Occurrence and characteristics of Bacillus cereus group bacterial atmospheric aerosols in Novosibirsk region

The microbial diversity of atmospheric bioaerosols involves microorganisms that can cause allergic and infectious diseases or toxic effects. They include bacteria of the Bacillus cereus group (B. cereus, B. thuringiensis, B. anthracis, B. mycoides, B. pseudomycoides, etc.), which can result in diarrhea, pneumonia, meningitis, septicemia, and other infectious diseases. Accordingly, monitoring the presence of Bacillus cereus group bacteria in aerosols is critical. However, practically no data exist on Bacillus cereus and other cereus-group bacteria in southwestern Siberia’s poorly investigated atmospheric aerosol environment. Bacteria of the cereus group are capable of effective production of various biologically active compounds, with important implications for biotechnology; microorganism strains with new capabilities are being investigated. This study aimed to determine the occurrence and characteristics of B. cereus group bacteria in ground-level and high-altitude atmospheric aerosols in Novosibirsk region of southwestern Siberia, and to evaluate the biotechnological potential of the obtained microbial isolates. High-altitude atmospheric samples were collected over Karakan Pine Forest, approximately 50 km south of Novosibirsk, at altitudes of 7000, 5500, 4000, 2000, 1500, 1000, and 500 m, by aircraft sounding. Вoundaries of the aircraft flight path: 54° 26'38'' N, 82° 30'47'' E; 54°10'55'' N, 81° 44'00'' E. Ground-level samples were collected at various sites in Koltsovo settlement, Novosibirsk region. Impingers with a flow rate of 50 L/min containing 50 ml of Hanks’ solution were used for air sampling. The obtained aerosol samples were sown on a set of nutrient media and incubated at 28–30°C and 6–10 °C. The titers of microorganisms in high-altitude and ground-level samples were determined in terms of 1 m3 of atmospheric air. Standard microbiological methods were employed to study the phenotypic characteristics of the identified microbial isolates. Lipolytic activity was determined on yolk agar and LB agarized medium containing fatty acid esters with 0.01% CaCl2 . The substrates used were 1.0% monolaurate (tween-20) and monooleate (tween-80). Amylolytic activity of the cultures was determined by their isolation on starch-ammonia agar, and proteolytic activity by their ability to hydrolyse milk gelatin and casein (Maniatis T et al., 1984). The ability to hemolysis was taken into account when cultures were plated on LB medium with the addition of ram’s blood. Nuclease activity was studied on LB medium with the addition of Sigma DNA (USA) (Maniatis T et al., 1984). The content of plasmid DNA in the isolates was determined by screening according to Maniatis T et al. (1984). The capacity for RNAase secretion in culture medium (peptone - 9.27 g/l, yeast extract - 5 g/l, NaCl - 3.00; 10 ml of 50% glycerin, 2 ml of 20% glucose; pH 7.07.2) during cultivation of bacteria at 30 °С, for 18-24 h, was determined by the accumulation of acid-soluble products, formed upon hydrolysis of high-polymer RNA of yeast (1 mg/ml). Antibiotic activity of the studied strains was determined by cross-strics (Yasuda T et al., 1992) on LB medium at 37 °C. The following pathogenic test strains were used: Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Candida albicans 620, Klebsiella pneumoniae B-4894, Escherichia coli ATCC 25922, Salmonella typhimurium 2606, and Shigella sonnei 32 (collection of FSBI State Research Centre Vektor of the Rospotrebnadzor). The genetic analysis of bacterial isolates was performed using PCR with specific primers on 16S rRNA. The calculation of the number of cultivated microorganisms in the samples was carried out according to the Kerber method [Bottone EJ, 2010], and the number of microorganisms was averaged over three parallels of the inoculated samples. The annual average numbers of cultivated microorganisms were calculated as a mean ± the confidence interval at a significance level of 95% of t-Student’s (p < 0.05). Percentages of spore-forming cultured bacteria in aerosol samples varied significantly across the years of observation (1998–present): in high-altitude samples, the minimum and maximum were 0.5% (in 2005) and 55% (in 2011), respectively, and, in ground-level samples, the minimum and maximum were 0.1% (in 2002) and 83% (in 2016), respectively (See Fig. 1 and 2). Annual averages of total concentration of microorganisms ranged from < 1 to 5×105 CFU/m3 . The number of cereus-group bacteria also varied significantly from sample to sample, with averages ranging from 0.01% to 6.5% of the total number of isolated microorganisms. A total of 2.025 bacterial isolates, of which 62 formed endospores, were isolated from ground-level and highaltitude aerosol samples collected during the predominance of south-westerly winds from Kazakhstan in autumn 2016, and were characterized by increased dust-component content. Spore-forming bacteria were identified as belonging to the genera Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus, and some others. Both high-altitude and ground-level aerosol samples were shown to contain bacteria of the cereus group: Bacillus cereus (Bc) and Bacillus thuringiensis (Bt), Bt ssp. kurstaki, Bt ssp. galleriae subspecies; Bt strains with indefinite serotype were also found. Notably, Bacillus anthracis species were not found (See Table 1). Screening for enzyme secretion revealed Bt and Bc strains with pronounced proteolytic, phosphatase, lipolytic, and amylolytic activities in a medium pH range from 5.0 to 9.0 (See Table 2). An atypical strain of B. thuringiensis Cb-527, which demonstrates high production of RNase, was isolated. All strains demonstrated hemolysis capability, were multi-resistant to antibiotics (resistant to 6-15 drugs (See Table 3), and suppressed the growth of the pathogenic yeast, Candida albicans, to varying degrees. The Bt Cb-527 strain, as well as several Bc strains, also effectively inhibited the growth of Gram-positive test strains of Staphylococcus aureus and Bacillus subtilis. Gram-negative bacterial test strains were low-sensitive to the action of metabolites of the studied Bc and Bt strains (See Table 4). In high-altitude and ground-level samples of the studied atmospheric aerosols, bacteria of the cereus group belonging to Bacillus cereus and Bacillus thuringiensis species were found in amounts ranging from 0.01 to 6.5% of the total number of cultured microorganisms isolated under experimental conditions. The presence of aggression enzymes such as phospholipases, hemolysins, proteases, and nucleolytic enzymes typical of representatives of these taxa, was found. We isolated Bc and Bt strains with high levels of secretion of enzymes and metabolites that possess antibiotic activity; these strains are promising as producers. The Bacillus thuringiensis Cb-527 strain (with a pronounced secretion of the RNase complex) can be used for the development of anti-RNA-containing virus drugs. The isolated Bc and Bt strains demonstrated multiple antibiotic resistance, which confirms literature data on the increasing prevalence of polyresistance among the identified natural microbial isolates. The paper contains 4 Figures, 4 Tables, and 41 References.