillium citrinum Penicillium oxalicum Penicillium chrysogenum verrucosum Trichoderma viride Curvularia lunata Phoma

+ + + + + + + + _ _ _ + + + + + + + +

+ + − + − + + − + + − − + + − + − − −

+ + − + − + + − + + + + + + − + + + −

Figure 6. Size and concentration distributions of airborne fluorescent particles detected using UVAPS inside a vehicle in Beijing with air conditioner off and on for 5, 10, and 15 min.

particle (viable bioaerosols) decreased significantly with time when the AC was turned on, especially pronounced after 10 min. However, an interesting peak was observed at 2.5 μm after use of the AC for 5 min, which was unexpected since the use of AC should reduce the particle concentration in all size ranges due to filtration of the particles from the outdoor air and the dilution of in-vehicle air by incoming filtered air. This can be also observed in SI Figure S6A in which a new fluorescent peak was observed around 2.5 μm after AC was turned on for 5 min. This unexpected peak can be only attributed to the reaerosolization of biological particles present in the filter dusts. After 10 and 15 min, the fluorescent particle concentration was reduced substantially for most size ranges as observed in Figure 6 and Figure S6. Accordingly, it is important that precautions be taken in order to avoid the microbial aerosol exposure risks when automobile AC is to be used, especially for a vehicle that has not been used for a long time. In a previous study, elevated microbial concentrations were also observed within 5−15 min after the use of an automobile or household AC, and subsequently these concentrations were found to decrease over time.1 In another study, 67% increase in bacterial counts and even doubling for fungal concentration were observed when the AC was turned on with an old filter.3 However, when the old filter was replaced with a new one, the in-vehicle microbiological air quality was significantly improved.3 This clearly implied that the old filter was an

Figure 5. Normalized size distributions (dN/dLogDp) of fluorescent particles detected using UVAPS from aerosolized dusts collected from vehicle-borne filters (AC and engine (E)) in different Chinese cities: B, Beijing; G, Guangzhou; H, Haikou.

generated fluorescent particle size distribution patterns were remarkably different from those of both indoors and outdoors obtained for Beijing. Differences in fluorescent particle size distribution could reveal the microbial characteristics for a specific geographical location. The fluorescence intensity levels, which reflect the stage of bacterial growth, shown in SI Figure S6 also suggest that bacteria and fungi present in the dust samples were more metabolically active than those of indoor or outdoor aerosol particles. This, on the other hand, suggests that vehicle filter dusts with a shield from atmospheric solar irradiation provide a better survival environment than the ambient air. Under high humidity levels, an automobile filter could be a hotbed for incubating many pathogens and presenting an important source of respiratory allergies or 10664

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important source of microbial agents. Since the AC filter is directly exposed to the outside air, those filter-borne biological agents could rapidly reproduce when the humidity level is high, e.g., during or after a rain. Similarly, it was shown that after installation of a new air conditioner, Acinetobacter spp. bloodstream infections (A-BSIs) occurred more frequently during the months of increased absolute humidity levels or environmental dew point.19 Automobile AC is identical to the indoor AC system with respect to filter system. In one study, it was found that respiratory allergies were also associated with automobile air conditioners.20 Kumar et al. also showed that after turning on the vehicle air conditioner the owners experienced exacerbation of allergic rhinitis and bronchial asthma.21 In another study, the automobile air conditioning system was shown to be contaminated by various types of fungi including Acremonium, Aspergillus, Alternaria, Aureobasidium, Cladosporium, and Penicillium, and the evaporator was also found to be colonized by odor-producing fungi Penicillium viridicatum.22 In addition, volatile organic compounds such as 4-methyl thiazole, terpenes alcohols, dimethyl disulfide, dimethyl trisulfide, and chlorophenol were also detected from a fungi-contaminated AC system.23 In a previous study, high levels of endotoxin (19.9−247.0 EU/ mg) and β-(1,3)-glucan (1.6−59.8 μg/g), which are of importance for asthmatics, were detected in the dusts collected from passenger seats of 40 automobiles.24 Related to air conditioning system, Legionella bacterial species were also detected in cooling towers as well as humidifiers. However, in this work no Legionella species were detected in the filter dust samples. Neglected in other studies, automobile air conditioning filter has been demonstrated in this work to be heavily contaminated with various microbial agents, including many human opportunistic pathogens and high levels of endotoxin. For those cars which have been dominant in a high humidity level and have not been used for a long time, use of air conditioning is not recommended when driving until it is properly cleaned, e.g., dumping the dusts and/or disinfecting the filter. For convenience, turning on AC with automobile cabin windows open for 15 min or longer before use could also avoid or minimize the microbial exposure risks. The results from this work suggest that relevant information as described above be provided in the user’s manual of an automobile to protect the occupant’s health.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

Additional tables and figures as mentioned in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: +86 010 6276 7282. Author Contributions §

M.L. and J.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Science Foundation of China (Grants 21277007 and 41121004). 10665

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(21) Kumar, P.; Lopez, M.; Fan, W.; Cambre, K.; Elston, R. C. Mold contamination of automobile air conditioner systems. Ann. Allergy 1990, 64, 174−177. (22) Simmons, R. B.; Noble, J. A.; Rose, L.; Price, D. L.; Crow, S. A.; Ahearn, D. G. Fungal colonization of automobile air conditioning systems. J. Ind. Microbiol. Biotechnol. 1997, 19, 150−153. (23) Rose, L. J.; Simmons, R. B.; Crow, S. A.; Ahearn, D. G. Volatile organic compounds associated with microbial growth in automobile air conditioning systems. Curr. Microbiol. 2000, 41, 206−209. (24) Wu, F. F.; Wu, M. W.; Chang, C. F.; Lai, S. M.; Pierse, N.; Crane, J.; Siebers, R. Endotoxin and β-(1,3)-glucan levels in automobiles: A pilot study. Ann. Agric. Environ. Med. 2010, 17, 327− 330.

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