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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: mzalikhan@gmail.com (M. Zeeshan).
https://doi.org/10.1016/j.buildenv.2018.03.010
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
69
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
Theatre
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
a
The hospital building was originally designed with a centralized HVAC system which was out of order.
b
Split air conditioning units of different capacities were being used in hospital for maintaining acceptable/comfortable temperature levels.
c
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.
d
1 patient and 10 hospital staff.
e
8 patients with 2–3 attendants/patient.
70
A. Asif et al. Building and Environment 135 (2018) 68–73
2500
(a).
Bacterial ConcentraƟon
2000
1500
(CFU/m3) 1000
500
0
OT1 OT2 OPD Emergency General General
Surgery Medicine
LocaƟon
Winter Spring
350
Fungal ConcentraƟon
300 (b).
250
(CFU/m3)
200
150
100
50
0
OT1 OT2 OPD Emergency General General
Surgery Medicine
LocaƟon
Winter Spring
Fig. 1. Seasonal variation of airborne (a) bacterial and (b) fungal levels.
71
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
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
a
Colony counts (percentage).
Table 5
Total isolates recovered (and percentage) of identified airborne fungal genera over each monitoring site for two seasons.a
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%)
a
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:
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A. Asif et al. Building and Environment 135 (2018) 68–73
73