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USING MATHEMATICAL METHODS OF ANALYSIS ANTIBIOTIC-STABILITY OF MICROORGANISMS OF LAKE BAIKAL Verkhozina E.V ., Institute of the Earth’s Crust Siberian Branch of the Russian Academy of Science;
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USING MATHEMATICAL METHODS OF ANALYSIS ANTIBIOTIC-STABILITY OF MICROORGANISMS OF LAKE BAIKAL Verkhozina E.V., Institute of the Earth’s Crust Siberian Branch of the Russian Academy of Science; Safarov A.S., L. A. Melentiev Energy Systems Institute of Siberian Branch of the Russian Academy of Science; Verkhozina V.A., National Research Irkutsk State Technical University.
It is shown that when analyzing a large number of data obtained, it is expedient to use the dispersion and correlation methods of analysis. This makes it possible to detect variability of antibiotic resistance of microorganisms in seasonal and inter-annual aspects, as well as to calculate pairwise correlation coefficients.
One of the most pressing problems in environmental studies is the detection of anthropogenic factor in the pollution of natural fresh water. Lake Baikal is a complex ecosystem consisting of several subsystems. Especially the ecosystems of pelagic and littoral lakes due to the huge difference in depth and temperature factor. These parameters determine the development of biota, the natural factor and the self-cleaning ability of ecosystems to anthropogenic factor.
At present, the relevance of microbiological monitoring of water bodies is becoming one of the most urgent problems. The study of microbial communities of water bodies and the determination of antibiotic resistance of bacteria showed that in reservoirs where anthropogenic influence is observed, favorable conditions are created for the formation and preservation of resistant strains of microorganisms. In addition, there is a substitution of antibiotic-sensitive microflora for resistance. It should also be noted that, from various environmental objects, water and, consequently, the waterway of transmission of infection are a particular epidemiological danger. The prevailing opinion among some epidemiologists that the external environment is a cemetery for pathogenic bacteria is currently undergoing significant changes.
In processing the results obtained, the correlation analysis method was applied using standard parametric and nonparametric criteria, as well as a package of computer programs "Statistica". This method is often used in the biomedical literature to identify cause-effect relationships and makes it possible to determine the strength and direction of variability between variables and is considered one of the promising, especially with a vast array of data obtained as a result of the studies. For statistical processing and data visualization, a freely distributed programming environment R was used. The evaluation of the data sampled in the work for compliance with the law of normal distribution was carried out using the Shapiro-Wheelk test.
When the cross-resistance of bacterial strains to different antibiotics was revealed, the correlation coefficient was calculated by Spearman's method. H0 hypothesis for the considered criterion - the value of the correlation coefficient does not differ significantly from zero (there is no reliable correlation). H0 deviated with the estimated probability of its acceptance P_value <α = 0.05. To determine the relationship between MNF and the averaged resistance of the bacterial community to antibiotics, an evaluation using the Spearman correlation coefficient was also used.
Discussion of the results Long-term studies have shown that in the conditions of active anthropogenic pollution of the littoral zone of the Baikal ecosystem, bacterial strains resistant to many antibiotics are observed. The data obtained are processed using dispersion and correlation analysis methods.
Analysis of samples of bacterial resistance to antibiotics studied averaged by the factor of belonging to a certain sampling month and separately by the factor of belonging to a certain year of sampling using the Shapiro-Wilk test showed that the samples under study are not distributed according to the normal law (P_value <α). In connection with this, the nonparametric Kraskel-Wallis criterion was used for the variance analysis, and for the correlation analysis the nonparametric correlation coefficient of Spearman. The variance analysis of the average resistance of the bacterial community to antibiotics, grouped by the factor of belonging to a certain sampling month, revealed that in different months of the year the average stability significantly differs from each other (P_value = 0.003 <α).
Dispersion analysis of the average bacterial resistance to antibiotics revealed practically no interannual differences in the close years (P_value = 0.34 <α). When calculating the pairwise correlation coefficients, it was possible to divide antibiotics into three groups. The first - the stability of pairs of antibiotics is formed independently of each other (values of the correlation coefficient r ≈ 0). The second group consists of pairs with reliable positive values of correlation coefficients (r> 0), i.e. the increase in resistance to a single antibiotic was accompanied by an increase in resistance to another antibiotic, the formation of cross-resistance. The third group are pairs of antibiotics with reliable negative values of correlation coefficients (r <0). In bacterial communities for such pairs of antibiotics, an increase in resistance to a single antibiotic was accompanied by a decrease in resistance to another.
Analysis of the actual material by season showed that strains of microorganisms resistant to antibiotics can be divided into 2 groups. The first group: antibiotics with relatively small values of the coefficients of variation in the resistance of bacterial communities to them (coefficient of variation <1). These drugs include: ampicillin, chloramphenicol, neogrammone, trimethoprim. The resistance to antibiotics of this group changes to a lesser extent in the transition from season to season. The second group: antibiotics with relatively high values of the coefficients of variation of the resistance of bacterial communities to them (1 <coefficient of variation <1.75). These are tetracycline, streptomycin, kanamycin, gentamicin, rifampicin, cefazolin, cefatoxime, pefloxacin. The resistance of the bacterial community to the antibiotics of this group is changing to a greater extent, during the five-month period from June to November. It was found that the resistance of bacterial strains to antibiotics varies significantly in different months of the year (P_value = 0.003 <α).
Long-term studies have shown that in conditions of active anthropogenic pollution of the littoral zone of the Baikal ecosystem, bacterial strains resistant to many antibiotics are observed. Analysis of samples of bacterial resistance to antibiotics studied averaged by the factor of belonging to a certain sampling month and separately by the factor of belonging to a certain year of sampling using the Shapiro-Wilk test showed that the samples under study are not distributed according to the normal law (P_value <α). In connection with this, the nonparametric Kraskel-Wallis criterion was used for the variance analysis, and for the correlation analysis the nonparametric correlation coefficient of Spearman. The variance analysis of the average resistance of the bacterial community to antibiotics, grouped by the factor of belonging to a certain sampling month, revealed that in different months of the year the average stability significantly differs from each other (P_value = 0.003 <α). For the estimated time periods from July to November, the minimum value of the average bacterial resistance to antibiotics is observed in July, then the resistance value begins to increase and reaches a maximum in September (Figure 1).
Thus, the littoral zone of the lake ecosystem. Baikal in the vicinity of Listvyanka settlement, located in the source of the Angara River, is experiencing a very strong anthropogenic load. The study of the arrival of antibiotic-resistant microorganisms in this region and affecting the formation of the quality of the southern basin of Lake Baikal and the source of the river. The hangars are undoubtedly relevant and socially significant. Ways of solving the problem:- prohibit the construction of hotels, campsites, restaurants, shops, kiosks, in the coastal zone of the lake,- strengthen control over the construction and operation of cesspools, toilets,- to put on the navigational commission the control of the receipt of sewage to the treatment plant,-to strengthen the control over the operation of treatment facilities, both for the quality of the treatment, and for the volume of incoming effluent for purification,- install dry closets on the ice of the lake during winter games, holidays and other mass events. Especially, this applies to the coastal zone of Lake Baikal, the valley of the river. Angara, as well as rivers, streams flowing into the Baikal and Angara. It is necessary to exclude or significantly reduce the way of domestic sewage entering the ecosystem of Lake Baikal.
