Characteristic and Concentration Distribution of Culturable Airborne Bacteria in Residential Environments in Beijing, China

The present investigation was conducted to assess the culturable concentration and distribution characteristics of airborne bacteria in 31 homes with children aged from 1 to 10 years in Beijing, China. The results showed that the concentration of culturable airborne bacteria in these homes ranged from 47 colony forming units (CFU)/m to 12341 CFU/m, with a mean and a median of 1821 and 877 CFU/m, respectively. A total of 632 bacterial isolates from the air in homes in different regions and different seasons were identified and distributed across 43 genera and 136 species of bacteria. Micrococcus (26.74%), Bacillus (14.56%), Kocuria (12.66%), and Staphylococcus (12.03%) were determined as the most common culturable airborne bacteria, and the dominant bacterial species were Micrococcus luteus (14.56%), Kocuria roseus (8.39%), Bacillus megaterium (4.75%), Staphylococcus cohnii (3.63%), and Micrococcus lylae (3.01%). Data analysis revealed that bacterial concentrations in homes with a male child were significantly higher than those with a female child (**P < 0.01), and a negative correlation was found between bacterial concentration and living area per person in the homes (**P < 0.01). Additionally, the mean bacterial concentration was highest in Spring, followed by Summer and Autumn, and lowest in Winter (**P < 0.01) in homes with child. The results provide an exposure database of airborne bacteria in family homes in Beijing, and suggest that the sex of children and living area per person in homes have a significant influence on the bacterial concentration in the air.


INTRODUCTION
Adverse health effects on residents provoked by exposure to various indoor air contaminants have been concerned increasingly all over the world during recent decades because contemporary people spend most of the time in confined indoor spaces (Kalogerakis et al., 2005;Lee et al., 2006;Srikanth et al., 2008;Qian et al., 2012).Adults spend almost 90% of their time indoors in a variety of enclosed spaces (Klepeis et al., 2001).Among the different spaces, the residential home is of vital importance due to the amount of time spent there, especially for the physically handicapped people such as the elderly and infants who stay at home even up to 95% of their total time (Dassonville et al., 2008;Nasir et al., 2012).Bioaerosol, defined as an airborne particle that is living or originate from living organisms, is one of the most important indoor air contaminants, and they contribute to about 5-34% of indoor air pollution (Srikanth et al., 2008).Exposure to bioaerosols might be responsible for a large part of respiratory diseases (such as rhinitis, asthma, and pneumonia) and other adverse health effects such as infections, hypersensitivity pneumonitis and toxic reaction (Gorny et al., 2002;Fracchia et al., 2006).As to airborne bacteria, they are important and abundant living component of atmospheric bioaerosols (Jaenicke, 2005).Potential health risk of exposure to airborne bacteria can occur in workplaces and residential spaces at any time, especially to those residents like patients, infants and old people who are physiologically sensitive to them (Douwes et al., 2003).It was reported that children on a per-bodyweight basis tent to inhale relatively more air than adults and elderly persons were more likely to have weak body defense systems, and people with compromised immunity (e.g., pregnant women, post-operative patients) or with existing respiratory conditions, such as allergies and asthma are at increased risk of exposure to airborne bacteria and their derivatives (Bunger et al., 2000;Nasir et al., 2012).
During recent decades, many studies have been conducted to investigate the level of airborne bacteria in residential settings that were closely related to human health (Pastuszka et al., 2000;Moschandreas et al., 2003;Aydogdu et al., 2005;Mentese et al., 2009;Nasir and Colbeck, 2010;Aydogdu et al., 2010).However, such studies face significant challenges, since there are too many tremendous influence factors such as seasonal effects, local climate, weather patterns, human activities etc. that might lead to enormous differences of airborne bacteria in different regions (Lee et al., 2006;Codina et al., 2008).In addition, different housing types in residential homes can have different ventilation performance depending on construction material, design and use of housing space which is largely influenced by political, social, environmental and economic factors (Nasir et al., 2012).It is necessary to investigate the concentration and composition of airborne bacteria indoors in different residential spaces across the globe and to further analyze its influence factors.Currently, China is a developing country in the world, and has about 1.3 billion inhabitants.Affordable houses remain narrow and insufficient in many cities due to the tremendous amount of population and the high price of housing.What's more, each family has only one child in most city homes because of the strict implementation of one-child policy in China.Recently, the occurrence of fog and haze weather at large area in China especially in large cities such as Beijing make us realize the importance and urgency to make systemic and scientific research on urban airborne microbial characterization both indoors and outdoors.Furthermore, the state of knowledge about the biological indoor air pollution in China remains relatively narrow and insufficient.Given the fact that housing conditions and living persons can have a considerable impact on bacterial exposure, a systematical investigation on the culturable airborne bacteria in 31 family homes with children aged from 1 to 10 years was carried out for one year in Beijing in the present study.It aims to describe the groups of culturable airborne bacteria and its variation characteristic, to reveal the overall baseline data and distribution characteristics of bacterial concentration, and to explore the relationship between bacterial concentration and housing condition or living persons in residential homes.

Selection of Residential Homes
Beijing is the political and cultural center of China, and is also one of the ten most populous cities in the world, with about 19.6 million inhabitants in 2011.In this study, 31 residential homes (16 with male child and 15 with female child), located in the east, south, west, north and center of Beijing in China were selected for bacterial sampling in the air.The selected residential homes (16 slab-type apartment buildings versus 15 tower buildings) need to meet the following criteria: with only one child (male or female) from 1 to 10 years old, no visible mold and no smell of mold in the apartment, no previous and present moisture damage at the home.The homes were selected from different area of Beijing city, and 3 apartments located in the east of Beijing (one with male child), 6 in the south (four with male child), 4 in the west (two with male child), 14 in the north (eight with male child), and 4 in the center (one with male child).The average age of children in each of the five areas (east, south, west, north, and center) was 4, 5, 6, 5, 6 years old, respectively.The largest area of the sampling apartment was 125 m 2 , and the smallest was 40 m 2 .

Description of Microbial Sampler
FA-1 sampler (imitated Andersen sampler, made by applied technical institute of Liaoyang, China) was utilized to collect culturable airborne bacteria inside the selected homes.Each stage of the sampler included a plate with 400 holes of uniform diameter through which air was drawn at 28.3 L/min to impact on petri dishes containing agar.Airborne particles were separated into six fractions, and the aerodynamic cut-size diameters in six stages were 7.0 µm (stage 1), 4.7-7.0µm (stage 2), 3.3-4.7 µm (stage 3), 2.1-3.3 µm (stage 4), 1.1-2.1 µm (stage 5), and 0.65-1.1µm(stage 6) respectively.In addition, the FA-1 sampler was sterilized in hot air oven at 180°C for 2 h before each 24 h measurement and washed with 5% bleach and 70% ethanol solution before each measurement in the sampling site.

Sampling Methods
Bacterial sampling in residential homes was performed from December 2009 to November 2010.Each indoor air sampling set-up was placed in the living room in order to collect a representative sample of the residential apartment.Each sampling device was operated at a sampling flow rate of 28.3 L/min, which was maintained with a platform at the height of 1.5 m and at a distance of approximately 2 m from the apartment wall.The indoor samplers were oriented vertically relative to the ground.The residents stayed at home and performed their normal activities, and HVACs (Heating, Ventilating and Air Conditioning) were normal running in Summer (air conditioning) and Winter (heating) during the sampling.Bacterial samples in each indoor site were collected for three minutes, one time each day, and continued for three consecutive days of each season in a year.For each bacterial sampling, the FA-1 sampler was loaded with 9.0 cm petri dishes containing nutrient agar (3 g beef extract, 10 g peptone, 5 g sodium chloride, 15 g agar, 1000 mL distilled water, pH = 7.2).Exposed culture dishes were incubated for 48 h at 37°C.

Bacterial Concentration Determination
After incubation, the colonies were counted and concentration of samples was expressed as CFU per cubic meter of air (CFU/m 3 ).However, since the superposition is unavoidable when the microbial particles impact the same spot through the same sieve pore (Wang et al., 2010), the colonies collected were revised by Eq. (1).CFU/m 3 was calculated by Eq. (2).(2) In the equations, P r is the revised colony in every stage; N is the number of sieve pore in every stage of the sampler; r is the number of sampling colonies; C is bacterial concentration in the air; P 1 , P 2 , P 3 , P 4 , P 5 , and P 6 are the revised colonies in every stage in the sampler; t is the sampling time; and F is the air flow rate of the sampler during sampling.

Bacterial Identification
In this study, approximately total 632 airborne bacteria were identified based on 16S rDNA sequence.The airborne bacterial colonies were isolated from different regions (east-136, south-141, west-122, north-116, and center-117) in Beijing and from different seasons  in a year in different sampling homes.A representative number of bacterial colonies were selected randomly from the sampler plates to quantitatively estimate the kinds of bacteria.Approximately 5% of the colonies were selected, with no less than 6 colonies and no more than 10, due to sampling constraints.Final analysis represents those bacteria that were culturable after initial plate counts.
Bacterial isolates selected from the sampling sites were further identified using the molecular method as described below.Each pure isolate was homogenized in liquid culture medium and then DNA was extracted using CATB method (Möller et al., 1992).The 16S rRNA gene was amplified using the following universal primer set: 27F: 5′-AGA GTT TGA TCC TGG CTC AG-3′ and 1492R: 5′-GGT TAC CTT GTT ACG ACT T-3′.The reaction mixture (50 μL) consisted of 0.3 μL Taq polymerase, 2 μL dNTP, 5 μL10 × PCR buffer, 2 μL each primer, and 1.0 μL (ca. 10 ng DNA) template.The amplification program was as follows: initial denaturation at 94°C for 5 min, 30 cycles of 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, and then final extension for 10 min at 72°C.The PCR products were purified and then detected by electrophoresis on a 1% agarose gel.The sequences were obtained using primer 27F by the Shanghai Majorbio Bio-technology Company, and were analyzed with the BLAST program of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/Blast.cgi).The sequences showing the highest similarity to those of the products were extracted from GenBank.

Statistical Analysis
All the experimental data were analyzed using Excel 2010 and SPSS Version 19.0 (SPSS.Inc., Standard Version).In this study, we collected all the experiment data from December 2009 to November 2010, which have a great variation in different sampling sites.Therefore, three kinds of bacterial concentration, including mean, median, and geometric mean, were calculated by SPSS Version 19.0 for the sake of reliable results.The percentage of different bacterial genera was counted by Microsoft Office Excel 2010.One-way analysis of variance (ANOVA) was used as the comparative method in different sampling sites and sampling time with SPSS Version 19.0.

Bacterial Groups and Variation Characterization Overall Bacterial Groups
The percentage of gram positive bacteria, accounting for 94.6%, was significantly higher than that of gram negative bacteria in the air in residential homes (P < 0.001), however, according Table 1 percentage of cocci was higher than rods.With regard to the bacterial groups, a total of 632 bacterial isolates distributed across 43 genera and 136 species of culturable airborne bacteria were identified from 31 residential homes in Beijing (Table 2).As a whole, the most common bacteria was Micrococcus (26.74%), followed by Bacillus (14.56%),Kocuria (12.66%),Staphylococcus (12.03%) , and they totally accounting for about 66% of culturable airborne bacteria in residential homes.The most common bacterial species identified in the air was Micrococcus luteus, followed by Kocuria roseus, Bacillus megaterium, Staphylococcus cohnii, and Micrococcus lylae, contributing to 14.56%, 8.39%, 4.75%, 3.63%, and 3.01% of the total airborne bacteria, respectively.

Bacterial Concentration and Variation Characterization Overall Concentration in Residential Homes
The survey on culturable bacteria concentration in these residential homes in Beijing was conducted detailedly from December 2009 to November 2010.Bacterial concentration in indoor air varied distinctly in 31 sampling sites.Highest bacterial concentration was 5573 CFU/m 3 at 31th sampling site, and lowest was 403 CFU/m 3 at 3th sampling site (Fig. 1).Table 5 demonstrated the bacterial concentration data in residential homes in Beijing.The range of culturable bacteria concentrations was from 47 to 12341 CFU/m 3 , and the mean and median concentration was 1821 CFU/m 3 and 877 CFU/m 3 , respectively.Significantly higher bacterial concentrations were found in residential homes with boy than those with girl (**P < 0.01).The mean concentration was 1946 CFU/m 3 (166-11202 CFU/m 3 ) in residential homes with boy, and for 1688 CFU/m 3 (47-12341 CFU/m 3 ) in those with girl.

Bacterial Concentration in Different Seasons
Significant differences in total bacterial concentrations were existed among seasons and the same seasonal variation pattern was found in residential homes with children.The mean bacterial concentrations in these residential homes were highest in Spring (months from Mar to May), followed by Summer (months from Jun to Aug), and Autumn (months from Sep to Nov), and lowest bacterial concentration was observed in Winter (months from Dec to Feb) (**P < 0.01) (Fig. 2).

Effects of Apartment Feature and Living Area Per Capita in Residential Homes on Bacterial Concentration
Effects of apartment feature on the bacterial concentration in residential homes were demonstrated in Fig. 3.No significant difference in bacterial concentration in the air was detected between slab-type building and tower building (P > 0.05).Fig. 4 showed the correlation between bacterial concentration and living area per capita in residential homes.The correlation coefficient was -0.667 calculated by SPSS19.0, and significant negative correlation was found between bacterial concentration and living area per capita in residential homes (P < 0.01).That is to say, bacterial concentration in the air increased with the decreasing of living area per capita in residential homes.

DISCUSSION
To reveal the adverse effects of airborne bacteria on human beings in indoor environment, we should firstly acknowledge the microbial profile of the atmosphere in residential homes, malls, supermarkets, hotels, subways, railway stations, and so on in different regions.Recently, many studies about bacterial investigation in the air have been carried out in such indoor environments as crowded and underground public concourse in Tokyo (Seino et al., 2005), public buildings of Korea (Kim and Kim, 2007), feedstuff-manufacturing factories (Kim et al., 2009), child day-care centers in Edirne city (Aydogdu et al., 2010), Mogao Grottoes (Wang et al., 2010), Polish libraries and archives (Karbowska-Berent et al., 2011), and occupied classroom (Qian et al., 2012).Our study indicated that airborne gram positive bacteria, accounting for about 94.6%, were dominated in residential homes, which was in agreement with other reports (Zhu et al., 2003;Awad, 2007;Aydogdu et al., 2010).However, great difference in the percentage of gram positive bacteria in the air between our result and previous study was observed.Aydogdu et al. (2010) reported that gram positive bacteria contributed to 97.16% and 95.04% indoors and outdoors in child day-care centers in Edirne City (Aydogdu et al., 2010).Shaffer and Lighthart (1997) found that the percentage of gram positive bacteria ranged from 70.0% to 85.0% at four diverse locations of urban, rural, forest, and coastal.It was reported that gram positive bacteria especially coccus, which can be isolated from the skin, mucous membrane, and other parts of human being and animal, are widely distributed in nature.Activities in residential homes with child might cause the full release of gram positive bacteria attached to the human body into the air, and then result in the substantial increasing of gram positive bacterial concentration (Koneman et al., 1995).
Micrococcus can be temporarily stranded on the skin, and Staphylococcus can be found on the skin and mucosa membrane of human and other mammalian.Micrococcus and Staphylococcus can be released into the ambient air from skin, mouths, nostrils, and hair of human being and pets by activities, and then result in the increasing of Micrococcus and Staphylococcus number (Aydogdu et al., 2010).Bacillus, closely following Micrococcus and occupying 12%-17%, was the dominant airborne bacteria in residential homes in this study, while Bacillus (6.61%) was the fourth dominant bacteria in child day-care centers in Edirne City (Aydogdu et al., 2010).The wide difference might be ascribed to different person composition and environmental condition between residential homes and child day-care centers.
The concentration of culturable airborne bacteria in residential homes in Beijing ranged from 47 to 12341 CFU/m 3 , with a mean concentration of 1821 CFU/m 3 .The bacterial concentration in the air varied greatly in residential homes in Beijing during a year, and the wide range could be attributed to the number of living person, housing types, ventilation feature, characteristics of human activities, etc. in different residential homes (ACGIH, 1999), and different meteorological parameters and climatic conditions in a year.Moreover, great differences of airborne bacterial concentration in residential environments were detected in different regions across the world (Pastuszka et al., 2000;Green et al., 2003;Lee and Jo, 2006;Nasir et al., 2012).It is unscientific to make direct comparison among various studies due to differences in housing types, household conditions, climatic and geographical parameters, but we find interestingly that the bacterial concentration in residential environments in Beijing was significantly higher than in developed countries such as USA and Korea (Green et al., 2003;Lee and Jo, 2006), and much lower than in other developing countries such as Pakistan (Nasir et al., 2012).It was reported that the living area per capita was about 67, 19.8 and 7 m 2 in USA, Korea and Pakistan, respectively (Nasir et al., 2012), whereas the living area per capita of selected residential homes in this study was ranged from 10 to 32 m 2 .On a large scale, all of these investigation data demonstrate that the number of living persons or living area per capita in residential homes has a significant influence on bacterial concentration in the indoor atmosphere, and bacterial concentration in the air increases with the decreasing of living area per capita.In the present study, significant negative correlation was found between bacterial concentration and living area per capita in residential homes.Our results were consistent with the finding reported by Qian et al. (2012) who observed the significant increases of total particle mass and bacterial genome concentrations during the occupied period compared to the vacant case, and emission rate per person-hour were 37 × 10 6 genome copies for airborne bacteria, and more bacteria in the air could be emitted from the skin, hair, nostrils, and mouths of occupants in a certain apartment with more living person (Qian et al., 2012).Therefore, the high bacterial load in residential homes could be contributed to the overcrowding and poor living conditions in developing countries.
There are many important factors that influence biological pollution in indoor environments.According to several studies, the moisture content of building materials, relative humidity and temperature (Pasanen et al., 2000;Ritchkoff et al., 2000), outdoor concentrations, air exchange rates (Kulmala et al., 1999), human activities (Buttner and Stetzenbach, 1993) and the number of people and pets (ACGIH, 1999) significantly affect the levels of indoor bioaerosols.In addition, housing conditions, the activities and lifestyle of occupants can contribute to the varying concentrations.In our study, the relationship between bacterial concentration and relevant factors that might significantly impact the distribution and quantity of bacteria such as apartment features, gender distribution, and living area per capita was analyzed detailedly.Interestingly, we found that bacterial concentration in the air was affected by child gender, and significantly higher bacterial concentrations were detected in residential homes with a male child than these with a female child (P < 0.01).A study, cited in an MSNBC article and published in the journal, showed that female participants exercised less than their male counterparts, and girls took part in 6% less vigorous playtime activity than boys.Females were also reported as almost half as physically active as males well before they reach their teens (Girls less sporty "from early age," BBC News, 25 July, 2001, 23:46 GMT 00:46 UK).In China, most of teen's activities were centered in residential homes due to parent's overprotection, and the more playtime activities performed by male child could motivate the emission of bacteria left on the floor, and then result in the increasing of bacterial concentration in the air in residential homes (Qian et al., 2012).Burge (1985Burge ( , 1990) also reported that the increase in airborne bacterial concentration was most probably due to the resuspension of previously settled particles indoors.
The season has been reported to influence the concentration of airborne bacteria indoors intensely.In this study, the bacterial concentration was highest in Spring, followed by Summer and Autumn, lowest in Winter in a year (**P < 0.01).However, Aydogdu et al. (2010) reported that no significant difference of bacterial concentration was observed in child day-care centers in Edirne city in a year.In Mogao Grottoes, higher bacterial concentration in the air was found in Summer than in Winter in the semi-closed cave and entrance (Wang et al., 2010).All of these differences in seasonal variation pattern of bacterial concentration in different regions might be resulted by geographical and environmental conditions.

SUMMARY
A one-year prospective investigation on culturable airborne bacteria was conducted to assess the background of culturable airborne bacteria in residential homes in China, and novel results between bacterial concentration in the air and living persons in residential home were explored in this study.Impressively, bacterial concentrations in residential homes with a male child were significantly higher than those with a female child, and negative correlation was found between bacterial concentration and living area per capita in residential homes.This is a new approach in the assessment of the factors that may affect the concentration of bacteria in the air indoor.

Fig. 3 .Fig. 4 .
Fig. 3. Bacterial concentration in residental homes with child in slab-type building and tower building in Beijing.

Table 1 .
Type composition of airborne bacteria in residential homes with child in Beijing.

Table 2 .
Bacterial groups in the air in residential homes with child in Beijing.

Table 3 .
Spatial variation pattern of culturable airborne bacteria in residential homes with child in Beijing.

Table 4 .
Seasonal variation pattern of culturable airborne bacteria in residential homes with child in Beijing.

Table 5 .
Concentration data on airborne bacteria in residential homes with child in Beijing (CFU/m 3 ).Seasonal variation pattern of culturable bacterial concentration in residential homes with child in Beijing.