Proteomic Analysis and Molecular Characterization of Airborne Bioaerosols in indoor and Outdoor environment in Al-Qassim Region, saudi Arabia

© The Author(s) 2019. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Elbehiry et al. J Pure Appl Microbiol, 13(2), 1041-1052 | June 2019 Article 5574 | https://dx.doi.org/10.22207/JPAM.13.2.42


INTRODUCTION
Air pollution is a serious problem not only in the Kingdom of Saudi Arabia (KSA) but also in many parts of the world. Albeit, this kind of contamination has different forms, some of which are unnoticeable by visualization. Pollution of air has been a grave problem since when the air we breathe carries contaminants, it can affect on our health in various approaches. Exposure to bioaerosols, holding airborne pathogens leads to various infections, including lung cancer, bronchial asthma, hypersensitivity pneumonitis, toxic reactions and various cardiac diseases 1,2,3 . This type of pollution can affect on people of various ages, but the incidence in elderly people and young children are frequently high. Consequently, the insufficient diagnosis and control of air quality creates significant health problems. Molds and bacteria are considered the most common microbes in indoor/outdoor areas 3 . The ability of microorganisms to circulate on indoor or outdoor subjects is based on the comparative moisture and the moistness content of the different objects. These may lead to obliteration, unfavorable effects on the health status 3 . For that reason, it is very important to characterize indoor and outdoor airborne microorganisms.
Indoor air plays a significant role as a health contributing factor, and controlling of the indoor air quality need strategies that vary from those utilized for outdoor air 4 . The microbial contamination in internal and external environmental regions is caused mainly by various types of bacteria and fungi, especially molds which represents a public health problem worldwide. The temperature, wind rapidity and relative humidity are considered important as the major environmental factors which effect on the feasibility of various pathogens 4,5 .
Bioaerosols are airborne particles of human, animal, plant or soil origin containing microorganisms or organic compounds 6,7,8 . These particles become airborne when floating in the air due to wind speed, turbulence, coughing, traffic etc. Gilbert and Duchaine 9 , Mandal and Brandl 10 , Despre´s et al. 11 and Fykse et al. 12 evaluated the microbial contamination of bioaerosols in numerous internal and external environments and they found that the bioaerosols were contaminated by various biological agents. Moreover, Gilbert and Duchaine 9 found that the viability of the airborne microbes is based on computable aspects, for example, relative humidity, temperature and solar irradiance, as well as the particular characters of the microbes themselves, as endospore forming bacteria and pigment production. In the last few decades, the microbial concentration in bioaerosols exposure has increased due to its relationship with a wideranging variety of health problems and the terror of bioterrorism (terrorism involving the intended release of biological agents as bacteria, viruses, or toxins).
In hospitals, it could be imperative to examine the air for the reason that it is a grave and extensive hazard that patients acquire nosocomial infections through the bioaerosols during staying in the hospital 8,13 . Therefore, examining the air at common places could assist in reducing the amount of possible bioaerosols assaults. To diminish the assault of infection, initial recognition of biological agents is compulsory. Frequent checking of the airborne for identification of definite microbes is potential 13 . Therefore, study the bioaerosol environment is very crucial as indicated by the National Research Council 14 .
In addition, airborne bacteria can be established in several surroundings, nevertheless in many conditions, they don't exist a public health danger to the unprotected persons. Therefore, the intensive care of present levels of out-ofdoors airborne biological agents is compulsory to assess the possible dangers affecting the health of individuals 15 .
Airborne fungi are also of public health importance, since they may cause serious diseases such as allergy and respiratory diseases 16 . On the other hand, Rotz et al. 17 indicated that bacteria, virus, fungi and toxins are considered the most common biological agents of bio-terrorism attack in accordance with several microorganisms that developed from the centers for prevention and monitoring of various diseases. Valen 8 found that bioaerosols can be established in both indoor and outdoor environment, and they frequently hold various microorganisms like bacteria, virus and fungi, which perhaps pathogenic or saprophytic. It has been recorded by previous study that few numbers (1%) of different microbes assembled from the surroundings can be cultured in various test centers by traditional methods. Nevertheless, molecular methods can identify approximately 99% of the remaining non-cultivable microbes by analysis of their nucleotide sequences and protein fingerprinting. Consequently, rapid and accurate identification of airborne biological agents using mass spectrometry technology has revealed a wellknown method nearly in all clinical laboratories 18 . Since the genetic methods used for recognition of various types of pathogens are time consuming and highly expensive, an alternative method using Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) has positively been utilized for recognition of various microorganisms recovered from various sources 19,20 . This method needs simple tools and comparatively rapid machinist training. It was noticed that using of the mass spectrometry technology represented by MALDI-TOF-MS needs small quantity of biological materials and comprises simple procedures without preliminary measurement, for instance gram staining, when compared with DNA analysis based technologies 21,22,23 .
To the best of our knowledge, there are few published researches related to the use of the MALDI Biotyper for identification of airborne pathogens 12,24 . From the previously mentioned data, our study aimed to rapid and precise identification of airborne pathogens isolated from the indoor and outdoor environment in certain areas in Al-Qassim region using mass spectrometry technology.

Location and time of sampling
Five hundred air samples were collected from three localities. The 1 st location (150 samples) was at the Qassim University (QU) classrooms and offices of the university which are located in Almulyda, North of Prince Nayef Airport (Al-Qassim region), next to the station for distribution of petroleum products (Aramco). The 2 nd sampling location (250 samples) was hospitals in Al-Qassim region, including King Fahad Specialist Hospital Buraidah (50 samples), Buraidah Central Hospital (50 samples), Qassim National Hospital Buraydah (50 samples), King Saud Hospital Unaizah (50 samples), Al Bukayriyah General Hospital (50 samples). The 3 rd sampling location was poultry slaughter houses (100 samples). All microbial air samples were collected by impactor air sampler from indoor and outdoor air in six replicates for each sample. The collection of air samples was carried out in the winter season of 2017, at

Journal of Pure and Applied Microbiology
Al-Qassim region, KSA. Throughout this season, there was an extensive utilization of environment insulation, which most probable rises the number of biological agents in the indoor and outdoor air, corresponding to high levels of breathing distress. The samples were collected by a six-stage impactor air sampler, therefore, the air was impacted in prepared plates with specific media for bacteria. The air sampler was stayed throughout the collection time, about two meters above floor level.

Isolation of bacteria
Tryptose Soy agar plus Benlate media was used for bacterial isolation. The petri dishes were incubated for 72 h at 28-30°C, after inoculation of the air samples in specific plates. The colonies of bacteria were then estimated after the 1 st and 3 rd day of incubation and the total of colony forming unit (CFU) m -3 air was also recorded. Moreover, the distinct colonies considered were isolated and compared with each other again. The isolated strains were stored at -70°C in glycerin 20% for further investigation. For studying of the morphological characters, the isolates were stained by particular staining technique and observed microscopically and morphological properties of the bacterial cells were demonstrated. Brain heart infusion agar (BHI) was used in our study as a suitable medium for growing of different types of microorganisms. Samples were collected using BHI agar for the enumeration of bacterial colony forming unit (CFU).

The hemolytic activity of isolated colonies
The isolated strains were examined for their degree of hemolysis. Blood agar plates supplemented with 5% sheep blood (Oxoid, UK) were used for this purpose. From each isolate, a fresh colony was inoculated onto agar medium and then preserved in 28-30°C for two successive days. After that, the colonies were categorized as α-hemolytic, β-hemolytic or γ-hemolytic.

Identification of airborne pathogens using protein fingerprinting (Microflex LT)
All air samples were analyzed using the MBT compass software (Bruker Daltonics, Germany). According to the instructions of the company, identification scores of ≥2.000 will be revealed as identification at the species level, whereas, the scores ranged from 1.700 to 1.999 will be indicated as identification at the genus levels. In contrast, the scores of 1.700 or less will be recognized as misidentification 25,26 .
Ethanol/formic acid extraction protocol was carried out according to the instructions provided by the Bruker Daltonics company. Briefly, one fresh colony was moved into sterilized tube containing 300 µl of highly purified water then mixed thoroughly. 900 ul of absolute ethanol was then added and then properly centrifuged at 13000 g for 2 min. The residue of the tube was left at room temperature for a couple of minutes. After that 50µl of 70% formic acid was added to the tube and strongly mixed. Fifty microliters of 70% acetonitrile were then added and centrifugation was carried out at 13000 rpm for a couple of min. One microliter of aliquot, was directly inoculated onto stainless steel target plate. Subsequently, 1µl of matrix solution (Sigma Aldrich, USA) was added to each hole. The target plate was consecutively placed in the MBT device for direct microbial identification. All examined samples were triplicated for precise identification.

Molecular assays
The commonly identified isolates were Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Acinetobacter baumanni and Escherichia coli. Therefore, we studied their virulence by real time PCR (RT-PCR, Applied Biosystem, USA). Firstly, DNA of identified isolates was extracted using a QIAamp tissue kit (Qiagen, Hilden, Germany) which was performed based on the manufacturer's recommendations. The designated primers were shown in Table 1.

RESULTS
A total of 1,900 isolates were grown on the air sample nutrient agar plates after one day of incubation. One hundred twenty-nine purified colonies were obtained after sub-culturing for the next investigation. As shown in Table 2, 36 (27.90%) of these isolates were from Qassim University, 26 (20.16%) were from Buraidah Hospitals, 10 (7.75%) were from King Saud Hospital in Unaizah, 17 (13.18%) were from Al Bukayriyah General Hospital and 40 (31%) were from a poultry slaughterhouse in Buraidah. Throughout the first day of air sampling, the temperature was 37°C and the humidity was 70%. While in the 2 nd and 3 rd day of sampling, the temperature was 39°C and the humidity was 50%.
Amongst the 129 colonies examined by MBT Compass software, respectable recognition of various types of airborne pathogens on the species levels was detected in 119 (92.25%) isolates with a score value ≥2.000 (Table 3). Whereas; 10 (7.75%) isolates were identified by score values ranged from 1.7000 to 1.999 ( Table  3). The MBT was able to identify 93 (72.10%) gram-positive bacterial and 36 (27.90%) gramnegative bacterial air isolates ( Table 4). The most common identified gram-positive genera were Staphylococcus (n = 43, 33.33%), Enterococcus (n = 15, 11.63%) and Bacillus (n = 15, 11.63%). The most common identified gram-negative genera were Escherichia (n = 16, 12.40%). As can be seen in Table 4 As shown in Fig. 1, Staphylococcus aureus and Escherichia coli were the most frequently identified species (n = 16, 12.40% for each),    (Fig. 2). We found a significant variation between the total numbers of airborne pathogens recovered from the Al-Qassim Hospitals compared with Qassim University and poultry slaughter house.  As can be seen in Fig. 3, the data analysis of MBT illustrated that numerous spectra for the most commonly isolated gram positive bacteria as Bacillus subtilis, Enterococcus faecalis and Staphylococcus aureus were scattered within the range from 2,000 to 15,000 m/z, and from 2,000 to 16,000 m/z for Acinetobacter baumanni with higher peak intensities were determined between 4,000 and 8,000 m/z for all identified gram positive bacteria. Likewise, for the commonly isolated gram negative bacteria as Citrobacter freundii, Srratia liquefaciens and Pseudomonas putida, several spectra were scattered within the range from 2,000 to 10,000 m/z and from 2,000 to 17,000 m/z for Escherichia coli with higher peak intensities were detected between 4,000 to 8,000 for all identified gram-negative bacteria (Fig. 4).

DISCUSSION
As community awareness of possible hazards arising from bioaerosols increases, the request for advanced and predominantly appropriate techniques for the recognition of bacterial portions in such releases is increasing congruently 32,33,34 . In the present investigation, it was aimed to establish MBT as an effective mass spectrometry technology for identification of various airborne bacteria with slight workload.
In the current study, a huge number of isolates recovered from air samples examined by MBT Compass software, and it was stated that MBT is considered as a suitable technique for the precise recognition of airborne pathogens at the genus and species levels. Throughout Al-Qassim region, samples of air were collected from different localities and showed a huge contamination by numerous environmental nonpathogenic and pathogenic bacterial species. One major restriction of the current research was that the samples were only examined for airborne bacteria, whereas, other types of microorganisms and their toxins were excluded from the study. Furthermore, the isolation was depended mainly on the culture technique, a restriction that perhaps reduced variety of the strains. Lastly, the amount of strains initially observed in the agar media didn't calculated to define which location was the greatest tainted with microorganisms.
MBT is used routinely in our lab. to recognize various types of bacteria at species and subspecies levels 15,35 . In our microbiology laboratory, we identify annually about 9,500 isolates by MBT. Since we examined the isolates recovered from air samples, the MBT database didn't comprise their spectra, and only 119 (92.25%) of the isolates were properly recognized with score values ≥2.000. Due to the existence of a large stored spectra in the Bruker Daltonics Journal of Pure and Applied Microbiology databank of certain types permitted the MBT to recognize a large number of bacteria within the species levels. Similarly, in a former study conducted Seng et al. 36 on samples isolated from patients, who found that MBT was able to identify approximately 95% of the bacterial isolates.
Proteomic identification of airborne bacteria by MBT confirmed by RT-PCR provided us with data regarding the bacterial pathogenicity in different localities at Al-Qassim region. Throughout our study, the identified bacteria seemed to be similar to the former studies reported by Papadopoulou et al. 37 and Martin et al. 38  In our study, among the three localities Al-Qassim Hospitals were the most commonly contaminated site with a huge number of bacteria (41.10%) and the genus Staphylococcus was the predominant isolated bacteria. Similar results were obtained by Kausar et al. 40 who indicated that a large number of Staphylococcus sp. were isolated from the air of Hail hospitals, Saudi Arabia. Our results, propose that hospitals have more microbial contamination than other sites, this is due to the presence of several kinds of patients. The presence of Staphylococcus, may has a possible hazard to the health of patients of these rooms 40 . This type of bacteria may effect on humans by different ways and are able to cause various illnesses such as shocking infections in these high-risk patients 41 . Moreover, surface contamination with settled microbes could also present a source of potential health risk. In the current study, we found a high incidence of airborne bacteria in Al-Qassim Hospitals, Qassim University and poultry slaughter house, Al-Qassim region, Saudi Arabia. As well, the RT PCR proven here was successfully applied to settle the recognition of nuc, gelE, bsub, recA and iss genes specific for Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Acinetobacter baumanni and Escherichia coli, respectively. A respectable link between MBT and PCR investigation was found.

CONCLUSIONS
The current study demonstrates that MBT is a powerful and rapid alternative technique to the conservative methods for airborne bacterial identification. Accurate and fast identification of bacterial contamination in bioaerosols is compulsory to avoid potential eruptions. Various types of bacteria (pathogenic and nonpathogenic) were identified in the indoor and outdoor environment of Qassim University, hospitals and poultry slaughterhouse, Al-Qassim region. The most commonly isolated bacteria were Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Acinetobacter baumanni. Therefore, students, employees, staff members, patients and doctors are potentially exposed to diverse airborne microorganisms, which may perhaps result in diversity of illnesses comprising respiratory distresses. Governmental organizations must increase the research activities to collect information concerning health influences that effect on both human and animal populations. We suggest that increasing the awareness regarding the possible health threats of indoor and outdoor bio-contaminants are considered as the first step in handling and eventually decreasing the diseases they persuade.