**3. Results**

(standard deviations) from the mean are given.

Based on characteristics of isolated microorganisms using the methods described above, four indices were calculated, which form the integral index of the hazard of microorganisms found out in atmospheric aerosols for humans. These indices were calculated both for each isolate and averaged for each year of research.

Let us compare the data presented in [28] with the results obtained in this research using the same methods. It should be noted that due to resource constraints for the company 2012–2016, not all the characteristics of the identified isolates were investigated, as it was done during the company 2006–2008. However, according to the authors' opinion, the results obtained on a truncated set of characteristics quite clearly demonstrate the changes occurring in the pool of cultured microorganisms isolated from atmospheric aerosols in the south of Western Siberia.

*3.1. Long-Term Trends of Average Annual Concentrations of Culturable Microorganisms in the Atmospheric Aerosol of the South of Western Siberia*

Figure 5 shows the long-term trends of average annual concentrations of culturable microorganisms in the atmospheric aerosol of southern Western Siberia. In constructing this graph, we used the results published in [65] and our own data obtained after the publication of this paper.

**Figure 5.** Trends of average annual concentrations of culturable microorganisms in the atmosphericaerosol in the south of Western Siberia. The average annual values with root mean square deviations

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Long-term observations of the concentrations of culturable microorganisms (bacteria, fungi and yeasts) in the atmosphere indicate that the average annual values of these concentrations have a pronounced tendency to decrease. In particular, from 2008 to 2016, this decrease is at least three times.

It should be noted that, for the concentration of cultuarable microorganisms in atmospheric aerosols in the south of Western Siberia, the intra-annual variability was revealed [65]. Figure 6 presents the data for 2001–2016 values averaged for each month of years 2001–2016, normalized to the corresponding annual average. As can be seen from this graph, the differences between the maximum and minimum concentrations are on average up to three times. Naturally, for each sampling, the concentration of microorganisms depends not only on the revealed dependence, but also on the specific meteorological conditions, the effect of which on this concentration can be very significant.

**Figure 6.** Intra-annual variability of concentrations of culturable microorganisms in the atmospheric aerosol of the south of Western Siberia. The average values for a given month for the entire observation period and the standard deviations from the average are given.

It should be noted that the data used to construct the integral index characterizing the concentration of isolates in the air make up only part of the raw data used to construct Figures 5 and 6. This is due to the fact that, to construct the integral index characterizing the concentration of specific isolates in the air, only data on the concentrations of bacteria and yeast, but not all culturable microorganisms, are used. To calculate the total concentration of culturable microorganisms in the air, data are used on all grown colonies in the seeded sample, but not all colonies succeed in obtaining isolates suitable for further studies, namely, isolate concentrations are used to construct the corresponding integral indices, and finally, Figures 5 and 6 are constructed for data for 2001–2016, and data obtained for a smaller number of years were used to construct integral indices.

#### *3.2. Sustainability of Culturable Microorganisms in the Environment*

The results of determining the integral index of resistance of microorganisms to adverse environmental factors are summarized in Table 1 for annual mean values and standard deviations from them.

**Table 1.** The annual average values and standard deviations from them of the integral index of the microorganisms' resistance to adverse environmental factors values.


As follows from the analysis of the data presented in Table 1 and in Supplementary Materials (see Tables S1–S3, S13) for the sustainability index of culturable microorganisms in the atmospheric aerosol of the south of Western Siberia, di fferently directed trends are observed for di fferent sites of observation. Changes in the sustainability index of culturable microorganisms in an atmospheric aerosol in the south of Western Siberia do not exceed 1.1–1.75 times for 10 years of observations. However, given the fairly large scatter of data for each year of measurement relative to average values, these changes cannot be considered non-zero.

There are those isolates among the isolates of bacteria and yeasts that have values of the microorganism sustainability index in the environment from 0 to 1 and the average annual values of the indexes are at the level of 0.2–0.45. Note that the maximum index values close to or equal to 1 exist for a small number of bacteria and yeasts, not in every sample, and not even in every year (Supplementary Materials, Tables S1–S3).

Thus, the obtained experimental data do not reliably reveal di fferences at a reliability level of 95% between the sustainability indexes of culturable microorganisms in the atmospheric aerosol of the south of Western Siberia determined for 2006–2008 and for 2012–2016. At a 95% reliability level, all three trends are significantly indistinguishable from zero.

#### *3.3. Potential Pathogenicity of Culturable Microorganisms in the Atmospheric Aerosol of the South of Western Siberia*

The results of determining the integral index of potential pathogenicity of pathogenicity of culturable bacteria and yeas<sup>t</sup> for humans in an atmospheric aerosol in the south of Western Siberia are summarized in Table 2 for annual mean values and standard deviations from them.


**Table 2.** Annual average values and standard deviations from them of the integral index of potential pathogenicity of culturable bacteria and yeas<sup>t</sup> in an atmospheric aerosol in the south of Western Siberia.

As follows from the analysis of the data shown in Table 2 and in Supplementary Materials (see Tables S4–S6, S13), there is no noticeable positive trend in the index of the potential pathogenicity of cultivated microorganisms in the atmospheric aerosol for air samples taken at the site of Klyuchi only. This increase from 2006 to 2016 is 1.5 times. The obtained experimental data do not reliably reveal di fferences at a reliability level of 95% in indices of potential pathogenicity of culturable microorganisms in the atmospheric aerosol of the south of Western Siberia determined in 2006–2008 and in 2012–2016 for the Klyuchi sampling site. At a 95% reliability level, all three trends are significantly indistinguishable from zero.

The average annual level of this index for all isolates was 0.2–0.4, while only a few of the microorganisms detected over the entire period had indices of the potential pathogenicity of culturable microorganisms in the atmospheric aerosol, approaching but not reaching 1.

#### *3.4. Culturable Microorganisms' Resistance to Antibiotics*

The results of determining the integral index of antibiotic resistance of culturable bacteria and yeas<sup>t</sup> in an atmospheric aerosol in the south of Western Siberia are summarized in Table 3 for annual mean values and standard deviations from them.


**Table 3.** Annual average values and standard deviations from them of the integral index of culturable microorganisms 'resistance to antibiotics bacteria and yeas<sup>t</sup> in an atmospheric aerosol in the south of Western Siberia.

The situation for this index is similar to the situation with the sustainability index of culturable microorganisms in the atmospheric aerosol of the south of Western Siberia. The trends of this index are multidirectional and over 8 years of observations of changes in the culturable microorganisms in the atmospheric aerosol in the south of Western Siberia resistance to the action of antibiotics do not exceed 1.5 times (Table 3 and Tables S4–S6, S13 in Supplementary Materials). Note that in 2015 and 2016, experimental studies on resistance to antibiotics of isolates from atmospheric aerosol have not been conducted. Given the fairly large scatter of data relative to the average values for each year of measurement, these changes can be considered indistinguishable from zero. Thus, the obtained experimental data do not reliably reveal di fferences at a reliability level of 95% between the indices of culturable microorganisms in the atmospheric aerosol in the south of Western Siberia resistance to antibiotics determined in 2006–2008 and in 2012–2014. At a 95% reliability level, all three trends are significantly indistinguishable from zero.

Among the identified isolates, there are both those that are susceptible and resistant to all studied antibiotics. It was found that the proportion of isolates sensitive to the action of all antibiotics or resistant to the action of no more than one antibiotic did not change significantly during the period studied. In 2006–2008, there were from 33% to 42% of such isolates, and in 2012–2014 there were from 28% to 42%. Two isolates, which are resistant to all studied antibiotics (11 out of 11) or 0.21%, were detected at all observation sites in 2006–2008 and in 2012–2014 there were three such isolates (8 out of 8, 9 out of 9, and 15 out of 15), or 0.95%, Supplementary Materials (Tables S10–S12). Consequently, the number of microorganisms that are most resistant to the action of antibiotics over 8 years has increased more than four times, while the indices of culturable microorganisms in the atmospheric aerosol in the south of Western Siberia resistance to the action of antibiotics have not changed.

The sources of atmospheric microorganisms, besides vegetation, animals, and human activity, are soil and water; most likely, bioaerosols should contain a similar percentage of bacteria resistant to the action of antibiotics. Indeed, about the same percentage of bacteria resistant to the action of at least one of the antibiotics studied was found for the water system [66–71] and in the soil [69,72].

#### *3.5. Integral Index*

The results of the determination of the integral index of hazard for humans of culturable bacteria and yeas<sup>t</sup> in an atmospheric aerosol in the south of Western Siberia are summarized in Table 4 for annual mean values and standard deviations from them.

As already noted, this integral index is the product of the 4 indices described above (see Tables S1–S12 in Supplementary Materials). However, for some isolates of microorganisms, some indices were not determined, therefore, the average annual indices of this index for the corresponding year were substituted for the calculation. These values in the relevant cells of Table S13 in Supplementary Materials are highlighted in light blue. In general, the average annual values of this index are not large and are in the range of 0.001–0.007. Recall that for individual pathogenic microbial isolates that are sustainable in the environment and do not possess antibiotic resistance, this index equals 1. Consequently, there are very few such pathogenic microorganisms in the atmospheric aerosol of the

south of Western Siberia (see Table S13 in Supplementary Materials). Moreover, the maximum value of this integral index over all the time of research for isolates from all observation sites is only 0.1074. At the same time, a fairly large number of isolates was found in the samples, for which the integral index is strictly equal to zero (see Table S13 in Supplementary Materials). Here is interesting situation: when the concentration of one of the isolates is very high (normalized index about 1) other indices are low; and when, for example, pathogenicity index is high, other indices are low. Hence, the integral index is not large for all studied isolates. The dependence of integral index for monthly averages is pronounced for 2006–2008 when the number of isolates is large for many months [28]. The isolates number for 2012–2016 is low, they are found not in every sample. So, integral index for monthly averages is not representative in 2012–2016 (Supplementary Materials, Tables S1–S13 for 2012–2016).


**Table 4.** The annual average values and standard deviations from them of the integral index of hazard of culturable bacteria and yeas<sup>t</sup> in an atmospheric aerosol in the south of Western Siberia.

As follows from the data presented in Table 4, all long-term trends of these integral indices are unidirectional, showing a tendency to decrease; however, the magnitude of this decrease is almost indistinguishable from zero at a reliability level of 95%. Based on this, one can conclude that in 10 years of research, the hazard to humans of microorganisms isolated from atmospheric aerosols of the south of Western Siberia remains almost unchanged.

As it was mentioned above, the large scatter of the experimentally determined values included in this index (see Table S13 in Supplementary Materials) makes it possible to speak only of the tendency of a small decrease in the integral index of the hazard of microorganisms isolated from atmospheric aerosols in the south of Western Siberia for humans. This decrease is primarily due to a decrease in the observed average annual number of culturable microorganisms in atmospheric aerosols in the south of Western Siberia.
