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Building and Environment 135 (2018) 68–73

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Microbial quality assessment of indoor air in a large hospital building during T

winter and spring seasons
Ayesha Asifa, Muhammad Zeeshana,∗, Imran Hashmia, Uzma Zahidb, Muhammad Faraz Bhattic
Institute of Environmental Sciences and Engineering (IESE), School of Civil and Environmental Engineering (SCEE), National University of Sciences and Technology
(NUST) H-12 Campus, Islamabad 44000, Pakistan
Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan
Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST) H-12 Campus, Islamabad 44000, Pakistan


Keywords: Sensitivity of hospital building environment due to presence of potential sources of wide range of airborne
Indoor air quality microbes, make it a complex environment. Present study aimed to investigate seasonal (winter & spring) var-
Airborne microbes iation in airborne microbial levels as well as species on various locations (i.e. operation theatres (OT1 & OT2),
Hospital building wards (GMW & SW), out-patient department (OPD) and emergency services (ES)) of a large hospital building. Air
Airborne fungi
samples were collected during peak hours, twice a week, covering one month of each season. Statistically sig-
Airborne bacteria
nificant variation (p > 0.05) in bacterial concentrations over two seasons was found only for OPD. However,
fungal concentrations significantly varied (p < 0.05) over two seasons for all sites except for OT1 and OT2.
Concentrations among most of sites were significantly different. Highest bacterial level was found in OPD (mean:
1649.7 CFU/m3) while lowest in the two OTs (mean: 221 CFU/m3 for OT1 and 236 CFU/m3 for OT2). Highest
fungal level was found in GMW (mean: 193.4 CFU/m3) while lowest in the two OTs (mean: 41.1 CFU/m3 for OT2
and 58 CFU/m3 for OT1). Bacterial identification showed dominancy of gram positive cocci (89.8%) followed by
gram positive rods (7.2%) and gram negative rods (3%). Identified bacterial strains belonged to genera sta-
phylococcus, micrococcus, kocuria, aerococcus, kytococcus, bacillus and pseudomonas. The most abundant
fungal genera included cladosporium (47%), aspergillus (17.1%), penicillium (7.1%), alterneria (6.2%), geo-
trichium (3.68%) and ulocladium (3.2%). Cleaning frequencies appeared to be important factor in maintaining
low microbial load in air.

1. Introduction (e.g. hospital, schools, food courts etc.) essential [7]. In hospital en-
vironment, airborne microbial population is present in diverse range
Tightening of buildings in the modern architectural trends for [8], where concentrations depend primarily on number and types of
achieving higher energy efficiency has affected the indoor air quality patients [9]. In addition, medical activity, cleaning frequency and
(IAQ) [1] for all buildings in general and hospital buildings in specific. cleaning procedures of hospitals [10], weather and ventilation rate [11]
Airborne micro-organisms, which are matter of great concern for public and building design [12] are also the decisive factors for airborne mi-
health, show variation in concentration with time, indoor as well as crobial concentration levels which combined to make the situation
outdoor conditions and geographic location [2]. Various natural and challenging to maintain satisfactory IAQ [10].
anthropogenic sources and factors contribute towards high concentra- Studies reported an increasing trend of infections caused by air-
tion buildup of micro-organisms in indoor air [3,4] e.g. biological (e.g. borne micro-organisms due to the recent concept of air-tight buildings
indoor plants), physical (e.g. temperature and humidity) and chemical [9]. Various health issues linked with the exposure of airborne micro-
factors (e.g. presence of airborne organic particulate matter) [5], out- organisms are infectious diseases, toxic reactions [6], pneumonia, hy-
door sources, number of occupants [4–6]. Lack of the availability of an persensitivity, bronchitis [13], tiredness, headache [14], asthma, al-
efficient mechanical Heating, ventilation and Air Conditioning (HVAC) lergies [15], alveolitis [4], rhinitis [16] and hay fever etc. where se-
control system, the indoor environment of the building is strongly in- verity of the symptoms being function of pathogenicity of micro-
fluenced by the fluctuations in its surroundings thus making the organisms, immune system of persons and environmental conditions
scheduled monitoring of the indoor environment of sensitive buildings [17]. In normal conditions, species of fungi are not supposed to cause

Corresponding author.
E-mail address: (M. Zeeshan).
Received 27 December 2017; Received in revised form 13 February 2018; Accepted 6 March 2018
Available online 07 March 2018
0360-1323/ © 2018 Published by Elsevier Ltd.
A. Asif et al. Building and Environment 135 (2018) 68–73

any infection, but they are found to spread diseases in im- temperature and relative humidity, recorded from nearest weather
munosuppressed patients of hospitals [11,12,18]. Although World station (33.62°N, 73.10°E), were 11 °C and 74% respectively while the
health organization (WHO) showed concern towards indoor biological spring sampling was carried out during April 2017 when average out-
agents and building moisture [19], majority of countries have no clear door temperatures and relative humidity were 26 °C and 46.5% re-
regulations or proposed guidelines for acceptable concentrations of spectively.
micro-organisms in indoor environments particularly [14]. Airborne microbial samples were collected twice a week during the
Keeping in view the sensitivity of hospital buildings, due to the peak hours of each sampling location using personal air sampler (Gilian
existence of airborne micro-organisms and patients with immune defi- 5000) operating at flow rate of 5 l/min for 10 min. To represent
ciencies, many researchers worked on airborne microbial indoor air breathing zone, sampling height was kept at 1.5 m above ground.
quality of hospitals. The main focus of these studies were operation Cellulose nitrate filter paper (Sartorius, 13107-47-CAN) with a pore size
theatres [8,20], orthopedic ward [12], hematological units [18], in- of 0.45 μm and diameter 47 mm was used as a collecting medium for
tensive care unit (ICU), patient rooms, and neonatal wards [9]. Some of microbes. Tryptone soy agar (TSA) (OXOID CM0131) for bacterial co-
the studies also focused on seasonal variation of airborne microbes lonies and potato dextrose agar (PDA) (OXOID CM0139) for fungal
[11,18]. There are studies on seasonal variation of airborne micro-or- colonies, autoclaved at 121 °C for 15–20 min, were used as culture
ganisms which showed the dominancy of gram-positive bacteria in in- media for the sampled microbes. After sampling, filter papers were
door air [3,21,22]. A few studies also reported the seasonal variation in placed directly [23] on the respective growth medium on plates under
fungal levels with no significant variation in bacterial levels [11,18]. sterile conditions. Plates were then sealed and transferred to laboratory
Sensitivity and complexity of hospitals vary from one place to an- where bacterial colonies were incubated at 37 °C for 24–48 h while
other. OTs are supposed to be the most sensitive places and require fungal colonies were incubated at 28.5 °C for 3–5 days. Colonies ob-
strict maintenance of acceptable levels of airborne micro-organisms. tained were then counted and expressed as CFU/m3 [11,24]. Samples
Similarly, other wards require control measures according to the sen- were collected in triplicate to ensure reproducibility of results and
sitivity of patients present there. Most of the previous research work on average values are reported.
microbial indoor air quality of hospitals has been focused on specific
locations like OTs, ICUs and wards. However, very few studies have 2.3. Isolation and identification of bacteria and fungi
assessed the seasonal variation of airborne microbial load of different
sections of a hospital. Thus, the aim of present study was to investigate Airborne bacterial colonies obtained on TSA plates were initially
airborne bacterial and fungal levels of different sites in a hospital along separated on the basis of their morphological characteristics (shape,
with their seasonal variations. For this purpose, six sites of a large size, color) and then identified up-to the genus level on the basis of their
publicly managed hospital of Islamabad, Pakistan was selected. The microscopic appearance and results of biochemical tests. Microscopic
most dominantly observed bacterial and fungal colonies were identified appearance of bacterial colonies was observed under a microscope with
up-to species level following the standard methods. Important factors oil immersion (1000× magnification) after gram-straining. Colonies
contributing towards buildup of higher airborne microbial levels were were grouped in classes of gram-negative and gram-positive according
identified so that the air quality may be managed more efficiently. to Bergey's Manual of determinative bacteriology [25]. Further bio-
chemical characterization of bacterial colonies was then performed by
2. Methodology modified oxidase test and catalase test. The most frequently observed
colonies were then identified up-to the specie level by sequencing the
2.1. Selected hospital information amplified regions of extracted 16s ribosomal DNA gene with paired
primers [26].
An 1100-bed publicly managed hospital of Islamabad, Pakistan, Airborne fungal colonies recovered on PDA plates were also initially
founded in 1985, covering area of 5.1 ha, was selected for airborne classified morphologically by their spores' color and shape.
microbial investigation during spring and winter seasons. Hospital was Identification of dominant colonies up-to genus level was performed by
selected for the study as it consists of 32 specialized clinics with 12 preparing wet-mount slides using lacto-phenol blue and then observed
critical units, catering for approximately 5000 patients per day with a under microscope of magnification 400×. Fungi were then identified
variety of medical histories, making the environment extremely com- according to their microscopic appearance.
plex. Six sites were selected for the purpose of study which include (i)
emergency operation theatre (OT1) (ii) general surgery operation 2.4. Statistical analysis
theatre (OT2), (iii) surgical ward (SW), (iv) general medicine ward
(GMW), (v) emergency services (ES) and (vi) out-patient department The data was analyzed in MS Excel (Microsoft Corporation, USA)
(OPD). and SPSS 14 (IBM Corp., USA). Normality of data was checked by
OT1 remains operational 24 h a day having 15–20 patients operated Kolmogrov-Smirnov test and Shapiro-Wilk test. One-Way ANOVA was
per day whereas in OT2, patients were operated from 8 h to 14 h, used to analyze the statistical difference among different sampling sites
having average 8 patients operated per day. Both OTs were washed and t-test was used to analyze the statistical difference between ob-
with disinfectants in morning and mopped with water before and after servations of two seasons for same site.
each surgery. Besides, periodic deep cleaning, including walls and
ceiling was performed on need basis. Selected wards for study purpose 3. Results and discussion
had a capacity for 8 patients each with 2–3 attendants. ES remains
operational 24 h while working hours for OPD were from 8 h to 14 h. 3.1. Airborne bacterial and fungal concentrations
Both locations had high occupation levels. Floors of both monitored
wards, OPD and ES were mopped with disinfectants twice a day. Indoor bacterial concentration in a hospital is supposed to be af-
Table 1 shows the complete description of monitored sites. fected by the type and number of patients in that particular area.
Moreover, indoor fungal concentration depends on the indoor moisture
2.2. Sampling duration and frequency conditions, cleaning frequency and outdoor atmospheric conditions of
that particular area. Table 2 shows descriptive statistics of indoor
Seasonal (winter and spring) assessment of airborne bacteria and concentration of airborne bacteria and fungus in six sites of the hos-
fungus was performed covering one month of each season. Winter pital.
sampling covered month of January 2017 when average outdoor Results showed higher range and mean values of airborne bacteria

A. Asif et al. Building and Environment 135 (2018) 68–73

Table 1
Sampling site description.

Location Symbol Floor Building Type Type of HVAC systema Facility Area Maximum Occupational Period
(m2) Capacity

Emergency Operation OT1 1st Floor Closed ACb on in Springc 31.1 11d 24 h
Surgical Operation Theatre OT2 1st Floor Closed ACb on in Springc 37.5 11d 8 h–14 h
Outpatient Department OPD Ground Floor Semi-closed Natural ventilation through windows 701.9 > 1200 8 h–14 h
with no AC
Emergency services ES Ground Floor Closed Natural Ventilation through doors with 177.1 > 200 24 h
ACb on in Spring
Surgical Ward SW 1st Floor Closed Natural Ventilation through windows 56.2 20–30e 24 h
With No AC
General medicine Ward GMW Ground Floor Closed Natural 56.2 20–30e 24 h
Ventilation through windows
With No AC

The hospital building was originally designed with a centralized HVAC system which was out of order.
Split air conditioning units of different capacities were being used in hospital for maintaining acceptable/comfortable temperature levels.
With out of order designed HVAC system, there was no specific provision for air renewal other than on-and-off door opening. OTs were equipped with UV light provision which was
being used for disinfection daily before and after operating times.
1 patient and 10 hospital staff.
8 patients with 2–3 attendants/patient.

Table 2 concentrations didn't show any significant difference. Although OPD

Descriptive statistics of indoor bacteria and fungus. was semi-closed location (Table 1), supposed to be having lower bac-
terial concentrations, higher occupancy appeared to be dominant
Location Bacteria (CFU/m3) Fungi (CFU/m3)
reason for higher bacterial concentrations.
OT1 Mean 221 58.4
Range 60–466.7 0–266.7 3.2. Seasonal variation
OT2 Mean 236.2 41.1
Range 60–460 0–153.8
OPD Mean 1649.7 176.5 Seasonal (winter and spring) variation of airborne bacteria and
Range 380–3577 20–500 fungus has been illustrated in Fig. 1 (a) and Fig. 1 (b) respectively. One-
ES Mean 1028.9 166.4 way ANOVA results showed no significant variation (p > 0.05) in
Range 280–2280 19.2–384.6
concentration levels of airborne bacteria between the two studied
SW Mean 369.9 135.2
Range 20–1038.5 20–307.7 seasons for any of the monitored locations except OPD, depicting var-
GMW Mean 383.9 193.4 ious other factors (discussed below) to be more significant contributors
Range 100–840 60–433.3 towards buildup of bacterial load than change in ambient conditions
over the two seasons. However, significant variation (p < 0.05) in
concentration levels of airborne fungal concentrations of OPD, ES,
as compared to fungal levels in all the studied sites. Highest con- GMW and SW has been observed showing seasonal variation as sig-
centration of bacteria was found in OPD (mean: 1649.7 CFU/m3) ex- nificant contributor. It is discussed in section 3.4 in details, indoor
plained by the fact that it was the site having large number of patients. fungal concentration levels were found to be associated with outdoor
At the time of sampling, the centralized HVAC system of the building plant activity which is at peak during spring and is assumed to be the
was out of order, OPD was directly connected with outdoor air through reason for significantly higher indoor fungal concentrations during this
open doors and hence there was uncontrolled/limited air renewal. season. Similar results have been reported earlier for effect of seasonal
Moreover, lowest concentration was found in OT1 (mean: 221 CFU/ factors on fungal and bacterial indoor levels in hospitals [11].
m3). Highest fungal load on the other hand was found in GMW (mean: OPD and ES are similar facilities, both having large number of at-
193.4 CFU/m3) while lowest in OT2 (mean: 41.1 CFU/m3). tendants and patients with diversity in disease types. Thus, airborne
Due to the difference in nature of activities and conditions at sites, microbial counts in both the areas were found to be highest among
large variation in concentrations of airborne micro-organisms was ob- monitored sites. Highest microbial counts in both the seasons were
served among the sites. Microbial concentrations observed at each site recorded in OPD ranging from 380 to 2480 CFU/m3 in winter and
were compared with other sites using One-way ANOVA test and results 1308–3577 CFU/m3 in spring. Moreover, airborne bacterial con-
showed significant statistical difference (p < 0.05) in 73% of bacterial centration of ES ranged from 280 to 2280 CFU/m3 in winter and
and 53% of fungal concentrations. As mentioned in Table 1, hospital 481–1519 CFU/m3 in spring. Fungal load of OPD was also highest
building was designed with a centralized HVAC system, which was out during winter season ranging from 20 to 500 CFU/m3 while that of ES
of order at the time of sampling (and remains nonfunctional) and hence ranged from 20 to 333 CFU/m3. However, during spring season, ES
there was no filtration of operation theater air in practice. However, showed the highest fungal load ranging from 19 to 385 CFU/m3.
since operation theaters are comparatively controlled environments Descriptive statistical analysis of seasonal variation in microbial con-
with less number of occupants and more cleaning frequency, overall centration levels is depicted in details in Table S1 (Supplementary In-
microbial load was found to be low and no significant difference formation, SI).
(p > 0.05) was observed in the airborne bacterial and fungal con- Wards, on the other hand, had less number of patients and atten-
centrations of the two locations. Similarly, both studied wards had dants as compared to OPD and ES facilities, thus comparatively less
more or less similar level of occupation, with same ambient conditions, microbial load was observed in wards. Airborne bacterial concentration
provided with limited air renewal through the windows (in absence of in SW during winter and spring ranged from 20 to 980 and
functional HVAC system), showed no significant difference (p > 0.05) 58–1038 CFU/m3 respectively. However, concentration levels in GMW
in microbial concentrations. Moreover, bacterial concentration of OPD ranged from 100 to 840 CFU/m3 in winter and 115–769 CFU/m3 in
and ES were significantly different (p < 0.05) while fungal spring season. Airborne fungal levels in SW during winter and spring

A. Asif et al. Building and Environment 135 (2018) 68–73


Bacterial ConcentraƟon


(CFU/m3) 1000


OT1 OT2 OPD Emergency General General
Surgery Medicine

Winter Spring

Fungal ConcentraƟon

300 (b).

OT1 OT2 OPD Emergency General General
Surgery Medicine

Winter Spring

Fig. 1. Seasonal variation of airborne (a) bacterial and (b) fungal levels.

Table 3 3.3. Exceedance from standards

Frequency (%) of exceedance to standards of microbial levels for monitored sites during
sampling period.
Pakistan has no national standards for airborne microbial con-
Location Bacterial Standards Fungal Standards centrations and there is no such proposed limit available which is ac-
cepted by all relevant scientific communities. Upper limit for fungal
ACGIH Portuguese WHO/Portuguese levels as suggested by World Health Organization (WHO) is 500 CFU/
(100 CFU/m3) (500 CFU/m3) (500 CFU/m3) m3 [27]. According to American Conference of Governmental Industrial
Winter Spring Winter Spring Winter Spring
Hygienists (ACGIH), for persons with immune deficiencies, limiting
value of bacterial concentration is 100 CFU/m3 [28]. A similar study
OT1 89 78 0 0 0 0 conducted earlier in Portugal [11] reported limiting value for both
OT2 89 89 0 0 0 0 airborne bacteria and fungus according to Portuguese National Stan-
OPD 100 100 77 100 0 0
ES 100 100 77 89 0 0
dards (PNS) as 500 CFU/m3 for winter season. Same standards are used
SW 66 83 22 50 0 0 in this study as reference. No location exceeded WHO/Portuguese Na-
GMW 100 100 22 16 0 0 tional Standards for fungal concentrations during monitoring period.
However, for the bacterial loads, OPD, ES and GMW showed 100%
observations beyond ACGIH limits for both seasons and 77 and 100%
ranged from 20 to 260 and 77–308 CFU/m3 while in GMW from 60 to (OPD) and 77 and 89% (ES) observations exceeding PNS limits for
433 and 77–269 CFU/m3 respectively. winter and spring seasons respectively. Table 3 shows detailed ex-
OTs are the most sensitive places of a hospital, thus requiring proper ceedance occurrences of airborne bacterial and fungal levels with
control of airborne microbes. Bacterial concentration in OT1 was found standard limits for all monitored locations during both seasons.
to be between 60 and 467 CFU/m3 in winter and 96–461 CFU/m3 in
spring season. However, in OT2 range was 60–460 CFU/m3 in winter
and 77–269 CFU/m3 in spring. Fungal load in OT1 in winter and spring 3.4. Identification of airborne microbes
was recorded as 0–267 CFU/m3 and 0–120 CFU/m3 while in OT2 as
0–96 CFU/m3 and 0–154 CFU/m3 respectively. Bacterial phenotypic identification through biochemical tests in-
dicated presence of three types of airborne micro-organisms (gram-
positive cocci, rods and gram negative rods) in both monitored seasons.
Gram-positive cocci were the most dominant group of genera (89.8%)

A. Asif et al. Building and Environment 135 (2018) 68–73

Table 4
Total isolates recovered (and percentage) of identified species of the airborne bacteria over each monitoring site for two seasons.a

Bacterial Colonies OT1 OT2 OPD ES SW GMW

Winter Spring Winter Spring Winter Spring Winter Spring Winter Spring Winter Spring

Gram positive cocci 76 (83.5) 64 (96.9) 89 (89.9) 57 (98.3) 391 (87.5) 330 (92.9) 274 (86.4) 282 (93.1) 150 (86.7) 86 (89.6) 138 (88.5) 89 (92.7)
Staphylococcus haemolyticus 38 (50) 33 (51.6) 48 (53.9) 31 (54.4) 164 (41.9) 75 (22.7) 143 (52.2) 79 (28) 64 (42.7) 32 (37.2) 73 (52.9) 36 (40.5)
Micrococcus luteus 17 (22.4) 8 (12.5) 13 (14.6) 7 (12.3) 124 (31.7) 83 (25.2) 60 (21.9) 52 (18.4) 49 (32.7) 14 (16.3) 27 (19.6) 11 (12.4)
Micrococcus terreus 6 (7.9) 3 (4.7) 13 (14.6) 4 (7) 28 (7.2) 37 (11.2) 9 (3.3) 15 (5.3) 12 (8) 10 (11.6) 13 (9.4) 10 (11.2)
Kocuria rosea 4 (5.3) 2 (3.1) 2 (2.5) 2 (3.5) 11 (2.8) 49 (14.8) 7 (2.6) 59 (20.9) 5 (3.3) 5 (5.8) 6 (4.4) 5 (5.6)
Staphylococcus aureus 0 7 (10.9) 1 (1.1) 7 (12.3) 4 (1) 26 (7.9) 9 (3.3) 24 (8.5) 2 (1.3) 12 (13.9) 3 (2.2) 15 (16.8)
Kocuria rhizophila 1 (1.3) 3 (4.7) 3 (3.4) 2 (3.5) 28 (7.2) 14 (4.2) 16 (5.8) 10 (3.5) 3 (2) 3 (3.5) 3 (2.2) 3 (3.4)
Kocuria kristinae 4 (5.3) 1 (1.5) 1 (1.1) 1 (1.7) 8 (2) 18 (5.4) 14 (5.1) 9 (3.2) 7 (4.7) 6 (6.9) 4 (2.9) 0
Aerococcus viridans 0 3 (4.7) 1 (1.1) 2 (3.5) 6 (1.5) 8 (2.4) 4 (1.5) 7 (2.5) 0 3 (3.5) 1 (0.7) 3 (3.4)
Kytococcus sedentarius 1 (1.3) 0 3 (3.4) 0 4 (1) 5 (1.5) 0 7 (2.5) 4 (2.7) 0 0 0
Staphylococcus cohnii 0 2 (3.1) 0 0 4 (1) 2 (0.6) 0 2 (0.7) 0 0 1 (0.7) 0
Others 5 (6.6) 2 (3.1) 4 (4.5) 1 (1.7) 10 (2.6) 13 (3.9) 12 (4.4) 18 (6.4) 4 (2.7) 1 (1.2) 7 (5.1) 6 (6.7)
Gram positive rod 3 (3.3) 2 (3) 9 (9.1) 1 (1.7) 50(11.2) 12 (3.4) 34(10.7) 10 (3.3) 19(10.9) 0 16(10.3) 7 (7.3)
Bacillus cereus 2 (66.7) 0 5(55.5) 0 21 (42) 0 17 (50) 0 8 (42.1) 0 10 (62.5) 0
Bacillus subtilis 0 0 4 (4.4) 1 (100) 17 (34) 2 (16.7) 3 (8.8) 0 8 (42.1) 0 4 (25) 4(57.1)
Gram negative rod 12 (13.2) 0 1 (1) 0 6 (1.3) 13 (3.7) 9 (2.8) 11 (3.6) 4 (2.3) 10 (10.4) 2 (1.3) 0
Pseudomonas stutzeri 12 (13.2) 0 1 (1) 0 6 (1.3) 13 (3.7) 9 (2.8) 11 (3.6) 4 (2.3) 10 (10.4) 2 (1.3) 0

Colony counts (percentage).

Table 5
Total isolates recovered (and percentage) of identified airborne fungal genera over each monitoring site for two seasons.a

Fungal Colonies OT1 OT2 OPD ES SW GMW

Winter Spring Winter Spring Winter Spring Winter Spring Winter Spring Winter Spring

Cladosporium Spp. 9 (45) 7 (70) 5 (38.5) 6 (60) 23(52.3) 28(49.1) 15(48.4) 33(62.3) 18 (40.9) 16 (40) 34(40.9) 10 (34.5)
Aspergillus fumigatus 7 (35) 0 3(23.1) 0 3 (6.8) 3 (5.3) 2 (6.5) 0 10 (22.7) 2 (5) 20(24.1) 1 (3.5)
Penicillium Spp. 0 0 0 0 6 (13.6) 2 (3.5) 3 (9.7) 1 (1.9) 6 (13.6) 4 (10) 3 (3.6) 6 (20.7)
Alterneria alternata 0 0 0 0 1 (2.3) 7 (12.3) 2 (6.5) 3 (5.7) 2(4.6) 8 (20) 3 (3.6) 1 (3.5)
Geotrichum Spp. 0 0 0 2 (20) 0 2 (3.5) 0 2 (3.8) 2(4.6) 1 (2.5) 5 (6) 2 (6.9)
Ulocladium chartarum 1 (5) 2 (20) 0 0 2 (4.5) 3 (5.3) 1 (3.2) 1 (1.9) 1 (2.3) 1 (2.5) 2 (2.4) 0
Aspergillus niger 0 0 2 (15.4) 0 0 3 (5.3) 2 (6.5) 0 1 (2.3) 1 (2.5%) 2 (2.4%) 0
Aspergillus flavus 0 0 1 (7.7%) 0 4(9.1%) 0 1 (3.2%) 0 0 0 6 (7.2%) 0
Others 3 (15%) 1 (10%) 2 (15.4%) 2 (20%) 5 (11.4%) 9 (15.8%) 5 (16.1%) 13 (24.5%) 4 (9.1%) 7 (17.5%) 8 (9.6%) 9 (31%)

Colony counts (percentage).

identified, followed by gram-positive rods (7.2%) and gram-negative 50.25%). Previously, presence of Cladosporium in the air has been re-
rods (3%) (Table 4 and Figs. S1 and SI). Seasonal assessment of re- ported in different parts of the world; e.g. Africa [34], Europe [35] and
covered airborne bacterial colonies also showed dominancy of gram- North America [36]. Abundance of cladosporium spp. may be explained
positive cocci (winter: 87.12%, spring: 93.22%) in both seasons. by the higher concentration levels of propagules in the outdoor en-
Bacterial genotypic identification of frequently observed colonies vironment due to presence of forests around the monitored sites as
characterized gram positive cocci into Staphylococcus such as reported earlier [18,22]. Cladosporium spp. are rarely associated with
Staphylococcus haemolyticus, Staphylococcus aureus and Staphylococcus human opportunistic infections [37] and are frequently reported as
cohnii, Micrococcus such as Micrococcus luteus and Micrococcus terreus, plant pathogens [38]. Three species of Aspergillus (Aspergillus fumigatus,
Kocuria such as Kocuria rosea, Kocuria rhizophila and Kocuria kristinae, Aspergillus niger and Aspergillus flavus) were identified from the re-
Aerococcus viridans and Kytococcus sedentarius. However, identified covered isolates. In previous studies, presence of Aspergillus species in
gram positive rods included Bacillus cereus and Bacillus subtilis and gram indoor air of hospitals was considered as a risk factor for patients due to
negative rods included Pseudomonas stutzeri. Detailed colony counts their ability to cause nosocomial infections and allergies [11,12]. Pre-
(and percentage) of the frequently observed bacterial species over the sence of Aspergillus in the hospital can be explained by extensive con-
monitoring sites for two seasons are given in Table 4. Staphylococcus struction activities ongoing around the hospital during monitoring
haemolyticus was found as the most abundantly prevalent bacterial period as previous studies [39] have reported hospital outbreaks of
specie on all monitored sites. Similar results have been reported for a invasive aspergillosis (disease caused by Aspergillus) associated with
study investigating airborne bacterial concentrations in Tunisian hos- demolition and building construction. Alterneria alternata and Ulocla-
pital [29]. Staphylococcus haemolyticus is commonly found on human dium chartarum were among the dominant species of Alterneria, and
skin [30] and is associated with the insertion of medical devices [31]. It Ulocladium. Detailed colony counts (and percentage) of the frequently
is known for its antibiotic resistant phenotype and as an opportunistic observed fungal colonies over each monitoring site are given Table 5
bacterial pathogen [32] which may cause meningitis, skin and pros- (and Figs. S2 and SI). It is to be noted here that TSA and PDA are widely
thetic join infections [33]. used and reported growth media for airborne bacteria and fungus re-
Overall, the most frequently observed airborne fungal genera in the spectively, due to their non-selective nature and suitability for sup-
six monitored sites were identified as Cladosporium (47%), Aspergillus porting a wide range of microbes, however there is no universal
(17.05%), Penicillium (7.14%), Alterneria (6.22%), Geotrichium (3.68%) medium that supports the growth of all microorganisms.
and Ulocladium (3.22%). For both, winter and spring, Cladosporium spp.
was the most abundantly found fungal isolate (winter: 44.25%, spring:

A. Asif et al. Building and Environment 135 (2018) 68–73

4. Conclusion R. Steinberg, et al., Characterization of indoor bioaerosols from a hospital ward in a

tropical setting, Afr. Health Sci. 12 (2) (2012) 217–225.
[13] M.F. Yassin, S. Almouqatea, Assessment of airborne bacteria and fungi in an indoor
Airborne microbial assessment of studied sites of a publicly man- and outdoor environment, Int. J. Environ. Sci. Technol. 7 (3) (2010) 535–544.
aged hospital showed dependency of microbial levels on the type of [14] H. Salonen, S. Lappalainen, O. Lindroos, R. Harju, K. Reijula, Fungi and bacteria in
area as well as ongoing activities and types of patients in those areas. mould-damaged and non-damaged office environments in a subarctic climate,
Atmos. Environ. 41 (32) (2007) 6797–6807.
Occupancy level showed significantly affecting the bacterial levels as [15] J. Madureira, I. Paciência, J. Rufo, E. Ramos, H. Barros, J.P. Teixeira, E. de Oliveira
two of the busiest locations (OPD and ES) showed maximum bacterial Fernandes, Indoor air quality in schools and its relationship with children's re-
concentrations during both seasons. However, outdoor climatic condi- spiratory symptoms, Atmos. Environ. 118 (2015) 145–156.
[16] J.H. Lee, W.K. Jo, Characteristics of indoor and outdoor bioaerosols at Korean high-
tions appeared to affect indoor fungal concentrations more sig- rise apartment buildings, Environ. Res. 101 (1) (2006) 11–17.
nificantly. Cleaning frequency, on the other hand showed significant [17] V. Kummer, W.R. Thiel, Bioaerosols–sources and control measures, Int. J. Hyg
impact on both, bacterial as well as fungal concentration levels as both Environ. Health 211 (3–4) (2008) 299–307.
[18] M. Sautour, N. Sixt, F. Dalle, C. L'Ollivier, V. Fourquenet, C. Calinon, et al., Profiles
OTs, having higher cleaning frequencies with provision of UV light
and seasonal distribution of airborne fungi in indoor and outdoor environments at a
sterilization, were observed to have lesser microbial loads. Gram posi- French hospital, Sci. Total Environ. 407 (12) (2009) 3766–3771.
tive cocci was found to be the most dominant bacterial group in all [19] J. Madureira, I. Paciência, J.C. Rufo, C. Pereira, J.P. Teixeira, E. de Oliveira
monitored sites followed by gram positive and gram negative rods. Fernandes, Assessment and determinants of airborne bacterial and fungal con-
centrations in different indoor environments: Homes, child day-care centres, pri-
Outdoor conditions appeared to affect indoor fungal concentrations as mary schools and elderly care centres, Atmos. Environ. 109 (2015) 139–146.
cladosporium sp. was found the most frequently observed fungal genera [20] C. Napoli, S. Tafuri, L. Montenegro, M. Cassano, A. Notarnicola, S. Lattarulo, et al.,
in indoor air. While other observed fungal genera included aspergillus, Air sampling methods to evaluate microbial contamination in operating theatres:
results of a comparative study in an orthopaedics department, J. Hosp. Infect. 80 (2)
penicillium, alterneria and ulocladium. Prevalence of alarmingly higher (2012) 128–132.
bacterial concentrations necessitates the provision of a functional and [21] K.J. Heo, B.U. Lee, Seasonal variation in the concentrations of culturable bacterial
well-maintained HVAC system capable of delivering adequate ventila- and fungal aerosols in underground subway systems, J. Aerosol Sci. 92 (2016)
tion rates as well as efficient filtration. To ensure stringent control, [22] E. Medrela-Kuder, Seasonal variations in the occurrence of culturable airborne
scheduled monitoring is essential. fungi in outdoor and indoor air in Cracow, Int. Biodeterior. Biodegrad. 52 (4)
(2003) 203–205.
[23] F. Pérez-Martín, S. Seseña, M. Fernández-González, M. Arévalo, M.L. Palop,
Acknowledgements Microbial communities in air and wine of a winery at two consecutive vintages, Int.
J. Food Microbiol. 190 (2014) 44–53.
The authors would like to acknowledge the National University of [24] B. Breza-Boruta, The assessment of airborne bacterial and fungal contamination
emitted by a municipal landfill site in Northern Poland, Atmos. Pollut. Res. 7 (6)
Sciences and Technology, Islamabad, Pakistan for the provision of
(2016) 1043–1052.
funding to carry out this research. The authors would also like to ac- [25] J.G. Holt, N.R. Kreig, P.H.A. Sneath, J.T. Staley, S.T. Williams, Bergey's Manual of
knowledge Dr. Iqbal Memon, Dean Surgery and Allied Specialties at Determinative Bacteriology, ninth ed., MD: Williams & Wilkins, Baltimore, 1994.
Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, for his [26] T. Maki, M. Kakikawa, F. Kobayashi, M. Yamada, A. Matsuki, H. Hasegawa,
Y. Iwasaka, Assessment of composition and origin of airborne bacteria in the free
unconditional guidance throughout the study. troposphere over Japan, Atmos. Environ. 74 (2013) 73–82.
[27] WHO, Indoor air quality: biological contaminants, WHO- Regional Publications.
Appendix A. Supplementary data European Series, World Health Organization, Copenhagen, 1990, p. 11031.
[28] ACGIH, American Conference of Governmental Industrial Hygienists, Guidelines for
the Assessment about Aerosols in the Indoor Environment, American Conference of
Supplementary data related to this article can be found at http://dx. Governmental Industrial Hygienists, Cincinnati, OH, USA, 1989. [29] R. Dziri, N. Klibi, C. Lozano, L.B. Said, R. Bellaaj, C. Tenorio, et al., High prevalence
of Staphylococcus haemolyticus and Staphylococcus saprophyticus in environ-
mental samples of a Tunisian hospital, Diagn. Microbiol. Infect. Dis. 85 (2) (2016)
References 136–140.
[30] C.E. Robertson, L.K. Baumgartner, J.K. Harris, K.L. Peterson, M.J. Stevens,
D.N. Frank, N.R. Pace, Culture-independent analysis of aerosol microbiology in a
[1] Lisa C. Ng, Andrew K. Persily, Steven J. Emmerich, IAQ and energy impacts of
metropolitan subway system, Appl. Environ. Microbiol. 79 (11) (2013) 3485–3493.
ventilation strategies and building envelope airtightness in a big box retail building,
[31] E.M. Barros, H. Ceotto, M.C.F. Bastos, K.R.N. Dos Santos, M. Giambiagi-deMarval,
Build. Environ. 92 (2015) 627–634.
Staphylococcus haemolyticus as an important hospital pathogen and Carrier of
[2] A. Rajasekar, R. Balasubramanian, Assessment of airborne bacteria and fungi in
methicillin resistance genes, J. Clin. Microbiol. 50 (1) (2012) 166–168.
food courts, Build. Environ. 46 (10) (2011) 2081–2087.
[32] F. Takeuchi, S. Watanabe, T. Baba, H. Yuzawa, T. Ito, Y. Morimoto, et al., Whole-
[3] W. Wang, Y. Ma, X. Ma, F. Wu, X. Ma, L. An, H. Feng, Seasonal variations of air-
genome sequencing of Staphylococcus haemolyticus uncovers the extreme plasticity
borne bacteria in the Mogao Grottoes, Dunhuang, China, Int. Biodeterior.
of its genome and the evolution of human-colonizing staphylococcal species, J.
Biodegrad. 64 (4) (2010) 309–315.
Bacteriol. 187 (21) (2005) 7292–7308.
[4] B. Mashat, Indoor and outdoor microbial aerosols at the holy mosque: a case study,
[33] M. Falcone, F. Campanile, M. Giannella, S. Borbone, S. Stefani, M. Venditti,
Atmospheric Pollution Research 6 (6) (2015) 990–996.
Staphylococcus haemolyticus endocarditis: clinical and microbiologic analysis of 4
[5] S.C. Lee, M. Chang, Indoor and outdoor air quality investigation at schools in Hong
cases, Diagn. Microbiol. Infect. Dis. 57 (3) (2007) 325–331.
Kong, Chemosphere 41 (1) (2000) 109–113.
[34] A.A. Hameed, M.I. Khoder, S. Yuosra, A.M. Osman, S. Ghanem, Diurnal distribution
[6] A. Forthomme, A. Joubert, Y. Andrès, X. Simon, P. Duquenne, D. Bemer, L. Le Coq,
of airborne bacteria and fungi in the atmosphere of Helwan area, Egypt, Sci. Total
Microbial aerosol filtration: growth and release of a bacteria–fungi consortium
Environ. 407 (24) (2009) 6217–6222.
collected by fibrous filters in different operating conditions, J. Aerosol Sci. 72
[35] D.J. O'Connor, M. Sadyś, C.A. Skjøth, D.A. Healy, R. Kennedy, J.R. Sodeau,
(2014) 32–46.
Atmospheric concentrations of alternaria, cladosporium, ganoderma and didymella
[7] J. Ferdyn-Grygierek, Monitoring of indoor air parameters in large museum ex-
spores monitored in Cork (Ireland) and Worcester (England) during the summer of
hibition halls with and without air-conditioning systems, Build. Environ. 107
2010, Aerobiologia 30 (4) (2014) 397–411.
(2016) 113–126.
[36] C. Calderón, J. Lacey, A. McCartney, I. Rosas, Influence of urban climate upon
[8] R. Bali, P. Sharma, S. Nagrath, P. Gupta, Microbial isolations from maxillofacial
distribution of airborne Deuteromycete spore concentrations in Mexico City, Int. J.
operation theatre and its correlation to fumigation in a teaching hospital in India, J.
Biometeorol. 40 (2) (1997) 71–80.
Maxillofac. Oral Surg. 13 (2) (2014) 128–132.
[37] M. Sandoval-Denis, D.A. Sutton, A. Martin-Vicente, J.F. Cano-Lira, N. Wiederhold,
[9] K. Qudiesat, K. Abu-Elteen, A. Elkarmi, M. Hamad, M. Abussaud, Assessment of
J. Guarro, J. Gené, Cladosporium species recovered from clinical samples in the
airborne pathogens in healthcare settings, Afr. J. Microbiol. Res. 3 (2) (2009)
United States, J. Clin. Microbiol., JCM (2015) 01482.
[38] M. Sadyś, R. Kennedy, C.A. Skjøth, An analysis of local wind and air mass directions
[10] C.C. Jung, P.C. Wu, C.H. Tseng, H.J. Su, Indoor air quality varies with ventilation
and their impact on Cladosporium distribution using HYSPLIT and circular statis-
types and working areas in hospitals, Build. Environ. 85 (2015) 190–195.
tics, Fungal Ecol. 18 (2015) 56–66.
[11] S.C. Verde, S.M. Almeida, J. Matos, D. Guerreiro, M. Meneses, T. Faria, et al.,
[39] B. Pilmis, V. Thepot-Seegers, C. Angebault, E. Weiss, I. Alaabouche, M.E. Bougnoux,
Microbiological assessment of indoor air quality at different hospital sites, Res.
J.R. Zahar, Could we predict airborne Aspergillus contamination during construc-
Microbiol. 166 (7) (2015) 557–563.
tion work? Am. J. Infect. Contr. 45 (1) (2017) 39–41.
[12] S. Sudharsanam, S. Swaminathan, A. Ramalingam, G. Thangavel, R. Annamalai,