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Characteristics of airborne bacteria in Mumbai urban environment
Science of the Total Environment 488–489 (2014) 70–74
Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Characteristics of airborne bacteria in Mumbai urban environment S. Gangamma ⁎ Department of Chemical Engineering, National Institute of Technology Karnataka, Surathkal, India
H I G H L I G H T S • • • •
Study demonstrated association between endotoxin and PM induced pro-inflammatory response Human or animal flora are identified as vital component of airborne culturable bacteria in Mumbai Study shows significant presence of pathogenic and opportunistic bacteria in the study area Study highlights the importance of airborne biological particles of ambient PM
a r t i c l e
i n f o
Article history: Received 9 February 2014 Received in revised form 14 April 2014 Accepted 16 April 2014 Available online xxxx Editor: Xuexi Tie Keywords: Airborne bacteria Endotoxin Proinflammatory Whole blood assay Tumor necrosis factor
a b s t r a c t Components of biological origin constitute small but a significant proportion of the ambient airborne particulate matter (PM). However, their diversity and role in proinflammatory responses of PM are not well understood. The present study characterizes airborne bacterial species diversity in Mumbai City and elucidates the role of bacterial endotoxin in PM induced proinflammatory response in ex vivo. Airborne bacteria and endotoxin samples were collected during April–May 2010 in Mumbai using six stage microbial impactor and biosampler. The culturable bacterial species concentration was measured and factors influencing the composition were identified by principal component analysis (PCA). The biosampler samples were used to stimulate immune cells in whole blood assay. A total of 28 species belonging to 17 genera were identified. Gram positive and spore forming groups of bacteria dominated the airborne culturable bacterial concentration. The study indicated the dominance of spore forming and human or animal flora derived pathogenic/opportunistic bacteria in the ambient air environment. Pathogenic and opportunistic species of bacteria were also present in the samples. TNF-α induction by PM was reduced (35%) by polymyxin B pretreatment and this result was corroborated with the results of blocking endotoxin receptor cluster differentiation (CD14). The study highlights the importance of airborne biological particles and suggests need of further studies on biological characterization of ambient PM. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Airborne particulate matter (PM) contain significant fraction (16%) of biological components (Jaenicke, 2005) and likely to play a significant role in health and climate. Airborne bacteria are one of the important components of airborne biological particles in natural and urban environment (Jaenicke, 2005; Brodie et al., 2007; Elbert et al., 2007; Jaenicke et al., 2007; Bowers et al., 2011). Bacteria present in the ambient atmosphere originate from natural and anthropogenic sources such as plants, animals, soil, water bodies, waste dumping ground, wastewater treatment plants and agricultural activities. Many human activities such as solid waste and sewage transport, processing and conducive humid environment may enhance the abundance of culturable airborne bacteria in an urban environment.
⁎ Tel.: +91 824 2474000; fax: +91 824 2474033. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.scitotenv.2014.04.065 0048-9697/© 2014 Elsevier B.V. All rights reserved.
Properties of urban airborne bacteria are studied over decades (Mancinelli and Shulls, 1978; Brodie et al., 2007; Fang et al., 2007; Bowers et al., 2011). These studies identified the dominant bacterial species in urban environments and have shown that the species properties and their proportions vary significantly among urban settings. Change in proportion of dominant airborne bacterial species with meteorological and micro-environmental properties was studied in various environmental settings (Lighthart et al., 2009; Wang et al., 2010). However, important factors leading to the variability in species composition, source contribution and survivability in urban air environment are not yet well understood. Health impacts of airborne bacteria and their components are also not well studied. Endotoxin is a cell wall component of Gram-negative bacteria (GNB). Initially, significant levels of airborne endotoxin was detected in several occupational environments such as cotton industry, agriculture processing, barns, sewage treatment, composting and waste handling. These studies have shown that in occupational environments, endotoxin is of one the most important component associated with
S. Gangamma / Science of the Total Environment 488–489 (2014) 70–74
health effects of PM exposure. Similarly, endotoxin in indoor environments such as in homes and offices has been characterized and linked to adverse health effects. Endotoxin is known to be present in ambient PM at low levels. Now it is known that ambient airborne endotoxin plays a significant role in particulate matter induced proinflammatory response. However, the exact role of low levels of endotoxin in inducing proinflammatory response is not well understood (Gangamma, 2012a, 2012b). Mumbai is one of the most densely populated cities in the world. Many human activities such as solid waste and sewage transport and processing may enhance the ambient concentration of bioaerosols in the city. However, the characterization of biological components of the ambient PM in the city has not been previously attempted. The present study characterizes species diversity of airborne bacteria in Mumbai. The paper adopts two methods to highlight the importance of airborne bioparticles in Mumbai. First method utilizes the species characterization information of airborne bacteria. Second method utilizes endotoxin which is an important fraction of airborne bioparticles to explain the inflammatory response of total particulate matter. Based on these methods, the study accentuates the importance of biological fraction of urban PM in Mumbai. 2. Materials and methods 2.1. Sampling of airborne bacteria and endotoxin The air samples were collected from the four ambient air quality monitoring sites (Fig. S1) in Mumbai during April–May 2010 with microbial Anderson impactor (Thermo Scientific USA, n = 22) and biosamplers (SKC USA, n = 34). The study planning and methods are given in the supplement. The bacterial colonies were separated from each impactor sample (n = 22) and were identified further to their species level (Gangamma et al., 2011) with Biolog Manual System-1 (Biolog, Inc., USA). 2.2. Whole blood assay for determination of TNF-α production Twenty randomly selected biosampler samples were lyophilized, and made up to 600 μL with endotoxin free water. The sample aliquots were stored at −20 °C for further analysis. The aliquots were subjected to the whole blood (ex vivo) assay for measuring the tumor necrosis factor (TNF-α). Venous blood was collected in EDTA coated vacutainer (BD Bioscience, India) from six healthy donors. The blood samples from donors were pooled and used within 2 h of withdrawal. The assay was conducted in a single batch. The endotoxin content of donor's blood sample was measured and was found to be below the detection limit of the assay. Details of methods are explained elsewhere (Gangamma et al., 2011). Briefly, 100 μL of fresh blood was incubated with 100 μL of the samples and 350 μL of 0.9% saline in a pyrogen free tube at 37 °C for 16 h. The cell free supernatant was used to measure the TNF-α produced by the cells as per the manufacturer's instruction (BD Bioscience, India). Samples of field blank were also incorporated in the ex vivo. No detectable amount of TNF-α was observed from the blanks. All samples and controls were analyzed in duplicate.
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determined as described above. The assay was conducted in a single batch. The specificity of polymyxin B to endotoxin was evaluated using recombinant interferon-gamma (rIFN-γ) and bacterial surface components (BSC) of Gram positive bacteria (GBP). Details of specificity test are discussed in the Supplementary material. No significant induction of TNF-α was observed after treatment with polymyxin B (Fig. S2). 2.4. Blocking of endotoxin receptors and TNF-α response The detailed experimental protocol is described in supplement. In brief, 100 μL of blood cells were incubated for 1 h with 20 μL of antiCD14 (BD Bioscience, India) prior to the ex vivo assay. The supernatant was collected for measurement of TNF-α. The assay with and without anti CD14 was conducted in single batch. 2.5. Statistical analysis Mann–Whitney-U (MWU) (Conover, 1971) test was used to verify the significant difference in the induction of TNF-α by air samples and air samples treated with polymyxin B. Principal component analysis (PCA) was performed for the identification of bacterial species groups (components) and their relative dominance in the samples (factor scores). Varimax rotation was used to improve the difference between the principal components. A program in FORTRAN was written for performing the above analysis (Reymend and Joreskog, 1996). Regression analysis was used to find the significance of the linear relationship between two variables. 3. Results 3.1. Spore forming bacterial species dominates airborne bacteria Gram positive bacteria were found to be predominantly present across all the sampling sites and represent about 82% of total culturable bacteria (Fig. 1). A total of 28 species belonging to 17 genera were identified (Table S1). The results show that the spore forming groups of bacteria (SFB) such as Bacillus species dominate the total culturable bacterial concentration. The SFB correlates well with the total concentration of bacteria (R2 = 0.884, p b 0.001) and may indicate the requirement of spore forming property for bacterial species to survive in the harsh air environment. To understand further characteristics of airborne bacterial species, principal component analysis (PCA) for the observed species concentration was carried out. The analysis shows that the total variability in species abundance among the samples can be significantly captured by three eigen factors (82%). Data variability explanation by three eigen factors suggests the existence of a few common characteristics, which
2.3. Endotoxin removal using polymyxin B sulfate Endotoxin is a stable and immune-sensitive component of Gram negative bacteria (GNB) and is a reasonable surrogate for bacteria in the inflammatory response assessment. The biosampler samples were measured for endotoxin content using kinetic limulus ambeocyte lysate (LAL) assay. Further the samples were treated with polymyxin B sulfate coated agarose beads (10 μg/mL; Sigma-Aldrich, St Louis) for 30 min to neutralize the endotoxin (Gangamma et al., 2011). The supernatant was collected and endotoxin concentration was measured again using LAL assay. The samples were subjected to ex vivo and TNF-α was
Fig. 1. Colonies collected in the Anderson impactor were isolated and the species were characterized. The fraction of Gram negative bacteria (GNB), Gram positive bacteria (GPB), spore forming bacteria (SFB) and Type II bacterial species are plotted in the figure. The GPB and SFB dominated the total identified culturable bacterial species. 16% of identified bacterial species belongs to Type II.
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are governing species abundance in the air environment. These characteristics could be the properties of the bacteria or characteristics of the sources or environmental factors such as temperature and humidity. The first factor (PC1) predominantly contains SFB and Micrococcus and Staphylococcus species that can survive in harsh environment (Shaffer and Lighthart, 1997; Fang et al., 2007). PC1 significantly correlated with SFB (p b 0.001). Since the sampling was conducted in a typical tropical summer season in Mumbai, the survival of the bacterial species could be a major screening factor for getting sampled as airborne culturable bacteria. This suggests that the ability to survive in the harsh environment is a major factor for occurrence of airborne bacteria in urban environment. 3.2. Bacterial species from human and animal flora present in the urban air environment The second factor (PC2) is associated with human and animal flora species. PC2 correlates significantly (p b 0.001) with bacterial species, human or animal flora species after exclusion of the species associated with PC1. The observed correlation suggests that human flora could be acting as a persistent source for these culturable bacteria. Analysis of frequency of occurrences (Table S1) of bacteria across the sampling sites gives corroborative evidence to previous observations of PC1 and PC2. One in six in Table S1 indicates that the specific bacterial species was identified only in one sample of six at the site. The most frequently observed bacteria were Enterobacter aerogenes, Stenotropomonas maltophilai, Bacillus licheniformis, Bacillus subtils, and Staphylococcus haemolyticus. The presence of a species at a site and its persistence in all samples is related to the temporal variability of the source and their survival or other environmental conditions. Fifty percent of all SFB is present in three or more of the sampling sites. Thirty three percent of non-spore forming human/animal flora is present in three or more of the sampling sites. Only fourteen percent of the species that is neither SFB nor belongs to human/animal flora are present in three or more sampling sites. The frequency distribution (spore forming N non-spore forming human/animal flora N non spore forming non-human/animal
flora) corroborates with the previous findings based on PCA that the survival and existence and human/animal flora are the two major factors influencing presence as culturable airborne bacteria in the urban environment (Fig. 2). 3.3. Pathogenic and opportunistic bacterial species present in the ambient air environment Whether the bacterial species identified in the air environment have an impact on human health is an important question to be addressed. To assess the health impacts of identified bacterial species, we classified the species based on their biosafety class suggested by cell culture repositories and health agencies. Type I safety requirements can be used to handle non-infectious bacteria whereas opportunistic and pathogenic bacteria are handled with Type II safety. This classification can give a rough estimate of the health hazard posed by these bacterial species. Of the total identified colonies 84% and 16% of the species could be classified as Type I and Type II, respectively (Fig. 1). The present analysis is based on culturable bacteria and 16% of Type II colonies may be of significant public health concern. Moreover, the fact that the culture based method used in the present study may not have identified all the bacterial species present in the environment adds importance of the data. However, further conclusions on health impacts are only possible through a more detailed study. 3.4. Airborne biological particles are important inducers of proinflammatory responses in ex vivo To understand the health impacts of bacteria, further we assessed the proinflammatory response of endotoxin in the air samples. Whole blood was stimulated with the lyophilized biosampler samples (see Supplementary material). To remove the endotoxin, the samples were pretreated with polymyxin B (Sigma, India). The supernatant concentration of proinflammatory cytokine (tumor necrosis factor-TNF-α) was measured for both the sets (Fig. 3a).
Fig. 2. Principal component analysis of the bacterial species concentration was used to identify the factors influencing their composition. Three factors could explain 82% of data variability. The first factor was associated with spore forming species that survives in harsh environments. The first factor score is plotted against the second factor score. Three separate groups of samples are formed. One group is high in factor 1 indicating high spore forming bacteria and high total culturable bacterial concentration. The second group has comparatively high factor 2 indicating low concentration (low factor 1) and high human and animal flora.
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Fig. 3. a Twenty lyophilized biosampler samples were used in stimulation experiments in ex vivo. The samples were pretreated with polymyxin B to neutralize endotoxin. The polymyxin treatment of PM reduced TNF-α induction by 35%. b Ten lyophilized biosampler samples were used in stimulation experiments in ex vivo. The samples were pretreated with antiCD14 to block the receptor prior to stimulation. The blocking of receptor reduced TNF-α inducted by PM.
Polymyxin B pretreatment resulted in significant reduction (35%) in the magnitude of TNF-α induction (p b 0.01). These results show the significant contribution of airborne bacteria and its components to inflammatory response. The significant contribution of endotoxin to TNF-α induction was further checked by blocking the endotoxin receptor CD14 (Wright et al., 1990; Ulevitch and Tobias, 1999). The CD14 antagonist can block the endotoxin signaling through toll like receptor (TLR4). AntiCD14 reduced TNF-α induction in ex vivo assay (Fig. 3b). This result corroborates with the previous results obtained by polymyxin B pretreatment. Endotoxin is a component of GNB and therefore, approximately represents the total GNB in the sample. Our data indicates that endotoxin is weakly associated with total culturable bacteria (p b 0.1). The airborne bacteria, in turn, are one of the components of a large variety of airborne biological particles such as fungi, pollen and animal/plant debris. Therefore, it can be deduced that the airborne biological particles, a fraction of which is endotoxin, contribute significantly to the proinflammatory response. This result is in concurrence with the previous studies reported (Monn and Becker, 1999; Ning et al., 2000). 4. Discussion The results have shown that 16% of the total bacterial species could be classified as Type II. Many of the bacterial species identified in the present study have potential implications on public health hazard. Several studies have also found similar species in the ambient environment (Fang et al., 2007; Wang et al., 2010). Acinetobacter species such as Acinetobacter johnsonii, Acinetobacter baumannii and Acinetobacter sp. ADPI are identified as nosocomial pathogens and can cause opportunistic infections including septicemia, meningitis, and endocarditis (Bergogne-Berezin and Towner, 1996). Serratia plymuthica
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and S. haemolyticus are identified as significant opportunistic pathogens to which a variety of infections including peritonitis, pneumonia and sepsis have been attributed (Flahaut et al., 2008; Goldman and Green, 2009). Species belonging to genus such as Enterobacter and Proteus is commonly present in the intestinal tract of human and other animals. Thus, the present study suggests that the population is exposed to opportunistic and infectious bacteria. PCA shows that the first factor includes Bacillus, Staphylococcus and Micrococcus species. These species dominates the airborne culturable bacteria. Moreover, qualitatively, these predominant bacterial species concentrations were higher at the site located near to unpaved roads and vehicular activity. These sites have also high ambient particulate matter concentration (unpublished data) indicating the possibility of soil origin for these bacteria. Previous studies have shown the dominance of Bacillus, Staphylococcus and Micrococcus in urban areas (Mancinelli and Shulls, 1978; Fang et al., 2007). Bacillus species are known to survive in harsh environment and retain their culturability in these environments (Shaffer and Lighthart, 1997). The above study reports that Bacillus species dominates the urban and rural environment. The human and animal flora are also reported to dominant in urban areas (Bowers et al., 2011). Presence of bacteria in the urban air environment is known for decades but information regarding their composition, survival, dispersion mechanisms and ultimately their health effects are still limited. Endotoxin concentration in the samples varied in the range of 0.06 to 2.15 ng/m3 with an average of 0.27 ng/m3. This corresponds approximately to 1.3 to 46 ng of endotoxin respiratory dose per day (Varghese and Gangamma, 2006, 2009). Actual endotoxin dose could be higher than the above estimate. Endotoxin dose could be high because endotoxin measurements in environmental samples are conservative (Rylander, 2002). Rylander (1997) have suggested 1:10 ratio for the measured and actual endotoxin in environmental samples. This result can be corroborated through calculation based on airborne bacterial concentration. Our measurement has shown that, airborne bacterial concentration (total viable and nonviable) is around 106 #/m3. This is not very high, as the bacteria in the ambient air environment are reported to vary in the range of 104 to 107 #/m3 (Bowers et al., 2010). If we assume it is 106 #/m3, the mass of bacteria (assuming size of 1 μm and density of water) in m3 of air is 0.52 μg/m3. It is established that around 7% bacterial mass is LPS (Darveau and Hancock, 1983) and we may assume that 50% of bacteria are Gram negative. This gives around 0.018 μg/m3 of endotoxin in ambient PM. This can be converted approximately to EU through a LAL potency of 13 EU/ng as 236.6 EU/m3 in ambient environment. This corresponds to 5062 ng of endotoxin respiratory dose per day. This estimation could be an upper bound for the ambient endotoxin dose but the above calculations suggest importance of airborne bacteria and their components such as endotoxin in ambient environment. Studies have proposed that airborne particle associated with inflammation in the lung and circulatory system leads to many disease pathogenesis (Seaton et al., 1999; Brook et al., 2010). Airborne particulate matter exposure increases pulmonary and airway inflammation. Thus, the results of the present study may suggest that inflammation produced by the biological fraction such as endotoxin may have an important role in pathogenesis of diseases associated with ambient airborne particles. Moreover, present study suggests the importance of pathogen pattern recognizing receptors and their downstream molecules in proinflammatory response induced by PM. These finding corroborates with previous results that the microbial pattern recognition pathways are involved in ambient PM induced cytokine response (Ning et al., 2000; Long et al., 2001; Becker et al., 2002; Shoenfelt et al., 2009; Kampfrath et al., 2011). Recent studies have suggested several mechanisms for activation of pattern recognizing receptors (Bauer et al., 2012; Gangamma, 2012b). But the exact pathways involved in such activation are still unknown (Gangamma, 2012a, 2012b). The results of present study re-emphasis the need for further experiments to
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understand the role of pattern recognizing receptors in adverse health effects associated with ambient particulate matter exposure. 5. Conclusion Human and animal microbial flora are the major source of airborne bacteria in Mumbai ambient environment. This study shows that airborne bacteria and their components are important fraction of ambient PM. The study highlights the importance of airborne biological particles and suggests need for further studies on biological composition of ambient PM. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2014.04.065. Conflict of interest The author declares no conflict of interest. Acknowledgment The author would like to express her sincere thanks to Department of Science and Technology, Ministry of Science and Technology, Govt. of India for providing financial support for carrying out this work (SR/ S4/AS:283/2007, SR/FTP/ES-40/2008). The author would also like to thank all officials of MCGM for extending their facilities and for their cooperation and also to Professor R.S. Patil and Professor Suparna Mukherji, IIT Bombay for providing their laboratory facilities. References Bauer RN, Diaz-Sanchez D, Jaspers I. Effects of air pollutants on innate immunity: the role of Toll-like receptors and nucleotide-binding oligomerization domain-like receptors. J Allergy Clin Immunol 2012;129:14–24. Becker S, Fenton MJ, Soukup JM. Involvement of microbial components and Toll-like receptors 2 and 4 in cytokine responses to air pollution particles. Am J Respir Cell Mol Biol 2002;27:611–8. Bergogne-Berezin E, Towner KJ. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev 1996;9:148–65. Bowers RM, McLetchie S, Knight R, Fierer N. Spatial variability in airborne bacterial communities across land-use types and their relationship to the bacterial communities of potential source environments. ISME J. 2010;5:601–12. Bowers RM, Sullivan AP, Costello EK, Collett Jr JL, Knight R, Fierer N. Sources of bacteria in outdoor air across cities in the midwestern United States. Appl Environ Microbiol 2011. http://dx.doi.org/10.1128/AEM.05498-11. Brodie EL, DeSantis TZ, Parker JPM, Zubietta IX, Piceno YM, et al. Urban aerosols harbor diverse and dynamic bacterial populations. Proc Natl Acad Sci U S A 2007;104: 299–304. Brook RD, Rajagopalan S, Pope CAIII, Brook JR, Bhatnagar A, Diez-Roux AV, et al. Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association. Circulation 2010;121:2331–78. Conover WJ. Practical nonparametric statistics. USA: John Wiley and Sons Inc.; 1971.
Darveau RP, Hancock RE. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol 1983;155:831–8. Elbert W, Taylor PE, Andreae MO, P¨oschl U. Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions. Atmos Chem Phys 2007;7:4569–88. Fang Z, Ouyang Z, Zheng H, Wang X, Hu L. Culturable airborne bacteria in outdoor environments in Beijing, China. Microb Ecol 2007;54:487–96. Flahaut S, Vinogradov E, Kelley KA, Brennan S, Hiramatsu K, Lee JC. Structural and biological characterization of a capsular polysaccharide produced by Staphylococcus haemolyticus. J Bacteriol 2008;190:1649–57. Gangamma S, Patil RS, Mukherji S. Characterization and proinflammatory response of airborne biological particles from wastewater treatment plants. Environ Sci Technol 2011;45:3282–7. Gangamma S. Airborne particulate matter associated endotoxin and proinflammatory responses. J Allergy Clin Immunol 2012a;130:1012–3. Gangamma S. Airborne particulate matter and innate immunity activation. Environ Sci Technol 2012b;46:10879–80. Goldman E, Green LH. Practical handbook of microbiology. Boca Raton: CRC Press; 2009. Jaenicke R, Matthias-Maser S, Gruber S. Omnipresence of biological material in the atmosphere. Environ Chem 2007;4:217–20. Jaenicke R. Abundance of cellular material and proteins in the atmosphere. Science 2005; 308:73. Kampfrath T, Maiseyeu A, Ying Z, Shah Z, Deiuliis JA, Xu X, et al. Chronic fine particulate matter exposure induces systemic vascular dysfunction via NADPH oxidase and TLR4 pathways. Circ Res 2011;108:716–26. Lighthart B, Shaffer B, Frisch A, Paterno D. Atmospheric culturable bacteria associated with meteorological conditions at a summer-time site in the mid-Willamette Valley, Oregon. Aerobiologia 2009;25:285–95. Long CM, Suh HH, Kobzik L, Catalano PJ, Ning YY, Koutrakis P. A pilot investigation of the relative toxicity of indoor and outdoor fine particles: in vitro effects of endotoxin and other particulate properties. Environ Health Perspect 2001;109:1019–26. Mancinelli RL, Shulls WA. Airborne bacteria in an urban environment. Appl Environ Microbiol 1978;35:1095–101. Monn C, Becker S. Cytotoxicity and induction of proinflammatory cytokines from human monocytes exposed to fine (PM2.5) and coarse particles (PM10-2.5) in outdoor and indoor air. Toxicol Appl Pharmacol 1999;155:245–52. Ning Y, Imrich A, Goldsmith CA, Qin G, Kobzik L. Alveolar macrophage cytokine production in response to air particles in vitro: role of endotoxin. J Toxicol Environ Health 2000;59:165–80. Reymend R, Joreskog KG. Applied factor analysis in natural sciences. UK: Cambridge university press; 1996. Rylander R. Criteria document on environmental endotoxin. Innate Immun 1997;4:380. Rylander R. Endotoxin in the environment—exposure and effects. J Endotoxin Res 2002;8: 241–52. Seaton A, Soutar A, Crawford V, Elton R, McNerlan S, Cherrie J, et al. Particulate air pollution and the blood. Thorax 1999;54:1027–32. Shaffer BT, Lighthart B. Survey of culturable airborne bacteria at four diverse locations in Oregon: urban, rural, forest, and coastal. Microb Ecol 1997;34:167–77. Shoenfelt J, Mitkus RJ, Zeisler R, Spatz RO, Powell J, Fenton MJ. Involvement of TLR2 and TLR4 in inflammatory immune responses induced by fine and coarse ambient air particulate matter. J Leukoc Biol 2009;86:303–12. Ulevitch RJ, Tobias PS. Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol 1999;11:19–22. Varghese SK, Gangamma S. Particle deposition in human respiratory system: deposition of concentrated hydroscopic aerosols. Inhal Toxicol 2009;2:1619–30. Varghese SK, Gangamma S. Particle deposition in human respiratory tract: effect of watersoluble fraction. Aerosol Air Qual Res 2006;6(4):360–79. Wang W, Ma Y, Ma X, Wu F, Ma X, An L, et al. Seasonal variations of airborne bacteria in the Mogao Grottoes, Dunhuang, China. Int Biodeterior Biodegrad 2010;64:309–15. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS-binding protein. Science 1990;249:1431–3.
Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Characteristics of airborne bacteria in Mumbai urban environment S. Gangamma ⁎ Department of Chemical Engineering, National Institute of Technology Karnataka, Surathkal, India
H I G H L I G H T S • • • •
Study demonstrated association between endotoxin and PM induced pro-inflammatory response Human or animal flora are identified as vital component of airborne culturable bacteria in Mumbai Study shows significant presence of pathogenic and opportunistic bacteria in the study area Study highlights the importance of airborne biological particles of ambient PM
a r t i c l e
i n f o
Article history: Received 9 February 2014 Received in revised form 14 April 2014 Accepted 16 April 2014 Available online xxxx Editor: Xuexi Tie Keywords: Airborne bacteria Endotoxin Proinflammatory Whole blood assay Tumor necrosis factor
a b s t r a c t Components of biological origin constitute small but a significant proportion of the ambient airborne particulate matter (PM). However, their diversity and role in proinflammatory responses of PM are not well understood. The present study characterizes airborne bacterial species diversity in Mumbai City and elucidates the role of bacterial endotoxin in PM induced proinflammatory response in ex vivo. Airborne bacteria and endotoxin samples were collected during April–May 2010 in Mumbai using six stage microbial impactor and biosampler. The culturable bacterial species concentration was measured and factors influencing the composition were identified by principal component analysis (PCA). The biosampler samples were used to stimulate immune cells in whole blood assay. A total of 28 species belonging to 17 genera were identified. Gram positive and spore forming groups of bacteria dominated the airborne culturable bacterial concentration. The study indicated the dominance of spore forming and human or animal flora derived pathogenic/opportunistic bacteria in the ambient air environment. Pathogenic and opportunistic species of bacteria were also present in the samples. TNF-α induction by PM was reduced (35%) by polymyxin B pretreatment and this result was corroborated with the results of blocking endotoxin receptor cluster differentiation (CD14). The study highlights the importance of airborne biological particles and suggests need of further studies on biological characterization of ambient PM. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Airborne particulate matter (PM) contain significant fraction (16%) of biological components (Jaenicke, 2005) and likely to play a significant role in health and climate. Airborne bacteria are one of the important components of airborne biological particles in natural and urban environment (Jaenicke, 2005; Brodie et al., 2007; Elbert et al., 2007; Jaenicke et al., 2007; Bowers et al., 2011). Bacteria present in the ambient atmosphere originate from natural and anthropogenic sources such as plants, animals, soil, water bodies, waste dumping ground, wastewater treatment plants and agricultural activities. Many human activities such as solid waste and sewage transport, processing and conducive humid environment may enhance the abundance of culturable airborne bacteria in an urban environment.
⁎ Tel.: +91 824 2474000; fax: +91 824 2474033. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.scitotenv.2014.04.065 0048-9697/© 2014 Elsevier B.V. All rights reserved.
Properties of urban airborne bacteria are studied over decades (Mancinelli and Shulls, 1978; Brodie et al., 2007; Fang et al., 2007; Bowers et al., 2011). These studies identified the dominant bacterial species in urban environments and have shown that the species properties and their proportions vary significantly among urban settings. Change in proportion of dominant airborne bacterial species with meteorological and micro-environmental properties was studied in various environmental settings (Lighthart et al., 2009; Wang et al., 2010). However, important factors leading to the variability in species composition, source contribution and survivability in urban air environment are not yet well understood. Health impacts of airborne bacteria and their components are also not well studied. Endotoxin is a cell wall component of Gram-negative bacteria (GNB). Initially, significant levels of airborne endotoxin was detected in several occupational environments such as cotton industry, agriculture processing, barns, sewage treatment, composting and waste handling. These studies have shown that in occupational environments, endotoxin is of one the most important component associated with
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health effects of PM exposure. Similarly, endotoxin in indoor environments such as in homes and offices has been characterized and linked to adverse health effects. Endotoxin is known to be present in ambient PM at low levels. Now it is known that ambient airborne endotoxin plays a significant role in particulate matter induced proinflammatory response. However, the exact role of low levels of endotoxin in inducing proinflammatory response is not well understood (Gangamma, 2012a, 2012b). Mumbai is one of the most densely populated cities in the world. Many human activities such as solid waste and sewage transport and processing may enhance the ambient concentration of bioaerosols in the city. However, the characterization of biological components of the ambient PM in the city has not been previously attempted. The present study characterizes species diversity of airborne bacteria in Mumbai. The paper adopts two methods to highlight the importance of airborne bioparticles in Mumbai. First method utilizes the species characterization information of airborne bacteria. Second method utilizes endotoxin which is an important fraction of airborne bioparticles to explain the inflammatory response of total particulate matter. Based on these methods, the study accentuates the importance of biological fraction of urban PM in Mumbai. 2. Materials and methods 2.1. Sampling of airborne bacteria and endotoxin The air samples were collected from the four ambient air quality monitoring sites (Fig. S1) in Mumbai during April–May 2010 with microbial Anderson impactor (Thermo Scientific USA, n = 22) and biosamplers (SKC USA, n = 34). The study planning and methods are given in the supplement. The bacterial colonies were separated from each impactor sample (n = 22) and were identified further to their species level (Gangamma et al., 2011) with Biolog Manual System-1 (Biolog, Inc., USA). 2.2. Whole blood assay for determination of TNF-α production Twenty randomly selected biosampler samples were lyophilized, and made up to 600 μL with endotoxin free water. The sample aliquots were stored at −20 °C for further analysis. The aliquots were subjected to the whole blood (ex vivo) assay for measuring the tumor necrosis factor (TNF-α). Venous blood was collected in EDTA coated vacutainer (BD Bioscience, India) from six healthy donors. The blood samples from donors were pooled and used within 2 h of withdrawal. The assay was conducted in a single batch. The endotoxin content of donor's blood sample was measured and was found to be below the detection limit of the assay. Details of methods are explained elsewhere (Gangamma et al., 2011). Briefly, 100 μL of fresh blood was incubated with 100 μL of the samples and 350 μL of 0.9% saline in a pyrogen free tube at 37 °C for 16 h. The cell free supernatant was used to measure the TNF-α produced by the cells as per the manufacturer's instruction (BD Bioscience, India). Samples of field blank were also incorporated in the ex vivo. No detectable amount of TNF-α was observed from the blanks. All samples and controls were analyzed in duplicate.
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determined as described above. The assay was conducted in a single batch. The specificity of polymyxin B to endotoxin was evaluated using recombinant interferon-gamma (rIFN-γ) and bacterial surface components (BSC) of Gram positive bacteria (GBP). Details of specificity test are discussed in the Supplementary material. No significant induction of TNF-α was observed after treatment with polymyxin B (Fig. S2). 2.4. Blocking of endotoxin receptors and TNF-α response The detailed experimental protocol is described in supplement. In brief, 100 μL of blood cells were incubated for 1 h with 20 μL of antiCD14 (BD Bioscience, India) prior to the ex vivo assay. The supernatant was collected for measurement of TNF-α. The assay with and without anti CD14 was conducted in single batch. 2.5. Statistical analysis Mann–Whitney-U (MWU) (Conover, 1971) test was used to verify the significant difference in the induction of TNF-α by air samples and air samples treated with polymyxin B. Principal component analysis (PCA) was performed for the identification of bacterial species groups (components) and their relative dominance in the samples (factor scores). Varimax rotation was used to improve the difference between the principal components. A program in FORTRAN was written for performing the above analysis (Reymend and Joreskog, 1996). Regression analysis was used to find the significance of the linear relationship between two variables. 3. Results 3.1. Spore forming bacterial species dominates airborne bacteria Gram positive bacteria were found to be predominantly present across all the sampling sites and represent about 82% of total culturable bacteria (Fig. 1). A total of 28 species belonging to 17 genera were identified (Table S1). The results show that the spore forming groups of bacteria (SFB) such as Bacillus species dominate the total culturable bacterial concentration. The SFB correlates well with the total concentration of bacteria (R2 = 0.884, p b 0.001) and may indicate the requirement of spore forming property for bacterial species to survive in the harsh air environment. To understand further characteristics of airborne bacterial species, principal component analysis (PCA) for the observed species concentration was carried out. The analysis shows that the total variability in species abundance among the samples can be significantly captured by three eigen factors (82%). Data variability explanation by three eigen factors suggests the existence of a few common characteristics, which
2.3. Endotoxin removal using polymyxin B sulfate Endotoxin is a stable and immune-sensitive component of Gram negative bacteria (GNB) and is a reasonable surrogate for bacteria in the inflammatory response assessment. The biosampler samples were measured for endotoxin content using kinetic limulus ambeocyte lysate (LAL) assay. Further the samples were treated with polymyxin B sulfate coated agarose beads (10 μg/mL; Sigma-Aldrich, St Louis) for 30 min to neutralize the endotoxin (Gangamma et al., 2011). The supernatant was collected and endotoxin concentration was measured again using LAL assay. The samples were subjected to ex vivo and TNF-α was
Fig. 1. Colonies collected in the Anderson impactor were isolated and the species were characterized. The fraction of Gram negative bacteria (GNB), Gram positive bacteria (GPB), spore forming bacteria (SFB) and Type II bacterial species are plotted in the figure. The GPB and SFB dominated the total identified culturable bacterial species. 16% of identified bacterial species belongs to Type II.
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are governing species abundance in the air environment. These characteristics could be the properties of the bacteria or characteristics of the sources or environmental factors such as temperature and humidity. The first factor (PC1) predominantly contains SFB and Micrococcus and Staphylococcus species that can survive in harsh environment (Shaffer and Lighthart, 1997; Fang et al., 2007). PC1 significantly correlated with SFB (p b 0.001). Since the sampling was conducted in a typical tropical summer season in Mumbai, the survival of the bacterial species could be a major screening factor for getting sampled as airborne culturable bacteria. This suggests that the ability to survive in the harsh environment is a major factor for occurrence of airborne bacteria in urban environment. 3.2. Bacterial species from human and animal flora present in the urban air environment The second factor (PC2) is associated with human and animal flora species. PC2 correlates significantly (p b 0.001) with bacterial species, human or animal flora species after exclusion of the species associated with PC1. The observed correlation suggests that human flora could be acting as a persistent source for these culturable bacteria. Analysis of frequency of occurrences (Table S1) of bacteria across the sampling sites gives corroborative evidence to previous observations of PC1 and PC2. One in six in Table S1 indicates that the specific bacterial species was identified only in one sample of six at the site. The most frequently observed bacteria were Enterobacter aerogenes, Stenotropomonas maltophilai, Bacillus licheniformis, Bacillus subtils, and Staphylococcus haemolyticus. The presence of a species at a site and its persistence in all samples is related to the temporal variability of the source and their survival or other environmental conditions. Fifty percent of all SFB is present in three or more of the sampling sites. Thirty three percent of non-spore forming human/animal flora is present in three or more of the sampling sites. Only fourteen percent of the species that is neither SFB nor belongs to human/animal flora are present in three or more sampling sites. The frequency distribution (spore forming N non-spore forming human/animal flora N non spore forming non-human/animal
flora) corroborates with the previous findings based on PCA that the survival and existence and human/animal flora are the two major factors influencing presence as culturable airborne bacteria in the urban environment (Fig. 2). 3.3. Pathogenic and opportunistic bacterial species present in the ambient air environment Whether the bacterial species identified in the air environment have an impact on human health is an important question to be addressed. To assess the health impacts of identified bacterial species, we classified the species based on their biosafety class suggested by cell culture repositories and health agencies. Type I safety requirements can be used to handle non-infectious bacteria whereas opportunistic and pathogenic bacteria are handled with Type II safety. This classification can give a rough estimate of the health hazard posed by these bacterial species. Of the total identified colonies 84% and 16% of the species could be classified as Type I and Type II, respectively (Fig. 1). The present analysis is based on culturable bacteria and 16% of Type II colonies may be of significant public health concern. Moreover, the fact that the culture based method used in the present study may not have identified all the bacterial species present in the environment adds importance of the data. However, further conclusions on health impacts are only possible through a more detailed study. 3.4. Airborne biological particles are important inducers of proinflammatory responses in ex vivo To understand the health impacts of bacteria, further we assessed the proinflammatory response of endotoxin in the air samples. Whole blood was stimulated with the lyophilized biosampler samples (see Supplementary material). To remove the endotoxin, the samples were pretreated with polymyxin B (Sigma, India). The supernatant concentration of proinflammatory cytokine (tumor necrosis factor-TNF-α) was measured for both the sets (Fig. 3a).
Fig. 2. Principal component analysis of the bacterial species concentration was used to identify the factors influencing their composition. Three factors could explain 82% of data variability. The first factor was associated with spore forming species that survives in harsh environments. The first factor score is plotted against the second factor score. Three separate groups of samples are formed. One group is high in factor 1 indicating high spore forming bacteria and high total culturable bacterial concentration. The second group has comparatively high factor 2 indicating low concentration (low factor 1) and high human and animal flora.
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Fig. 3. a Twenty lyophilized biosampler samples were used in stimulation experiments in ex vivo. The samples were pretreated with polymyxin B to neutralize endotoxin. The polymyxin treatment of PM reduced TNF-α induction by 35%. b Ten lyophilized biosampler samples were used in stimulation experiments in ex vivo. The samples were pretreated with antiCD14 to block the receptor prior to stimulation. The blocking of receptor reduced TNF-α inducted by PM.
Polymyxin B pretreatment resulted in significant reduction (35%) in the magnitude of TNF-α induction (p b 0.01). These results show the significant contribution of airborne bacteria and its components to inflammatory response. The significant contribution of endotoxin to TNF-α induction was further checked by blocking the endotoxin receptor CD14 (Wright et al., 1990; Ulevitch and Tobias, 1999). The CD14 antagonist can block the endotoxin signaling through toll like receptor (TLR4). AntiCD14 reduced TNF-α induction in ex vivo assay (Fig. 3b). This result corroborates with the previous results obtained by polymyxin B pretreatment. Endotoxin is a component of GNB and therefore, approximately represents the total GNB in the sample. Our data indicates that endotoxin is weakly associated with total culturable bacteria (p b 0.1). The airborne bacteria, in turn, are one of the components of a large variety of airborne biological particles such as fungi, pollen and animal/plant debris. Therefore, it can be deduced that the airborne biological particles, a fraction of which is endotoxin, contribute significantly to the proinflammatory response. This result is in concurrence with the previous studies reported (Monn and Becker, 1999; Ning et al., 2000). 4. Discussion The results have shown that 16% of the total bacterial species could be classified as Type II. Many of the bacterial species identified in the present study have potential implications on public health hazard. Several studies have also found similar species in the ambient environment (Fang et al., 2007; Wang et al., 2010). Acinetobacter species such as Acinetobacter johnsonii, Acinetobacter baumannii and Acinetobacter sp. ADPI are identified as nosocomial pathogens and can cause opportunistic infections including septicemia, meningitis, and endocarditis (Bergogne-Berezin and Towner, 1996). Serratia plymuthica
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and S. haemolyticus are identified as significant opportunistic pathogens to which a variety of infections including peritonitis, pneumonia and sepsis have been attributed (Flahaut et al., 2008; Goldman and Green, 2009). Species belonging to genus such as Enterobacter and Proteus is commonly present in the intestinal tract of human and other animals. Thus, the present study suggests that the population is exposed to opportunistic and infectious bacteria. PCA shows that the first factor includes Bacillus, Staphylococcus and Micrococcus species. These species dominates the airborne culturable bacteria. Moreover, qualitatively, these predominant bacterial species concentrations were higher at the site located near to unpaved roads and vehicular activity. These sites have also high ambient particulate matter concentration (unpublished data) indicating the possibility of soil origin for these bacteria. Previous studies have shown the dominance of Bacillus, Staphylococcus and Micrococcus in urban areas (Mancinelli and Shulls, 1978; Fang et al., 2007). Bacillus species are known to survive in harsh environment and retain their culturability in these environments (Shaffer and Lighthart, 1997). The above study reports that Bacillus species dominates the urban and rural environment. The human and animal flora are also reported to dominant in urban areas (Bowers et al., 2011). Presence of bacteria in the urban air environment is known for decades but information regarding their composition, survival, dispersion mechanisms and ultimately their health effects are still limited. Endotoxin concentration in the samples varied in the range of 0.06 to 2.15 ng/m3 with an average of 0.27 ng/m3. This corresponds approximately to 1.3 to 46 ng of endotoxin respiratory dose per day (Varghese and Gangamma, 2006, 2009). Actual endotoxin dose could be higher than the above estimate. Endotoxin dose could be high because endotoxin measurements in environmental samples are conservative (Rylander, 2002). Rylander (1997) have suggested 1:10 ratio for the measured and actual endotoxin in environmental samples. This result can be corroborated through calculation based on airborne bacterial concentration. Our measurement has shown that, airborne bacterial concentration (total viable and nonviable) is around 106 #/m3. This is not very high, as the bacteria in the ambient air environment are reported to vary in the range of 104 to 107 #/m3 (Bowers et al., 2010). If we assume it is 106 #/m3, the mass of bacteria (assuming size of 1 μm and density of water) in m3 of air is 0.52 μg/m3. It is established that around 7% bacterial mass is LPS (Darveau and Hancock, 1983) and we may assume that 50% of bacteria are Gram negative. This gives around 0.018 μg/m3 of endotoxin in ambient PM. This can be converted approximately to EU through a LAL potency of 13 EU/ng as 236.6 EU/m3 in ambient environment. This corresponds to 5062 ng of endotoxin respiratory dose per day. This estimation could be an upper bound for the ambient endotoxin dose but the above calculations suggest importance of airborne bacteria and their components such as endotoxin in ambient environment. Studies have proposed that airborne particle associated with inflammation in the lung and circulatory system leads to many disease pathogenesis (Seaton et al., 1999; Brook et al., 2010). Airborne particulate matter exposure increases pulmonary and airway inflammation. Thus, the results of the present study may suggest that inflammation produced by the biological fraction such as endotoxin may have an important role in pathogenesis of diseases associated with ambient airborne particles. Moreover, present study suggests the importance of pathogen pattern recognizing receptors and their downstream molecules in proinflammatory response induced by PM. These finding corroborates with previous results that the microbial pattern recognition pathways are involved in ambient PM induced cytokine response (Ning et al., 2000; Long et al., 2001; Becker et al., 2002; Shoenfelt et al., 2009; Kampfrath et al., 2011). Recent studies have suggested several mechanisms for activation of pattern recognizing receptors (Bauer et al., 2012; Gangamma, 2012b). But the exact pathways involved in such activation are still unknown (Gangamma, 2012a, 2012b). The results of present study re-emphasis the need for further experiments to
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understand the role of pattern recognizing receptors in adverse health effects associated with ambient particulate matter exposure. 5. Conclusion Human and animal microbial flora are the major source of airborne bacteria in Mumbai ambient environment. This study shows that airborne bacteria and their components are important fraction of ambient PM. The study highlights the importance of airborne biological particles and suggests need for further studies on biological composition of ambient PM. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2014.04.065. Conflict of interest The author declares no conflict of interest. Acknowledgment The author would like to express her sincere thanks to Department of Science and Technology, Ministry of Science and Technology, Govt. of India for providing financial support for carrying out this work (SR/ S4/AS:283/2007, SR/FTP/ES-40/2008). The author would also like to thank all officials of MCGM for extending their facilities and for their cooperation and also to Professor R.S. Patil and Professor Suparna Mukherji, IIT Bombay for providing their laboratory facilities. References Bauer RN, Diaz-Sanchez D, Jaspers I. Effects of air pollutants on innate immunity: the role of Toll-like receptors and nucleotide-binding oligomerization domain-like receptors. J Allergy Clin Immunol 2012;129:14–24. Becker S, Fenton MJ, Soukup JM. Involvement of microbial components and Toll-like receptors 2 and 4 in cytokine responses to air pollution particles. Am J Respir Cell Mol Biol 2002;27:611–8. Bergogne-Berezin E, Towner KJ. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev 1996;9:148–65. Bowers RM, McLetchie S, Knight R, Fierer N. Spatial variability in airborne bacterial communities across land-use types and their relationship to the bacterial communities of potential source environments. ISME J. 2010;5:601–12. Bowers RM, Sullivan AP, Costello EK, Collett Jr JL, Knight R, Fierer N. Sources of bacteria in outdoor air across cities in the midwestern United States. Appl Environ Microbiol 2011. http://dx.doi.org/10.1128/AEM.05498-11. Brodie EL, DeSantis TZ, Parker JPM, Zubietta IX, Piceno YM, et al. Urban aerosols harbor diverse and dynamic bacterial populations. Proc Natl Acad Sci U S A 2007;104: 299–304. Brook RD, Rajagopalan S, Pope CAIII, Brook JR, Bhatnagar A, Diez-Roux AV, et al. Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association. Circulation 2010;121:2331–78. Conover WJ. Practical nonparametric statistics. USA: John Wiley and Sons Inc.; 1971.
Darveau RP, Hancock RE. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol 1983;155:831–8. Elbert W, Taylor PE, Andreae MO, P¨oschl U. Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions. Atmos Chem Phys 2007;7:4569–88. Fang Z, Ouyang Z, Zheng H, Wang X, Hu L. Culturable airborne bacteria in outdoor environments in Beijing, China. Microb Ecol 2007;54:487–96. Flahaut S, Vinogradov E, Kelley KA, Brennan S, Hiramatsu K, Lee JC. Structural and biological characterization of a capsular polysaccharide produced by Staphylococcus haemolyticus. J Bacteriol 2008;190:1649–57. Gangamma S, Patil RS, Mukherji S. Characterization and proinflammatory response of airborne biological particles from wastewater treatment plants. Environ Sci Technol 2011;45:3282–7. Gangamma S. Airborne particulate matter associated endotoxin and proinflammatory responses. J Allergy Clin Immunol 2012a;130:1012–3. Gangamma S. Airborne particulate matter and innate immunity activation. Environ Sci Technol 2012b;46:10879–80. Goldman E, Green LH. Practical handbook of microbiology. Boca Raton: CRC Press; 2009. Jaenicke R, Matthias-Maser S, Gruber S. Omnipresence of biological material in the atmosphere. Environ Chem 2007;4:217–20. Jaenicke R. Abundance of cellular material and proteins in the atmosphere. Science 2005; 308:73. Kampfrath T, Maiseyeu A, Ying Z, Shah Z, Deiuliis JA, Xu X, et al. Chronic fine particulate matter exposure induces systemic vascular dysfunction via NADPH oxidase and TLR4 pathways. Circ Res 2011;108:716–26. Lighthart B, Shaffer B, Frisch A, Paterno D. Atmospheric culturable bacteria associated with meteorological conditions at a summer-time site in the mid-Willamette Valley, Oregon. Aerobiologia 2009;25:285–95. Long CM, Suh HH, Kobzik L, Catalano PJ, Ning YY, Koutrakis P. A pilot investigation of the relative toxicity of indoor and outdoor fine particles: in vitro effects of endotoxin and other particulate properties. Environ Health Perspect 2001;109:1019–26. Mancinelli RL, Shulls WA. Airborne bacteria in an urban environment. Appl Environ Microbiol 1978;35:1095–101. Monn C, Becker S. Cytotoxicity and induction of proinflammatory cytokines from human monocytes exposed to fine (PM2.5) and coarse particles (PM10-2.5) in outdoor and indoor air. Toxicol Appl Pharmacol 1999;155:245–52. Ning Y, Imrich A, Goldsmith CA, Qin G, Kobzik L. Alveolar macrophage cytokine production in response to air particles in vitro: role of endotoxin. J Toxicol Environ Health 2000;59:165–80. Reymend R, Joreskog KG. Applied factor analysis in natural sciences. UK: Cambridge university press; 1996. Rylander R. Criteria document on environmental endotoxin. Innate Immun 1997;4:380. Rylander R. Endotoxin in the environment—exposure and effects. J Endotoxin Res 2002;8: 241–52. Seaton A, Soutar A, Crawford V, Elton R, McNerlan S, Cherrie J, et al. Particulate air pollution and the blood. Thorax 1999;54:1027–32. Shaffer BT, Lighthart B. Survey of culturable airborne bacteria at four diverse locations in Oregon: urban, rural, forest, and coastal. Microb Ecol 1997;34:167–77. Shoenfelt J, Mitkus RJ, Zeisler R, Spatz RO, Powell J, Fenton MJ. Involvement of TLR2 and TLR4 in inflammatory immune responses induced by fine and coarse ambient air particulate matter. J Leukoc Biol 2009;86:303–12. Ulevitch RJ, Tobias PS. Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol 1999;11:19–22. Varghese SK, Gangamma S. Particle deposition in human respiratory system: deposition of concentrated hydroscopic aerosols. Inhal Toxicol 2009;2:1619–30. Varghese SK, Gangamma S. Particle deposition in human respiratory tract: effect of watersoluble fraction. Aerosol Air Qual Res 2006;6(4):360–79. Wang W, Ma Y, Ma X, Wu F, Ma X, An L, et al. Seasonal variations of airborne bacteria in the Mogao Grottoes, Dunhuang, China. Int Biodeterior Biodegrad 2010;64:309–15. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS-binding protein. Science 1990;249:1431–3.