Isolation and Identification of Airborne Bacteria Inside Swiftlet Houses in Sarawak, Malaysia

Air consists of such microorganisms as bacteria, fu ngi, and viruses. Exposure to these airborne bacter i indoors may cause infectious and noninfectious adverse health e ffects. However, the sources and origins of bacteri a a e not fully understood. The aim of this study was to isolate an d identify the bacteria present in the air inside s wiftlet houses located in Kota Samarahan, Saratok, Betong, Maludam, Miri, Kuching, Semarang, Sepinang, Sarikei, and Sibu in S arawak, Malaysia. A total of 100 bacterial isolates from 20 samples were collected from swiftlet houses. The b acteria present in the air were collected using Plate Count Agar. Two plates were exposed at the front and back inside sw iftlet houses for 15 sec and then incubated at 37 ± 1 C for 24 h. The 16S rRNA analysis method was used t o i entify the isolates from the samples. The air inside the swiftlet houses had a total mean airborne bacteria colony count of 2.0 2 ± 0.72 log10 cfu/m/sec; the highest was in Miri (3.08 ± 0.29 log10 cfu/m /sec), and the lowest was in Sibu (1.05 ± 0.85 log10 cfu/m/sec). Twenty-seven bacteria species were identifie d, and Lysinibacillus sp. B4 (16%) was most frequently isolated.


Introduction
Air consists of tiny organisms such as bacteria, fungi, mycotoxins, and viruses.These small groups of organisms clump and survive in the air under high humidity [1].Microorganisms are ubiquitous and can be transferred from the environment to everyday objects especially humans.Pathogenic microbes can be transmitted through air, skin, food, water, and other interpersonal contact [2].Researchers have reported that exposure to pathogenic microbes may cause respiratory disorders, infections, hypersensitivity pneumonitis, and toxic reaction in infected humans [3].Bacterial infection has been receiving increased attention in recent years due to the increase in mortality and morbidity [4].Swiftlet farming is defined as a modern and commercialized method of building birds' nests differently from the traditional cave-harvesting method.Malaysia is the third largest producer of edible birds' nests in the world, after Indonesia and Thailand [5].An estimated 30,000 shop houses and commercial premises had been converted into swiftlet farms by June 2005 throughout Malaysia, especially northern Malaysia [6].Researchers have reported that indoor air is mostly full of bacteria [4].No research related to airborne bacteria inside swiftlet houses has been conducted.There are many methods for collecting airborne bacteria such as collection plates, electrostatic collectors and impactors, and mass spectrometers, which are suitable for research purposes [7].More research should be done on airborne bacteria that may pose health risks to humans.Therefore, this study was conducted to isolate and identify the bacteria present in the air inside swiftlet houses.

Sample collection.
Sampling and sample processing procedures for isolating airborne bacteria were carried out according to Malaysia Veterinary Health Air quality sampling [8].Two Plate Count Agar plates (Oxoid, England) used to collect the airborne bacteria were placed at the front and back of each sampling site.The medium of the Plate Count Agar was exposed in the air for 15 sec.Then the cap of the agar plate was re-joined, sealed, and labeled.The agar plates were incubated at 37 ± 1 o C for 24 h.The airborne bacteria colony was counted according to Nyakundi and Mwangi (2011) and Tsai et al. (2002) [9][10].The bacterial colonies formed on the medium were reported as the number of colonyforming units (log 10 cfu/m 2 /sec) [11].Colonies were randomly picked for further identification.

Conventional biochemical tests.
The bacteria isolated from the air samples were identified phenotypically using conventional biochemical tests according to Bergey's manual [12] DNA extraction.After the bacterial genera were detected, bacterial DNA was extracted using the boiling method as described by Maria et al. (2008) [13] with minor modifications.The bacterial cultures were prepared by growing the bacteria in Luria broth (Scharlau, Spain) at 37 ± 1 °C for 24 h.One thousand and five hundred microliters of overnight culture was transferred into a 1.5 ml microcentrifuge tube and centrifuged at 10,000 rpm for 5 min.Then the supernatant was discarded.Five hundred microliters of sterile distilled water was added and vortexed to resuspend the cell pellet.The content of the microcentrifuge tubes was then boiled for 10 min together with the tubes, and then the tubes were immediately placed on ice for 5 min.After 5 min, the microcentrifuge tubes were centrifuged at 10,000 rpm for 10 min, and the supernatant was collected.
Sequencing analysis.The bacteria DNA was purified using the Qiagen sequencing purification kit (QIAquick Gel Extraction Kit, USA).The purified DNA product was sent to First Base Laboratories Sdn.Bhd. in Selangor, Malaysia, for the DNA sequencing process.All 16S rRNA sequences were compared using the BLAST program to determine the closest identity matches.

Statistical analysis.
Statistical analysis of the data was conducted using the SPSS Statistics version 21.0 program.All data was analyzed for analysis of variance (ANOVA).All differences between the means were compared using the Tukey multiple range test after a significant F-test at P < 0.05.

Results and Discussion
Bacterial colony count.The mean total of the airborne viable bacteria counts inside the swiftlet houses is shown in Table 1.The table shows that the interior of the swiftlet houses had a total mean airborne bacteria colony count of 2.02 ± 0.72 log 10 cfu/m 2 /sec.The mean airborne bacteria count was highest in Miri (3.08 ± 0.29 log 10 cfu/m 2 /sec) and lowest in Sibu (1.05 ± 0.85 log 10 cfu/m 2 /sec).The mean total bacterial colony counts collected from Miri were significantly (P < 0.05) higher than those collected from Sibu.
Occurrence of airborne bacteria inside swiftlet houses.The biochemical results showing the occurrence of the bacteria isolated and the 16S rRNA gene sequencing results of the bacteria isolated from the indoor air of the swiftlet houses are shown in Tables 2  and 3.The biochemical results showed 12 main bacterial genera among the indoor air bacteria isolated.The 16S rRNA gene sequencing results revealed that 27 bacterial species were isolated in the indoor air in the swiftlet houses: Bacillus amyloliquefaciens (8%), Bacillus cereus (10%), Bacillus subtilis (8%), Bacillus sp. 1 (5%), Bacillus sp.  1).
The types and number of airborne bacteria were investigated in ten areas in Sarawak.Airborne bacteria colonies were counted to measure the total number of microorganisms present in the air inside swiftlet houses.The total mean bacteria viable counts in the air inside swiftlet houses at the sampling sites was 2.02 ± 0.72 log 10 cfu/m 2 /sec (Table 1).The mean total airborne bacteria count was highest in Miri (3.08 ± 0.29 log 10 cfu/m 2 /sec) (Table 1).Miri is situated in northern Sarawak, which has higher temperatures than the southern and central Sarawak regions [17].Temperature can affect the growth rate of various bacterial species [18].According to Chan et al. (2005) [18], the growth rate of microorganisms doubled as the temperature increased.Airborne bacteria count could be a standard for determining the hygiene levels and housing regulations [19].
A total of 100 airborne bacterial were isolated, and 27 bacteria species were identified using 16S rRNA gene sequence analysis.Results of this preliminary microbiological examination of the airborne bacteria inside the swiftlet houses revealed that most of the bacteria isolated were a mixture of Gram-positive andnegative bacteria.Figure 1 shows that Lysinibacillus sp.(16%) was the most frequently isolated, followed by B. cereus (8%), B. subtilis (8%), B. amyloliquefaciens (8%), S. aquimarina (8%), and others.Lysinibacillus sp. is a member of the Bacillaceae family and produces endospores [20].Bacillus sp. was the most frequently isolated airborne bacteria from all areas.
Bacillaceae family bacteria are facultative anaerobe bacteria that can grow aerobically and anaerobically [21].In addition, Bacillus sp.produces endospores that remain dormant under stressful environments for long periods [11] and thus can survive even in dry swiftlet droppings and spread further in the air.According to Kocijan 2009 [22], contamination of bird feed in the droppings sample could also be an explanation for the occurrence of various Staphylococcus sp. in the air.
Researchers have isolated Bacillus sp. and E. coli from the air inside houses in Zaria [4].Additional airborne bacteria are being investigated to determine their risk.

Conclusions
This preliminary study, using

Table 1 . Mean Total Airborne Bacteria Viable Counts Inside the Swiftlet Houses at the Sampling Sites
* The means with the same letter superscript are not significantly different at the 5% level.

Table 2 . The Occurrence of Bacterial Isolates in Indoor Air in the Swiftlet Houses at the Sampling Sites
Corynebacterium sp.