Isolation and characterization of air microbiota from indoor and outdoor environments

Introduction

The study of air microbiota, which involves examining the composition and diversity of microorganisms in the atmosphere, is a critical aspect of aerobiology. Understanding these microbial communities is essential, as they represent the organisms we are exposed to through daily inhalation. Investigating and characterizing air microbiota from both indoor and outdoor environments provides insights into microbial diversity, community dynamics, and potential health risks. Airborne microbial populations are influenced by various factors: indoor microbiota can be shaped by occupancy, ventilation, and building materials, whereas outdoor microbiota is affected by seasonal variations, pollution levels, and geographical location. Analyzing these communities enhances our understanding of microbial ecology, bioaerosol transmission, and environmental impacts on microbial dynamics. Characterization of air microbiota employs a combination of approaches, including culturebased techniques, microscopy, and molecular methods such as polymerase chain reaction (PCR) and metagenomics. These techniques allow identification of microbial species, assessment of their abundance and diversity, and inference of their potential ecological functions. Insights gained from such studies are valuable for improving indoor air quality, monitoring environmental health, and implementing strategies to control airborne diseases. In this study, we used the settle plate method to isolate and compare microbial diversity across different indoor and outdoor environments. The findings aim to advance our understanding of the factors shaping air microbiota composition and dynamics, and their implications for environmental and human health. [11] .

Sampling

Airborne microbial samples were collected by settle plate method at four sites in Hazra, Kolkata: (i) Microbiology laboratory, Jogamaya Devi College; (ii) Garbage van (Hazra crossing); (iii) Traffic-congested Hazra road; (iv) Chittaranjan National Cancer Institute premises. PDA and TSA plates were exposed for 10 min and 1 h, then incubated at 37 °C for 24-48 h (Flores, et al., 2011) [5] .

Isolation and Pure Culture

Eleven representative strains (SJ-1, SJ-3, SJ-4, SJ-5, SJ-6, SJ-7, SJ-9, SJ-10, SJ-13, SJ-15, SJ-16) were subcultured on TSA plates and purified by repeated streaking (Fakunle, et al., 2021) [4] .

Biochemical Characterization

Gram staining was performed to differentiate Gram-positive and Gram-negative isolates. Motility was tested using TTCsupplemented semi-solid agar (Beef extract 3 g, Casein digest 10 g, NaCl 5 g, Agar 4 g/L). Mannitol Salt Agar (MSA) test assessed mannitol fermentation (NaCl 75 g, Mannitol 10 g/L, phenol red indicator). Urease test was performed on Christensen's agar (Urea 20 g/L, phenol red indicator). Oxidase test used 1% p-amino-N,Ndimethylaniline oxalate reagent on nutrient broth-grown cultures. Starch hydrolysis was examined on starch agar plates after iodine flooding (Ruokolainen, et al., 2017) [12] .

Antibiotic Susceptibility Test

Minimum Inhibitory Concentration (MIC) of kanamycin was determined in Mueller Hinton broth using two-fold serial dilutions (4096-16 µg/ml). Isolates were adjusted to OD600 = 0.5-1.9, inoculated into test tubes, and incubated at 37 °C for 24-48 h. MIC was defined as the lowest concentration with no visible growth (Ekhaise, et al., 2010) [3] .

Results and Discussion

Characterization of different bacterial morphologies

Settle-plates with Trypticase soy agar (TSA) and Potato Dextrose agar (PDA) were used for sampling air (10 min and 1 hr). Air samples were taken from 4 different places (laboratory, garbage van, traffic congested road, hospital) to analyse the air microbiota in different environments which could play role on air quality and public health. Many colonies were observed on each plate. Some common morphological characters were white, cream and yellow colored, with mucoid or chalky consistency, and irregular or smooth margins. Fungus with white mycelia was also obtained mainly in Potato dextrose agar (PDA) medium (Table 1, Fig. 12345678910111213) similar as found Napoli, et al., 2012) [10].

Subculture and establishment of pure culture

In microbiology, streaking is a technique used to isolate a pure strain from single species of microorganisms, often bacteria. Samples can be then taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested. 11 strains (SJ-1,SJ-3,SJ-4,SJ-5,SJ-6,SJ-7,SJ-9,SJ-10,SJ-13,SJ-15,SJ-16) were chosen from the mother plates based on their striking colony morphologies and sample sites. Then, the pure cultures were established by multiple subculture by streaking on pre-prepared Tryptone soy agar (TSA) plate (Table 2). Subculturing is a common practice in microbiology that involves transferring a small amount of cells from one culture vessel to a fresh growth medium to continue their growth and maintain a healthy culture. Isolation of pure culture by streak plating technique is shown below (Fig. 5) closed to result fund by Berg, et al., 2014) [2] .

Colony morphology and CFU/m3

A CFU stands for colony forming unit. CFU is a unit that we used as a measure of viable bacterial or fungal cells. When a microbiologist plates a sample onto a growth medium, each live cell has the potential to multiply and form a visible colony. Each colony that arises from a single cell or group of cells is counted as one CFU. In some plates, fungus with white mycelia was also obtained. CFU/m3 was estimated to be 0.0424, 0.328, 0.167 & 0.099 (10 min). To explore the effect of haze pollutants on the concentration of culturable bacteria and fungi, we compared the numbers of CFU/m3 (Tables 1213) (Mohamad, 2023).

Formula: CFU/m3=CFU/t*k where, CFU-mean of colony forming unit; t-total sampling time expressed in minutes; K-a conversion factor for cubic feet to cubic meters (k=35.3) (Yang, et al., 2023) [19] .

Gram staining

The gram staining is one of the most crucial staining techniques in microbiology to differentiation gram positive and gram negative bacteria based on their cell wall composition. The basic principle of gram staining is the properties of certain bacteria cell wall to retain the crystal violet dye. The cell walls of gram positive microorganisms have a higher peptidoglycan and lower lipid content than gram negative bacteria. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of thick layer of peptidiglycan in their walls, which retains the crystal violet these cells are stained with. The final step in gram staining is to use safranin to give decolorized gram-negative bacteria reddish pink color for easier identification, it is also known as counter stain (Tripathi, and Sapra, 2023) [16] . In the present study, all the bacterial strains were gram positive, with majority coccus in shape, a few were rod (Table 5, Fig. 67).

Catalase test

The catalase test is performed in microbiology to differentiate bacteria based on their ability to produce the enzyme catalase, which catalyzes the breakdown of hydrogen peroxide into water and oxygen. In the present study we used catalase test for differentiating catalase positive microbes from catalase negative ones. It was also valuable in separation of certain gram positive. In case of positive reaction, oxygen was liberated as effervescence when the drop of diluted hydrogen peroxide was added to the bacterial culture taken on the slide (Aryal, et al., 2023) [1] . After performing the catalase test aseptically, data was recorded for each of the colonies. Rate of effervescence (fast, delayed and no effervescence) was noted and shown in Table 6 and Fig. 8. Majority of strains were catalase positive except SJ-3 & SJ-10 (Tankeshwar, 2022) [5] .

Mannitol salt agar (MSA) test

Mannitol salt agar (MSA) is a selective and differential medium used in microbiology to isolate and identify Staphylococcus aureus from other Staphylococci. Staphylococci that can ferment mannitol produce acidic byproducts, causing the phenol red indicator in the medium to turn yellow. The medium mainly used for this purpose was Mannitol salt agar medium (Kateete, et al., 2010) [7] . In this study, after performing the MSA test based on morphology and color, we have selected only 4 strains and performed the test. By this test we have 3 (SJ-9, 15, 16) positive and SJ-10 was negative result. Data were recorded (Table-16, Figure -9) similar as result found Kumurya, 2017) [8] .

Motility test

The motility test is performed in microbiology to determine if a microorganism is capable of movement, which can be important for identifying certain species of bacteria. The test involves inoculating a semisolid agar medium with the microorganism and observing for the presence of growth away from the point of inoculation, indicating motility. This test is particularly useful in distinguishing between motile and non-motile species, which can be relevant for clinical diagnosis, environmental monitoring, and research purposes (Shields, and Cathcart, 2011) [13] . After performing the test, all the bacterial strains were motility positive after 24 & 48 h. except SJ-10 & SJ-13 were negative after 24 h only (Tables 9-10 & Fig. 1011).

Urease test

Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid ureasepositive organisms turn the entire medium pink within 24 to 72 hours. Christensen's agar medium was used to detect urease activity in a variety of microorganisms similar as Uotani,and Graham, 2015) [17] . This test was primarily used to differentiate organisms based on their ability to produce urease and the positive result was given by SJ-4, SJ-7, SJ-15, SJ-16 another left strains were negative for this result. Data was recorded in Table 19 and Fig. 12.

Oxidase test

The oxidase test is performed in microbiology to quickly identify bacteria that produce cytochrome c oxidase, an enzyme involved in the electron transport chain. In presence of the enzyme cytochrome oxidase (gram-negative bacteria), the N, N-dimethyl-p-phenylenediamine oxalate and αnaphthol react to indophenol blue. The test involves applying a drop of oxidase reagent to a bacterial colony, and if the colony turns purple within seconds, it indicates the presence of the oxidase enzyme. I have used Nutrient broth medium to study oxidase test (Shields, and Cathcart, 2011) [13] . So I was performed this test properly, and results the majority of strains were negative for this test except SJ-4 & 13 (Table 11, Fig. 13).

Starch hydrolysis test

Starch hydrolysis test is use to determine if a bacterium can break down starch using the enzyme amylase. In this test, bacteria were grown on a starchcontaining medium and after incubation, iodine was added. According to test, if the starch is hydrolyzed using the enzyme amylase then the medium around the bacterial growth will remain clean and make a zone showing the starch is absent if the starch is not hydrolyzed, then the iodine will turn the medium blue-black colour (Sigmon, 2008) [14] . In this study, after adding the iodine solution I found that some strains were positive for this test and some were negative. And the positive strains are SJ-1, 4, 6, 7 & 9. (Table 12).

Antibiotic susceptibility test by Minimum Inhibitory Concentration (MIC) method

The Antibiotic Susceptibility Test by Minimum Inhibitory Concentration (MIC) method is performed to determine the lowest concentration of an antibiotic that inhibits the growth of a specific bacterium. This information is crucial for selecting the most effective antibiotic treatment for bacterial infections. By determining the MIC, healthcare providers can identify the most appropriate antibiotic and dosage regimen to achieve optimal therapeutic outcomes while minimizing the risk of antibiotic resistance. Firstly, primary culture must be performed to do this test (Table 13). I used the kanamycin for this test and done by MIC method. I have choosen the range of concentration between 2048 to 16 цg/ml to check in which concentration the bacteria will inhibited and I was observed that the MIC value for some strains were 32 цg/ml and some strains have the MIC value between 128-256 цg/ml except one strain that was SJ-5, the MIC value for this was greater than 2048 цg/ml. The final results data are given below (Table 14, Fig. 16-26) similar as found by Kadeřábková, et al., 2024).

Conclusion

Air microbiota from laboratory, garbage van, trafficcongested road, and hospital environments were isolated and characterized using TSA and PDA media. Bacterial colonies displayed diverse morphologies, while fungi with white mycelia were mainly observed on PDA. Eleven representative bacterial strains were selected for pure culture establishment. All strains were Gram-positive, mostly cocci, and exhibited variable biochemical profiles: most were catalase-and motility-positive, three strains were identified as Staphylococcus aureus on Mannitol Salt Agar, and urease, oxidase, and starch hydrolysis tests further distinguished the isolates. Our tests also suggested that SJ-6, 7 belong to Bacillus subtilis or Bacillus cereus. SJ-3, 10 were gram positive cocci, non-motile, catalase and oxidase negative, suggesting presence of Streptococcus sp., which is a common bacterial species in any air sample. Antibiotic susceptibility testing via MIC revealed varying kanamycin resistance, from 32 μg/ml to >2048 μg/ml.

These results demonstrate the heterogeneity of airborne microbial communities across different environments and provide important insights into their morphological, biochemical, and antibiotic resistance traits. The findings underscore the significance of monitoring air microbiota for environmental and public health management.

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